More than 80R2R3MYB regulatory genes in the genome of Arabidopsis

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Regulatory T cells in cancer

Review articleRegulatory T cells in cancer Marc Beyer and Joachim L.SchultzeIncreasing evidence supports the existence of elevated numbers of regulatory T cells (T reg cells)in solid tumors and hematologic malignancies.Whereas the biology of CD4؉CD25؉FOXP3؉T reg cells in murine models seems to be rather straightforward, studies in human diseases are more difﬁcult to interpret due to expression of CD25on activated effector T cells as well as T reg cells.More importantly,early studies in humantumors were mainly focused on CD4؉CD25؉T reg cells lacking interrogation of more spe-ciﬁc markers such as FOXP3expression.Although the increase of T reg cells seemsto be a characteristic feature in mosttumors,little is known about the molecu-lar and cellular mechanisms respon-sible for the increase and maintenanceof elevated levels of T reg cells in cancer.We will discuss earlier data in the con-text of recentﬁndings in T reg-cell biol-ogy with a particular emphasis onCD4؉CD25high FOXP3؉T reg cells in hu-man malignancies.(Blood.2006;108:804-811)©2006by The American Society of HematologyIntroductionIn1971,Gershon and Kondo identiﬁed so-called“suppressor”cells when they transferred antigen-speciﬁc tolerance to naive animals by transferring antigen-experienced T cells.1Due to conﬂicting results,the concept of T-cell suppression vanished into relative obscurity in the late1980s.However,reports describing murine T cells responsible for suppression of antitumor immune responses2 and the identiﬁcation of human CD4ϩT-cell clones suppressing autologous cytotoxic antitumor immune responses3clearly sug-gested that in vivo mechanisms of tumor-driven cellular immune suppression must exist.Sakaguchi et al were theﬁrst to stir up again the interest in now termed“regulatory”T cells(T reg cells)by identifying a population of CD4ϩT cells highly expressing CD25and preventing autoimmu-nity in a murine model.4Numerous reports in the following years enlightened major aspects of T reg-cell biology,characterizing different T-cell subpopulations with regulatory properties including naturally occurring CD4ϩCD25high T reg cells,induced T reg cells,eg Tr1and TH3cells,as well as CD4ϩCD25high T reg cells developing in the periphery by conversion of CD4ϩCD25ϪT cells.All these different T-cell populations with regulatory function coexist and contribute to immune suppression.5-8In the mouse,CD25is a good marker for T reg cells,as animals are held under pathogen-free conditions.However,humans are constantly exposed to foreign antigens,leading to a signiﬁcant fraction of recently activated CD25ϩeffector T cells.In search of more speciﬁc T reg-cell markers,the transcription factor FOXP3has been identiﬁed as uniquely expressed in T reg cells in the mouse9-11 and expression has been proposed as a lineage marker already in developing T reg cells.12,13However,caution about its speciﬁcity still is recommended because recent reports in humans demonstrated induction of FOXP3in activated conventional T cells without suppressive activity.14-16Characteristics of CD4ϩCD25high FOXP3ϩT reg cells are their anergic state,their ability to actively inhibit CD4ϩCD25ϪT cells,CD8ϩT cells,dendritic cells(DCs),natural killer(NK) cells,natural killer T(NKT)cells,and B cells in a cell-to-cell contact and dose-dependent manner.17-22Phenotypically CD4ϩCD25high FOXP3ϩT reg cells are characterized as antigen-experienced memory T cells,although lately some reports have described naive CD4ϩCD25high FOXP3ϩT cells in mice as well as humans.23,24Among the cell-surface markers associated with T reg-cell phenotype and function,cytotoxic T lymphocyte-associated protein4(CTLA-4)and glucocorticoid-induced TNFR-related protein(GITR)are the most prominent mol-ecules.25-28Additionally,IL-10and TGF-␤,although rarely expressed in vitro,might have functional importance for T reg cells in vivo,particularly in the context of disease.29,30Major topics of current research are the characterization of T reg-cell defects in autoimmune diseases and their role in infectious diseases and transplantation tolerance,particularly after alloge-neic bone marrow transplantation.5,6,31-34Although research in T reg-cell biology is intensifying,it is still unclear whether T reg cells in human tumors are primed mainly in the thymus or emerge in the periphery due to antigen-speciﬁc stimulation.The lack of more speciﬁc cell-surface markers is a major reason that many functionally relevant aspects of T reg cells are still un-known.In this review,we focus mainly on naturally occurring CD4ϩCD25high FOXP3ϩT reg cells with particular emphasis on novel aspects in malignant disease as well as potentials for improving antitumor immunity by targeting T reg cells. Increase of T reg cells in cancer-bearing miceT reg cells protect the host from autoimmune disease by suppress-ing self-reactive cells.As such,T reg cells may also block antitumor immune responses.Particularly in the context of cancer,T reg-cell frequencies and function are important becauseFrom Molecular Tumor Biology and Tumor Immunology,Clinic I for Internal Medicine,University of Cologne,Cologne,GermanySubmitted February14,2006;accepted March16,2006.Prepublished online as Blood First Edition Paper,April6,2006;DOI10.1182/blood-2006-02-002774. Supported by the Sofja Kovalevskaja Award of the Alexander von Hum-boldt-Foundation,the Wilhelm-Sander Stiftung,and the Nationales Genomforschungsnetz(J.L.S.).Reprints:Joachim L.Schultze,Molecular Tumor Biology and Tumor Immunology Clinic I for Internal Medicine,University of Cologne, Joseph-Stelzmann Str9/Haus16,50931Cologne,Germany;e-mail: joachim.schultze@uk-koeln.de.©2006by The American Society of Hematology804BLOOD,1AUGUST2006⅐VOLUME108,NUMBER3increased numbers might favor tumor development or growth and inﬂuence the course of the disease.Currently,a number of important questions are under intense investigation.Is the increase of T reg-cell frequencies an early event at the onset of disease or more likely a response of the immune system during tumor progression?Do organ-speciﬁc and more importantly,do tumor-speciﬁc T reg cells,play a role?How does therapy inﬂuence T reg-cell numbers,particularly in already established tumors?Is there a possibility of long-term depletion of T reg cells and is this connected to induction of autoimmunity?The development of T reg cells during tumor progression has been addressed in aﬁbrosarcoma model in C57BL/6N mice as well as in a colon adenocarcinoma model in BALB/c mice.Transfer of unfractionated tumor-draining lymph node(LN)cells isolated on day9after tumor challenge achieved complete rejection of established tumors,whereas even4-fold higher numbers of cells harvested on day12seldom prevented lethal tumor progression. This treatment failure was due to cotransfer of tumor-induced T reg cells,indicating that during this relatively short time span of tumor development the induction of a highly suppressive T reg-cell popula-tion occurred.35Relatively early induction of T reg cells during tumor development has signiﬁcant impact in human disease as the time point of T reg-cell induction in cancer patients would certainly precede the time of diagnosis in the majority of patients.The suppressive effect of naturally occurring T reg cells against tumor-speciﬁc CD8ϩT cells was established in a poorly immuno-genic B16melanoma model.T reg cells efﬁciently suppressed cytotoxic T lymphocyte(CTL)–mediated concomitant immunity against a rechallenge with the same tumor,demonstrating that precursor T reg cells in naive hosts gave rise to effective suppressors during tumor development.Theseﬁndings clearly suggest that T reg cells are major regulators of concomitant tumor immunity.36 Further evidence for the interference of T reg cells with CD8ϩT cell-mediated antitumor immune responses in vivo was established in a transgenic murine colon carcinoma model where T reg cells abrogated CD8ϩT cell–mediated tumor rejection by speciﬁcally suppressing cytotoxicity of CTLs.37Selective accumulation of T reg cells in the tumor environment was studied in a murineﬁbrosarcoma model where the majority of tumor-inﬁltrating lymphocytes(TILs)at late stage of tumor progression were T reg cells.Their depletion during the effector rather than priming phase successfully enhanced antitumor immu-nity.Blockade of IL-10and TGF-␤partially reversed the suppres-sion imposed by CD4ϩT cells.Furthermore,local depletion of CD4ϩT cells inside the tumor led to eradication of well-established tumors and development of long-term antitumor memory.This study suggested that suppression of antitumor immunity by T reg cells occurs predominantly at the tumor site and that local reversal of suppression,even late during tumor development,can be an effective treatment.38This has been conﬁrmed in a murine pancre-atic cancer model,suggesting that the tumor actively promotes the accrual of T reg cells through several mechanisms involving activa-tion of naturally occurring T reg cells as well as conversion of non-T reg cells into T reg cells.39Analysis of tumor-draining LNs demonstrated that both antitu-mor effector T cells and FOXP3ϩT reg cells are primed in the same LNs during tumor progression.These tumor antigen-speciﬁc T reg cells possessed the same functional properties as T reg cells that arise naturally in the thymus.40Whether there is a systemic increase of T reg cells in cancer-bearing mice is not yet clearly deﬁned because conﬂicting data have been reported.In a colon carcinoma model,an expansion of T reg cells was observed only in the spleen but not in peripheral blood(PB).41In contrast,in a murine transgenic model of prostate dysplasia,increased frequencies of T reg cells and enhanced production of inhibitory cytokines were observed in PB, resulting in impaired T-cell function,which also correlated with tumor progression.42Although these experiments focused on accumulation and function of T reg cells,the trafﬁcking behavior of T reg cells and their cellular interactions and localization to and within the tumor microenvironment and in tumor-draining LNs were not studied in these models.Immunohistochemistry revealed FOXP3ϩT reg cells in close proximity to CD11cϩDCs,FOXP3ϪCD4ϩT cells,and CD8ϩT cells in the T-cell regions of lymphoid tissues in normal and tumor-bearing mice.43Further insights into the signals in-volved in T reg-cell attraction to tumor sites came from studies in a lung cancer model.Tumor-derived prostaglandin E2(PGE2)re-sulted in an increase of T reg-cell activity and Foxp3expression (Figure1).Assessment of E-prostanoid(EP)receptor requirements revealed that absence of EP4receptor led to reduced induction of Foxp3,whereas absence of EP2ablated expression.In vivo,COX2 inhibition reduced T reg-cell frequency and activity,attenuated Foxp3expression,and decreased tumor burden.Transfer of T reg cells or administration of PGE2to mice receiving COX2inhibitors reversed these effects,indicating that COX2inhibition suppressed T reg-cell activity and might be useful to enhance antitumor re-sponses.44Interestingly,PGE2also enhanced the in vitro inhibitory function of human T reg cells and induced a regulatory phenotype in CD4ϩCD25ϪT cells.Furthermore,PGE2exposure induced FOXP3 in CD4ϩCD25ϪT cells and up-regulated its expression in T reg cells. Similarly,incubation with supernatants from COX2-overexpress-ing lung cancer cells secreting PGE2signiﬁcantly induced FOXP3, indicating that PGE2can indeed modulate FOXP3expression and T reg function.45T reg-cell depletion leads to restoration of antitumor immunityEven before the identiﬁcation of CD4ϩCD25ϩT reg cells,early data indicated that nonspeciﬁc depletion of CD4ϩT cells can lead tothe Figure1.Model of the accumulation of T reg cells in human tumors.One of the possible scenarios for how T reg-cell attraction to the tumor site and expansion of T reg cells occurs may be the release of CCL22as well as H-ferritin by tumor cells and tumor-inﬁltrating macrophages leading to the accumulation of CCR4ϩnaive T reg cells in the tumor microenvironment.Interaction with PGE2-induced tolerogenic DCs then gives rise to the differentiation and peripheral expansion of naive T reg cells into memory T reg cells.Together with tolerogenic DCs,these T reg cells than inhibit the generation of effector T cells,resulting in the induction of tolerance against the tumor.REVIEW OF REGULATORY T CELLS805BLOOD,1AUGUST2006⅐VOLUME108,NUMBER3induction of efﬁcient antitumor immunity (Figure 2).46More speciﬁcally targeting T reg cells by administration of CD25monoclo-nal antibody (mAb)abrogated immunologic unresponsiveness to tumors and induced spontaneous development of tumor-speciﬁc CD8ϩeffector T cells and NK cells.47Interestingly,depletion of T reg cells led to cross-reactive tumor immunity against tumors of diverse origins.48Timing of T reg -cell elimination also seems to be an important aspect.Administration of CD25mAb later than 2days after inoculation of myeloma cells caused no tumor regression,irrespective of T reg -cell depletion.49As already outlined,this might be due to the induction of antitumor tolerance at a relatively early time point of tumor development,resulting in inefﬁcient activation of effector cells.Furthermore,the number of T reg cells after CD25depletion is restored over time and the capacity to mount an antitumor response progressively diminishes.50Depletion of T reg cells together with other immunostimulatory approaches,for example,CTLA-4blockade,has also been bination of T reg -cell depletion and CTLA-4blockade was synergistic and resulted in maximum tumor rejection.The observed synergism indicates that both pathways represent 2alternatives for suppression of autoreactive T cells so that simultaneous interven-tion might be a promising concept for the induction of therapeutic antitumor immunity.51Because immune responses to malignant tumors often are weak and ineffective,solely depleting T reg cells might not always result in tumor regression.Approaches combin-ing T reg -cell depletion with other immunologic interventions,for example,transfer of activated T cells or DC-based vaccinations,therefore might be more beneﬁcial.52-54Reduction of T reg cells is associated with the immunostimulatory effect of cyclophosphamideIt has long been recognized that cyclophosphamide exerts an immunostimulatory effect.55Early data indicated that cyclophospha-mide preferentially destroys CD4ϩsuppressor T cells causing immunologically mediated regression of immunogenic lymphomas in mice.56In a rat colon cancer model,administration of cyclophos-phamide depleted T reg cells and delayed the outgrowth of tumors.57Combining cyclophosphamide and immunotherapy even cured themice,whereas both strategies applied alone had no curative effect.57,58Low-dose cyclophosphamide not only decreases num-bers of T reg cells but also leads to decreased function,enhanced apoptosis,and decreased homeostatic proliferation.59This suggests that cyclophosphamide might be successfully integrated into chemoimmunotherapy,as recently shown by Dudley et al.60The combination of adoptive transfer of ex vivo activated tumor-speciﬁc T cells to patients with lymphopenic melanoma after chemotherapy with cyclophosphamide and ﬂudarabine induced tumor regression in up to 50%of patients treated.Collateral damage needs to be accounted for after T reg -cell depletionBecause T reg cells are an important cellular mechanism suppressing autoantigen-speciﬁc conventional T cells from attacking self tis-sues,nonspeciﬁc depletion of these cells might be a too crude approach to be used without leading to signiﬁcant collateral damage.The ﬁne balance between beneﬁt and harm of manipulat-ing T reg cells was elegantly demonstrated in the following experi-ment:transfer of a mixture of CD4ϩCD25Ϫand CD4ϩCD25ϩT cells prevented effective adoptive immunotherapy of established melanoma.In contrast,adoptive transfer of CD4ϩCD25ϪT cells together with tumor-as well as self-reactive CD8ϩT cells into CD4ϩT cell–deﬁcient hosts followed by vaccination induced both regression of established melanoma but also severe and undesired autoimmunity.61Similarly,depletion of T reg cells with CD25mAb in a mammary gland tumor model resulted in tumor regression but signiﬁcantly increased susceptibility to autoimmune thyroiditis.This in vivo priming to both tumor-and self-antigens attests to the presence of otherwise undetectable immune effectors that are under negative regulation and demonstrates that modulation of T reg cells is a powerful strategy in cancer therapy,but may also signiﬁcantly increase autoimmune complications.62,63Broadly expressed self-antigens arerecognized by tumor-associated T reg cellsFor most current tumor models the antigens recognized by T reg cells are not known.In a series of elegant experiments,Nishikawa et al demonstrated that immunization with tumor-associated self-antigens and tumor-speciﬁc CTL epitopes heightened CD8ϩT-cell responses and increased resistance to tumor challenge in a CD4ϩT cell–dependent manner.In contrast,immunization with self-antigens alone increased the susceptibility to tumor challenge,64leading to the development of highly active T reg cells with enhanced expression of Foxp3.Induction of T reg cells was also associated with acceleration of tumor development,which was abolished by depletion of CD4ϩT cells or CD25ϩT cells.Acceleration of tumorigenesis was not only observed in self-antigen vaccinated mice but could also be adoptively transferred with T reg cells derived from immunized mice.65Human T reg cells in cancer:current knowledge and open questionsEven in the early 1990s,T cells with regulatory function were reported in patients with cancer;however,these reports werenotFigure 2.Inﬂuence of treatment of T reg -cell frequency.Different approaches affecting T reg -cell frequencies and function have been proposed in recent years and tested either in murine models or ﬁrst clinical trials.Targeting of T reg cells in human tumors,however,does not always result in reduced T reg -cell numbers but may also,under given circumstances,be linked to the induction of autoimmunity or the expansion or development of T reg cells.806BEYER and SCHULTZEBLOOD,1AUGUST 2006⅐VOLUME 108,NUMBER 3followed up until the identiﬁcation of CD4ϩCD25ϩT reg cells in the mid-1990s.4Since then,an increase of T reg cells in cancer patients has been reported by numerous investigators.In contrast to the murine system,deﬁnition of human T reg cells has been more difﬁcult,and assessment of the most speciﬁc marker—namely, FOXP3—has not been performed in many of the early studies. Although human CD4ϩCD25high T cells are most enriched for FOXP3ϩT cells,there are still signiﬁcant numbers of FOXP3ϩcells within the CD4ϩCD25low T-cell population.In the absence of more speciﬁc cell-surface markers,it is not yet possible to study human FOXP3ϩT reg cells irrespective of their CD25expression. These limitations also explain why T reg cells in humans currently need to be characterized by a combination of FOXP3and CD25 expression as well as analysis of inhibitory function of T-cell populations enriched for FOXP3ϩcells,mainly by sorting CD25high T cells.Comparability of previous reports is further challenged by use of different antibodies to detect CD25or different gating strategies when assessing CD25ϩ/CD25high cells.Similarly,function of T reg cells has been assessed with numerous in vitro approaches,making it rather difﬁcult to compare results of different studies.To reconcile our recentﬁndings about T reg cells,it is most important to identify speciﬁc cell-surface markers for T reg cells that allow us to isolate these cells and functionally test them in the context of malignant disease.Increased frequencies of T reg cells in solid cancersWoo et al66were theﬁrst to report increased percentages of CD4ϩCD25ϩT reg cells in TILs in non–small-cell lung cancer and ovarian cancer.These T reg cells were shown to secret TGF-␤, providingﬁrst evidence that T reg cells contribute to immune dysfunction in patients with cancer.66Further characterization of these cells showed constitutive high-level expression of CTLA-4. More importantly,T reg cells mediated potent inhibition of T-cell proliferation.67Supporting this initial report,a larger study con-cluded that prevalence of CD4ϩCD25ϩT reg cells is increased not only in the tumor microenvironment of patients with invasive breast or pancreas cancers but also in PB,suggesting that the increase of T reg cells is a generalized phenomenon.68In malignant melanoma,an increase of functional CD4ϩCD25ϩT reg cells was observed,69which was further linked to increases in the serum level of H-ferritin.70Release of H-ferritin by melanoma cells led to activation of IL-10–producing functional T reg cells as a potential mechanism of T reg-cell induction in cancer patients.71A more recent study demonstrated FOXP3mRNA expression in the increased subset of CD4ϩCD25high T reg cells in patients with melanoma,conﬁrming the previous reports.72In patients with gastrointestinal malignancies,the relative increase of T reg cells might actually be explained by a signiﬁcant reduction of CD4ϩCD25ϪT cells.Interestingly,in patients with gastric carcinoma,poor prognosis and decreased survival rates were closely correlated with higher T reg-cell frequencies.73,74After curative resections,previously elevated T reg cells numbers were signiﬁcantly reduced.In contrast,prevalence of T reg cells increased again in patients having a relapse after tumor resection.75These ﬁndings underline the close correlation of tumor growth and T reg-cell frequencies.Curiel et al demonstrated that CD4ϩCD25ϩFOXP3ϩT reg cells suppress tumor-speciﬁc T-cell immunity in ovarian cancer,contrib-ute to tumor growth,and accumulate during progression.76Further-more,increased frequencies of T reg cells were associated with a high death hazard ratio and reduced survival.T reg cells preferen-tially moved to and accumulated in tumors and ascites,but rarely entered draining LNs in later cancer stages.Tumor cells and surrounding macrophages produced the chemokine CCL22,which mediated trafﬁcking of T reg cells to the tumor via CCR4(Figure1). This speciﬁc recruitment of T reg cells might represent a mechanism by which tumors may foster immune privilege.For patients with squamous-cell carcinoma of the head and neck,a signiﬁcantly elevated frequency of FOXP3ϩGITRϩT reg cells was shown.77These T reg cells were signiﬁcantly more sensitive to apoptosis than non-T reg cells,which might hint at a rapid turnover in the peripheral circulation.77How the higher sensitivity to apoptosis inﬂuences T reg-cell frequencies,however, has not been addressed yet.Increased numbers of T reg cells have also been reported in PB and TILs of patients with hepatocellular carcinoma.78Although the increase of T reg cells seems to be a common theme in solid tumors,there are clear but yet unexplained differences between individual tumor entities.In a comparative study,differences in T reg-cell frequencies were shown for malignant pleural effusions from patients with mesothelioma compared with carcinomatous pleural effusions from patients with non–small-cell lung cancer or breast cancer.79Overall,previous work has clearly established that T reg cells are increased in most human solid tumors.Furthermore,there seems to be a stage-dependent increase of T reg cells with frequencies of T reg cells probably correlated to overall survival.However,little is known about the mechanisms leading to this increase.Aﬁrst study by Wolf et al might help us to understand the underlying molecular mechanisms.80This study showed that increased frequencies of T reg cells in PB of cancer patients are due to active proliferation rather than redistribution from other compartments(ie,secondary lym-phoid organs or bone marrow).Thisﬁnding,in combination with the proposed attraction of T reg cells to the tumor via CCL22/CCR4 and induction of T reg cells by PGE2or H-ferritin,might be one possible mechanism responsible for expansion of T reg cells in cancer patients(Figure1).T reg cells in hematologic malignanciesWhereas the question of T reg cells in solid tumors sparked interest relatively early,studies addressing T reg cells in hematologic malig-nancies have been conducted only recently.Theﬁrst study by Marshall et al demonstrated large populations of both IL-10–secreting Tr1and CD4ϩCD25ϩT reg cells in Hodgkin lymphoma(HL)inﬁltrating lymphocytes and peripheral-blood mononuclear cells(PBMCs).Suppressive function was mediated by IL-10secretion,cell-to-cell contact,and CTLA-4expression.81 The difﬁculty of applying FOXP3as a T reg cell–speciﬁc marker in human diseases is exempliﬁed by a second study in HL.82The frequency of FOXP3ϩcells was determined in lymphoma-afﬂicted LNs.Low frequencies of FOXP3ϩcells and high frequencies of CTLs in the reactive background of LNs were correlated with poor overall survival.However,costaining for FOXP3and CD4or CD25was not performed,limiting the signiﬁcance of thisﬁnding because FOXP3expression in humans might not be conﬁned to T reg cells only.14Alternatively,T reg cells might actually play a beneﬁcial role in HL,which is characterized by a chronic inﬂammatory response,similar to Helicobacter-associated lymphoma.In patients with B-cell chronic lymphocytic leukemia(CLL) we recently established a stage-dependent increase of CD4ϩCD25high FOXP3ϩCTLA4ϩGITRϩT reg cells with full sup-pressive capacity.However,when patients with CLL were treated withﬂudarabine,frequencies of T reg cells decreased andREVIEW OF REGULATORY T CELLS807BLOOD,1AUGUST2006⅐VOLUME108,NUMBER3T reg cells showed impaired function.Ongoing studies are addressing the question of howﬂudarabine mediated this effect.83The increase of CTLA-4ϩT reg cells in untreated CLL patients,which correlated with advanced disease stage and unfavorable cytogenetics,was recently conﬁrmed by others.84 Similarly,in patients with B-cell non-Hodgkin lymphomas (B-NHLs)increased frequencies of FOXP3ϩCTLA4ϩT reg cells have been observed.PD1expression was partly responsible for the suppressive activity of these LN-inﬁltrating T reg cells. Furthermore,as reported for ovarian cancer,the tumor cells released CCL22and thereby attracted CCR4ϩT reg cells into the area of the lymphoma(Figure1).85For patients with acute myeloid leukemia(AML)higher frequencies of CD4ϩCD25high T reg have been observed.Similar to observations in solid tumors, T reg cells of patients with AML were less resistant to apoptosis but showed higher proliferation compared with healthy individuals.86Comparable to other hematologic malignancies,we were also able to demonstrate increased frequencies of CD4ϩCD25high FOXP3ϩT reg cells in patients with monoclonal gammopathy of undetermined signiﬁcance(MGUS)or multiple myeloma(MM).24Independent of prior therapy or stage of disease T reg cells exhibited a strong inhibitory capacity.Moreover,the increase of T reg cells was also dependent on stage and resulted from peripheral expansion.We also established for theﬁrst time that naive CD4ϩCD25high FOXP3ϩT reg cells coexpressing CD45RA and CCR7are expanded in MM patients,further supporting the concept of peripheral expansion of this T-cell compartment.The importance of identifying more speciﬁc markers as well as more standardized functional assays is supported by a recent report on FOXP3ϩcells in PB from patients with MM.87Due to an alternative experimental approach only assessing FOXP3in context of CD4ϩT cells but not CD25ϩcells, these data are difﬁcult to compare with other studies on naturally occurring CD4ϩCD25high T reg cells.Although this report came to the conclusion that T reg cells are dysfunctional in MM patients,the assays chosen to assess T reg-cell function allowed for alternative explanations of the observed results including already described defects in conventional autologous T cells in MM patients.88 Taken together,these data established the concept of increased T reg cells in solid tumors as well as hematologic malignancies. However,some of the early studies need to be validated by using more speciﬁc markers such as FOXP3as well as more sophisti-cated and standardized functional assays.Speciﬁcity of T reg cells in human tumorsSo far,little is known about the antigen-speciﬁcity of human T reg cells.Wang et al reported the identiﬁcation of LAGE1-speciﬁc CD4ϩCD25ϩGITRϩfully functional T reg-cell clones in cancer patients.89Ligand-speciﬁc activation and cell-to-cell contact were required for T reg cells to exert suppressive activity,suggesting that the presence of tumor-speciﬁc T reg cells at tumor sites may have profound effects on the inhibition of T-cell responses against cancer.In a second study,a CD4ϩCD25ϩT reg-cell line was established from a patient with colorectal carcinoma.This T-cell line was tumor-cell dependent in its growth but did not lyse autologous tumor cells and suppressed proliferative responses of allogeneic lymphocytes and autologous CTLs as well as the induction of CTLs from autologous PBMCs.These effects were mediated by TGF-␤and did not require cell-to-cell contact,which would be in line with induced regulatory capacity.90Clearly,further work is needed to understand how the enrichment of T reg cells in cancer patients occurs and if accumulation or preferential induction of clonal,oligoclonal,or polyclonal tumor-speciﬁc T reg cells plays a role during tumor progression.The question of whether induction of T reg cells might be inversely correlated with the induction of tumor-antigen speciﬁc immunity during cancer development in vivo was addressed by Nishikawa et al.91Using NY-ESO-1as a model,the authors demonstrated that the in vitro generation of NY-ESO1-speﬁcic TH1cells in NY-ESO-1–seropositive cancer patients was indepen-dent of T reg-cell depletion,indicating that tumor antigen-associated T reg cells were not enriched in patients naturally mounting an immune response against this antigen.In contrast,T reg-cell deple-tion in NY-ESO1–seronegative patients was always required for induction of NY-ESO-1–speciﬁc TH1cells,which suggests the existence of T reg cells speciﬁcally suppressing the expansion of tumor-antigen–speciﬁc T cells.Moreover,NY-ESO-1–speciﬁc TH1 cells were derived from naive precursors in seronegative patients, whereas preexisting memory populations were detectable exclu-sively in NY-ESO-1–seropositive patients.The memory popula-tions were also less sensitive than naive populations toward T reg cell–mediated suppression.These results strongly support the hypothesis that tumor-speciﬁc T reg cells exist in patients with tumors and actively suppress antigen-speciﬁc antitumor immunity. Inﬂuence of treatment on T reg-cell frequency and functionAs already outlined,a correlation of increased T reg cells with greater disease burden and poorer overall survival has been reported.In CLL,we have observed reduced frequencies of functionally impaired T reg cells afterﬂudarabine treatment;how-ever,not every chemotherapeutic agent seems to induce this effect, because in CLL or MM no other treatment including autologous stem cell transplantation induced similar effects.In line with this observation,frequency and suppressive function of T reg cells in tumor-draining LNs derived from patients with cervical cancer were not inﬂuenced by chemotherapy or combined chemoradiation.92Recent work has demonstrated that IL-2signaling is required for thymic development,peripheral expansion,and suppressive activity of T reg cells.93During immune reconstitution after chemo-therapy,IL-2therapy led to a homeostatic peripheral expansion of T reg cells and to a markedly increased T reg-cell compartment.IL-2 therapy induced expansion of existent T reg cells in healthy hosts and this expansion was further augmented by lymphopenia.T reg cells generated by IL-2therapy expressed FOXP3at levels observed in healthy individuals and these T reg cells also were of similar potency, suggesting that IL-2and lymphopenia are modulators of T reg-cell homeostasis.94Similarly,in patients with melanoma or renal-cell carcinoma(RCC),the frequency of fully functional T reg cells was signiﬁcantly increased after IL-2treatment,which was also accom-panied by an increase of FOXP3,demonstrating that administration of high-dose IL-2increases the frequency of circulating FOXP3ϩT reg cells.95This might also explain why therapy with IL-2in patients with RCC has not yet fully lived up to expectations because signiﬁcant induction of T reg cells might counteract poten-tial antitumor effects of IL-2.Surprisingly,vaccination of melanoma patients with DCs either loaded with synthetic peptides or tumor lysates was also shown to induce increased frequencies of T reg cells,concomitant with the expansion of tumor-speciﬁc CTLs.Whether this enhances antitumor tolerance and negatively inﬂuences the induction of clinically efﬁcient antitumor immune responses needs further attention,because the mechanisms of this phenom-enon are not yet understood.96808BEYER and SCHULTZE BLOOD,1AUGUST2006⅐VOLUME108,NUMBER3。

DNA点阵杂交鉴定逆境应答的转录因子新基因

将它们列为候选基因，为更深入的基因功能分析奠定了基础。

本研究所发现的这些新的逆境相关转录因子和相关信息为今后进一步研究植物非生物逆境下的基因表达调控提供了许多有价值的线索，对促进水稻的抗逆育种也具有一定的意义。

关键词：水稻；转录因子；cDNA点阵技术；非生物逆境；差异表达AbstractDrought and salt loading are two major dehydration stresses that cause adverse effects on plant growth and the productivity of crops. In order to reduce the threat to crops posed by environmental stresses, an essential step towards understanding plant responses to environmental stresses is to identify genes involved in the stress responses.Numerous studies have shown that array of transcription factors have roles in regulating plant responses to environmental stresses. A small portion of them, however, have been identified or characterized. In this study, a matured technology—DNA array was employed to identify novel transcription factors involved in plant responses to abiotic stresses in a high throughput fashion.Based on the annotation, more than 2300 putative transcription factors were predicted in rice genome and classified by gene family. More than half of them were supported by expressed sequences. We made a comprasion between rice and Arabidopsis in the distribution of gene families of transcription factors. It was suggested that the percentage of transcription factor genes in different families in rice was generally similar to that in Arabidopsis.With an attempt to identify novel stress responsive transcription factors, a DNA array containing 753 putative rice transcription factors was generated to analyze the transcript profiles of these genes under drought and salinity stresses and ABA treatment at seedling stage of rice. About 80% of these transcription factors showed detectable level of transcript in seedling leaves, and no transcription factor gene family showed predominance. A total of 18 up-regulated transcription factors and 29 down-regulated transcription factors were detected with the folds of changes ranging from 2.0 to 20.5 in at least one of the stress treatments. A total of 44 transcription factors were involved. Most of these stress-responsible genes have not been reported.To validate the stress-responsive TFs identified from array hybridization and further analyze their expression pattern under stresses, five TF genes, representing low (2-3 folds), mediate (4-6 folds) and high level (>8 folds) of differential expression, were chosen for Northern analysis. All the selected genes showed stress-induced or suppressed expression and were in agreement with the array results. More detailed information abouttheir expression pattern was provided by Northern analysis, suggesting their possible roles in plant responses to abiotic stresses.In addition, the full length cDNAs were isolated for part of the stress responsive transcription factors as candidate genes for further analysis of gene function.These novel stress-responsible transcription factors provide new opportunities both to the study of gene expression and regulation in plant under stress conditions and to the genetic improvement of stress tolerance of economically important crops such as rice .Key words: rice (Oryza sativa L.); transcription factor; cDNA array; abiotic stress; differential expression缩略词表缩写形式英文全称中文意义 TIGRThe Institute of Genomic Research 美国的基因组研究专业网站 DDBJDNA Databank of Japan 日本核酸序列数据库 KOME Knowledge-based Oryza MolecularBiological Encyclopedia 日本水稻生物信息学平台TF Transcription factor 转录因子ZF Zinc finger 锌指蛋白RWC Relative water content 相对含水量ABA Abscisic acid 脱落酸JA Jasmonic acid 茉莉酸MH63 Minghui63 明恢63ORF Open reading frame 开放读码框BLAST Basic Local Alignment Search Tool 局部比对基本检索工具cDNA Complementary DNA 互补DNAhr hour 小时min minute 分钟sec second 秒钟bp Base Pair 碱基对EST Expressed Sequence Tag 表达序列标签PCR Polymerase Chain Reaction 聚合酶链式反应RT Reverse Transcription 反转录RT-PCR Reverse Transcript RCR 反转录聚合酶链式反应ddH 2O DoubleDistilled Water 双蒸水前言海洋孕育了生命。

植物Myb转录因子的研究进展

ｓ铂Ｏ量ｅ嘲ｎｏｍｌ警ｃｓ与ａｎ擘ｄＡ乏ｐ嵩ｐｌ跫ｅｄＢｍ—ｌｏ—ｇｙｗｗｗ．ｇｅｎｏａｐｐｉｂｉ０１．ｏｒｇＩＸ）Ｉ：１０．３９６９／ｇａｂ．０２８．０００３６５有超过８０个Ｍ），ｂ转录因子（ｇａｂｉｎｏｗｉｃｚｅｔａ１．．１９９９），而棉花中发现大约有２００个Ｍｙｂ转录因子（Ｃｅｄｒｏｎｉｅｔａ１．，２００３）。

功能研究表明，Ｍ如参与了植物次生代谢（ＵｉｍａｒｉａｎｄＳｔｒｏｍｍｅｒ，１９９７；杜海等，２００８），激素和环境因子应答（Ｃｈｅｎｅｔａｌ。

２００３；Ｈｏｅｒｅｎｅｔａｌ．，１９９８；Ｌｅａｅｔａｌ．，２００７），并对细胞分化、细胞周期（Ｐａｙｎｅｅｔａ１．，２０００；Ｓｕｏｅｔａ１．。

２００３）以及叶片等器官形态建成（ＬｅｅａｎｄＳｃｈｉｅｆｅｌｂｅｉｎ，２００２；Ｌｅｇａｙｅｔａ１．，２００７；Ｙａｎｇｅｔａ１．，２００７）具有重要的调节作用。

最近的研究发现，Ｍｙｂ转录因子参与了植物积累花色素过程，对果皮、果肉、叶片和花器官等各种颜色的形成具有重要作用（Ａｚｕｍａｅｔａ１．．２００８；Ｂａｎｅｔａ１．．２００７；Ｅｓｐ．１ｅｙｅｔａ１．，２００７；Ｔａｋｏｓｅｔａ１．，２００６）。

本文就Ｍｙｂ转录因子的特点和最新功能研究进展进行了综述，以期为该因子的研究和利用提供参考。

１Ｍｙｂ类转录因子的发现Ｍｙｂ基因序列早在１９４１年便从引起禽急性成髓细胞白血病病毒ＡＭＶ和Ｅ２６中成功鉴定出来（Ｇｒａｆ，１９９２）。

Ｋｌｅｍｐｎａｕｅｒ等（１９８２）又从禽成髓细胞瘤病毒（ａｖｉａｎｍｙｅｌｏｂｌａｓｔｏｓｉｓｖｈａｔｓ）中鉴定出一个ｃｏｒｎ．ｍａｎｔｒａｎｓｆｏｒｍｉｎｇ基因，称为影一ｍｙｂ癌基因。

不久后发现，在正常动物细胞中也存在相应的原癌基因ｃ—ｍｙｂ，而且具有调控细胞增值和分化作用的ｃ．ｍｙｂ等位基因Ａ一，扎伯和曰—ｍ伯已从人类肿瘤细胞中被成功鉴定出来（Ｇｏｌａｙｅｔａｌ．，１９９６）。

无核荔枝MYB转录因子基因克隆及进化分析

无核荔枝MYB转录因子基因克隆及进化分析摘要为获得在无核荔枝中胚珠退化过程中差异表达全长MYB转录因子基因，并为其功能研究打下基础，根据从已构建的无核荔枝胚珠败育SSH消减文库中得到的差异表达的MYB转录因子EST序列，提取高质量的总RNA，运用SMART RACE技术，获得一个1177bp的MYB基因核苷酸序列。

生物信息学分析表明，该序列含有879 bp的开放阅读框，推测的蛋白质为292个氨基酸，具有2个SANT结构功能域，在系统发育树上与大豆MYB基因亲缘关系最近。

该基因为MYB基因家族中的新成员。

Abstract To obtain the full length of MYB gene and lay the foundation its function research that was differentially expressed in ovule degeneration of seedless litchi. The EST encoding MYB was obtained from young abortive ovule of seedless litchi via SSH（suppression subtractive hybridization），high quality total RNA was extracted and obtained a 1 177 bp full length cDNA sequence by SMAT-RACE method，bioinformatics analysis showed that it included an 879 bp open reading frame，encoded a deduced protein with 292 amino acids，and contained 2 SANT domains，phylogenetic tree showed it got the closest relatives with Glycine max. The results showed that it’s a new member of the MYB family.Key words MYB；seedless litchi；gene cloning；phylogenic treeMYB（myeloblastosis）基因家族是一类广泛存在于植物、动物和真菌中的转录因子家族[1]，在植物生长发育过程中起着非常重要的作用[2-3]。

苹果愈伤过表达MdMYB1对花色素苷合成的影响

苹果愈伤过表达MdMYB1对花色素苷合成的影响CHEN Li;YAO Yuncong【摘要】[目的]苹果生产是北京市都市型农业的支柱产业之一.果实色泽是苹果果实重要的外观品质,主要由花色素苷的含量决定.MdMYB1转录因子在调控花色素苷及类黄酮的生物合成中发挥着重要的作用.[方法]将MdMYB1转录因子在‘王林’(Malus domestica cv.‘Orin')愈伤组织进行过表达,进行持续光照和低温处理,观察愈伤组织类黄酮类物质变化,同时检测花色素苷生物合成基因表达水平的变化,确定其对花色素苷生物合成的调控功能.[结果]愈伤组织中过表达MdMYB1后,在持续光照和低温处理下花色素苷含量显著提高,并能够促进花色素苷生物合成基因的表达.[结论]该研究为深入了解MdMYB1调控花色素苷作用机理提供技术支撑.【期刊名称】《北京农学院学报》【年(卷),期】2019(034)003【总页数】5页(P37-41)【关键词】苹果;花色素苷;愈伤;转基因【作者】CHEN Li;YAO Yuncong【作者单位】;【正文语种】中文【中图分类】S661.4苹果为蔷薇科(Rosaceae)苹果属(Malus)落叶乔木，是世界四大水果之首[1]。

苹果产业在农业结构调整，农民增收和生态改良方面具有重要意义。

经过多年发展，目前中国苹果栽培面积、总产量和出口量均居世界首位[2-4]。

在苹果产量大幅提升的同时，苹果品质较低已经成为阻碍中国苹果产业发展的重要因素[5-7]。

苹果品质的提升对于苹果产业的发展显得尤为重要。

果实色泽是苹果果实重要的外观品质，能够在一定程度上影响其市场价格。

而红色果皮主要由花色素苷的含量决定[8]。

花色素苷生物合成的分子机理研究表明影响花色素苷生物合成的基因有两类：一类是不同植物共同具有的花色素苷生物合成基因，它们直接编码花色素苷生物合成酶类[9]；另一类是调节基因，调节花色素苷生物合成基因表达的强度和过程及色素在空间和时间上的积累[10]。

VIGS技术在辣椒基因功能研究中的应用进展

收稿日期 :2020 - 12 - 30 基金项目:国家自然科学基金项目(31760592) ;红河学院科研基金博硕资助项目( XJ17B07,XJ17B08) 作者简介:李杰( 1987-) ,男,甘肃天水人,讲师,博士,主要从事设施作物栽培与环境调控研究。 E-mail:gsau23@ 126. com 通信作者:杨萍( 1985-) ,女,甘肃定西人,讲师,博士,主要从事植物营养研究。 E-mail:gsau123@ 163. com
Application Progress of VIGS Technology in the Research of Pepper Gene Function
LI Jie,LUO Jianghong,WAN Zilong,YANG Ping
( College of Life Science and Technology,Honghe University,Mengzi 661100,China)
生长发育、代谢调控、生物胁迫及非生物胁迫应答等诸多方面,重点介绍在辣椒激素调节及生物和非生物胁迫应答等方面利用 VIGS 技术的研究进展 (表 2)。
第6期
李杰等:VIGS 技术在辣椒基因功能研究中的应用进展
11
靶基因 Target gene Pun1 Comt、Kas、pAmt MYB Psy、Lcyb、Ccs、Crtz CYP1 CYP450A LIP1 LRR51 GLP1 WRKY1 PMEI1 WRKY40 CaPHL8 WRKYd Btf3 GLP CaLOX2 SRC2 - 1 MLO2 AIR1 CaDHN5 DIN1 RAV1、OXR1 CaMADS PO2 CaATBZ1

翻译MtPARMYB转录因子作为一个开关参与苜蓿原花色素合成

MtPAR MYB转录因子作为一个开关参与苜蓿原花色素的合成摘要：MtPAR（蒺藜苜蓿原花色素调节子）是myb蛋白家族的转录因子，在模式豆科植物苜蓿的原花色素（PA）的合成中起关键调控作用。

MtPAR在种皮中表达，pa积累的部位。

功能缺陷的Par突变体种皮中的pa含量比野生型低很多，然而花色素和其他专门的代谢物水平是正常的。

相反，当MtPAR在转基因苜蓿的毛状根中异常表达时，pa却大量积累。

对par突变体和MtPAR表达的毛状根进行转录分析，结合酵母单杂交分析，发现MtPAR 很可能通过激活WD40-1，从而正向调控编码类黄酮到pa合成途径中的酶的基因。

MtPAR 在牲畜饲料的紫花苜蓿中表达，导致根中含有可观察到的pa含量，突出了这一潜在增加牧草植物中pa含量的基因生物技术的战略，降低以此为食的反刍动物的气胀。

原花色素（pa，也成缩合单宁）是黄烷-3 -醇单元的低聚物，pa复合物在种皮，叶，果实，花，许多植物的树皮中都有分布。

Pa，儿茶素，表儿茶素是对人体健康有益的抗氧化剂，包括保护心脏，抗癌，消炎。

牧草植物中的Pa与蛋白结合，减慢在反刍动物的瘤胃的发酵，降低了微生物产生的甲烷含量，从而使动物免受可能致死的气胀病的危害。

牧草植物中等水平的pa含量，可以改善氮素营养，减少尿氮排泄，有利于抵抗肠道寄生虫。

不幸的是，很多豆科植物，包括世界上最重要的牧草植物，紫花苜蓿，没有足够多的pa来抵抗食草动物的胀气病。

因此，提高苜蓿和其他饲料豆科植物根中pa含量成为生物技术的一个重要目标。

Pa的合成和调控在非豆科植物的模式植物拟南芥中已研究清楚。

拟南芥中pa的合成知识大部分来自于tt突变体，表现为种子色素沉着较少。

20个tt基因已被鉴定，它们编码参与pa 合成及储存或者调控pa产量的蛋白的酶或者转运子。

转运子包括一系列的转录因子：tt2，myb家族的转录因子；tt8，bHLH转录因子；ttg1，WD40蛋白，共同组成一个调控花色素还原酶转录的三聚体。

CRISPRCas-mediated genome editing in the rat via direct injection of one-cell embryos

2Center for Cancer and Stem Cell Biology, Institute of
Published online 25 September 2014; doi:10.1038/nprot.2014.171
© 2014 Nature America, Inc. All rights reserved.
protocol
CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos
Yanjiao Shao1,3, Yuting Guan1,3, Lu1, Yuting Chen1, Lijuan Wu1, Yongmei Li1, Xueyun Ma1, Mingyao Liu1,2 & Dali Li1
engineered meganucleases14 and the CRISPR/Cas system34–40 is greatly accelerating the development of genetic engineering technologies in the rat, and it is providing benefits to the research community. Engineered nucleases are guided to specific genomic loci to make site-specific DNA double-strand breaks (DSBs). Distinct from the other engineered nuclease-based technologies, the CRISPR/Cas system is composed of a Cas9 nuclease and a guide RNA that directs the nuclease to the target DNA through base-pairing rules41,42 (Fig. 1). We have adapted the CRISPR/Cas system to modify both mouse and rat genomes and demonstrated that it is an efficient, reliable and convenient technology for multiple gene knockout in mice and rats15. The CRISPR/Cas system The CRISPR/Cas system is an RNA-mediated adaptive immune system found in bacteria and archaea that protects against the invasion of viruses and plasmids41. On the basis of locus organization and signature Cas gene composition, three major types of CRISPR systems (types I–III) have been identified43. The type II CRISPR/Cas system derived from Streptococcus pyogenes has been modified and widely used for gene editing43,44. The main components of this system are the CRISPR RNA (crRNA) array, the trans-activating crRNA (tracrRNA) and the Cas9 nuclease. The crRNA array comprises direct repeats that flank different variable elements (protospacers) derived from the exogenous target genome. In the target genome, the sequence complementary to the protospacer must be directly upstream of a protospaceradjacent motif (PAM) for cleavage to occur. The PAM sequence varies depending on the host system and Cas9 nuclease present43; the PAM for Cas9 in S. pyogenes is NGG (where N is any nucleotide)37. The crRNA forms a duplex with the tracRNA, and it is processed so that the protospacer sequence (20 bases at the 5′ end of the processed crRNA) can direct the Cas9 nuclease to the complementary target DNA, where Cas9 mediates site-specific cleavage37. Theoretically, by replacing the crRNA protospacer

支持转基因的英文作文

支持转基因的英文作文英文：Genetic engineering, specifically in the form of genetically modified organisms (GMOs), has been a contentious issue for decades. However, I firmly supportthe use of GMOs for various reasons.First and foremost, GMOs have the potential to address global food security challenges. With a rapidly growing population, especially in developing countries, traditional agricultural methods may not be sufficient to meet the increasing demand for food. GMOs offer solutions such as crops engineered to resist pests and diseases, tolerate extreme weather conditions, and enhance nutritional content. For instance, genetically modified rice enriched withvitamin A, also known as "golden rice," has the potentialto alleviate vitamin A deficiency in regions where rice isa staple food.Moreover, GMOs can contribute to sustainableagriculture by reducing the need for chemical pesticidesand fertilizers. This not only minimizes environmental pollution but also promotes the health of farmers who are frequently exposed to these harmful chemicals. Additionally, GMOs can enable farmers to practice conservation tillage, which helps prevent soil erosion and maintains soilfertility.Critics often raise concerns about the safety of consuming GMOs. However, extensive scientific research and regulatory assessments have consistently demonstrated that approved GMOs are as safe for human consumption as theirnon-modified counterparts. Furthermore, advancements in genetic engineering technologies, such as gene editingusing CRISPR-Cas9, offer precise and targeted modifications, minimizing unintended effects on the organism.In addition to agricultural benefits, GMOs have the potential to revolutionize other sectors, includingmedicine and industry. For example, genetically modified bacteria can produce insulin and other life-saving drugsmore efficiently and affordably than traditional methods.In the industrial sector, GMOs can be engineered to produce biofuels and biodegradable plastics, reducing our dependence on fossil fuels and mitigating environmental degradation.In conclusion, the widespread adoption of GMOs has the potential to address pressing global challenges related to food security, environmental sustainability, and human health. While acknowledging the need for rigorous safety assessments and regulatory oversight, I believe that embracing genetic engineering technologies is essential for advancing agriculture and promoting a more sustainable future.中文：转基因技术，特别是转基因生物（GMO）的使用，长期以来一直备受争议。

The R2R3-MYB gene family in Arabidopsis thaliana

447MYB factors represent a family of proteins that include the conserved MYB DNA-binding domain. In contrast to animals, plants contain a MYB-protein subfamily that is characterised by the R2R3-type MYB domain. ‘Classical’ MYB factors, which are related to c-Myb, seem to be involved in the control of the cell cycle in animals, plants and other higher eukaryotes. Systematic screens for knockout mutations in MYB genes, followed by phenotypic analyses and the dissection of mutants with interesting phenotypes, have started to unravel the functions of the 125 R2R3-MYB genes in Arabidopsis thaliana. R2R3-type MYB genes control many aspects of plant secondary metabolism, as well as the identity and fate of plant cells. AddressessMax-Planck-Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, D-50829Köln, Germany*e-mail: weisshaa@mpiz-koeln.mpg.deCurrent Opinion in Plant Biology2001, 4:447–4561369-5266/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.AbbreviationsAGI Arabidopsis Genome InitiativeAS1ASYMMETRICAL LEAVES1CDC5cell division cycle5GL1GLABROUS1KNOX KNOTTEDPAP1PRODUCTION OF ANTHOCYANIN PIGMENT1PCR polymerase chain reactionPHAN PHANTASTICATT2TRANSPARENT TESTA2WER WEREWOLFIntroductionRegulation of gene expression at the level of transcription controls many crucial biological processes. A number of different factors are required for the process of transcription. These include factors required for chromatin remodelling and DNA unwinding, as well as proteins of the pre-initiation complex and the RNA polymerase II complex. In addition to this general inventory, other factors control promoter strength. These factors are referred to as ‘transcription factors’, a term usually used to describe proteins that recognise DNA in a sequence-specific manner and that regulate the frequency of initiation of transcription upon binding to specific sites in the promoter of target genes. T ranscription factors—which can be activators, repressors, or both—display a modular structure. On the basis of similarities in one of the modules, namely the DNA-binding domain, transcription factors have been classified into families [1]. In plants, MYB factors comprise one of the largest of these families [2,3•].The first MYB gene identified was the ‘oncogene’v-Myb derived from the avian myeloblastosis virus [4]. Evidence obtained from sequence comparisons indicates that v-Myb may have originated from a vertebrate gene, which mutated once it became part of the virus. Many vertebrates contain three genes related to v-Myb—c-Myb, A-Myb and B-Myb [5]—and other similar genes have been identified in insects, plants, fungi and slime moulds [6]. The encoded proteins are crucial to the control of proliferation and differentiation in a number of cell types, and share the con-served MYB DNA-binding domain. This domain generally comprises up to three imperfect repeats, each forming a helix-turn-helix structure of about 53 amino acids. Three regularly spaced tryptophan residues, which form a trypto-phan cluster in the three-dimensional helix-turn-helix structure, are characteristic of a MYB repeat [7,8]. The three repeats in c-Myb are referred to as R1, R2 and R3; and repeats from other MYB proteins are categorised according to their similarity to either R1, R2 or R3. Here, we discuss the MYB gene inventory of the Arabidopsis thaliana genome, and review the conservation of sequence and function among A. thaliana MYB proteins by including several well-known examples from other species.MYB genes in plantsMYB proteins can be classified into three subfamilies depending on the number of adjacent repeats in the MYB domain (one, two or three) [9,10]. We refer to MYB-like proteins with one repeat as ‘MYB1R factors’, with two as ‘R2R3-type MYB’ factors, and with three repeats as ‘MYB3R’ factors. The MYB-like proteins with a single repeat(or sometimes just a partial one)are fairly divergent and include factors that bind the consensus sequence of plant telomeric DNA (TTTAGGG) [11]. It has also been shown that MYB1R factors (e.g.MYBST1 or StMYB1R1) can act as transcriptional activators [12], and some are associated closely with the activity of the circadian clock (A1 [CIRCADIAN CLOCK ASSOCIATED1] and LHY [LATE ELONGATED HYPOCOTYL]) [13]. CCA1 and LHY1 bind DNA, indicating that they might act by modulating transcription [14,15].Primarily through genome sequencing, genes encoding three Myb repeats have been detected in A. thaliana[16]. MYB3R genes have also been detected in all major lineages of land plants [17]. These data show that the genes encoding R2R3-MYB factors (see below) are not the equivalents of c-Myb from animals, as was initially thought before the detection of plant MYB3R genes.A.thaliana contains five AtMYB3R genes (see T able1). It has been shown recently that plant MYB3R factors similar to MYB proteins in animals are involved in controlling the cell cycle [18]. Like B-MYB in blood cells [19], MYB3R factors are involved in regulating the transcription of cyclin genes via MYB recognition elements in cyclin promoters [18]. This may indicate functional conservation amongThe R2R3-MYB gene family in Arabidopsis thaliana Ralf Stracke, Martin Werber and Bernd Weisshaar*448Cell signalling and gene regulationThe R2R3-MYB gene family in Arabidopsis thaliana Stracke, Werber and Weisshaar 449MYB3R genes from plants to humans. However, the exact contribution of MYB3R factors and proteins such as AtMYBCDC5 [20] in cell cycle control in plants requires further elucidation. AtMYBCDC5 is related to the yeast cell cycle protein CDC5+ and the human regulator of mitotic entry HsCDC5. These are potentially multifunctional MYB proteins that are involved in transcript splicing [21] and transcriptional regulation[22].We have so far been unable to find knockout alleles for AtMYB3R genes, although we have screened several of the available insertion-mutagenesis populations. AtMYB3R genes may play a role in cell cycle control, and so, it is possible that mutations in these genes are lethal, thus the respective mutant alleles are under-represented in knockout populations. The map positions of the A.thaliana MYB3R genes were compared with those of embryonic lethal mutations (emb), but candidates for mutant alleles were not revealed conclusively. This might be because of the high number of emb genes and the difficulties involved in integrating physical and genetic maps [23].450Cell signalling and gene regulationR2R3-MYB genes in A.thalianaMYB genes containing two repeats (i.e. R2R3-MYB) con-stitute the largest MYB gene family in plants. The large size of this gene family was apparent from the work of Romero et al.[2] in A.thaliana and was also confirmed in Zea mays[24]. About 80 different A.thaliana genes were described initially [2], and this number increased to 97 through the combined efforts of six European laboratories in an European Community funded consortium.Our listing herein is based on the systematic names previ-ously assigned to R2R3-MYB genes [2,25,26]. In some instances, synonyms are listed for genes bearing different names in the literature. The list includes AtGL1,the first R2R3-MYB gene identified in A.thaliana as AtMYB0(the designation ‘AtMYB1’ was assigned to the first A.thaliana R2R3-type MYB gene identified by polymerase chain reac-tion [PCR]-based methods) [25]. As new MYB genes were disclosed through sequencing by the Arabidopsis Genome Initiative (AGI), we generated Genbank/EMBL database entries for the deduced cDNAs of AtMYB and AtMYB3R genes (see T able 1). Upon inspection, and in some cases correction, of the predicted splicing patterns, we con-firmed the existence of the respective transcript by reverse transcription (RT)-PCR using gene-specific primers, resulting in an amplicon spanning at least one intron posi-tion (R Stracke, B Weisshaar, unpublished data). In an attempt to complete the listing, we screened the TIGR and MIPS whole-genome datasets (version 03202001) with various sequence-similarity search tools. T able1 lists 125 distinct AtMYB genes of the R2R3 type that were detected on the basis of the (near) complete sequence ofThe R2R3-MYB gene family in Arabidopsis thaliana Stracke, Werber and Weisshaar 451452Cell signalling and gene regulationA.thaliana[27•]. The systematic designations, and the gene identifiers (e.g. At3g27920) assigned to genes in the AGI annotation, may help to avoid the confusion that often results when multiple names are used for the same gene in such a large gene family.There are two unusual genes encoding MYB proteins with two or more repeats: AtMYBCDC5and AtMYB4R1. AtMYBCDC5 contains a MYB domain consisting of two repeats that are only distantly related to those of the R2R3-type MYB domain (having 31% identity to a typical R2R3-type MYB domain, whereas R2R3-type MYB domains generally display at least 40% identity to the consensus); AtMYB4R1 (T able1) is a putative MYB protein containing four R1R2-like repeats.Riechmann et al.[3•] calculated that of the almost 26,000 A.thaliana genes, about 1600 genes (6%) encode transcription factors, and classified 131 of them as factors of the R2R3-MYB type. The InterPro MYB domain signature patterns—PS00037 (W-[ST]-x[2]-E-[DE]-x[2]-[LIV]) and PS00334 (W-x[2]-[LI]-[SAG]-x[4,5]-R-x[8]-[YW]-x[3]-[LIVM]; accession IPR001005)[28] —used to detect MYB factors in the Riechmann study do, as already stated in the ‘Supplementary Material’ of that study, retrieve ‘false-positives’ resulting in an over-estimation of the total number of AtMYB genes. We have tried to exclude such false positives by manual inspection of the deduced amino-acid sequence of every gene shown in T able1. This may explain the difference between the number of R2R3-type MYB genes detected (131 versus 125). Clarification of this discrepancy will require information, which is currently unavailable,on which open reading frames were considered by Riechmann et al.[3•]. We cannot exclude, however, the existence of a small number of additional MYB proteins with two or more repeats in A.thaliana. This is because not all of the genome has been sequenced yet, and because a potential candidate gene with an unusual gene structure and/or very small exons might have escaped our detection. All 125 A.thaliana R2R3-type MYB domain sequences were used to deduce a consensus sequence and to determine the frequency of the most prevalent amino acids at each position within the repeats. Figure1 shows clearly the high level of conservation not only of the tryptophan residues but also of the phenylalanine residue that replaces the first tryptophan in the R3 repeat. In R2, 25 (of 53) positions are occupied by a single, or two very similar, residues in more than 80% of the proteins. For R3, the equivalent number is 31 (out of 51), indicating that R3 is slightly more conserved than R2. It is worth noting that there are five additional amino-acid positions conserved immediately amino-terminal to R2 (consensus: CDKAG) of the R2R3-type MYB domain (M Werber, B Weisshaar, unpublished data). However, it is not known what function this sequence might have.R2R3-MYB genes mainly regulateplant-specific processesNo functional data are available for most of the 125 R2R3-type AtMYB genes. However, systematic searches for knockouts have been initiated recently [29] and the number of AtMYB genes for which functional information has become available has grown significantly during the past year. R2R3-type MYB genes have been shown to regulate phenylpropanoid metabolism in A.thaliana. Overexpression of AtMYB75/PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1) and AtMYB90/PAP2[30•] results in accumulation of anthocyanins, and AtMYB4 represses the synthesis of sinapoyl malate [31••]. The analysis of AtMYB4 also demonstrated that R2R3-type MYB proteins can act as transcriptional activators as well as repressors. Recently, the TRANSPARENT TESTA2(TT2) gene has been shown to encode an R2R3-type MYB factor (AtMYB123; N Nesi, L Lepiniec, personal communication). This is not a surprise as several R2R3-type MYB genes, such as ZmMYBC1from Zea mays[32] or PhMYBAN2from Petunia hybrida [33] (which also control phenylpropanoid metabolism [9,34]), are known to be derived from other plant species. AtMYB34/ATR1(ALTERED TRYPTOPHAN REGULATION1) is a regulator of tryptophan biosynthesis, which demonstrates that pathway control by such factors is not limited to secondary metabolism[35].Another important function for R2R3-type MYB factors is the control of development and determination of cell fate and identity. AtMYB0/GLABROUS 1(GL1) [36] and AtMYBB66/WEREWOLF(WER) [37•] are involved in epidermal cell patterning. In fact, both genes encode very similar proteins (subgroup 15, Figure2; [26]). The classical A.thaliana genetic locus ASYMMETRICAL LEAVES1 (AS1)was shown to encode a MYB factor—AtMYB91/AS1 [38•]—and is orthologous to the cell differentiation genes AmMYBPHAN[39] from Anthirrhinum majus and ZmMYBRS2 [40,41]. AtMYB0/GL1 interacts with a basic helix-loop-helix (bHLH) factor encoded by AtbHLH1/GL3[42••] —similar to the the combined action of ZmMYBC1 and ZmR, which together regulate anthocyanin production in Zea mays[43]. This evidence suggests that R2R3-type MYB factors often are involved in the combinatorial interactionThe R2R3-MYB gene family in Arabidopsis thaliana Stracke, Werber and Weisshaar 453of transcription factors for the generation of highly specific expression patterns.R2R3-type MYB factors also participate in plant responses to environmental factors and in mediating hormone actions, examples of which have been discussed in detail recently[9]. In A.thaliana, AtMYB2 has been found to regulate the AtADH1(ALCOHOL DEHYDROGENASE1) gene promoter, and it might also be involved in the response to low oxygen [44]. AtMYB30expression is strongly correlated with cell death during the hypersensitive response upon pathogen attack or elicitor treatment [45•]. Elicitor-responsive R2R3-type MYB genes have also been described from Nicotiana tabacum[46], and an AtMYB78-related MYB gene from Oryza sativa has been shown to be expressed in response to fungal attack [47]. T aken as a whole, it seems that R2R3-type MYB genes are involved predominantly in controlling ‘plant-specific’ processes[9,34]. This finding is especially interesting in the light of the observation that,to the best of our current knowledge,MYB genes of the R2R3 type are only present in plants [3•]. It is tempting to speculate that R2R3-type MYB genes have, during evolution, contributed to plant speciation.None of the known mutations in R2R3-type MYB genes, including those detected so far in various knockout pop-ulations [29], result in strong phenotypes that are expected for essential genes. More data are required to determine if any R2R3-type MYB genes are absolutely essential, or whether functional redundancy often masks the effects of mutations in crucial R2R3-type MYB genes. Complexity of the R2R3-MYB gene familyThe R2R3-type MYB factors encoded by the AtMYB genes have been categorised into 22 subgroups on the basis of conserved amino-acid sequence motifs present carboxy-terminal to the MYB domain [26]. Re-evaluation of these motifs, using the newly extended dataset, confirmed most of these subgroups (Figure2). Despite the divergence of the amino-acid sequence outside of the MYB domain, there are some conserved motifs that may contribute to function. Evidence for the importance of these motifs comes from AtMYB0/GL1 and the nonfunctional gl1 protein encoded by the gl1-2allele that lacks the motif-defining subgroup15 [26]. The conserved motifs may facilitate the identification of functional domains outside of the DNA-binding domain of R2R3-type MYB factors.It is also interesting to note that there are several cases of functional conservation of genes that cluster together in the dendrogram. AtMYB91/AS1and AmMYBPHAN have both been shown to negatively regulate KNOX(KNOTTED) expression in organ primordia [38•]. AtMYB66/WER and AtMYB0/GL1, both clustering together in subgroup15 (Figure2), can functionally complement each other and display different biological functions only because of their different spatial expression patterns [48••]. AtMYB23 is closely related to AtMYB66/WER and AtMYB0/GL1, and it would be interesting to see if the AtMYB23 gene is also able to complement wer and gl1 mutants when expressed under the control of the respec-tive promoter. A third case for correlation between sequence similarity and function might be AtMYB123, because this factor is sequence-related to ZmMBC1 and is identical to the R2R3-type MYB factor encoded by TT2 (see above). Both, AtMYB123/TT2 and ZmMYBC1, con-trol the pigmentation of seed or kernals, respectively. AtMYB75/PAP1 and AtMYB90/PAP2 are also closely related to ZmMYBC1 (Figure2) and appear to have a somehow related function[30•].ConclusionsThe clear examples of functional conservation described above support the optimistic view that data from the model plant A.thaliana will contribute to a better understanding of MYB gene functions in other species, and vice versa. Part of the apparent redundancy observed among the MYB genes at the amino-acid sequence level is due to genes that have similar molecular functions but still display different biological phenotypes when mutated because of differential temporal and/or spatial expression characteristics. Consequently, the genes are not redundant in terms of the developmental biology of the plant. This concept can explain the existence, and maintenance during evolution, of genes encoding a number of closely related factors in the same species. When transferred to the level of the target genes, this interpretation suggests that a single target gene is regulated by a fairly high number of factors from the same gene family, which all transmit their signal via the same cis-acting element. There is no evidence that any of the 125 R2R3-type MYB genes might represent a pseudogene, inferring that selection might maintain intact MYB genes. This indicates that there must be a phenotype associated with any mutation on which selection can act. The challenge is to find the correct conditions and screens to force the plant to display this phenotype, thereby helping to reveal the function of the remaining 90% of the MYB genes in A.thaliana. AcknowledgementsWe would like to thank all members of the EC MYB consortium, particularly Javier Paz-Ares, Chiara T onelli, Mike Bevan, Sjef Smeekens and Cathie Martin, for stimulating discussions on plant MYB proteins; Loic Lepiniec and Masaki Ito for communicating results prior to publication; AGI for sequencing and annotation; and Heiko Schoof for updating gene identifiers in MATDB. We also thank Norddeutsche Pflanzenzucht (NPZ), Hohenlieth, for funding the PhD work of MW; John Doonan, Imre Somssich and Cathie Martin for critical reading of the manuscript; and Klaus Hahlbrock and Francesco Salamini for continuous support and laboratory space. References and recommended readingPapers of particular interest, published within the annual period of review, have been highlighted as:•of special interest••of outstanding interest1.Pabo CO, Sauer RT: Transcription factors: structural families andprinciples of DNA recognition.Annu Rev Biochem 1992,61:1053-1095.2.Romero I, Fuertes A, Benito MJ, Malpical JM, Leyva A, Paz-Ares J:More than 80 R2R3-MYB regulatory genes in the genome ofArabidopsis thaliana.Plant J 1998, 14:273-284.454Cell signalling and gene regulation3.Riechmann JL, Heard J, Martin G, Reuber L, Jiang CZ, Keddie J, •Adam L, Pineda O, Ratcliffe OJ, Samaha RR et al.: Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes.Science 2000, 290:2105-2110.The authors evaluated the sequenced eukaryotic genomes in terms of transcription factor genes and gene families. About 45% of the approximately 1600 Arabidopsis transcription factors seem to belong to families that are specific to plants.4.Klempnauer KH, Gonda TJ, Bishop JM: Nucleotide sequence of theretroviral leukemia gene v-myb and its cellular progenitor c-myb: the architecture of a transduced oncogene.Cell 1982,31:453-463.5.Weston K: Myb proteins in life, death and differentiation.Curr OpinGenet Dev 1998, 8:76-81.6.Lipsick JS: One billion years of Myb.Oncogene 1996, 13:223-235.7.König P, Giraldo R, Chapman L, Rhodes D: The crystal structure ofthe DNA-binding domain of yeast RAP1 in complex with telomeric DNA.Cell 1996, 85:125-136.8.Ogata K, Hojo H, Aimoto S, Nakai T, Nakamura H, Sarai A, Ishii S,Nishimura Y: Solution structure of a DNA-binding unit of Myb: ahelix-turn-helix-related motif with conserved tryptophans forminga hydrophobic core.Proc Natl Acad Sci USA 1992, 89:6428-6432.9.Jin H, Martin C: Multifunctionality and diversity within the plantMYB-gene family.Plant Mol Biol 1999, 41:577-585.10.Rosinski JA, Atchley WR: Molecular evolution of the Myb family oftranscription factors: evidence for polyphyletic origin.J Mol Evol1998, 46:74-83.11.Yu EY, Kim SE, Kim JH, Ko JH, Cho MH, Chung IK:Sequence-specific DNA recognition by the Myb-like domain ofplant telomeric protein RTBP1.J Biol Chem 2000, 275:24208-24214.12.Baranowskij N, Frohberg C, Prat S, Willmitzer L: A novel DNAbinding protein with homology to Myb oncoproteins containingonly one repeat can function as a transcriptional activator.EMBO J 1994, 13:5383-5392.13.Schaffer R, Landgraf J, Accerbi M, Simon V, Larson M, Wisman E:Microarray analysis of diurnal and circadian-regulated genes inArabidopsis.Plant Cell 2001, 13:113-123.14.Wang ZY, Kenigsbuch D, Sun L, Harel E, Ong MS, Tobin EM:A Myb-related transcription factor is involved in the phytochromeregulation of an Arabidopsis Lhcb gene.Plant Cell 1997,9:491-507.15.Schaffer R, Ramsay N, Samach A, Corden S, Putterill J, Carre IA,Coupland G: The late elongated hypocotyl mutation ofArabidopsis disrupts circadian rhythms and the photoperiodiccontrol of flowering.Cell 1998, 93:1219-1229.16.Braun EL, Grotewold E: Newly discovered plant c-myb-like genesrewrite the evolution of the plant myb gene family.Plant Physiol1999, 121:21-24.17.Kranz H, Scholz K, Weisshaar B: c-MYB oncogene-like genesencoding three MYB repeats occur in all major plant lineages.Plant J 2000, 21:231-235.18.Ito M, Araki S, Matsunaga S, Itoh T, Nishihama R, Machida Y,Doonan JH, Watanabe A: G2/M-phase-specific transcription during the plant cell cycle is mediated by c-Myb-like transcription factors.Plant Cell 2001, 13:in press.19.Muller C, Yang R, Idos G, Tidow N, Diederichs S, Koch OM,Verbeek W, Bender TP, Koeffler HP: c-myb transactivates thehuman cyclin A1 promoter and induces cyclin A1 geneexpression.Blood 1999, 94:4255-4262.20.Hirayama T, Shinozaki K: A cdc5+ homolog of a higher plant,Arabidopsis thaliana.Proc Natl Acad Sci USA 1996,93:13371-13376.21.Burns CG, Ohi R, Krainer AR, Gould KL: Evidence that Myb-relatedCDC5 proteins are required for pre-mRNA splicing.Proc Natl Acad Sci USA 1999, 96:13789-13794.22.Lei XH, Shen X, Xu XQ, Bernstein HS: Human Cdc5, a regulator ofmitotic entry, can act as a site-specific DNA binding protein.J Cell Sci 2000, 113:4523-4531.23.Meinke DW, Cherry JM, Dean C, Rounsley SD, Koornneef M:Arabidopsis thaliana: a model plant for genome analysis.Science 1998, 282:662-682.24.Rabinowicz P, Braun E, Wolfe A, Bowen B, Grotewold E: Maize R2R3Myb genes: sequence analysis reveals amplification in the higher plants.Genetics 1999, 153:427-444.25.Shinozaki K, Yamaguchi-Shinozaki K, Urao T, Koizumi M: Nucleotidesequence of a gene from Arabidopsis thaliana encoding a mybhomologue.Plant Mol Biol 1992, 19:493-499.26.Kranz HD, Denekamp M, Greco R, Jin H, Leyva A, Meissner RC,Petroni K, Urzainqui A, Bevan M, Martin C et al.: Towards functional characterisation of the members of the R2R3-MYB gene familyfrom Arabidopsis thaliana.Plant J 1998, 16:263-276.27.The Arabidopsis Genome Initiative: Analysis of the genome •sequence of the flowering plant Arabidopsis thaliana.Nature 2000, 408:796-815.This paper summarises years of highly coordinated work from laboratories all over the world to obtain the first complete genome sequence of a plant. Within 115.4 Megabases of sequenced DNA, 25,500 genes were identified and annotated.28.Apweiler R, Attwood TK, Bairoch A, Bateman A, Birney E, Biswas M,Bucher P, Cerutti T, Corpet F, Croning MDR et al.: The InterProdatabase, an integrated documentation resource for protein families, domains and functional sites.Nucleic Acids Res 2001, 29:37-40. 29.Meissner RC, Jin H, Cominelli E, Denekamp M, Fuertes A, Greco R,Kranz HD, Penfield S, Petroni K, Urzainqui A et al.: Function search ina large transcription factor gene family in Arabidopsis: assessingthe potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.Plant Cell 1999, 11:1827-1840.30.Borevitz JO, Xia YJ, Blount J, Dixon RA, Lamb C: Activation tagging •identifies a conserved MYB regulator of phenylpropanoid biosynthesis.Plant Cell 2000, 12:2383-2393.An A.thaliana mutant line with intensive purple pigmentation in many vegetative organs was identified from an activation-tagging population. The dominant phenotype resulted from overexpression of AtMYB75/PAP1, which caused a massive activation of phenylpropanoid biosynthetic genes and enhanced accumulation of lignin, hydroxycinnamic acid esters, and purple anthocyanins. AtMYB75/PAP1, and the closely related factor AtMYB90/PAP2, are both similar to Petunia hybrida AtMYBAN2.31.Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C, ••Weisshaar B, Martin C: Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis.EMBO J 2000, 19:6150-6161.The R2R3-type MYB factor AtMYB4 was shown to act as a negative regulator of cinnamate 4-hydroxylase gene expression. It also negatively regulates other steps of phenylpropanoid metabolism in a dose-dependent way. This is the first example of a plant MYB protein that acts as a transcriptional repressor. Functionally, AtMYB4 controls the synthesis of sinapate esters, which provide tolerance to UV-B irradiation.32.Paz-Ares J, Wienand U, Peterson PA, Saedler H: Molecular cloningof the c1 locus of Zea mays: a locus regulating the anthocyaninpathway.EMBO J 1986, 5:829-834.33.Quattrocchio F, Wing J, van der Woude K, Souer E, de Vetten N,Mol J, Koes R: Molecular analysis of the anthocyanin2gene ofpetunia and its role in the evolution of flower color.Plant Cell1999, 11:1433-1444.34.Martin C, Paz-Ares J: MYB transcription factors in plants.TrendsGenet 1997, 13:67-73.35.Bender J, Fink GR: A Myb homologue, ATR1, activates tryptophangene expression in Arabidopsis.Proc Natl Acad Sci USA 1998,95:5655-5660.36.Oppenheimer DG, Herman PL, Sivakumaran S, Esch J, Marks MD:A MYB gene required for leaf trichome differentiation inArabidopsis is expressed in stipules.Cell 1991, 67:483-493.37.Lee MM, Schiefelbein J: WEREWOLF, a MYB-related protein in •Arabidopsis, is a position-dependent regulator of epidermal cell patterning.Cell 1999, 24:473-483.The authors isolated the AtMYB66/WER gene. Plants mutated at WER display a hairy root phenotype. AtMYB66/WER is required for normal epidermal-cell patterning, and regulates the position-dependent expression of GL2.38.Byrne ME, Barley R, Curtis M, Arroyo JM, Dunham M, Hudson A, •Martienssen RA: Asymmetric leaves1mediates leaf patterning and stem cell function in Arabidopsis.Nature 2000,408:967-971.The paper describes the cloning of AtMYB91/AS1, which was found to be a homologue of AmMYBPHAN from A.majus and ZmMYBRS2 from Z.mays. Molecular and genetic analyses define conserved interactions between AS1 and the KNOX homeobox genes.The R2R3-MYB gene family in Arabidopsis thaliana Stracke, Werber and Weisshaar 455。

R2R3-MYB转录因子GmMYB184调节大豆异黄酮合成

作物学报ACTA AGRONOMICA SINICA 2018, 44(2): 185 196/ISSN 0496-3490; CN 11-1809/S; CODEN TSHPA9E-mail: xbzw@ DOI: 10.3724/SP.J.1006.2018.00185R2R3-MYB转录因子GmMYB184调节大豆异黄酮合成朱莹1,**褚姗姗2,**张培培1程浩1喻德跃1王娇1,*1南京农业大学大豆研究所 / 作物遗传育种与种质创新国家重点实验室 / 江苏省现代作物生产协同创新中心 / 国家大豆改良中心,江苏南京210095; 2河南农业大学农学院 / 河南省粮食作物协同创新中心, 河南郑州 450002摘要: 异黄酮是一类主要含在豆科植物中的次生代谢物, 在植物防御体系中发挥重要作用, 并与人类健康密切相关。

大豆异黄酮含量受多基因和复杂代谢网络控制, 调控代谢途径上的结构基因不能显著改变大豆异黄酮含量, 与异黄酮代谢途径相关的转录因子的鉴定和应用可能会有效解决这个问题。

本研究克隆了一个与大豆异黄酮合成相关的R2R3类型MYB转录因子GmMYB184, 并进行了初步的功能验证。

亚细胞定位研究结果表明GmMYB184转录因子定位于细胞核。

组织表达分析结果表明该转录因子基因与IFS2 (异黄酮合酶2编码基因)的表达模式相同。

同时, GmMYB184和IFS2的表达模式与异黄酮的积累模式相似。

谷胱甘肽诱导表达分析表明该转录因子基因与IFS2共同被诱导, 说明这两个基因可能参与同一或相似的生物过程。

采用双荧光素酶报告系统分析其对异黄酮合成途径关键基因的转录激活活性影响, 发现GmMYB184能够促进IFS2和CHS8启动子表达活性分别提高5倍和7倍。

最后, 通过发根农杆菌介导的遗传转化系统, 找到该转录因子在异黄酮合成调控中的直接作用证据。

沉默GmMYB184导致大豆毛状根异黄酮含量的显著下降。

但是, 过表达GmMYB184不足以显著提高毛状根中异黄酮的含量。

Biotransformation of the mycotoxin zearalenone by fungi of the genera Rhizopus and Aspergillus

R E S E A R C H L E T T E RBiotransformation of the mycotoxin zearalenone by fungi of the genera Rhizopus and AspergillusAntje Brodehl 1,2,Anne M €oller 1,Hans-J €o rg Kunte 2,Matthias Koch 1&Ronald Maul 1,31Department of Analytical Chemistry,Reference Materials,BAM Federal Institute for Materials Research and Testing,Berlin,Germany;2Department of Materials and Environment,BAM Federal Institute for Materials Research and Testing,Berlin,Germany;and 3Department of Quality,Leibniz-Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V.,Großbeeren,GermanyCorrespondence:Ronald Maul,Department of Analytical Chemistry,Reference Materials,BAM Federal Institute for Materials Research and Testing,Richard-Willst €a tter-Straße 11,12489Berlin,Germany.Tel.:+493081045960;fax:+493081041177;e-mail:ronald.maul@bam.deReceived 8May 2014;revised 31July 2014;accepted 18August 2014.Final version published online 11September 2014.DOI:10.1111/1574-6968.12586Editor:Matthias BrockKeywordsmicrobial conversion;metabolites;fermentation;a -zearalenol;conjugation.AbstractZearalenone (ZEN)is a nonsteroidal estrogenic mycotoxin biosynthesized by various Fusarium fungi.These fungal species frequently infest grains;therefore,ZEN represents a common contaminant in cereal products.The biotransforma-tion of ZEN differs signiﬁcantly from species to species,and several metabolites are known to be formed by animals,plants,and microorganisms.The aim of the present study was to investigate the microbial conversion of ZEN by species of the genera Rhizopus and Aspergillus representing relevant fungi for food pro-cessing (e.g.fermentation).To monitor the ZEN metabolism,ZEN was added to liquid cultures of the different fungal species.After a period of 3days,the media were analyzed by HPLC-MS/MS for metabolite formation.Two Aspergillus oryzae strains and all seven Rhizopus species were able to convert ZEN into various metabolites,including ZEN-14-sulfate as well as ZEN-O -14-and ZEN-O -16-glucoside.Microbial transformation of ZEN into the signiﬁcantly more estrogenic a -zearalenol (a -ZEL)was also observed.Additionally,a novel fungal metabolite,a -ZEL-sulfate,was detected.Semi-quantiﬁcation of the main metabolites indicates that more than 50%of initial ZEN may be modiﬁed.The results show that fungal strains have the potential to convert ZEN into various metabolites leading to a masking of the toxin,for example in fermented food.IntroductionZearalenone (ZEN),a b -resorcylic acid lactone,is pro-duced by several species of Fusarium ,including Fusarium graminearum and Fusarium crookwellense (Di Menna et al.,1991;Plasencia &Mirocha,1991),which infests grain cereals such as maize and wheat (Schollenberger et al.,2006).The infected host plant as well as other fun-gal species can transform ZEN into different conjugated forms which are primarily sulfate and glucoside esters.A recent study of Kovalsky Paris et al.(2014)has reported the conversion of ZEN into a mixture of ZEN-14-O -b -glucoside (ZEN-14-Glc)and ZEN-16-O -b -glucoside (ZEN-16-Glc)in barley.Studies on the metabolism of ZEN by Rhizopus and Thamnidium species have shown the formation of ZEN-14-Glc and ZEN-14,16-O -b -dig-lucoside (Kamimura,1986;El-Sharkawy,1989).ZEN-14-sulfate (ZEN-14-S)was found to be a natural metabolite of Fusarium graminearum ,Rhizopus arrhizus,andAspergillus niger (El-Sharkawy et al.,1991;Plasencia &Mirocha,1991;Jard et al.,2010).The occurrence of these conjugated ZEN derivatives has been described for a variety of food and feed matri-ces,including corn and wheat products (Vendl et al.,2010;De Boevre et al.,2012).The total amount of conju-gated forms can even exceed the content of the parental mycotoxin (Berthiller et al.,2013).In addition to natural ZEN conjugate contamination in feed and food,biotech-nological fermentation of food can also lead to ZEN masking.Aspergillus and Rhizopus are common fungal species that are used in biotechnological production of tempeh and soy products (Hering et al.,1991;Wiesel et al.,1997;Chancharoonpong et al.,2012).For soy sauce production,soybeans or a mixture of soybeans and wheat is used as raw material for fermentation by Aspergillus oryzae (A.oryzae )(Chancharoonpong et al.,2012).Tempeh and tempeh-like products are made by fermenta-tion of soybeans and cereal grains with Rhizopus sp.M I C R O B I O L O G Y L E T T E R S(Hachmeister&Fung,1993;Larsson Alminger et al., 2012).Because these unfermented cereal and soybean raw materials can contain ZEN(Lee et al.,1991;Schollenber-ger et al.,2006;De Boevre et al.,2012),conjugated ZEN could be present.Due to the fact that conjugated ZEN derivatives can be efﬁciently hydrolyzed into their parent compound by human colonic microbiota(Dall’Erta et al.,2013;Koval-sky Paris et al.,2014),the exposure of ZEN might be underestimated.Because of its estrogenic effects that may lead to severe hormonal disorder and infertility,a tolera-ble daily intake(TDI)for ZEN of0.25l g kgÀ1body weight has been established(Shier et al.,2001;EFSA, 2011).Besides conjugated ZEN products,reductive metabolites are formed in plant and fungal metabolism, for example a-and b-ZEL.Whereas b-ZEL is less toxic than ZEN,a-ZEL possesses an about10-fold higher estr-ogenicity than ZEN(Metzler et al.,2010).For further toxicological investigations of ZEN and ZEL conjugates, the availability of pure standard substances is required. The objective of this study was to investigate the for-mation of ZEN conjugates by molds using different Rhizopus species and A.oryzae strains.Especially,fungal strains that are used in biotechnological fermentation processes are analyzed for their potential of ZEN conjuga-tion.Because fermentation of ZEN contaminated raw materials could lead to conjugated ZEN derivatives,an underestimation of toxicological effects of ZEN and its derivatives in theﬁnal food product is possible.There-fore,this study gives aﬁrst qualitative overview about ZEN metabolite formation by A.oryzae and Rhizopus spe-cies and enables estimating the possibility to utilize those fungal strains for facilitated biosynthesis.Materials and methodsChemicals and mediaPotato dextrose agar(PDA)and potato dextrose broth (PDB)were purchased from Carl Roth GmbH+Co.KG (Karlsruhe,Germany).ZEN was acquired from Tocris Bioscience(Bristol,England).A stock(1mg mLÀ1)and working(5l g mLÀ1)solution of ZEN was prepared as methanolic solution and stored atÀ20°C.a-ZEL and b-ZEL were purchased from Sigma-Aldrich Chemie GmbH(Steinheim,Germany).Stock solutions of a-ZEL (101.8l g mLÀ1)and b-ZEL(261.6l g mLÀ1)were pre-pared in acetonitrile and stored atÀ20°C.ZEN-14-S, ZEN-14-Glc,and ZEN-16-Glc were kindly provided by Prof.Franz Berthiller(University of Natural Resources and Life Sciences,Vienna,Austria).Stock solutions of ZEN-14-Glc and ZEN-16-Glc of approximately 5l g mLÀ1were prepared in acetonitrile.Acetonitrile and methanol were of HPLC-grade and obtained from POCH S.A.(Gliwice,Poland).Ammonium acetate was purchased from Mallinckrodt Baker Inc.(Griesheim,Germany). Ultrapure water was obtained from a Seralpur PRO90CN puriﬁcation system by Seral(Ransbach-Baumbach,Germany).For enzymatic hydrolysis of ZEN-14-S and a-ZEL-sulfate(a-ZEL-S),a solution con-taining sulfatase(Type H-1isolated from Helix pomatia, Sigma-Aldrich,≥10000units gÀ1solid)dissolved in potassium phosphate buffer(10mM,pH7.1)was used. Fungal strains and growth conditionsNine different fungal strains of the genera Rhizopus and Aspergillus were used for this study:Rhizopus oryza e (R.oryzae)DSM906,R.oryzae DSM907,R.oryzae DSM 908,R.stolonifer DSM855,R.microsporus var.chinensis DSM1834,R.microsporus var.oligosporus DSM1964, and A.oryzae DSM1864were obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig,Germany).Rhizopus oligosporus CD(LMH 1133T)was isolated from Hering et al.(1991)from a commercial tempeh inoculum on the basis of riceﬂour produced by LIPI(Lembaga Ilmu Pengetahuan Indonesia –Indonesian institute of sciences)and kindly provided by Prof.Bernward Bisping.Aspergillus oryzae NBRC 100959was purchased by NBRC(Nite Biological Resource Center,Tokyo,Japan).Stock cultures were grown on PDA medium for7days at30°C.The organ-isms R.oryzae DSM906,DSM907,and R.stolonifer DSM855have been chosen based on their ability to transform and produce steroids,respectively(Holland, 1981;Jong&Birmingham,1989).For A.oryzae NBRC 100959,the genome sequence is known(Machida et al., 2005).In accordance with the speciﬁcations of the DSMZ R.oryzae DSM908,R.microsporus DSM1834,DSM 1964,and R.oligosporus CD(Hering et al.,1991)have been chosen,because of their importance for biotechnol-ogy as strains applied in tempeh or soy sauce fermenta-tion.Cultivation for biotransformationLiquid cultures(50mL PDB)in250-mL Erlenmeyer ﬂasks were inoculated with pieces of mycelia and incu-bated at30°C for5days in a New Brunswick Scientiﬁc Innova TM4230rotary shaker set to170r.p.m.For metab-olism studies,1mL working solution of ZEN was added to each liquid culture of the fungal strains,and incuba-tion was continued for3days.After the incubation per-iod,an aliquot of1mL from each liquid media was transferred to a1.5-mL Eppendorf tube and centrifuged at16200g for10min.For protein precipitation,500l LFEMS Microbiol Lett359(2014)124–130ª2014Federation of European Microbiological Societies. Fungal biotransformation of zearalenone125icecold acetonitrile was added to500l L supernatant, stored overnight at4°C and centrifuged at11500g for 5min.The supernatant was transferred to a HPLC(high-performance liquid chromatography)vial and analyzed by HPLC hyphenated to tandem mass spectrometry(HPLC-MS/MS).Liquid chromatography–mass spectroscopy HPLC-MS/MS was performed on an API4000mass spec-trometer(AB Sciex,MA)connected to an Agilent1100 series HPLC(Agilent Technologies GmbH,B€o blingen, Germany).The analytical column was a ProntoSIL120-5-C18AQ(150mm93mm,5l m;Bischoff Chroma-tography,Leonberg,Germany),preceded by a ProntoSIL 120-5-C18AQ guard column(10mm93mm,5l m). Mobile phase A was water with5mM ammonium acetate and as mobile phase B acetonitrile/water(99:1v/v)with 5mM ammonium acetate was used.The gradient used was as follows:0–2min10%B,2–5.5min10–30%B, 5.5–7min30–60%B,7–12min60–65%B,12–13.5min 65–100%B,13.5–15.5min100%B,15.5–16min100–10%B,and16–19min10%B.The column oven was set to30°C.Theﬂow rate of the mobile phase was 0.7mL minÀ1and the injection volume was10l L.The ESI interface was operated in negative ionization mode at 450°C with the following settings:curtain gas pressure, nitrogen,1.38bar(20psi);nebulizer gas pressure,nitro-gen, 4.14bar(60psi);heater gas pressure,nitrogen, 4.14bar(60psi);and ionspray voltageÀ4500V.Two selected reaction monitoring(SRM)transitions were recorded for each analyte:ZEN m/z317.0?130.8/174.8, ZEN-sulfate m/z397.1?317.1/175.0,ZEN-glucoside m/z 479.1?317.0/130.8,ZEL m/z319.2?174.0/160.0, and ZEL-sulfate m/z399.2?319.2/275.2.Additionally, potential mass transitions for ZEN di-glucosides,di-sul-fates and a combination of sulfate and glucoside conju-gates of ZEN were checked in selected fungal strains. However,no peaks were visible for these SRM transitions. For obtaining product ion spectra additionally,the prod-uct ion scan mode(MS2)was used to acquire mass spec-tra between75and400amu.Enzymatic hydrolysis of sulfate estersAliquots(100l L)of the liquid media samples were evap-orated to dryness at30°C under a gentle stream of nitro-gen.After adding500l L of sulfatase solution (≥20U mLÀ1),the closed tubes were shaken for24h at 700r.p.m.on a thermo shaker(Labor-Brand,Gießen, Germany)at37°C.The hydrolysis was stopped by add-ing500l L of ice-cold acetonitrile,and the solution was centrifuged at11500g for3min.The supernatant was used for direct analysis by HPLC-MS/MS.A reference sample was treated as described above without the addi-tion of sulfatase.Semi-quantiﬁcation of ZEN metabolitesSemi-quantitative measurements were conducted for ZEN-14-S,a-ZEL-S,ZEN-14-Glc,and ZEN-16-Glc.ZEN-14-S and a-ZEL-S were determined using relative response factors of ZEN-14-S to ZEN and a-ZEL-S to a-ZEL.Response factors were estimated by comparing the MS/MS peak area before and after quantitative sulfate ester cleavage.ZEN and a-ZEL were determined by exter-nal calibration using the commercially available standard substances.Matrix-matched calibration was applied for semi-quantiﬁcation of ZEN-14-Glc and ZEN-16-Glc. Results and discussionRhizopus and Aspergillus-mediated biotransformation of ZENZEN biotransformation was investigated using representa-tives of the genera Aspergillus and Rhizopus that are of importance for biotechnology,that are known to produce and transform steroids,or that have a sequenced genome, which would allow an investigation of the ZEN metabo-lism in fungi on a genomic level.All of the seven tested Rhizopus spp.and two A.oryzae strains have the capabil-ity to metabolize ZEN into conjugated derivatives.The added ZEN was almost completely removed from the media within the incubation period of3days.The results are in accordance with Varga et al.(2005)who observed a decrease in ZEN concentration in liquid culture of numerous strains of the species R.stolonifer,R.oryzae, and R.microspo rus(Varga et al.,2005).A reduction of detectable ZEN caused by adsorption on cell material of the investigated fungal strains also has to be considered. Pure yeast as well as yeast cell wall products is able to adsorb ZEN(Yiannikouris et al.,2004;Fr€u hauf et al., 2012).Therefore,a control experiment using autoclaved (121°C;20min)liquid culture material of inactivated Rhizopus spp.and A.oryzae was conducted in a like man-ner with ZEN(0.1l g mLÀ1).After an incubation period of3h,the remaining ZEN amount was measured.In the different incubated fungal cultures,detectable free ZEN decreased to<20%.The pronounced loss of ZEN due to passive adsorption may be caused by the fact that a rela-tively low initial concentration was chosen.Nevertheless, in all incubations except the inactivated control experi-ments,distinct signals for ZEN and ZEL derivatives were visible in the LC-MS/MS analysis.Thus,both metabolism and adsorption of ZEN occur simultaneously.126 A.Brodehl et al.ZEN metabolite pattern of Rhizopus and Aspergillus speciesFor elucidation of ZEN metabolite pattern,the focus was set on the investigation of known ZEN conjugates:glu-cosides and sulfates.Additionally,we searched for reduc-tive metabolites such as the two isomers of ZEL and their sulfated products.The main focus of this study was on the comparison of the investigated fungal cultures regarding to the occurrence of predominant ZEN metab-olites and also to identify promising strains for biosyn-thesis purposes.The formation of b-ZEL was not observed,but the appearance of a-ZEL was shown for four of the investi-gated fungi(Fig.1).The glycosylation of ZEN could be observed for eight of the nine strains,whereas all selected fungi catalyzed the formation of ZEN-14-S.ZEN-14-Glc is known to be a natural metabolite formed by Rhizopus species(Kamimura,1986).Also,the formation of ZEN-16-Glc,a substance discovered only recently by Berthiller and coworkers(Kovalsky Paris et al.,2014),could be conﬁrmed as a fungal metabolite in the present study for theﬁrst time.ZEN-16-Glc is predominantly formed by the Rhizopus strains R.oryzae DSM907,DSM908, R.stolonifer DSM855,and R.oligosporus LMH1133T, whereas glycosylation of ZEN to ZEN-14-Glc was obtained for R.oryzae DSM906and DSM908in consid-erable amounts.Qualitative identiﬁcation of the detected ZEN-sulfate is based on a comparison of the retention time of a ZEN-14-S standard with the sulfate ester metabolite of ZEN formed in our experiments.We suggest that the identiﬁed signal in the HPLC chromatogram can be attributed to ZEN-14-S exclusively,because the selected HPLC gradient can separate both ZEN glucosides.Thus, the potential for separation of ZEN sulfates can be assumed.However,co-elution of ZEN-14-S and ZEN-16-S cannot be excluded.Also for ZEL,the formation of a sulfate ester could be conﬁrmed as a novel fungal metabolite formed in eight of the nine strains.With regard to the signal intensity of the various ZEN metabolites in the MS analysis,the investigated fungi can be divided into three groups.While the Rhizopus strains R.oryzae DSM907and R.stolonifer DSM855predomi-nantly catalyze the glycosylation of ZEN,R.oryzae DSM 906and R.oligosporus DSM1964,as well as A.oryzae DSM1864and A.oryzae NBRC100959,form sulfated ZEN metabolites on a larger scale.Rhizopus strains DSM 908,DSM1834,and R.oligosporus LMH1133T have shown a homogeneous pattern of both metabolite classes (exemplarily shown in Fig.2).a-ZEL-S:a novel metabolite from fungiFor theﬁrst time,the formation of a-ZEL-S as a fungal metabolite was observed,as indicated by monitoring the mass transitions of ZEL-sulfate[M–H]À:399.2amu(pre-cursor ion)to ZEL with m/z319.0(product ion)at a retention time of8.7min in the HPLC-MS/MS(Fig.3, solid line).The identity of the novel sulfate conjugatewasFig.1.Zearalenone(ZEN)metabolites formed by different fungal species72h after supplementation of the fungal culture in liquid media PDB with ZEN.All metabolites showing a peak intensity exceeding a threshold area of1000counts have been included for the evaluation.FEMS Microbiol Lett359(2014)124–130ª2014Federation of European Microbiological Societies. Fungal biotransformation of zearalenone127conﬁrmed by a single signal at the same retention time for a second mass transition of m/z 399.2to m/z 275.2;this fragment corresponds to the most abundant product ion of ZEL in ESI-negative-MS (Pfeiffer et al.,2011).By analyzing the ZEL-sulfate metabolite in product ion scan mode,the fragmentation pattern was recorded (Fig.4):The mass spectrum showed one major signal at m/z 319.0of [ZEL-H]Àresulting from the loss of a sulfonic group (SO 3À)of 80amu.The mass difference between [ZEL-H]Àand the fragment ion at m/z 300.9was 18amu which represents the elimination of a water molecule [ZEL-H-H 2O]À.The fragment ion at m/z 275.2corre-sponds to [ZEL-H-CO 2]À.The fragment ions at m/z 174.1and m/z 159.7are speciﬁc for ZEL and indicate bond cleavages and rearrangements in the ring system (Z €ollner et al.,2003).Subsequently,the detected ZEL-sulfate has been exam-ined by sulfatase treatment of PDB media containing ZEL-sulfate.To a media sample of R.oryzae DSM 908also containing ZEN-14-S,sulfatase was added,and after incubation (3h,37°C),the resulting metabolite spec-trum was measured (Fig.3).The HPLC-MS chromato-gram recorded after the cleavage reveals two new signals corresponding to ZEN and a -ZEL,while the signals of the sulfate esters have disappeared.The formation of a -ZEL from the conjugate of interest due to sulfatase treatment provides strong evidence that the unknown metabolite formed by various fungal strains is a -ZEL-S.However,no information of the site of the sulfation of a -ZEL can be given at the moment.To date,a -ZEL-S has not been described as a microbial metabolite.As the cleavage of ZEN from its sulfate andglucose conjugates was shown recently (Dall’Erta et al.,2013;Kovalsky Paris et al.,2014),also a more compre-hensive investigation of the novel a -ZEL-S seems advisable.Quantitative estimation of metabolite formationAn assessment of the formed metabolite quantity is ham-pered by the lack of analytical standards of sufﬁcient pur-ity.To get an approximate idea of the quantitative dimensions of ZEN conversion,a semi-quantiﬁcation of the formed ZEN metabolites was carried out for two of the fungal incubations.For ZEN-14-S and a -ZEL-S,rela-tive response factors have been determined for theusedFig.2.Reconstructed total ion chromatogram (TIC)with mass transitions of a -zearalenol (a -ZEL),a -zearalenol-sulfate (a -ZEL-S),zearalenone (ZEN),zearalenone-O -14-glucoside (ZEN-14-Glc),zearalenone-O -16-glucoside (ZEN-16-Glc),and zearalenone-14-sulfate (ZEN-14-S)analyzed in liquid media from Rhizopus microsporus var.chinensis DSM 1834incubated with ZEN for 72h.bined ion chromatogram of the mass transitions for a -zearalenol (a -ZEL),a -zearalenol-sulfate (a -ZEL-S),zearalenone (ZEN),and zearalenone-14-sulfate (ZEN-14-S)obtained for the analysis of liquid media incubated with Rhizopus oryzae DSM 908before (solid line)and after (dotted line)sulfatase catalyzed hydrolysis of ZEN-14-S and a -ZEL-S to ZEN and a-ZEL.Fig.4.Product ion scan spectrum of the selected precursor ion a -zearalenol-sulfate ([M-H]À,m/z 399.2)in the liquid media of Rhizopus oryzae DSM 908.128 A.Brodehl et al.ESI-MS method by applying an enzymatic cleavage with sulfatase and subsequent quantiﬁcation of the released ZEN and a-ZEL.The resulting response factors(i.e.peak area of conjugate divided by peak area of the free form) are11for ZEN-14-S compared to ZEN and16for a-ZEL-S compared to a-ZEL.These data are in accor-dance with previously publishedﬁndings,stating that for ESI-MS analysis,ZEN-sulfate is detectable even at very low levels caused by its high ionization efﬁciency(Vendl et al.,2010).Semi-quantiﬁcation of the ZEN glucosides was conducted using standards that are not of certiﬁed purity and concentration.For R.oligosporus LMH1133T after the3-day incubation period,6%of the initially added ZEN amount remained;53%were recovered in the form of the metabolites ZEN-14-S(2%),ZEN-16-Glc (42%),a-ZEL(8%),and a-ZEL-S(1%).Thus,the metab-olism of R.oligosporus LMH1133T directly isolated from tempeh production(Hering et al.,1991)may lead to a conjugation of ZEN in these fermented foods.Addi-tionally,the metabolite formation of A.oryzae NBRC 100959has been investigated.The sum of formed ZEN metabolites is about58%which is composed of ZEN-14-S(52%)and a-ZEL-S(6%).The remained ZEN amount was1%.This fully sequenced genome of the Aspergillus strain possesses blocks of speciﬁc sequences which are enriched for genes involved in secondary metabolism (Machida et al.,2005).This will allow for subsequent molecular analysis of genes and their corresponding enzymes that are involved in ZEN metabolism.The results also indicate that the adsorbed ZEN(about80%) is readily available for biotransformation in vital fungi, because more than50%of ZEN was metabolized and present in an extractable form in the media or even metabolized before being adsorbed to the fungal matrix. Analytical studies of ZEN contamination of fermented foods must be extended to conjugated mycotoxins.To enable a quantitative monitoring of conjugated ZEN derivatives,the preparation of pure reference standards is mandatory.Additionally,formation of ZEN adducts with fungal matrix or media components are of particular interest,because a signiﬁcant proportion of approximately 50%of the initially spiked ZEN remain undetected.In sum,this study showed that the investigated fungal strains have a high potential for ZEN conjugation by metabolic processes.Five different metabolites of ZEN have been detected to be formed by Rhizopus and Asper-gillus species which were co-incubated with ZEN.The formation of a-ZEL-S and ZEN-16-Glc as fungal myco-toxin conjugates is described for theﬁrst time.All these derivatives are not covered by most routine analysis due to the lack of the pure compounds.The development of reference standards is required to allow for the quantita-tive measurement of ZEN metabolites.We consider the exploitation of the metabolic capabilities of the fungi demonstrated in our study as an option for providing such standards.AcknowledgementsThe authors thank Jenny Straßner and Ines Wedell for support during the microbiological investigations.We are grateful to Prof.Franz Berthiller for providing us with ZEN-14-S,ZEN-14-Glc,and ZEN-16-Glc.Our thanks also go to Prof.Bernward Bisping for the supply of R.oli-gosporus CD(LMH1133T).ReferencesBerthiller F,Crews C,Dall’Asta C,De Saeger S,Haesaert G, Karlovsky P,Oswald IP,Seefelder W,Speijers G&Stroka J (2013)Masked mycotoxins:a review.Mol Nutr Food Res57: 165–186.Chancharoonpong C,Hsieh P-C&Sheu S-C(2012) Production of enzyme and growth of Aspergillus oryzae S. on soybean koji.Int J Biosci Biochem Bioinforma2:228–231. Dall’Erta A,Cirlini M,Dall’Asta M,Del Rio D,Galaverna G& Dall’Asta C(2013)Masked mycotoxins are efﬁciently hydrolyzed by human colonic microbiota releasing their aglycones.Chem Res Toxicol26:305–312.De Boevre M,Di Mavungu JD,Landschoot S,Audenaert K, Eeckhout M,Maene P,Haesaert G&De Saeger S(2012) Natural occurrence of mycotoxins and their masked forms in food and feed products.World Mycotoxin J5:207–219. Di Menna ME,Lauren DR&Smith WA(1991)Effect of incubation temperature on zearalenone production by strains of Fusarium crookwellense.Mycopathologia116:81–86.EFSA(2011)Scientiﬁc opinion on the risk for public health related to the presence of zearalenone in food.EFSA J9: 2197.El-Sharkawy SH(1989)Microbial transformation of zearalenone III.Formation of2,4-O-b-diglucoside.Acta Pharm Jugosl39:303–310.El-Sharkawy SH,Selim MI,AﬁﬁMS&Halaweish FT(1991) Microbial transformation of zearalenone to a zearalenone sulfate.Appl Environ Microbiol57:549–552.Fr€u hauf S,Schwartz H,Ottner F,Krska R&Vekiru E(2012) Yeast cell based feed additives:studies on aﬂatoxin B-1and zearalenone.Food Addit Contam Part A Chem Anal Control Expo Risk Assess29:217–231.Hachmeister KA&Fung DYC(1993)Tempeh:a moldmodiﬁed indigenous fermented food made from soybeans and/or cereal grains.Crit Rev Microbiol19:137–188. Hering L,Bisping B&Rehm HJ(1991)Patterns and formation of fatty-acids at tempe fermentation by several strains of Rhizopus sp.Fett Wiss Technol93:303–308. Holland HL(1981)Microbial hydroxylation of steroids.7. hydroxylation of b-nortestosterone and related-compoundsFEMS Microbiol Lett359(2014)124–130ª2014Federation of European Microbiological Societies. Fungal biotransformation of zearalenone129by Rhizopus-arrhizus ATCC-11145,and C-13nuclear magnetic-resonance spectra of some b-norsteroids.Can J Chem59:1651–1655.Jard G,Liboz T,Mathieu F,Guyonvarc’h A,Andr e F, Delaforge M&Lebrihi A(2010)Transformation of zearalenone to zearalenone-sulfate by Aspergillus spp.World Mycotoxin J3:183–191.Jong SC&Birmingham JM(1989)United-States patents and fungal cultures.Mircen J Appl Microbiol5:411–450. Kamimura H(1986)Conversion of zearalenone to zearalenone glycoside by Rhizopus sp.Appl Environ Microbiol52:515–519. Kovalsky Paris MP,Schweiger W,Hametner C,St€u ckler R, Muehlbauer GJ,Varga E,Krska R,Berthiller F&Adam G (2014)Zearalenone-16-O-glucoside:a new masked mycotoxin.J Agric Food Chem62:1181–1189.Larsson Alminger M,Eklund-Jonsson C,Kidman S&Langton M (2012)Starch microstructure and starch hydrolysis in barley and oat tempe during in vitro digestion.Food Dig3:53–62. Lee YW,Kim JG,Chung DH,Roh PU&Pestka JJ(1991) Natural occurrence of zearalenone in rice and soybean produced in Korea.Mycotoxin Res7:69–72.Machida M,Asai K,Sano M et al.(2005)Genome sequencing and analysis of Aspergillus oryzae.Nature438:1157–1161. Metzler M,Pfeiffer E&Hildebrand AA(2010)Zearalenone and its metabolites as endocrine disrupting chemicals.World Mycotoxin J3:385–401.Pfeiffer E,Kommer A,Dempe JS,Hildebrand AA&Metzler M (2011)Absorption and metabolism of the mycotoxin zearalenone and the growth promotor zeranol in Caco-2 cells in vitro.Mol Nutr Food Res55:560–567.Plasencia J&Mirocha CJ(1991)Isolation and characterization of zearalenone sulfate produced by Fusarium spp.Appl Environ Microbiol57:146–150.Schollenberger M,M€u ller HM,R€uﬂe M,Suchy S,Plank S& Drochner W(2006)Natural occurrence of16Fusarium toxins in grains and feedstuffs of plant origin from germany.Mycopathologia161:43–52.Shier WT,Shier AC,Xie W&Mirocha CJ(2001)Structure-activity relationships for human estrogenic activity in zearalenone mycotoxins.Toxicon39:1435–1438.Varga J,P e teri Z,T a bori K,T e ren J&V a gv€o lgyi C(2005) Degradation of ochratoxin A and other mycotoxins by Rhizopus isolates.Int J Food Microbiol99:321–328.Vendl O,Crews C,MacDonald S,Krska R&Berthiller F (2010)Occurrence of free and conjugated Fusarium mycotoxins in cereal-based food.Food AdditContam Part A Chem Anal Control Expo Risk Assess27: 1148–1152.Wiesel I,Rehm HJ&Bisping B(1997)Improvement of tempe fermentations by application of mixed cultures consisting of Rhizopus sp.and bacterial strains.Appl Microbiol Biotechnol 47:218–225.Yiannikouris A,Francßois J,Poughon L,Dussap CG,Bertin G, Jeminet G&Jouany JP(2004)Adsorption of zearalenone by beta-D-glucans in the Saccharomyces cerevisiae cell wall.J Food Prot67:1195–1200.Z€o llner P,Leitner A,Jodlbauer J,Mayer BX&Lindner W (2003)Improving LC-MS/MS analyses in complex food matrices,part II–mass spectrometry.LC-GC Eur16:354–362.130 A.Brodehl et al.。

菜豆R2R3-MYB_基因家族鉴定及其在BCMV_侵染后的表达分析

山西农业科学 2023，51（9）：1020-1033Journal of Shanxi Agricultural Sciences菜豆R2R3-MYB 基因家族鉴定及其在BCMV 侵染后的表达分析王古悦，唐慕宁，牛静雅，冯雪（山西农业大学植物保护学院，山西太谷 030801）摘要：为了明确菜豆R2R3-MYB 基因家族的成员及受到BCMV 侵染后的表达情况，利用生物信息学方法对菜豆R2R3-MYB 基因家族成员进行鉴定和系统分析，同时利用BCMV 侵染菜豆后在显症期的转录组数据筛选可能与BCMV 侵染调控相关的R2R3-MYB 基因家族成员，并对这些基因在病毒侵染不同阶段的表达模式进行实时荧光定量PCR 分析。

结果表明，菜豆R2R3-MYB 基因家族共有159个成员，不均匀分布于菜豆11条染色体上，多含有2个内含子，编码184~554个氨基酸，均为亲水性蛋白。

系统进化关系将菜豆R2R3-MYB 基因家族成员分为32个亚组（P1~P32），同一亚组成员具有高度保守的基因结构和基序。

菜豆基因组内共线性分析表明，片段复制事件和串联复制事件均进行了纯化选择。

转录组数据显示，BCMV 侵染前后菜豆接种叶和系统叶分别有20、33个差异表达基因；qRT -PCR 分析表明，这些基因在病毒侵染后表达均有变化，其中，PvMYB18、PvMYB33、PvMYB92、PvMYB93在侵染早期和显症期均呈现先升高后降低的趋势，而PvMYB29、PvMYB97、PvMYB124、PvMYB128、PvMYB134仅在侵染早期剧烈响应，这些基因的表达特点揭示其可能参与了菜豆对BCMV 侵染不同阶段应答反应的调控。

关键词：菜豆；R2R3-MYB 基因家族；BCMV ；表达分析中图分类号：S643.1 文献标识码：A 文章编号：1002‒2481（2023）09‒1020‒14Identification of R2R3-MYB Gene Family in Common Bean （Phaseolusvulgaris L.）and Expression Analysis after BCMV InfectionWANG Guyue ，TANG Muning ，NIU Jingya ，FENG Xue（College of Plant Protection ，Shanxi Agricultural University ，Taigu 030801，China ）Abstract ：The purpose of this study is to clarify the members of R2R3-MYB gene family in common bean and their expression after being infected by BCMV. Identification and systematic analysis of R2R3-MYB gene family members in common bean was conducted by bioinformatics method. Meanwhile, R2R3-MYB gene family members that may be related to the regulation of BCMV infection were screened by transcriptome data obtained at symptomatic stage after BCMV infection of common bean, and the expression patterns of these genes at different stages of virus infection were analyzed by qRT -PCR. The results showed that total there were 159 members of R2R3-MYB gene family in common bean, they were unevenly distributed on 11 chromosomes of common bean, most of them contained 2 introns, and encoded 184-554 amino acids, all of them were hydrophilic proteins. R2R3-MYB gene family members in common bean were divided into 32 subgroups(P1-P32) according to the phylogenetic relationship, and the same subgroup members had highly conserved gene structures and motifs. The collinearity analysis in the genome of common bean showed that both segmental replication events and tandem replication events had evolved under purifying selection. Transcriptome data showed that there were 20 and 33 differentially expressed genes in the inoculated leaves and system leaves of common bean before and after BCMV infection, respectively. qRT -PCR analysis showed that the expression of these genes changed after virus infection, among which PvMYB18, PvMYB33, PvMYB92, and PvMYB93 showed a trend of increasing first and then decreasing at the early and symptomatic stages of viral infection. However, PvMYB29, PvMYB97, PvMYB124, PvMYB128, and PvMYB134 only responded strongly at the early stage of infection. The expression characteristics of these genes suggested that they might be involved in the regulation of common bean response to BCMV infection at different stages.Key words ：common bean; R2R3-MYB gene family; BCMV; expression analysisdoidoi:10.3969/j.issn.1002-2481.2023.09.07收稿日期：2023-01-28基金项目：国家自然科学基金青年项目（32202247）；山西省自然科学研究面上项目（20210302123367）；山西省回国留学人员科研资助项目（2020-063）作者简介：王古悦（1997-），女，山西长治人，在读硕士，研究方向：植物病毒基因功能及寄主互作关系。

基于转录组的漆树MYB转录因子的筛选及分析

西北林学院学报2021,36(1)：108116Journal of Northwest Forestry Universitydoi：10.3969/j.issn.1001-7461.2021.01.15基于转录组的漆树MYB转录因子的筛选及分析谢冬冬】，王武萍2,何学高】，黄晓华1*(1.西北农林科技大学林学院，陕西杨陵712100；.中华全国供销合作总社西安生漆涂料研究所，陕西西安710000)摘要:MYB转录因子家族是植物最大的转录因子家族之一，在植物生长发育、形态建成、次生代谢等的调控中起着重要作用。

本研究在漆树转录组测序的基础上，应用生物信息学方法，筛选到48个漆树MYB转录因子，依据MYB保守域的特点将其分为3类：1条R-MYB序列，21条R2R3-MYB序列和26条R]-MYB序列。

结果表明，蛋白序列分析显示，48条漆树MYB转录因子大部分属疏水性蛋白，且无规则卷曲和a螺旋在二级结构中占有较大比例。

GO分析发现漆树R2R-MYB蛋白共注释到生物学过程、细胞组分和分子功能3大类功能的27个亚类。

系统进化分析将48个漆树MYB转录因子分为了8个亚组。

表达谱数据分析表明，漆树MYB基因的表达具有组织特异性，11个在叶片中高水平表达，21个在茎中的表达量较高。

研究结果为今后进一步解析漆树MYB转录因子的结构和生物学功能奠定基础，有助于进一步研究漆树MYB转录因子在次生代谢产物中的调控功能。

关键词：漆树；MYB转录因子；基因家族；生物信息学；GO功能注释;基因表达中图分类号:S794.2文献标志码:A文章编号10017461(021)01010809Screening and Analysis of MYB Transcription Factors Based on Transcriptome Datain Toxicodendron vernicfluumXIE Dong-dong1,WANG Wu-ping2,HE Xuegao1,HUANG Xiao-hua1*(1.College of Forestry,Northwest A&-F University犢angling712100^Shaanxi,China；2.Xian Research Instituts of Chinese Lacquer All China Federabion of Supply and'Marketing CooperaLiuss^Xi7an710000, Shaanxi,China/Abstract:The transcription factor family of MYB is one of the most abundant families in plant.It plays im-portantrolesinregulatinggrowthanddevelopment，physiologicalmetabolism，ce l morphologyandpa t ern formation in plant.In this study,a total of48MYB transcription factors of Toxicodendron vemicifluum were screened by bioinformatics methods on the basis of transcriptome sequencing,which were classified into3distinct groups according to the structure of MYB domain,,e.,1R?-MYB,21R2R3-MYB and26 R1-MYB.Protein sequence analysis showed that most MYB transcription factors were hydrophobic proteins withalargeproportionofrandomcoilsandalphahelicesinthesecondarystructure GO(GeneOntology) annotation showed that those proteins could be categorized into27function subgroups of three main categories:biological processes,cellular components and molecular function.Phylogenetic analysis showed that 48MYBtranscriptionfactorsweredividedinto8distinctgroups Expressionprofilesanalysisshowedthat the expression of T.vemicifluum MYB had tissues specificity,11MYB genes were high-level expression in the leaves while21were higher in the stems.The research wi ll lay the foundation for further analysis of thestructureandbiologicalfunctionsof T vernicifluum MYBtranscriptionfactorsinthefuture，andwi l helpthefurtherstudyofMYBtranscriptionfactorsintheregulationofsecondary metabolites收稿日期：2020-03-04修回日期：2020-04-11基金项目：国家重点研发计划项目：漆树丰产经营与精细产品开发技术研究(017YFD0600705)作者简介：谢冬冬。

我对转基因生物的看法英语作文

我对转基因生物的看法英语作文Genetically modified organisms or GMOs have been a topic of much debate and controversy in recent years. As an individual, I have given this issue a lot of thought and have developed my own perspective on it. In this essay, I will share my views on GMOs and the various considerations surrounding this complex topic.To begin, it is important to understand what exactly genetically modified organisms are. GMOs are living organisms whose genetic material has been artificially manipulated in a laboratory through genetic engineering. This technology allows for the transfer of genetic traits from one organism to another, including the transfer of genes between different species. The primary purpose of genetic modification is to introduce new characteristics or traits into an organism, such as increased resistance to pests, diseases, or environmental stresses.Proponents of GMOs argue that this technology has the potential to offer a wide range of benefits to both human society and the environment. One of the most commonly cited advantages of GMOsis their ability to improve food security and nutrition. By creating crops that are more resilient to pests, diseases, and harsh environmental conditions, genetic modification can help to increase agricultural yields and ensure a more reliable food supply, especially in regions where food scarcity is a pressing issue. Additionally, some GMO crops have been engineered to have enhanced nutritional profiles, such as increased vitamin or mineral content, which could help to address issues of malnutrition and micronutrient deficiencies.Another potential benefit of GMOs is their ability to reduce the environmental impact of agriculture. Certain GMO crops have been designed to require fewer chemical inputs, such as pesticides and fertilizers, which can have detrimental effects on the surrounding ecosystems. By reducing the reliance on these harmful chemicals, GMOs may contribute to more sustainable and environmentally-friendly agricultural practices.Furthermore, proponents argue that genetic modification can be a powerful tool in the fight against various diseases and medical conditions. For example, some research has explored the use of genetically engineered organisms, such as bacteria or viruses, as potential treatments for certain cancers or infectious diseases. Additionally, the production of certain pharmaceuticals and medical treatments may be facilitated by the use of GMOs, potentially leading to more affordable and accessible healthcare solutions.However, it is important to acknowledge that the use of GMOs is not without its critics and concerns. One of the primary criticisms is the potential risk of unintended consequences. The long-term effects of introducing genetically modified organisms into the environment and food supply are not yet fully understood, and there are concerns about the potential for unpredictable and irreversible consequences. For instance, the transfer of genes from GMOs to wild or non-genetically modified organisms could have unforeseen ecological impacts, potentially disrupting delicate natural balances and ecosystems.Another concern is the potential for GMOs to have negative impacts on human health. While proponents argue that GMOs are safe for consumption, there is ongoing scientific debate and uncertainty surrounding the potential health risks associated with the consumption of genetically modified foods. Some studies have suggested potential links between GMO consumption and various health issues, such as allergic reactions, organ damage, and even the development of antibiotic-resistant bacteria. However, the scientific consensus on the safety of GMOs remains divided, and more long-term research is needed to fully understand the potential health implications.Additionally, there are ethical and social concerns surrounding theuse of GMOs. Some argue that the patenting and corporate control of genetically modified organisms raise issues of fairness, accessibility, and the equitable distribution of the potential benefits of this technology. There are also concerns about the impact of GMOs on traditional farming practices and the livelihoods of small-scale farmers, who may be unable to compete with the large-scale production of genetically modified crops.Furthermore, the issue of GMO labeling and consumer choice is a contentious one. Many consumers argue that they have a right to know whether the food they are purchasing and consuming contains genetically modified ingredients, and that the lack of clear and mandatory labeling requirements is a violation of their right to make informed choices. Proponents of GMOs, on the other hand, argue that such labeling requirements may be unnecessary and could potentially mislead consumers by implying that GMOs are inherently unsafe or undesirable.In conclusion, my views on genetically modified organisms are complex and nuanced. I recognize the potential benefits that GMOs can offer in terms of improving food security, reducing environmental impact, and advancing medical research. However, I am also cognizant of the valid concerns and uncertainties surrounding the long-term consequences of genetic modification, both in terms of human health and the environment. I believe thatfurther scientific research, transparent regulatory oversight, and inclusive public dialogue are necessary to ensure that the development and use of GMOs are done in a responsible and ethical manner that prioritizes the well-being of both people and the planet.Ultimately, I believe that the decision to embrace or reject GMOs should be based on a careful consideration of the available evidence, the potential risks and benefits, and the broader societal and ethical implications. As an individual, I strive to stay informed on this issue and to form my own opinions based on a balanced and critical assessment of the available information. I encourage others to do the same, and to engage in constructive discussions and debates that can help to shape the future of this important and complex technology.。

Multiple Repeats of a Promoter Segment Causes Transcription

Multiple Repeats of a Promoter Segment Causes Transcription Factor Autoregulation in Red Apples WRichard V.Espley,a,b Cyril Brendolise,a David Chagne´,c Sumathi Kutty-Amma,a Sol Green,a Richard Volz,dJo Putterill,b Henk J.Schouten,e Susan E.Gardiner,c Roger P.Hellens,a and Andrew C.Allan a,1a New Zealand Institute for Plant and Food Research Limited,Mt Albert Research Centre,Auckland1025,New Zealandb School of Biological Sciences,University of Auckland,Auckland1142,New Zealandc New Zealand Institute for Plant and Food Research Limited,Palmerston North4442,New Zealandd New Zealand Institute for Plant and Food Research Limited,Havelock North4157,New Zealande Plant Research International,6700AA Wageningen,The NetherlandsMutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to color phenotypes.Generally,this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin.Here,we describe a rearrangement in the upstream regulatory region of the gene encoding an apple(Malus3 domestica)anthocyanin-regulating transcription factor,MYB10.We show that this modiﬁcation is responsible for increas-ing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit ﬂesh.This rearrangement is a series of multiple repeats,forming a minisatellite-like structure that comprisesﬁve direct tandem repeats of a23-bp sequence.This MYB10rearrangement is present in all the red foliage apple varieties and species tested but in none of the whiteﬂeshed varieties.Transient assays demonstrated that the23-bp sequence motif is a target of the MYB10protein itself,and the number of repeat units correlates with an increase in transactivation by MYB10protein.We show that the repeat motif is capable of binding MYB10protein in electrophoretic mobility shift assays.Taken together, these results indicate that an allelic rearrangement in the promoter of MYB10has generated an autoregulatory locus,and this autoregulation is sufﬁcient to account for the increase in MYB10transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant.INTRODUCTIONAntioxidants are a key component of the health properties of fruit,and there is mounting epidemiological evidence that com-pounds such asﬂavonoids and anthocyanins can signiﬁcantly reduce the incidence of chronic illnesses when part of a regular healthy diet(Knekt et al.,2002).It has been proposed that the combination of phytochemicals in fresh apple(Malus3domes-tica),includingﬂavonoids and anthocyanins,produces the strong antioxidant activity essential to maintain good health (Eberhardt et al.,2000),and there has been a surge of interest in varieties with higher levels of antioxidants,such as phenolics. Recently,genetically engineered tomatoes(Solanum lycopersi-cum)showing high levels of anthocyanins have been shown to increase the longevity of cancer-susceptible mice(Butelli et al., 2008),while a study using anthocyanin-rich maize(Zea mays) showed an increase in protection against ischemia-reperfusion injury in rats(Toufektsian et al.,2008).The fruitﬂesh(cortex)of most apple varieties is white or off-white in color.The skin is usually green or red,with the red anthocyanins accumulating in response to developmental,hor-monal,and light signals(Ubi et al.,2006).However,there are a number of high anthocyanin,red-ﬂeshed apple varieties origi-nating from the wild-apple forests of Central Asia,including Malus pumila var Niedzwetzkyana(Harris et al.,2002)and Malus3domestica‘Red Field’Open Pollinated(OP)(‘Red Field’; Espley et al.,2007).These high anthocyanin varieties possess a dramatic phenotype with highly pigmented vegetative,ﬂoral, and fruit tissues.Many steps in the plant anthocyanin pathway have been described through analysis of natural mutants with several examples of biosynthetic gene-related mutations leading to phenotypic changes.In particular,studies onﬂower color poly-morphism have shown that mutations can determineﬂower color.However,these are mostly caused by the insertion of transposons into genes or deletion of transposons from genes, such as the anthocyanin structural genes chalcone synthase (Habu et al.,1998),dihydroﬂavonol4-reductase(Inagaki et al., 1994),and anthocyanin synthase(also know as leucoanthocya-nidin dioxygenase;Hisatomi et al.,1997).There are many examples of regulation of anthocyanin bio-synthesis by MYB transcription factors in diverse plant species (Allan et al.,2008).Small changes to these MYB proteins can have a marked effect on phenotype(Schwinn et al.,2006). Disruption of MYB gene expression can result in more severe phenotypes.For example,in grape(Vitis vinifera),a retrotrans-poson-induced mutation in the promoter region of mybA1leads1Address correspondence to aallan@.The author responsible for distribution of materials integral to theﬁndings presented in this article in accordance with the policy describedin the Instructions for Authors()is:Andrew C.Allan(aallan@).W Online version contains Web-only data./cgi/doi/10.1105/tpc.108.059329The Plant Cell,Vol.21:168–183,January2009,ã2009American Society of Plant Biologiststo a loss of anthocyanin accumulation in berry skin(Kobayashi et al.,2004),although further investigation has revealed that multiple mutations in an adjacent MYB gene also account for the difference in grape berry color(Walker et al.,2007).Mutations in genes encoding another major family of anthocyanin regulators, the basic helix-loop-helix(bHLH)transcription factors,also pro-duce anthocyanin-related phenotypic changes.These include the Rc mutation in rice(Oryza sativa),which accounts for the white pericarp of most rice varieties(Sweeney et al.,2007).These characterized anthocyanin regulator mutations result in a loss or a restricted distribution of anthocyanin.In some instances within the phenylpropanoid pathway,mutations can result in gain of function.TT2(a MYB transcription factor),TT8(a bHLH tran-scription factor),and TTG1(contains a WD40domain)directly regulate both BANYULS,encoding a core enzyme in proantho-cyanidin biosynthesis,and TT8expression at the transcriptional level in a self-activated feedback loop(Debeaujon et al.,2003; Baudry et al.,2004,2006).Recently,there have been a number of studies of the tran-scriptional regulation of the anthocyanin pathway in apple(Takos et al.,2006;Ban et al.,2007;Espley et al.,2007).Two genes, MYB1(Takos et al.,2006)and MYBA(Ban et al.,2007),have been described as being responsible for apple skin color,while we previously demonstrated that the MYB10gene regulated ﬂesh color(Espley et al.,2007).MYB1,MYB10,and MYBA share at least98%identity at the nucleotide residue level.At the deduced amino acid level,MYB1and MYBA are identical and differ from MYB10in three amino acids(Ban et al.,2007),but it is not clear whether these genes are alleles or tightly linked genes. In the anthocyanin-accumulating red-ﬂeshed variety‘Red Field,’anthocyanin levels are regulated by MYB10,with elevation of transcript levels of MYB10correlated with higher levels of the anthocyanin biosynthetic gene transcripts(Espley et al.,2007). MYB10has also been shown to cosegregate with the redﬂesh and foliage phenotype(Chagne´et al.,2007),suggesting that the Rni locus that described the redﬂesh phenotype may be an allelic variant at the MYB10locus.Overexpression of MYB10in a white-ﬂeshed,green-leaved apple variety results in transgenic plants with a red phenotype(Espley et al.,2007).We found that the white-ﬂeshed‘Paciﬁc Rose’and red-ﬂeshed‘Red Field’varieties carry MYB10alleles encoding identical proteins(Espley et al.2007),suggesting that differences in transcriptional regu-lation of MYB10are responsible for the ectopic accumulation of anthocyanin in‘Red Field.’Here,we report a23-bp repeat motif in the upstream regulatory region of alleles of MYB10found only in red-ﬂeshed apples.This allele is autoregulatory;the MYB10protein is able to bind and transactivate its own promoter,leading to an increase in transcript levels of MYB10and the level of anthocyanin throughout the plant. RESULTSIsolation of the Upstream Regulatory Region of MYB10To investigate further the molecular basis of the red-ﬂeshed phenotype,we used genome walking to isolate the upstream regulatory sequence of MYB10.This sequence was cloned from both white-ﬂeshed apple varieties,Malus3domestica‘Sciros’(‘Paciﬁc Rose’),Malus3domestica‘Granny Smith,’and Malus3domestica‘Royal Gala’and red-ﬂeshed Malus3domestica‘RedField’open pollinated(a cross between‘Wolf River’and Maluspumila var Niedzwetzkyana).Apples are outcrossing,and as aresult they are highly heterozygous.Our isolated DNA fragmentsrevealed two sequences of different sizes for the same region.One sequence was present in all four varieties and showedlimited variation between varieties.As an example,a comparisonof the sequence from‘Granny Smith’with‘Paciﬁc Rose’revealedsix single nucleotide polymorphisms(SNPs),ﬁve single basedeletions,and four single base insertions over the1700bp ofpromoter analyzed.The other fragment contained an insertion of ;100bp.This latter version was only found in the promoter region of the red-ﬂeshed,red-foliaged variety Malus3domes-tica‘Red Field’OP.The insertion consisted of a23-bp sequence,duplicated in a complex pattern ofﬁve near-perfect tandemrepeats(Figure1A).We deﬁne this duplication as a minisatellitebased on the size of the duplicated units.A single version of the23-bp sequence is found in all varieties tested(both red andwhite-ﬂeshed)located;30bases downstream of the mini-satellite insertion site.Between this potential donor sequence and the minisatellite lies a dinucleotide microsatellite.Figure1B shows a schematic of these elements in both the promoters isolated from white-ﬂeshed varieties with one repeat unit(R1)and those from red-ﬂeshed varieties with six repeat units(R6).Se-quence alignment of the longest cDNA clones identiﬁed through 59rapid ampliﬁcation of cDNA ends(Espley et al.,2007)with genomic sequence locates a transcription start site at the G nucleotide at position262from the translational start ATG (indicated by an arrow in Figure1B).A Minisatellite Is Associated with the EctopicAnthocyanin PhenotypePreviously,we showed that MYB10cosegregates with Rni,a locus associated with redﬂesh and red foliage phenotypes in apple(Chagne´et al.,2007).PCR ampliﬁcation of the promoter region from red and white-ﬂeshed varieties consistently showed that the R6minisatellite repeat motif was ampliﬁed in all the plants with red-ﬂeshed fruit(Figure2).The genealogy of these apple varieties is detailed in the Supplemental Methods online.We determined the association of the minisatellite with the red-ﬂeshed phenotype by sequencing the region encompassing the minisatellite over19apple varieties with diverse fruit phenotypes (11red and eight whiteﬂeshed;Table1).A number of sequence variations were found in the upstream region(e.g.,SNPs at positions2612and2245from the ATG start site;Table1),but only the minisatellite polymorphism is associated with the ele-vated accumulation of anthocyanins that causes redﬂesh and red foliage.The same region was PCR ampliﬁed from a further set of77apple varieties taken from two collections of Malus species,and in each case the product corresponding to the minisatellite motif was absent in the green foliage,white-ﬂeshed varieties(see Supplemental Table1and Supplemental Figure 1online).All the white-ﬂeshed varieties tested contained only the R1version,while the red-ﬂeshed apple varieties contained both R1and R6,or R6only(Table1).Autoregulation of an Apple MYB169Induction of Anthocyanin Pigmentation by R 6:MYB10in Tobacco and ApplePrevious studies have shown that when MYB10was fused to the cauliﬂower mosaic virus 35S promoter (35S)and coinﬁltrated into tobacco (Nicotiana tabacum )with a 35S:bHLH3construct encoding a potential apple bHLH cofactor,a strong increase in anthocyanin pigment could be detected at the inﬁltration site (Espley et al.,2007).We therefore compared the level of antho-cyanin that accumulated in tobacco leaves inﬁltrated with Agro-bacterium tumefaciens suspensions with the MYB10gene driven by either the R 1or R 6promoter sequences.Similar levels of anthocyanin were observed when either R 6:MYB10or 35S:MYB10was coinﬁltrated with 35S:bHLH3(Figure 3A).However,we were unable to detect anthocyanin accumulation in leaves inﬁltrated with the R 1:MYB10,both with and without 35S:bHLH3.To investigate the properties of the R 6promoter in apple,we transformed ‘Royal Gala’(green leaves and white ﬂesh)with MYB10driven by the R 6promoter.While the R 1promoter is found in ‘Royal Gala,’R 6is not (Figure 2).We have already shown that when ‘Royal Gala’was transformed with 35S:MYB10,red callus is produced and regenerates to produce red plants (Espley et al.,2007).We observed a similar callus phenotype when ‘Royal Gala’is transformed with R 6:MYB10,with bright red areas on regenerating callus (Figure 3B),while no pigmentation was seen on regenerating apple callus transformed with R 1:MYB10.Similarly,callus transformed with an empty vector cassetteshowed no pigmentation.To further study the development of the ‘Royal Gala’R 6:MYB10transgenic lines,transformed plant-lets were micrografted onto ‘M9’rootstock,adapted from a previously described protocol (Lane et al.,2003).The grafted plants remained highly pigmented (Figure 3C),with a phenotype similar to the red foliaged,red-ﬂeshed variety Malus 3domestica ‘Red Field’OP and to the previously described 35S:MYB10transgenic lines (Espley et al.,2007)(Figure 3D).These results strongly suggest that the R 6promoter allele is responsible for the increased accumulation of anthocyanins in red-ﬂeshed apple types.Autoregulation of the MYB10Promoter in the Dual Luciferase Transient Tobacco AssayOne possible result of the minisatellite insertion within the pro-moter of MYB10is an increase in the basal activity of the promoter.A dual luciferase assay was used to quantify the activity of the two versions of the MYB10promoter.R 1and R 6promoter sequences were fused to LUCIFERASE (LUC )and transactivation of the LUC gene measured relative to 35S:RENILLA (REN )by measurement of luminescence after transient expression in Nicotiana benthamiana .The R 1and R 6promoters showed little difference in activity,as determined by the ratio of luminescence produced by MYB10-promoter-LUC to 35S-REN (Figure 4A).However,when 35S:MYB10was coinﬁltratedwithFigure 1.Sequence and Schematic of the Minisatellite Region in the MYB10Promoter in Red-Fleshed Apple Varieties.(A)Relative positions of the repeat units in the R6promoter allele of the MYB10gene.Unit 1lies downstream of the microsatellite and is predicted to be the origin of the repeat units in the minisatellite.Units 2to 5form the minisatellite and are found only in the MYB10promoter from red-ﬂeshed varieties.They are near-perfect copies of unit 1except that unit 3is dissected by units 4and 5.(B)Schematic of the differences between the R 1(found in both white-and red-ﬂeshed apple varieties)and the R 6promoter showing positions of the ATG translation start site,repeat unit 1,the microsatellite,and the minisatellite in R 6.To the right of the schematics are representative phenotypes:(i)the white-ﬂeshed Malus 3domestica ‘Sciros’(Paciﬁc Rose)and (ii)the red-ﬂeshed Malus 3domestica ‘Red Field’OP.The predicted transcription start site (TSS)is indicated by an arrow at position À62relative to the ATG start site.170The Plant CellMYB10-promoter-LUC constructs,it transactivated the pro-moters.When 35S:MYB10was coinﬁltrated with R 1:LUC ,there was a slight elevation in transactivation (Figure 4A),while 35S:MYB10transactivated R 6:LUC >30-fold compared with back-ground promoter activity.There was a sevenfold increase in the effect of 35S:MYB10on R 6:LUC compared with R 1:LUC (0.6966SE 0.02compared with 0.0986SE 0.001)(Figure 4A).This level of transactivation suggests that the presence of the repeat motifs in R6:LUC act as an enhancer of MYB10-induced transcription,resulting in the elevated LUC levels.To further investigate the effect of the promoter on MYB10transcript accumulation and predicted protein levels,we re-peated this assay,replacing the 35S:MYB10with either the R 1or R 6promoter fused to MYB10.Results indicated that the high transcript abundance of MYB10fused to the R 6promoter en-ables transactivation of the reporter,particularly when the re-porter is fused to R 6(Figure 4B).The results show a similar level of activity to the 35S promoter.With the R 1:LUC fusion,R 6:MYB10appears to be more effective at transactivating the promoter than 35S:MYB10.The R 1:MYB10fusion did not inﬂu-ence transactivation to the same extent.Repeat Number Inﬂuences Transactivation in the Dual Luciferase Transient Tobacco AssayA series of constructs were built to test the inﬂuence of the number of 23-bp repeat units in the upstream region of MYB10on MYB10-induced transcriptional activity.These constructs were based on the native promoter sequences,but with repeat units ranging from one (R 1)to six (R 6),and were then fused to the LUC reporter (Figure 5A)and assayed as above.To test the spatial effect that the presence of the repeat-containing minisatellite sequence might exert on other unidentiﬁed regulatory regions,a further construct (R 1+)was built where the minisatellite sequence from R 6was replaced with nonspeciﬁc DNA of the same length from a cloning vector (Promega).The results show a strong positivecorrelationFigure 2.PCR Ampliﬁcation of the MYB10Promoter Region from 10Apple Varieties.PCR Ampliﬁcation of the MYB10promoter region gave two fragments:a 496-bp fragment corresponding to R 6that is present only in red-ﬂeshed varieties (lanes 1to 6)and is absent in white-ﬂeshed varieties (lanes 7to 10)and a 392-bp fragment corresponding to R 1,present in both types.Images of fruit and corresponding gel lanes are as follows.1,open-pollinated (OP)Malus 3domestica ‘Mildew Immune Seedling’;2,M.3purpurea ‘Aldenhamen s i s’;3,M.3domestica var Niedzwetzkyana ;4,M.3domestica ‘Prairieﬁre’;5,OP M.3domestica var ‘Geneva’;6,OP M.3domestica ‘Pomme Grise’;7,M.3domestica ‘Granny Smith’;8,M.3domestica ‘Royal Gala’;9,M.3domestica ‘Fuji’;10,M.3domestica ‘Braeburn;11,no template control.’Autoregulation of an Apple MYB 171between the number of repeat units and the activation of the promoter by35S:MYB10(Figure5B).When the promoter con-structs were coinﬁltrated with35S:MYB10,there is basal activity from both R1and R1+and an increasing activation from R2to R6. We have previously shown the dependency of MYB10in activat-ing promoters of the anthocyanin biosynthetic pathway on a cofactor bHLH(Espley et al.,2007).In this assay,activation by MYB10of all the promoters(R1through to R6)is enhanced with the addition of35S:bHLH3(Figure5B).Deletion Analysis of the MYB10Promoter Using the Dual Luciferase Transient Tobacco AssayResults from the dual luciferase transient tobacco assays de-scribed above suggest that the MYB10transcription factor positively autoregulates its own transcription by interacting with the DNA repeat present in a single copy in the R1promoter and in six copies in the R6promoter.In addition,a putative cofactor bHLH3enhances the ability of MYB10to transactivate these promoters.To further deﬁne regions in the promoter of MYB10responsi-ble for transcriptional regulation,deletion variants of the pro-moters(R1and R6)were analyzed.Deletions were designed to deﬁne the distal end of the R1and R6promoters and the possible role of a59G-box in bHLH-related transactivation(Figure6).A second set of deletions(Figure7)was designed to delete the native R1repeat from the R6promoter and to probe the role of the microsatellite,and data are presented in the next section. Figure6A shows the proximal promoter fragments of834and 405bp(R1)and934and505bp(R6)(D a and D b,respectively)selected for testing.The G-box identiﬁed at the extreme59end of the isolated promoter fragments was absent from these variants.A third variant(R1D c and R6D c)with362-and463-bp proximal fragments,respectively,and with a restored distal region,in-cluding the G-box,was tested.A fourth variant,D d,contained just the distal fragment,including the G-box,and was included as a control for transactivation levels.The combinatorial interaction of cis-acting elements that are recognized by transcription factors,such as bHLH and MYB, produce a cooperative regulation of the spatial distribution of ﬂavonoids(Hartmann et al.,2005).Analysis of the promoter regions using the database PLACE(Higo et al.,1999)predicted many cis-acting elements.Flavonoid-related MYB binding elements were identiﬁed,including MYB26PS(Uimari and Strommer,1997),MYB core(Planchais et al.,2002),MYBPLANT (Tamagnone et al.,1998),and MYBPZM(Grotewold et al.,1994). It was also noted that the23-bp repeat motif starts with GTTAG,a partial sequence from reported MYB binding domains(Grotewold et al.,1994;Uimari and Strommer,1997;Romero et al.,1998)and that this sequence is also adjacent to the microsatellite.Further bHLH-related cis-acting elements,E-boxes(CACATG)(Atchley and Fitch,1997;Heim et al.,2003),and G-boxes(CACGTG) (Giuliano et al.,1988;Williams et al.,1992)were identiﬁed,and their location is shown in Figure6A.All of the R1variants showed a reduction in the relative level of transactivation.In the presence of MYB10,transactivation was barely detectable for any of the deletion constructs.The coinﬁl-tration of35S:MYB10and35S:bHLH3partly restored this activity for R1D a,although it remained at half the level of the full-length R1 promoter.This suggests that elements in the distal region areTable1.Association of the Minisatellite Motif with the Red-Fleshed PhenotypeVariety Flesh Color G/T SNP PosÀ612Minisatellite Motif A/T SNP PosÀ245Malus3‘Babine’Red G:G R1:R6A:TMalus3‘Okanagan’a Red G:G R1:R6A:TMalus3‘Simcoe’a Red G:G R6:R6T:TMalus3‘Slocan’a Red G:T R1:R6A:TMalus3‘Formosa’a Red G:T R1:R6A:TM.sieversii629319a Red G:G R6:R6T:TM.sieversii FORM35(33-01)a Red G:T R1:R6A:TM.sieversii01P22a Red G:G R6:R6T:TM.sieversii3563.q a Red G:G R6:R6T:TM.aldenhamii Red T:T R1:R6A:TMalus3domestica‘Red Field’OP Red G:T R1:R6A:TMalus3domestica‘Close’White G:T R1:R1A:TMalus3domestica‘Mr Fitch’White T:T R1:R1A:AMalus3domestica‘Guldborg’White G:T R1:R1A:TMalus3domestica‘Alkmene’White T:T R1:R1A:AMalus3domestica‘Red Melba’White T:T R1:R1A:AMalus3domestica‘Rae Ime’White G:G R1:R1T:TMalus3domestica‘Lady Williams’White T:T R1:R1A:AMalus3domestica‘Granny Smith’White G:T R1:R1A:AAssociation test(r2)0.18510.491a Plant material and DNA samples supplied by Charles J.Simon and Philip L.Forsline(Agricultural Research Service,USDA).A number of sequence variations were found in the promoter region,but only the minisatellite polymorphism was associated with the elevated accumulation of anthocyanins.All11red-ﬂeshed varieties tested have the duplicated repeat motifs found in the MYB10R6promoter,while these were absent from the white-ﬂeshed varieties.The positions shown are relative to the ATG translation start site.172The Plant Cellcritical for transactivation.This was more evident with R 1D b,which showed no transactivation,indicating that there are key elements in both the distal (2834to 21704from the ATG translation start site)and middle regions (2405and 2834)of the promoter.The loss of these elements from both regions in R 1D b prevented transactivation.The restoration of the distal region,including the G-box,in R 1D c does partly restore trans-activation but at a lower level than R 1D a.A low level of trans-activation was observed with R 1D d.The results from the inﬁltration assay for R 6D a and R 6D b with 35S:MYB10showed a similar level of transactivation,indicating that the R 6promoter can function in the most extensively deleted version.However,both variants were capable of only half the transactivation level of the intact R 6promoter,suggesting that upstream regulatory elements,beyond the minisatellite,are important for the very high level of transactivation previously seen with the R 6promoter and MYB10.Various MYB-related cis -acting elements were deleted in these variants,including MYB-CORE,MYBPZM,and MYBPLANT elements.Restoration of the distal promoter region in R 6D c did not elevate transactivation.Low levels of transactivation were detected with R 6D d.The coinﬁltration of 35S:MYB10and 35S:bHLH3with the R 6variants resulted in little difference to transactivation levels,although restoration of the distal region R 6D c partly restored activity.It is clear from the data that transactivation by R 1is dependent on distal upstream elements that encompass the predicted bHLH binding sites.It is also clear that the R 6promoter is partly dependent on distal upstream elements but that good levels of transactivation can be initiated by just the proximal 505-bp region.This version differs from the equivalent R 1version primarily by the presence of the minisatellite.Deletion of the Primary Repeat Unit and Microsatellite in the MYB10PromoterAnother set of constructs was built to test the importance of the primary repeat unit and the adjacent microsatellite on autoacti-vation of the MYB10promoter by MYB10(Figure 7A).The primary repeat unit,designated repeat 1(Figure 1B),was deleted from both the R 1:LUC and R 6:LUC constructs,producing a version of the MYB10promoter lacking the 23-bp unit (R 0)and a version with the ﬁve units from the minisatellite-like insertion from R 6(R 5).In addition,the microsatellite was independentlydeletedFigure 3.The Native Apple Promoter Containing the Minisatellite In-duces Ectopic Anthocyanin Accumulation.(A)Red coloration around the inﬁltration site in the leaves of N.tabacum 8d after transient transformation with (i)R 6:MYB10and (ii)35S:MYB10but not with (iii)R 1:MYB10.All three patches were coinﬁltrated with 35S:bHLH3.(B)Photographs of regenerating ‘Royal Gala’callus transformed with (i)R 6:MYB10and (ii)R 1:MYB10.Red pigmentation was observed only on callus transformed with R 6:MYB10.Emerging shoots showed a similar pigmented phenotype as shown for 35S:MYB10in Espley et al.(2007).(C)Representative R 6:MYB10plantlet (left),micrografted onto ‘M9’rootstock shown next to ‘Royal Gala’control (right).Pigmentation levels of the R 6:MYB10lines remained high.(D)Leaves at same developmental stage taken from representative lines of (i)35S:MYB10(Espley et al.,2007),(ii)‘Royal Gala’control,and (iii)R 6:MYB10.Autoregulation of an Apple MYB 173from R 1and R 6,producing R 1-MS and R 6-MS ,respectively.The partial MYB binding site (GTTAG)adjacent to the microsatellite was retained in these constructs.A construct was also built that contained neither a repeat unit nor a microsatellite,R 0-MS .For the R 1deletions (R 0,R 1-MS ,and R 0-MS ),there was little detectable transactivation activity with the coinﬁltration of 35S:MYB10(Figure 7B).A similar result was seen when R 0and R 0-MS were coinﬁltrated with both 35S:MYB10and 35S:bHLH3.How-ever,transactivation was evident when R 1and R 1-MS were coinﬁltrated with both MYB10and bHLH3.This result suggests that the repeat unit is necessary for autoregulation of the pro-moter with MYB10/bHLH3but that the presence of the micro-satellite is not critical.Similar deletions of the R 6version of the promoter showed a large reduction in transactivation for both R 5and R 6-MS when inﬁltrated with MYB10.Coinﬁltration with both 35S:MYB10and 35S:bHLH3appeared to restore transactivation with R 6-MS but to a much lesser extent with R 5.For the R 6promoter,it appears that the ﬁrst repeat unit is critical to enable high levels of trans-activation and that the microsatellite is required for the highest level of transactivation.Analysis of MYB10Protein:DNA Interaction Using Electrophoretic Mobility Shift AssaysTo test directly MYB10binding to DNA,we analyzed in vitro binding using electrophoretic mobility shift assays (EMSAs).Recombinant His-tagged MYB10protein was puriﬁed from Escherichia coli and incubated with DNA probes representing the 23-bp repeated unit or mutations of the unit (Figure 8A).EMSA showed a band shift when the oligonucleotide probe,corresponding to the repeat motif found in the native promoter,and recombinant MYB10were bound (Figure 8B).In control EMSA experiments,a nonspeciﬁc His-tagged protein did not complex with the repeat motif (see Supplemental Figure 2online).When cold competitor DNA of the same sequence (r1)was added in 200-fold excess,this binding was reduced,indi-cating that the reaction was speciﬁc.To further determine active sites important for the oligonucleotide-protein binding,we used mutated versions of the motif as cold competitors (Figure 8B).In these versions,nucleotides were substituted across ﬁve different areas of the R 1repeat unit (Figure 8A).The results indicated that the extreme 39and 59of the repeat unit are less important for binding as both m1,which no longer contains the partial se-quence from predicted MYB binding domains (GTTAG),and m5were able to compete off the r1probe to a similar extent as the native r1competitor.Three other competitors carrying mutations in the inner part of the sequence (m2,m3,and m4)were less able to compete,suggesting that this region,comprising the se-quence ACTGGTAGCTATT,is critical for binding.To conﬁrm this ﬁnding,we substituted areas of the repeat unit sequence with randomly selected sequence from the apple actin gene (accession number CN938023)and performed binding assays.For these,substitutions were made at both the outer 39and 59sequences of the repeat unit (m6)and the entire inner sequence (m7)(Figure 8A).The signal was reduced when m6was used as a competitor,but m7was not able to compete off the r1probe (Figure 8C).When the m6and m7oligos were themselves radiolabeled and used as probes for binding to MYB10protein,the m7probe failed to bind (Figure 8D),while the m6probe bound to the protein and was partially competed off by the inclusion of r1or m6competitor DNA.In the m6assay,an additional band was observed that may be due to nonspeciﬁc binding of the m6probe with partially degraded MYB10protein.DISCUSSIONOur data suggest that a minisatellite in the upstream regulatory DNA region of a gene encoding an apple transcriptionfactor,Figure 4.Interaction of Native Apple Promoters and MYB10in a Dual Luciferase Transient Tobacco Assay.(A)Leaves of N.benthamiana were inﬁltrated with R 1and R 6promoter-LUC fusions on their own or coinﬁltrated with 35S:MYB10,and then luminescence of LUC and REN was measured 3d later and expressed as a ratio of LUC to REN.(B)Comparison of the transactivation activity of R 1and R 6promoter-driven MYB10to 35S:MYB10,when coinﬁltrated with the R 1and R 6promoter-LUC fusions.The results provide a measure for the potential activity of the apple promoters,which shows a signiﬁcant increase in the case of the R 6-driven MYB10.Data in both panels are presented as means (6SE )of six replicate reactions.174The Plant Cell。

Myb转录因子

• In contrast to the highly conserved MYB domain, the other regions of R2R3-MYB proteins are highly variable. • Based on the conservation of the DNAbinding domain and of amino acid motifs in the C terminal domains, R2R3-MYB proteins have been divided into subgroups.
differences in the intensity of shifts, depending on the boxes and Myb proteins used .
• Expression analysis of the PAL, GS1b, PAT, Myb1, Myb4 and Myb8 genes in maritime pine
• Isolation of maritime pine GS1b, PAL and PAT promoters.In silico analysis of putative cis elements
• R2R3Myb proteins bind to the PAL, PpGS1b and PAT AC-II boxes • An in vitro analysis of protein–DNA interactions using an electrophoretic mobility shift assay (EMSA).
• Other MYB factors for which direct targets have been identified seem to target primarily non-transcription factor genes.

3个澳洲坚果MYB蛋白靶基因的克隆与生物信息学分析

3个澳洲坚果MYB蛋白靶基因的克隆与生物信息学分析作者：李冰，李季东，杨祥燕，蔡元保，李穆，曾黎明，黄思婕来源：《农业研究与应用》2021年第04期摘要：【目的】为阐明澳洲坚果MYB（v-myb avian myeloblastosis viral oncogene homolog）蛋白靶基因的功能及分子作用机制提供了科学的理论依据。

【方法】基于澳洲坚果转录组数据和RT-PCR方法克隆澳洲坚果MYB蛋白靶基因，并利用生物信息学分析这些基因编码蛋白质的同源性、系统进化、理化性质、亚细胞定位、跨膜域和信号肽。

【结果】获得了3个新的澳洲坚果MYB蛋白靶基因MiTOM1、MiTOM2和MiTOM3，GenBank登录号分别为MT319120、MT319121和MT319122。

MiTOM1、MiTOM2和MiTOM3都包含典型结构域VHS-ENTH-ANTH，與其它植物来源的TOM蛋白具有极高的序列相似度，与荷花TOM蛋白（XP_010260421.1、XP_010244847.1和XP_010260421.1）的序列相似度分别高达68.93%、75.76%和68.80%。

MiTOM1、MiTOM2和MiTOM3蛋白均为亲水性蛋白。

MiTOM1可能定位于细胞核，为非分泌蛋白和非跨膜蛋白;MiTOM2可能定位于细胞质，为跨膜分泌蛋白;MiTOM3可能定位于细胞核或微体，为非分泌蛋白和非跨膜蛋白。

蛋白二级结构预测显示，MiTOM1、MiTOM2和MiTOM3二级结构主要含有无规则卷曲和α螺旋。

【结论】MiTOM1、MiTOM2和MiTOM3包含TOM家族典型结构域VHS-ENTH-ANTH，可能在澳洲坚果体内物质运输及控制形态发育方面起重要作用。

关键词：澳洲坚果;TOM基因;基因克隆;生物信息学分析中图分类号：S664.9 文献标志码：ACloning and Bioinformatics Analysis of Three MYB Protein Target Genes from Macadamia （Macadamia integrifolia）LI Bing，LI Jidong，YANG Xiangyan，CAI Yuanbao*，LI Mu，ZENG Liming，HUANG Sijie（Guangxi Subtropical Crops Research Institute， Guangxi Academy ofAgricultural Sciences， Nanning， Guangxi 530001， China）Abstract：【Objective】This study provides a scientific theoretical basis for elucidating the function and molecular mechanism of macadamia MYB （v-myb avian myeloblastosis viral oncogene homolog） protein target genes.【Method】Based on the macadamia transcriptome data， 3 MYB protein target genes of macadamia were cloned by RT-PCR method， and the homology，phylogeny， physicochemical properties， subcellular localization， transmembrane domains， and signal peptides of these genes encoding proteins were analyzed by bioinformatics.【Result】Three new MYB target genes MiTOM1， MiTOM2 and MiTOM3 were cloned. The GenBank accession numbers are MT319120， MT319121 and MT319122 respectively. MiTOM1， MiTOM2 and MiTOM3 which contain the typical domain VHS-ENTH-ANTH， have high sequence similarity with other plant TOM proteins. The amino acid sequence similarity between MiTOM1 and the lotus TOM proteins （XP_010260421.1， XP_01024847.1， XP_010260421.1） reach 68.93%， 75.76%，and 68.80%， respectively. MiTOM1， MiTOM2 and MiTOM3 are hydrophilic proteins. MiTOM1 which might be located in the nucleus is non-secretory and non-transmembrane proteins; MiTOM2 which might be located in the cytoplasm is transmembrane secretory proteins; MiTOM3 which might be located in the nucleus or microbodies is non-secretory and non-transmembrane proteins. Protein secondary structure prediction shows that MiTOM1， MiTOM2 and MiTOM3 secondary structures mainly contained irregular crimp and α helix. 【Conclusion】MiTOM1， MiTOM2 and MiTOM3 which include TOM family typical domain VHS-ENTH-ANTH， may play an important role in material transportation and morphological development in macadamia.Key words： Macadamia （Macadamia integrifolia）; TOM genes; gene cloning; bioinformatics analysis转录因子通过识别靶基因上游启动子区高度保守的特异碱基序列并与之结合，对靶基因的表达进行调控，进而调控植物生长发育、生理代谢以及各种逆境胁迫的应答机制。