DsRed and GFP double innoculated

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chapter5-ledger accounts and double entry

FINANCIAL STATEMENTS
Ledger accounts and nominal ledger
1.

The records of transactions, assets and liabilities should be kept in the following ways.
In chronological order, and dated so that transactions can be related to a particular period of time. Build up in cumulative totals. Books of original entry then to summarize these records.
The
wages of employee is the deduction of profit. Any amount paid by a business to its proprietor is treated by accountants as withdrawals of profit (the usual term is appropriations of profit) and not as expenses incurred by the business. The drawings of owner are the deduction of capital.
2.

The entries in the books of prime entry are posted to ledgers.
The books of prime entry are totaled up and two entries will be made in these accounts with each of these totals – this is called double entry. Ledger accounts summarise all the individual transactions listed in the books of pd general ledger, is an accounting record which summarizes the financial affairs of a business. It is the double entry books of account.

形容多个传感器之间可以相互独立的英文单词

形容多个传感器之间可以相互独立的英文单词Title: Describing Sensors that Can Operate IndependentlyIntroductionIn the world of technology and IoT (Internet of Things), sensors play a crucial role in collecting and transmitting data for various applications. These sensors can operate independently, performing their designated functions without the need for constant human intervention. In this article, we will explore and describe various terms used to depict sensors that can function autonomously.1. AutonomousAutonomous sensors refer to devices that can operate or function independently without the need for external control. These sensors are self-sufficient in terms of power supply, data collection, processing, and transmission. The autonomy of these sensors allows them to perform their tasks efficiently without relying on a central system or operator.2. StandaloneStandalone sensors are those that are self-contained and do not rely on other devices for operation. These sensors aredesigned to function independently, collecting and processing data on their own. Standalone sensors are commonly used in applications where mobility, flexibility, or remote operation is required.3. DecentralizedDecentralized sensors operate independently and can communicate with each other without the need for a centralized control system. This decentralized approach allows sensors to collaborate and exchange information to achieve a common goal. Decentralized sensors are often used in distributed sensor networks for monitoring and control applications.4. Self-organizingSelf-organizing sensors have the ability to form networks and coordinate their activities without human intervention. These sensors can establish connections, assign roles, and adapt to changing environments autonomously. Self-organizing sensors are commonly used in dynamic and unpredictable settings where traditional communication methods may not be feasible.5. Ad-hocAd-hoc sensors are capable of forming temporary networks on the fly, without the need for pre-established infrastructure. These sensors can spontaneously connect with each other to share data and collaborate on tasks. Ad-hoc sensors are often used in scenarios where quick deployment and flexibility are essential.ConclusionIn conclusion, sensors that can operate independently play a vital role in various applications, from smart homes and industries to healthcare and environmental monitoring. Understanding and describing the characteristics of these sensors, such as autonomy, standalone capability, decentralization, self-organization, and ad-hoc networking, are essential for designing and implementing efficient sensor systems. By harnessing the power of independent sensors, we can create smarter, more responsive, and interconnected systems for a wide range of practical applications.。

新型双幅板压制滑轮及其制造技术

新型双幅板压制滑轮及其制造技术陈创业朱国和上海振华重工（集团）股份有限公司长兴分公司上海 201913摘要：文中针对原有技术存在的不足，通过理论分析和现场型式试验，自主研发开发了一种新型双幅板压制滑轮，并形成了独特的双幅板压制滑轮制造技术。

压制滑轮的轮缘采用冷加工方式成型,避免了热加工成型如铸造、锻造和热轧滑轮制造过程中不可避免的表面氧化和脱碳问题，而表面氧化和脱碳直接影响滑轮轮缘的表面硬度和服役寿命，且压制加工方式简单、制造成本低，具有独特的优势。

研究结果表明，该款新型双幅板压制滑轮具有结构设计合理、强度和刚度好、自重轻、轮槽耐磨使用寿命长的特点，能够完全替代常规的热轧滑轮。

使用该技术制造的滑轮，已在多个项目中得到应用和验证，创造了良好的经济效益和社会效益，新型双幅板压制滑轮发展前景良好，市场前景广阔。

关键词：起重机；滑轮；压制；双幅板中图分类号：TG457. 25 文献标识码：B 文章编号：1001-0785（2023）13-0077-04Abstract: Considering the shortcomings of the original technology, through theoretical analysis and field type test, a new type of double-plate pressed pulley was independently developed, and a unique manufacturing technology of double-plate pressed pulley was developed. The flange of pressed pulley is formed by cold working, which avoids the inevitable surface oxidation and decarbonization in hot working, such as casting, forging and hot rolling pulley manufacturing. Surface oxidation and decarbonization will directly affect the surface hardness and service life of pulley flange, and the pressed method is simple and the manufacturing cost is low, with unique advantages. The research results show that this new type of double-plate pressed pulley has the characteristics of reasonable structure and design, good strength and stiffness, light weight, long service life of wheel groove wear resistance and so on, and can completely replace the conventional hot rolling pulley. Pulleys manufactured with this technology have been applied and verified in many projects, which has brought good economic and social benefits. Therefore, this new double-plate pressed pulley has a good development prospect and a broad market prospect.Keywords:crane; pulley; suppress; double plate0 引言滑轮作为承载部件之一，广泛应用在各种起重运输机械中。

Development and Applications of CRISPR-Cas9 for Genome Engineering

Leading EdgeReviewDevelopment and Applications ofCRISPR-Cas9for Genome EngineeringPatrick D.Hsu,1,2,3Eric nder,1and Feng Zhang1,2,*1Broad Institute of MIT and Harvard,7Cambridge Center,Cambridge,MA02141,USA2McGovern Institute for Brain Research,Department of Brain and Cognitive Sciences,Department of Biological Engineering, Massachusetts Institute of Technology,Cambridge,MA02139,USA3Department of Molecular and Cellular Biology,Harvard University,Cambridge,MA02138,USA*Correspondence:zhang@/10.1016/j.cell.2014.05.010Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9are enabling the systematic interrogation of mammalian genome function.Analogous to the search function in modern word processors,Cas9can be guided to speciﬁc locations within complex genomes by a short RNA search ing this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice.Cas9-mediated genetic perturbation is simple and scalable,empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review,we describe the development and applications of Cas9for a variety of research or translational applications while highlighting challenges as well as future directions.Derived from a remarkable microbial defense system,Cas9is driving innovative applications from basic biology to biotechnology and medicine.IntroductionThe development of recombinant DNA technology in the1970s marked the beginning of a new era for biology.For theﬁrst time,molecular biologists gained the ability to manipulate DNA molecules,making it possible to study genes and harness them to develop novel medicine and biotechnology.Recent advances in genome engineering technologies are sparking a new revolution in biological research.Rather than studying DNA taken out of the context of the genome,researchers can now directly edit or modulate the function of DNA sequences in their endogenous context in virtually any organism of choice, enabling them to elucidate the functional organization of the genome at the systems level,as well as identify causal genetic variations.Broadly speaking,genome engineering refers to the process of making targeted modiﬁcations to the genome,its contexts (e.g.,epigenetic marks),or its outputs(e.g.,transcripts).The ability to do so easily and efﬁciently in eukaryotic and especially mammalian cells holds immense promise to transform basic sci-ence,biotechnology,and medicine(Figure1).For life sciences research,technologies that can delete,insert, and modify the DNA sequences of cells or organisms enable dis-secting the function of speciﬁc genes and regulatory elements. Multiplexed editing could further allow the interrogation of gene or protein networks at a larger scale.Similarly,manipu-lating transcriptional regulation or chromatin states at particular loci can reveal how genetic material is organized and utilized within a cell,illuminating relationships between the architecture of the genome and its functions.In biotechnology,precise manipulation of genetic building blocks and regulatory machin-ery also facilitates the reverse engineering or reconstruction of useful biological systems,for example,by enhancing biofuel production pathways in industrially relevant organisms or by creating infection-resistant crops.Additionally,genome engi-neering is stimulating a new generation of drug development processes and medical therapeutics.Perturbation of multiple genes simultaneously could model the additive effects that un-derlie complex polygenic disorders,leading to new drug targets, while genome editing could directly correct harmful mutations in the context of human gene therapy(Tebas et al.,2014). Eukaryotic genomes contain billions of DNA bases and are difﬁcult to manipulate.One of the breakthroughs in genome manipulation has been the development of gene targeting by homologous recombination(HR),which integrates exogenous repair templates that contain sequence homology to the donor site(Figure2A)(Capecchi,1989).HR-mediated targeting has facilitated the generation of knockin and knockout animal models via manipulation of germline competent stem cells, dramatically advancing many areas of biological research.How-ever,although HR-mediated gene targeting produces highly pre-cise alterations,the desired recombination events occur extremely infrequently(1in106–109cells)(Capecchi,1989),pre-senting enormous challenges for large-scale applications of gene-targeting experiments.To overcome these challenges,a series of programmable nuclease-based genome editing technologies havebeen1262Cell157,June5,2014ª2014Elsevier Inc.developed in recent years,enabling targeted and efﬁcient modi-ﬁcation of a variety of eukaryotic and particularly mammalian species.Of the current generation of genome editing technolo-gies,the most rapidly developing is the class of RNA-guided endonucleases known as Cas9from the microbial adaptive im-mune system CRISPR (clustered regularly interspaced short palindromic repeats),which can be easily targeted to virtually any genomic location of choice by a short RNA guide.Here,we review the development and applications of the CRISPR-associated endonuclease Cas9as a platform technology for achieving targeted perturbation of endogenous genomic ele-ments and also discuss challenges and future avenues for inno-vation.Programmable Nucleases as Tools for Efﬁcient and Precise Genome EditingA series of studies by Haber and Jasin (Rudin et al.,1989;Plessis et al.,1992;Rouet et al.,1994;Choulika et al.,1995;Bibikova et al.,2001;Bibikova et al.,2003)led to the realization that tar-geted DNA double-strand breaks (DSBs)could greatly stimulate genome editing through HR-mediated recombination events.Subsequently,Carroll and Chandrasegaran demonstrated the potential of designer nucleases based on zinc ﬁnger proteins for efﬁcient,locus-speciﬁc HR (Bibikova et al.,2001,2003).Moreover,it was shown in the absence of an exogenous homol-ogy repair template that localized DSBs can induce insertions or deletion mutations (indels)via the error-prone nonhomologous end-joining (NHEJ)repair pathway (Figure 2A)(Bibikova et al.,2002).These early genome editing studies established DSB-induced HR and NHEJ as powerful pathways for the versatileand precise modiﬁcation of eukaryotic genomes.To achieve effective genome editing via introduction of site-speciﬁc DNA DSBs,four major classes of customizable DNA-binding proteins have been engineered so far:meganucleases derived from microbial mobile genetic elements (Smith et al.,2006),zinc ﬁnger (ZF)nucleases based on eukaryotic transcrip-tion factors (Urnov et al.,2005;Miller et al.,2007),transcription activator-like effectors (TALEs)from Xanthomonas bacteria (Christian et al.,2010;Miller et al.,2011;Boch et al.,2009;Mos-cou and Bogdanove,2009),and most recently the RNA-guided DNA endonuclease Cas9from the type II bacterial adaptive im-mune system CRISPR (Cong et al.,2013;Mali et al.,2013a ).Meganuclease,ZF,and TALE proteins all recognize speciﬁc DNA sequences through protein-DNA interactions.Although meganucleases integrate its nuclease and DNA-binding domains,ZF and TALE proteins consist of individual modules targeting 3or 1nucleotides (nt)of DNA,respectively (Figure 2B).ZFs and TALEs can be assembled in desired combi-nations and attached to the nuclease domain of FokI to direct nucleolytic activity toward speciﬁc genomic loci.Each of these platforms,however,has unique limitations.Meganucleases have not been widely adopted as a genome engineering platform due to lack of clear correspondence between meganuclease protein residues and their target DNA sequence speciﬁcity.ZF domains,on the other hand,exhibit context-dependent binding preference due to crosstalk between adjacent modules when assembled into a larger array (Maeder et al.,2008).Although multiple strategies have been developed to account for these limitations (Gonzaelz et al.,2010;Sander et al.,2011),assembly of functional ZFPs with the desired DNA binding speciﬁcity remains a major challenge that requires an extensive screening process.Similarly,although TALE DNA-binding monomers are for the most part modular,they can still suffer from context-dependent speciﬁcity (Juillerat et al.,2014),and their repetitive sequences render construction of novel TALE arrays labor intensive and costly.Given the challenges associated with engineering of modular DNA-binding proteins,new modes of recognition would signiﬁ-cantly simplify the development of custom nucleases.The CRISPR nuclease Cas9is targeted by a short guide RNA that recognizes the target DNA via Watson-Crick base pairing (Figure 2C).The guide sequence within these CRISPR RNAs typically corresponds to phage sequences,constituting the nat-ural mechanism for CRISPR antiviral defense,but can be easily replaced by a sequence of interest to retarget the Cas9nuclease.Multiplexed targeting by Cas9can now be achieved at unprecedented scale by introducing a battery of short guideFigure 1.Applications of Genome EngineeringGenetic and epigenetic control of cells with genome engineering technologies is enabling a broad range of applications from basic biology to biotechnology and medicine.(Clockwise from top)Causal genetic mutations or epigenetic variants associated with altered biological function or disease phenotypes can now be rapidly and efﬁciently recapitulated in animal or cellular models (Animal models,Genetic variation).Manipulating biological circuits could also facilitate the generation of useful synthetic materials,such as algae-derived,silica-based diatoms for oral drug delivery (Materials).Additionally,precise genetic engineering of important agricultural crops could confer resistance to envi-ronmental deprivation or pathogenic infection,improving food security while avoiding the introduction of foreign DNA (Food).Sustainable and cost-effec-tive biofuels are attractive sources for renewable energy,which could be achieved by creating efﬁcient metabolic pathways for ethanol production in algae or corn (Fuel).Direct in vivo correction of genetic or epigenetic defects in somatic tissue would be permanent genetic solutions that address the root cause of genetically encoded disorders (Gene surgery).Finally,engineering cells to optimize high yield generation of drug precursors in bacterial factories could signiﬁcantly reduce the cost and accessibility of useful therapeutics (Drug development).Cell 157,June 5,2014ª2014Elsevier Inc.1263RNAs rather than a library of large,bulky proteins.The ease of Cas9targeting,its high efﬁciency as a site-speciﬁc nuclease,and the possibility for highly multiplexed modiﬁcations have opened up a broad range of biological applications across basic research to biotechnology and medicine.The utility of customizable DNA-binding domains extends far beyond genome editing with site-speciﬁc endonucleases.Fusing them to modular,sequence-agnostic functional effector domains allows ﬂexible recruitment of desired perturbations,such as transcriptional activation,to a locus of interest (Xu and Bestor,1997;Beerli et al.,2000a;Konermann et al.,2013;Maeder et al.,2013a;Mendenhall et al.,2013).In fact,any modular enzymatic component can,in principle,be substituted,allowing facile additions to the genome engineering toolbox.Integration of genome-and epigenome-modifying enzymes with inducible protein regulation further allows precise temporal control of dynamic processes (Beerli et al.,2000b;Konermann et al.,2013).CRISPR-Cas9:From Yogurt to Genome EditingThe recent development of the Cas9endonuclease for genome editing draws upon more than a decade of basic research into understanding the biological function of the mysterious repetitive elements now known as CRISPR (Figure 3),which are found throughout the bacterial and archaeal diversity.CRISPR loci typically consist of a clustered set of CRISPR-associated (Cas)genes and the signature CRISPR array—a series of repeat sequences (direct repeats)interspaced by variable sequences (spacers)corresponding to sequences within foreign genetic elements (protospacers)(Figure 4).Whereas Cas genes are translated into proteins,most CRISPR arrays are ﬁrst tran-scribed as a single RNA before subsequent processing into shorter CRISPR RNAs (crRNAs),which direct the nucleolytic activity of certain Cas enzymes to degrade target nucleic acids.The CRISPR story began in 1987.While studying the iap enzyme involved in isozyme conversion of alkaline phosphatase in E.coli ,Nakata and colleagues reported a curious set of 29nt repeats downstream of the iap gene (Ishino et al.,1987).Unlike most repetitive elements,which typically take the form of tandem repeats like TALE repeat monomers,these 29nt repeats were interspaced by ﬁve intervening 32nt nonrepetitive sequences.Over the next 10years,as more microbial genomes were sequenced,additional repeat elements were reported from genomes of different bacterial and archaeal strains.Mojica and colleagues eventually classiﬁed interspaced repeat sequences as a unique family of clustered repeat elements present in >40%of sequenced bacteria and 90%of archaea (Mojica et al.,2000).These early ﬁndings began to stimulate interest in such micro-bial repeat elements.By 2002,Jansen and Mojica coined the acronym CRISPR to unify the description of microbial genomic loci consisting of an interspaced repeat array (Jansen et al.,2002;Barrangou and van der Oost,2013).At the same time,several clusters of signature CRISPR-associated (cas )genes were identiﬁed to be well conserved and typically adjacent to the repeat elements (Jansen et al.,2002),serving as a basis for the eventual classiﬁcation of three different types of CRISPR systems (types I–III)(Haft et al.,2005;Makarova et al.,2011b ).Types I and III CRISPR loci contain multiple Cas proteins,now known to form complexes with crRNA (CASCADE complex for type I;Cmr or Csm RAMP complexes for type III)to facilitate the recognition and destruction of target nucleic acids (BrounsFigure 2.Genome Editing Technologies Exploit Endogenous DNA Repair Machinery(A)DNA double-strand breaks (DSBs)are typically repaired by nonhomologous end-joining (NHEJ)or homology-directed repair (HDR).In the error-prone NHEJ pathway,Ku heterodimers bind to DSB ends and serve as a molecular scaffold for associated repair proteins.Indels are introduced when the complementary strands undergo end resection and misaligned repair due to micro-homology,eventually leading to frameshift muta-tions and gene knockout.Alternatively,Rad51proteins may bind DSB ends during the initial phase of HDR,recruiting accessory factors that direct genomic recombination with homology arms on an exogenous repair template.Bypassing the matching sister chromatid facilitates the introduction of precise gene modiﬁcations.(B)Zinc ﬁnger (ZF)proteins and transcription activator-like effectors (TALEs)are naturally occurring DNA-binding domains that can be modularly assembled to target speciﬁc se-quences.ZF and TALE domains each recognize 3and 1bp of DNA,respectively.Such DNA-binding proteins can be fused to the FokI endonuclease to generate programmable site-speciﬁc nucleases.(C)The Cas9nuclease from the microbial CRISPR adaptive immune system is localized to speciﬁc DNA sequences via the guide sequence on its guide RNA (red),directly base-pairing with the DNA target.Binding of a protospacer-adjacent motif (PAM,blue)downstream of the target locus helps to direct Cas9-mediated DSBs.1264Cell 157,June 5,2014ª2014Elsevier Inc.et al.,2008;Hale et al.,2009)(Figure 4).In contrast,the type II system has a signiﬁcantly reduced number of Cas proteins.However,despite increasingly detailed mapping and annotation of CRISPR loci across many microbial species,their biological signiﬁcance remained elusive.A key turning point came in 2005,when systematic analysis of the spacer sequences separating the individual direct repeats suggested their extrachromosomal and phage-associated ori-gins (Mojica et al.,2005;Pourcel et al.,2005;Bolotin et al.,2005).This insight was tremendously exciting,especially given previous studies showing that CRISPR loci are transcribed (Tang et al.,2002)and that viruses are unable to infect archaeal cells carrying spacers corresponding to their own genomes (Mojica et al.,2005).Together,these ﬁndings led to the specula-tion that CRISPR arrays serve as an immune memory and defense mechanism,and individual spacers facilitate defense against bacteriophage infection by exploiting Watson-Crick base-pairing between nucleic acids (Mojica et al.,2005;Pourcel et al.,2005).Despite these compelling realizations that CRISPR loci might be involved in microbial immunity,the speciﬁc mech-anism of how the spacers act to mediate viral defense remained a challenging puzzle.Several hypotheses were raised,including thoughts that CRISPR spacers act as small RNA guides to degrade viral transcripts in a RNAi-like mechanism (Makarova et al.,2006)or that CRISPR spacers direct Cas enzymes to cleave viral DNA at spacer-matching regions (Bolotin et al.,2005).Working with the dairy production bacterial strain Strepto-coccus thermophilus at the food ingredient company Danisco,Horvath and colleagues uncovered the ﬁrst experimental evidence for the natural role of a type II CRISPR system as an adaptive immunity system,demonstrating a nucleic-acid-based immune system in which CRISPR spacers dictate target speci-ﬁcity while Cas enzymes control spacer acquisition and phage defense (Barrangou et al.,2007).A rapid series of studies illumi-nating the mechanisms of CRISPR defense followed shortly and helped to establish the mechanism as well as function of all three types of CRISPR loci in adaptive immunity.By studying the type I CRISPR locus of Escherichia coli ,van der Oost and colleagues showed that CRISPR arrays are transcribed and converted into small crRNAs containing individual spacers to guide Cas nuclease activity (Brouns et al.,2008).In the same year,CRISPR-mediated defense by a type III-A CRISPR system from Staphylococcus epidermidis was demonstrated to block plasmid conjugation,establishing the target of Cas enzyme activity as DNA rather than RNA (Marrafﬁni andSontheimer,Figure 3.Key Studies Characterizing and Engineering CRISPR SystemsCas9has also been referred to as Cas5,Csx12,and Csn1in literature prior to 2012.For clarity,we exclusively adopt the Cas9nomenclature throughout this Review.CRISPR,clustered regularly interspaced short palindromic repeats;Cas,CRISPR-associated;crRNA,CRISPR RNA;DSB,double-strand break;tracrRNA,trans -activating CRISPR RNA.Cell 157,June 5,2014ª2014Elsevier Inc.12652008),although later investigation of a different type III-B system from Pyrococcus furiosus also revealed crRNA-directed RNA cleavage activity(Hale et al.,2009,2012).As the pace of CRISPR research accelerated,researchers quickly unraveled many details of each type of CRISPR system (Figure4).Building on an earlier speculation that protospacer-adjacent motifs(PAMs)may direct the type II Cas9nuclease to cleave DNA(Bolotin et al.,2005),Moineau and colleagues high-lighted the importance of PAM sequences by demonstrating that PAM mutations in phage genomes circumvented CRISPR inter-ference(Deveau et al.,2008).Additionally,for types I and II,the lack of PAM within the direct repeat sequence within the CRISPR array prevents self-targeting by the CRISPR system.In type III systems,however,mismatches between the50end of the crRNA and the DNA target are required for plasmid interference(Marraf-ﬁni and Sontheimer,2010).By2010,just3years after theﬁrst experimental evidence for CRISPR in bacterial immunity,the basic function and mecha-nisms of CRISPR systems were becoming clear.A variety of groups had begun to harness the natural CRISPR system for various biotechnological applications,including the generation of phage-resistant dairy cultures(Quiberoni et al.,2010)and phylogenetic classiﬁcation of bacterial strains(Horvath et al., 2008,2009).However,genome editing applications had not yet been explored.Around this time,two studies characterizing the functional mechanisms of the native type II CRISPR system elucidated the basic components that proved vital for engineering a simple RNA-programmable DNA endonuclease for genome editing. First,Moineau and colleagues used genetic studies in Strepto-coccus thermophilus to reveal that Cas9(formerly called Cas5,Csn1,or Csx12)is the only enzyme within the cas gene cluster that mediates target DNA cleavage(Garneau et al.,2010).Next,Charpentier and colleagues revealed a key component in the biogenesis and processing of crRNA in type II CRISPR systems—a noncoding trans-activating crRNA(tracrRNA)that hybridizes with crRNA to facilitate RNA-guided targeting of Cas9(Deltcheva et al.,2011).This dual RNA hybrid,together with Cas9and endogenous RNase III,is required for processing the CRISPR array transcript into mature crRNAs(Deltcheva et al.,2011).These two studies suggested that there are at least three components(Cas9, the mature crRNA,and tracrRNA)that are essential for recon-stituting the type II CRISPR nuclease system.Given the increasing importance of programmable site-speciﬁc nucleases based on ZFs and TALEs for enhancing eukaryotic genome editing,it was tantalizing to think that perhaps Cas9could be developed into an RNA-guided genome editing system. From this point,the race to harness Cas9for genome editing wason.Figure4.Natural Mechanisms of Microbial CRISPR Systems in Adaptive Immunity Following invasion of the cell by foreign genetic elements from bacteriophages or plasmids(step 1:phage infection),certain CRISPR-associated (Cas)enzymes acquire spacers from the exoge-nous protospacer sequences and install them into the CRISPR locus within the prokaryotic genome (step2:spacer acquisition).These spacers are segregated between direct repeats that allow the CRISPR system to mediate self and nonself recognition.The CRISPR array is a noncoding RNA transcript that is enzymatically maturated through distinct pathways that are unique to each type of CRISPR system(step3:crRNA biogenesis and processing).In types I and III CRISPR,the pre-crRNA transcript is cleaved within the repeats by CRISPR-asso-ciated ribonucleases,releasing multiple small crRNAs.Type III crRNA intermediates are further processed at the30end by yet-to-be-identiﬁed RNases to produce the fully mature transcript.In type II CRISPR,an associated trans-activating CRISPR RNA(tracrRNA)hybridizes with the direct repeats,forming an RNA duplex that is cleaved and processed by endogenous RNase III and other unknown nucleases.Maturated crRNAs from type I and III CRISPR systems are then loaded onto effector protein complexes for target recognition and degradation.In type II systems, crRNA-tracrRNA hybrids complex with Cas9to mediate interference.Both type I and III CRISPR systems use multi-protein interference modules to facilitate target recognition.In type I CRISPR,the Cascade com-plex is loaded with a crRNA molecule,constituting a catalytically inert surveillance complex that rec-ognizes target DNA.The Cas3nuclease is then recruited to the Cascade-bound R loop,mediatingtarget degradation.In type III CRISPR,crRNAs associate either with Csm or Cmr complexes that bind and cleave DNA and RNA substrates,respectively.In contrast,the type II system requires only the Cas9nuclease to degrade DNA matching its dual guide RNA consisting of a crRNA-tracrRNA hybrid.1266Cell157,June5,2014ª2014Elsevier Inc.In2011,Siksnys and colleaguesﬁrst demonstrated that the type II CRISPR system is transferrable,in that transplantation of the type II CRISPR locus from Streptococcus thermophilus into Escherichia coli is able to reconstitute CRISPR interference in a different bacterial strain(Sapranauskas et al.,2011).By 2012,biochemical characterizations by the groups of Charpent-ier,Doudna,and Siksnys showed that puriﬁed Cas9from Strep-tococcus thermophilus or Streptococcus pyogenes can be guided by crRNAs to cleave target DNA in vitro(Jinek et al., 2012;Gasiunas et al.,2012),in agreement with previous bacte-rial studies(Garneau et al.,2010;Deltcheva et al.,2011;Sapra-nauskas et al.,2011).Furthermore,a single guide RNA(sgRNA) can be constructed by fusing a crRNA containing the targeting guide sequence to a tracrRNA that facilitates DNA cleavage by Cas9in vitro(Jinek et al.,2012).In2013,a pair of studies simultaneously showed how to suc-cessfully engineer type II CRISPR systems from Streptococcus thermophilus(Cong et al.,2013)and Streptococcus pyogenes (Cong et al.,2013;Mali et al.,2013a)to accomplish genome editing in mammalian cells.Heterologous expression of mature crRNA-tracrRNA hybrids(Cong et al.,2013)as well as sgRNAs (Cong et al.,2013;Mali et al.,2013a)directs Cas9cleavage within the mammalian cellular genome to stimulate NHEJ or HDR-mediated genome editing.Multiple guide RNAs can also be used to target several genes at once.Since these initial studies,Cas9has been used by thousands of laboratories for genome editing applications in a variety of experimental model systems(Sander and Joung,2014).The rapid adoption of the Cas9technology was also greatly accelerated through a com-bination of open-source distributors such as Addgene,as well as a number of online user forums such as http://www. and . Structural Organization and Domain Architecture ofCas9The family of Cas9proteins is characterized by two signature nuclease domains,RuvC and HNH,each named based on homology to known nuclease domain structures(Figure2C). Though HNH is a single nuclease domain,the full RuvC domain is divided into three subdomains across the linear protein sequence,with RuvC I near the N-terminal region of Cas9and RuvC II/IIIﬂanking the HNH domain near the middle of the pro-tein.Recently,a pair of structural studies shed light on the struc-tural mechanism of RNA-guided DNA cleavage by Cas9. First,single-particle EM reconstructions of the Streptococcus pyogenes Cas9(SpCas9)revealed a large structural rearrange-ment between apo-Cas9unbound to nucleic acid and Cas9in complex with crRNA and tracrRNA,forming a central channel to accommodate the RNA-DNA heteroduplex(Jinek et al., 2014).Second,a high-resolution structure of SpCas9in complex with sgRNA and the complementary strand of target DNA further revealed the domain organization to comprise of an a-helical recognition(REC)lobe and a nuclease(NUC)lobe consisting of the HNH domain,assembled RuvC subdomains,and a PAM-interacting(PI)C-terminal region(Nishimasu et al.,2014) (Figure5A and Movie S1).Together,these two studies support the model that SpCas9 unbound to target DNA or guide RNA exhibits an autoinhibited conformation in which the HNH domain active site is blocked by the RuvC domain and is positioned away from the REC lobe (Jinek et al.,2014).Binding of the RNA-DNA heteroduplex would additionally be sterically inhibited by the orientation of the C-ter-minal domain.As a result,apo-Cas9likely cannot bind nor cleave target DNA.Like many ribonucleoprotein complexes,the guide RNA serves as a scaffold around which Cas9can fold and orga-nize its various domains(Nishimasu et al.,2014).The crystal structure of SpCas9in complex with an sgRNA and target DNA also revealed how the REC lobe facilitates target binding.An arginine-rich bridge helix(BH)within the REC lobe is responsible for contacting the308–12nt of the RNA-DNA het-eroduplex(Nishimasu et al.,2014),which correspond with the seed sequence identiﬁed through guide sequence mutation ex-periments(Jinek et al.,2012;Cong et al.,2013;Fu et al.,2013; Hsu et al.,2013;Pattanayak et al.,2013;Mali et al.,2013b). The SpCas9structure also provides a useful scaffold for engi-neering or refactoring of Cas9and sgRNA.Because the REC2 domain of SpCas9is poorly conserved in shorter orthologs, domain recombination or truncation is a promising approach for minimizing Cas9size.SpCas9mutants lacking REC2retain roughly50%of wild-type cleavage activity,which could be partly attributed to their weaker expression levels(Nishimasu et al., 2014).Introducing combinations of orthologous domain re-combination,truncation,and peptide linkers could facilitate the generation of a suite of Cas9mutant variants optimized for different parameters such as DNA binding,DNA cleavage,or overall protein size.Metagenomic,Structural,and Functional Diversity of Cas9Cas9is exclusively associated with the type II CRISPR locus and serves as the signature type II gene.Based on the diversity of associated Cas genes,type II CRISPR loci are further subdivided into three subtypes(IIA–IIC)(Figure5B)(Makarova et al.,2011a; Chylinski et al.,2013).Type II CRISPR loci mostly consist of the cas9,cas1,and cas2genes,as well as a CRISPR array and tracrRNA.Type IIC CRISPR systems contain only this minimal set of cas genes,whereas types IIA and IIB have an additional signature csn2or cas4gene,respectively(Chylinski et al.,2013). Subtype classiﬁcation of type II CRISPR loci is based on the architecture and organization of each CRISPR locus.For example,type IIA and IIB loci usually consist of four cas genes, whereas type IIC loci only contain three cas genes.However, this classiﬁcation does not reﬂect the structural diversity of Cas9proteins,which exhibit sequence homology and length variability irrespective of the subtype classiﬁcation of their parental CRISPR locus.Of>1,000Cas9nucleases identiﬁed from sequence databases(UniProt)based on homology,protein length is rather heterogeneous,roughly ranging from900to1600 amino acids(Figure5C).The length distribution of most Cas9 proteins can be divided into two populations centered around 1,100and1,350amino acids in length.It is worth noting that a third population of large Cas9proteins belonging to subtype IIA,formerly called Csx12,typically contain around1500amino acids.Despite the apparent diversity of protein length,all Cas9pro-teins share similar domain architecture(Makarova et al.,2011a;Cell157,June5,2014ª2014Elsevier Inc.1267。

日粮蛋白水平对单胃动物肠道消化吸收功能及健康影响的研究进展

Digital PCR Technology and Its Application in Animal ProductionHONG Qihua 1, CHEN Xueqiu 1, JIN Miaoren 2, HU Caihong 1*（1. College of Animal Sciences, Zhejiang University, Zhejiang Hangzhou 310058, China; 2. Healthy (Hangzhou) Husbandry Sci-Tech Co., Ltd., Zhejiang Hangzhou 311261, China ）Abstract: Digital PCR (dPCR) has been the third generation of PCR technology and developed rapidly in recent years. The mixed PCR reaction system is divided into a large number of partitions where a single molecule is amplified independently. The fluorescence signals are then collected, and the results are calculated according to the Poisson distribution and positive ratio. This technique can realize absolute quantification of target nucleic acid in extremely low concentration, and possesses various advantages such as high sensitivity, high accuracy, good reproducibility, and no need of standard curve. In this paper, the basic principle, main advantages, developed platforms of dPCR and its applications in the detection of animal pathogens, genes related to growth and reproduction, feed ingredients, as well as quality analysis of animal products are reviewed.Keywords: Digital PCR; Detection and quantification; Animal production（责任编辑：郑本艳）日粮蛋白水平对单胃动物肠道消化吸收功能及健康影响的研究进展燕磊1,2,3，安沙1,2，蒋梦宇1,2，吕尊周1,2，周桂莲1,2*，呙于明3*（1.山东新希望六和集团有限公司，山东青岛 266061；2.农业部饲料及畜禽产品质量安全控制重点实验室，四川新希望六和科技创新有限公司，畜禽饲料与畜产品质量安全控制四川省重点实验室，四川成都 610101；3.中国农业大学动物科学技术学院，北京 100193）摘要：动物肠道具有消化吸收和免疫防御的双重作用，肠道健康受肠道的营养物质吸收、黏膜免疫以及微生物等多方面影响。

论新加坡的“红绿灯”等食品包装正面标签系统的意义和启示

食安管理论新加坡的“红绿灯”等食品包装正面标签系统的意义和启示张蕊，黄玉坤，马嫄，张广峰，陈晟*（西华大学食品与生物工程学院，四川成都 610039）摘要：本文介绍了新加坡在食品包装正面标签（Front of Package，FOP）方面所做的努力，包括被称为“红绿灯”的食品营养分级标签和“更健康的选择”标签，并分析了这些标签的设计逻辑。

介绍了新加坡的“本地生产的良好农产品”标签、食品安全管理优良标签等其他辅助性的食品标签，并探讨了这些标签的积极作用和借鉴意义，为提高我国的食品安全、食品营养管理水平提供参考。

关键词：食品安全；食品营养；食品标签；新加坡The Significance and Enlightenment of Singapore’s “Traffic Light” and Other Food Front-of-Package Labeling Systems ZHANG Rui, HUANG Yukun, MA Yuan, ZHANG Guangfeng, CHEN Sheng*(School of Food and Bio-Engineering, Xihua University, Chengdu 610039, China) Abstract: This article describes Singapore’s efforts in front of package (FOP), including food nutri-grade labelling known as “trafﬁc lights” and “healthier choice” labeling, and analyzes the design logic of these labels. It also introduced other complementary food labels such as Singapore’s “locally produced good produce” label, food safety management excellent label, and so on. The positive role of these labels and the possibility of reference are discussed, which provides reference for improving the management level of food safety and food nutrition in China.Keywords: food safety; food nutrition; food labelling; Singapore预包装食品的标签直接影响了消费者的知情权、选择权，也间接影响了消费者的饮食健康。

Neutrino oscillations and neutrinoless double-beta decay

a rXiv:h ep-ph/9912427v12Dec1999DFTT 71/99hep-ph/9912427Neutrino oscillations and neutrinoless double-βdecay C.Giunti INFN,Sezione di Torino,and Dipartimento di Fisica Teorica,Universit`a di Torino,Via P.Giuria 1,I–10125Torino,Italy Abstract We consider the scheme with mixing of three neutrinos and a mass hierarchy.We shown that,under the natural assumptions that massive neutrinos are Majorana particles and there are no unlikely ﬁne-tuned cancellations among the contributions of the diﬀerent neutrino masses,the results of solar neutrino experiments imply a lower bound for the eﬀective Majorana mass in neutri-noless double-βdecay.We also discuss brieﬂy neutrinoless double-βdecay in schemes with mixing of four neutrinos.We show that one of them is favored by the data.Presented at TAUP’99,6–10September 1999,College de France,Paris,France.Typeset using REVT E XNeutrino oscillations[1]have been observed in solar and atmospheric neutrino experi-ments.The corresponding neutrino mass-squared diﬀerences are∆m2sun∼10−6−10−4eV2(MSW),(0.1)in the case of MSW transitions,or∆m2sun∼10−11−10−10eV2(VO),(0.2)in the case of vacuum oscillations,and∆m2atm∼10−3−10−2eV2.(0.3)These values of the neutrino mass-squared diﬀerences and the mixing required for the ob-served solar and atmospheric oscillations are compatible with the simplest and most natural scheme with three-neutrino mixing and a mass hierarchy:∆m2sunm1≪m2≪m3∆m2atm.(0.4)This scheme is predicted by the see-saw mechanism[1],which predicts also that the three light massive neutrinos are Majorana particles.In this case neutrinoless double-βdecay (ββ0ν)is possible and its matrix element is proportional to the eﬀective Majorana mass| m |=k U2ek m k,(0.5)where U is the neutrino mixing matrix and the sum is over the contributions of all the mass eigenstate neutrinosνk(k=1,2,3).In principle the eﬀective Majorana mass(0.5)can be vanishingly small because of can-cellations among the contributions of the diﬀerent mass eigenstates.However,since the neutrino masses and the elements of the neutrino mixing matrix are independent quanti-ties,if there is a hierarchy of neutrino masses such a cancellation would be the result of an unlikelyﬁne-tuning,unless some unknown symmetry is at work.Here we consider the possibility that no such symmetry exist and no unlikelyﬁne-tuning operates to suppress the eﬀective Majorana mass(0.5)[2].In this case we have| m |≃maxk| m |k,(0.6) where| m |k is the absolute value of the contribution of the massive neutrinoνk to| m |:| m |k≡|U ek|2m k.(0.7) In the following we will estimate the value of| m |using the largest| m |k obtained from the results of neutrino oscillation experiments.Let us consider ﬁrst | m |3,which,taking into account that in the three-neutrino scheme under consideration m 3≃ ∆m 2atm ,is given by| m |3≃|U e 3|2 21− ∆m 2atm .One can seefrom Fig.1that theresults ofthe CHOOZ experiment imply that | m |3 2.7×10−2eV,the results of the Super-Kamiokande experiment imply that | m |3 3.8×10−2eV,and the combination of the results of the two experiments drastically lowers the upper bound to| m |3 2.5×10−3eV .(0.9)Since there is no lower bound for |U e 3|2from experimental data,| m |3could be much smaller than the upper bound in Eq.(0.9).Hence,the largest contribution to | m |could come from | m |2≡|U e 2|2m 2.In the scheme (0.4)m 2≃ ∆m 2sun and,since |U e 3|2is very small,|U e 2|2≃11−sin 22ϑsun [8],where ϑsun is the two-neutrino mixing angle used in the analysis of solar neutrino data.Therefore,| m |2is given by| m |2≃11−sin 22ϑsun ∆m 2sun .From Fig.2one can see that the LMA solution of thesolar neutrino problem implies that7.4×10−4eV | m |2 6.0×10−3eV .(0.13)Assuming the absence ofﬁne-tuned cancellations among the contributions of the three neu-trino masses to the eﬀective Majorana mass,if|U e3|2is very small and| m |3≪| m |2, from Eqs.(0.6)and(0.13)we obtain7×10−4eV | m | 6×10−3eV.(0.14) Hence,assuming the absence of an unlikelyﬁne-tuned suppression of| m |,in the case of the LMA solution of the solar neutrino problem we have obtained a lower bound of about 7×10−4eV for the eﬀective Majorana mass inββ0νdecay.Also the small mixing angle MSW(SMA)and the vacuum oscillation(VO)solutions of the solar neutrino problem imply allowed ranges for| m |2,but their values are much smaller than in the case of the LMA ing the99%CL allowed regions obtained in[10]from the analysis of the total rates measured in solar neutrino experiments we have 5×10−7eV | m |2 10−5eV(SMA),(0.15)10−6eV | m |2 2×10−5eV(VO).(0.16) If futureββ0νexperiments willﬁnd| m |in the range shown in Fig.2and future long-baseline experiments will obtain a stronger upper bound for|U e3|2,it would mean that| m |2gives the largest contribution to the eﬀective Majorana mass,favoring the LMA solution of the solar neutrino problem.On the other hand,if futureββ0νexperiments willﬁnd| m |in the range shown in Fig.2and the SMA or VO solutions of the solar neutrino problem will be proved to be correct by future solar neutrino experiments,it would mean that| m |3gives the largest contribution to the eﬀective Majorana mass and there is a lower bound for the value of|U e3|2.Finally,let us consider brieﬂy the two four-neutrino mixing schemes compatible with all neutrino oscillation data[1],including the indications in favor ofνµ→νe oscillations found in the short-baseline(SBL)LSND experiment[11]:(A)∆m2atmm1<m2<∆m2sunm3<m4∆m2SBL,(0.17)(B)∆m2sunm1<m2<∆m2atmm3<m4∆m2SBL.(0.18)Since the mixing ofνe with the two massive neutrinos whose mass-squared diﬀerence gener-ates atmospheric neutrino oscillations is very small[1],the contribution of the two“heavy”mass eigenstatesν3andν4to the eﬀective Majorana mass(0.5)is large in scheme A and very small in scheme B.Hence,the eﬀective Majorana mass is expected to be relatively large in scheme A and strongly suppressed in scheme B.In particular,in the scheme A the SMA solution of the solar neutrino problem implies a value of| m |larger than the the present upper bound obtained inββ0νdecay experiments[12]and is,therefore,disfavored. Furthermore,since the measured abundances of primordial elements produced in Big-Bang Nucleosynthesis is compatible only with the SMA solution of the solar neutrino problem[13], we conclude that the scheme A is disfavored by the present experimental data and there is only one four-neutrino mixing scheme supported by all data:scheme B[2].REFERENCES[1]See:S.M.Bilenky,C.Giunti,and W.Grimus,Prog.Part.Nucl.Phys.43,1(1999).[2]C.Giunti,hep-ph/9906275(Phys.Rev.D).[3]M.Apollonio et al.(CHOOZ Coll.),Phys.Lett.B466,415(1999).[4]M.Nakahata,these proceedings.[5]G.L.Fogli,these proceedings.[6]S.M.Bilenky,C.Giunti,C.W.Kim and M.Monteno,Phys.Rev.D57,6981(1998).[7]S.M.Bilenky et al.,Phys.Lett.B465,193(1999).[8]S.M.Bilenky and C.Giunti,Phys.Lett.B444,379(1998).[9]Y.Fukuda et al.,Phys.Rev.Lett.82,1810(1999).[10]J.N.Bahcall,P.I.Krastev and A.Yu.Smirnov,Phys.Rev.D58,096016(1998).[11]D.H.White(LSND Coll.),Nucl.Phys.B(Proc.Suppl.)77,207(1999).[12]L.Baudis et al.,Phys.Rev.Lett.83,41(1999).[13]S.M.Bilenky,C.Giunti,W.Grimus and T.Schwetz,Astropart.Phys.11,413(1999).Figure2。

2001 Fluorescent transformation markers for insect transgenesis

been successfully transformed (recently reviewed in: Handler and James, 2000; Handler, 2001; Atkinson et al., 2001. In order to apply these germ-line transformation systems to a broad range of insect species, the development of new marker systems that allow easy and reliable identiﬁcation of transgenic individuals will now be of key importance. 2. Eye color genes as transformation markers The ﬁrst efﬁcient germ-line transformations in nondrosophilid insect species utilized marker genes that allowed the identiﬁcation of transformants by mutantrescue selection similar to typical transformation experiments in D. melanogaster (Rubin and Spradling, 1982). In these cases the marker gene represents the wild-type allele of a gene that, when mutated, causes a recessive, visible but viable phenotype. After introduction of the wild-type gene into the corresponding mutant insect strain by transformation, the wild-type allele complements the recessive mutation and the visible phenotype is rescued. Commonly used marker genes are eye color genes responsible for the production of screening pigments in the compound eye. Mutations in these genes cause obvious phenotypes that are readily distinguished

Characterization of DFR allelic variations and their associations with

Characterization of DFR allelic variations and their associations with pre-harvest sprouting resistance in a set of red-grained Chinese wheat germplasmH.H.Bi •Y.W.Sun •Y.G.Xiao •L.Q.XiaReceived:26April 2013/Accepted:31July 2013/Published online:15August 2013ÓSpringer Science+Business Media Dordrecht 2013Abstract Pre-harvest sprouting (PHS)of wheat greatly reduces the quality and economic value of grain,and PHS resistance is one of the most important traits in wheat breeding.Red-grained wheat varieties are generally more resistant to PHS than white-grained ones;however,some are still susceptible.The red pigment of red-grained wheat is synthesized through the ﬂavonoid biosynthetic path-way,in which the dihydroﬂavonol-4-reductase gene (DFR )is one of the genes involved in anthocyanin synthesis.In this study,a set of 120red-grained Chinese wheat cultivars and lines with distinct PHS resistance were used to characterize TaDFR geno-type variations and their association with PHS resistance.Whereas no variation or functional var-iation of TaDFR genes was detected on chromo-somes 3A and 3D,a novel TaDFR allele,designated TaDFR -Bb ,was explored on chromosome pared with TaDFR -Ba ,an 8bp insertion (CTCTAGGA)was identiﬁed in the promoter region of TaDFR -B in most of the PHS resistant red-grained wheat varieties and advanced lines.Based on this,aCAPS marker was designed and validated with a set of Chinese red-grained wheat cultivars and lines with distinct PHS resistance.In most cases,TaDFR -Bb was associated with higher PHS resistance.An association study indicated that wheat varieties with the 8bp insertion (average seed germination index 23.6%)were signiﬁcantly more resistant (P \0.01)to PHS than those without the insertion (average seed germination index 69.5%).Further study on gene expression demonstrated that the insertion led to increased TaDFR -B expression in cultivars with PHS resistance.Transient expression of TaDFR -B in coleoptiles of wheat cv.Chinese Spring revealed that increasing TaDFR gene expression did not induce the synthesis of anthocyanins.Keywords Triticum aestivum ÁGrain color ÁDihydroﬂavonol-4-reductase gene ÁPre-harvest sprouting ÁGene expressionAbbreviations PHS Pre-harvest sprouting CHS Chalcone synthase CHI Chalcone ﬂavanone isomerase F3H Flavanone 3-hydroxylase DFR Dihydroﬂavonol-4-reductase DPA Days post-anthesis GI Germination indexqRT-PCR Quantitative reverse-transcript polymerasechain reactionCAPS Cleaved ampliﬁed polymorphic sequenceH.H.Bi and Y.W.Sun have contributed equally to this work.H.H.Bi ÁY.W.Sun ÁY.G.Xiao ÁL.Q.Xia (&)The National Key Facility for Crop Gene Resources and Genetic Improvement,Institute of Crop Sciences,Chinese Academy of Agricultural Sciences (CAAS),12Zhongguancun South Street,Beijing 100081,China e-mail:xialanqin@Euphytica (2014)195:197–207DOI 10.1007/s10681-013-0986-zIntroductionPre-harvest sprouting(PHS)of grains while they are still in the ear usually occurs in response to damp conditions(Bewley1997;Lenton2001;Appels et al. 2003).The main effects of PHS are lower yields due to harvest losses and,more importantly,reductions in end-product quality.In particular,the synthesis of a-amylase results in starch breakdown and production of breads with lower volumes and compact and sticky crumb structures(Gale and Lenton1987).Conse-quently,the value of the grain is reduced as is the quality of products(Derera et al.1977;Lenton2001). PHS is a worldwide natural problem which mainly occurs in the USA,Australia,Canada,Japan,UK and China(Lenton2001;Xia et al.2008).Many factors contribute to PHS,including seed dormancy,a-amylase activity,spike characteristics,grain color, and germination inhibitors in glumes and testa(Gale et al.1983;Gale and Lenton1987;Adkins et al.2002).Red-grained wheat varieties are generally more tolerant to PHS than their white counterparts(Flintham 2000;Warner et al.2000;Himi et al.2011).Although red grain color is associated with grain dormancy,it does not guarantee dormancy(Flintham et al.1999; Flintham2000;Himi et al.2002).The pigment of red grain color is composed of anthocyanin,proanthocy-anidin and catechin which are synthesized by the enzymes chalcone synthase(CHS),chalconeﬂava-none isomerase(CHI),ﬂavanone3-hydroxylase(F3H) and dihydroﬂavonol-4-reductase(DFR)in theﬂavo-noid synthesis pathway(Holton and Cornish1995; Chopra et al.1996;Mol et al.1998).The wheat genes encoding these enzymes have been isolated;they express mainly in the immature grain coat of red grain and are almost totally suppressed in white-grained lines(Himi et al.2005).Among the factors affecting PHS,Viviparous1(Vp1),is a key element that plays an important role in the seed maturation processes,such as seed dormancy and seed desiccation(McCarty et al. 1989,1991;Giraudat et al.1992).It was reported that Vp1/ABA/GA coordinated the control of grain color and PHS via Myb-dependent and Myb-independent pathways.Mutations in Vp1inhibit anthocyanin synthesis(Robertson1955;McCarty et al.1989). Myb/c1are transcriptional activators ofﬂavonoid synthesis genes(Himi and Noda2005),which are located on the long arms of chromosomes3A,3B and 3D,approximately30cM proximal to the Vp1locus, consistent with observed linkage between grain dor-mancy and red grain(Groos et al.2002;Himi and Noda 2004).Vp1interacts with the Sph cis-element in the promoter region of the Myb/c1gene and regulates its expression(Hattori et al.1992;Carson et al.1997), whereas the Myb/c1transcription factor binds to the P element in the promoter region of the DFR gene which is an important structural gene in theﬂavonoid biosynthetic pathway(Winkel-Shirley2001;Pola-shock et al.2002),and functions in the anthocyanin biosynthesis pathway to generate grain color(Himi and Noda2004).Oligomicroarray analysis indicated that the genes involved inﬂavonoid biosynthesis,including DFR,respond to both Vp1and ABA(Suzuki and Ketterling2003).Also,it is interesting to note that Chinese Spring wheat lines with deletions of the long arm of chromosome3D produce seeds with white color (Himi and Noda2004),indicating that deletion of either the DFR gene or Myb/c or TaVp-1D or all of these three genes on chromosome3D have loss or gain functions in grain color(Xia et al.2008).DFR functions at an important regulatory point upstream of anthocyanin,proanthocyanidin,and phlob-aphene products in theﬂavonoid synthesis pathway (Winkel-Shirley2001;Polashock et al.2002).However, direct evidence for DFR involvement in PHS resistance in red grained wheat remains unclear.In order to reveal the potential role of DFR gene in PHS resistance of red-grained wheat,we cloned three DFR homologues and their promoter regions from several red-grained wheat cultivars with distinct PHS resistance,and allelic varia-tions were found in the promoter region of TaDFR-B.We then made a preliminary association study with a set of 120red-grained Chinese wheat cultivars and advanced lines varying in PHS response.The expression of each TaDFR-B allele in immature seeds was investigated. Transient expression of the TaDFR-B in the coleoptiles of wheat cv.Chinese Spring was studied to investigate its role in anthocyanin synthesis.Materials and methodsPlant materialsOne hundred and twenty red-grained Chinese wheat cultivars and advanced lines with distinct PHSresponses were used to determine the association between PHS resistance(average germination index) and DFR genotypes(Table1).Among them,ten cultivars differing in PHS response;that is,ﬁve PHS resistant cultivars,viz.Longmai13(average seed germination index10.11%),Kenjiu1(6.77%),Long 94-4081(1.44%),Longfumai10(4.11%)and Kef-eng10(11.75%);two intermediate PHS response cultivars,Jilimai(45%)and Kefeng9(37.0233%); and three PHS susceptible cultivars,Taiyuan566 (67%),Fengkang8(83%)and Jingshuang16(95%) were used to amplify the full genomic sequences of TaDFR-A,TaDFR-B and TaDFR-D.In addition,PHS susceptible cultivars Nongda45,Nongda311and Jingdong6,intermediate PHS response cultivars Aiganzao,Yangmai3and Hongtuzi,and PHS resistant cultivar05-5061were used to investigate the expres-sion levels of different TaDFR-B alleles.These wheat materials were sown at the CAAS Experimen-tal Station in late September2010and2011under normalﬁeld management.Spikes were tagged at anthesis and grains were harvested from spikes at 5days post anthesis(DPA)and ten DPA,respec-tively,then frozen in liquid nitrogen and stored at -80°C.PHS response assayPHS response was assessed by germination index. Spikes from each cultivar or line were harvested at the dough-yellow ripening stage(around40DPA in China),hand-threshed,sterilized with HgCl2,and placed crease down in plastic Petri dishes on moist ﬁlter paper at room temperature.Germinated seeds were counted daily and removed.Germination index was calculated according to GI value(Walker-Sim-mons1988):GI¼7Ân1þ6Ân2þ5Ân3þ4Ân4þ...þ1Ân77Âtotal grainswhere n1,n2…n7are the number of seeds germinated on theﬁrst,second,and the subsequent days until the 7th day,respectively.Three replications for each sample were performed with100seeds per test.Final GI values were averaged data obtained over2years (2000–2001and2001–2002wheat growing seasons) across two locations(Anyang,Henan province and Beijing,China)(Xiao et al.2004).Primer designGenome-speciﬁc primers DFRAF1/R1,DFRAF2/R2, DFRBF1/R1,DFRBF2/R2,DFRDF1/R1and DFRDF2/ R2were designed based on a DNA sequence alignment of three wheat DFR homologues TaDFR-A,TaDFR-B and TaDFR-D available in GenBank(www.ncbi.nlm. )under the accession numbers AB162138, AB162139and AB162140,respectively.Two pairs of primers were used to obtain the full length of each DFR homologue(Fig.1;Table2).DNA extraction and PCR ampliﬁcationGenomic DNA was extracted from ten seeds of each wheat line using a DNAquick Plant System Kit(TIAN-GEN,China).PCR were performed in an MJ Research PTC-200thermal cycler in total volumes of50l l including5l l109PCR buffer,125l M of each dNTP,8pmol of each primer,2.0units of proofreading rTaq polymerase(Takara Ltd,China)and100ng of template DNA.PCR conditions were as follows:94°C for5min,followed by36cycles of94°C for30s, 53–66°C for30s and72°C for30s to2min (depending on the size of PCR product),with aﬁnal extension of72°C for10min.Ampliﬁed PCR frag-ments were separated on1.5%agarose gels,stained with ethidium bromide,and visualized using UV light. Development of a CAPS markerDFR-B-F/R primers(Table2)were used to amplify the DNA fragments for restriction endounclease digestion analysis.A20l l restriction endonuclease digestion system was used,including2l l109buf-fer,0.5l l Hin f I(restriction site:GANTC,10U/l l, Promega),and17.5l l PCR product.After incubation at37°C for3h,fragments were separated on1.5% agarose gels,stained with ethidium bromide,and visualized under UV light.Expression analysis of the TaDFR-B geneTotal RNA was extracted from5DPA and10DPA grains using a RNAprep Pure Plant Kit(TIANGEN Co Ltd,China).The concentration and quality of RNA were determined spectrophotometrically by absorbance at260nm and by A260/A280ratio,respectively.RNA integrity was assessed by the relative intensities of28STable1Polymorphisms of TaDFR-B alleles in120red-grained Chinese wheat cultivars and lines with distinct PHS resistance Cultivar or advanced line Average GI9100%a TaDFR-B Cultivar or advanced line Average GI9100%TaDFR-B02-2083 3.82H Kenjianmai2 4.09H02-2460 3.65H Kenjiu1 6.77H03-32458.18H Kenjiu9 1.49H03-3313 1.02H Kenjiu100.00H03-33390.27H Keyi2981.50903-3351 1.47H Leting183183.00903-3365 1.73H Liaochun431.00H03-3679 3.46H Long03-3266 2.48H03-3769 1.12H Long05-5061 2.80H04-43100.61H Long79-775916.64H04-439312.42H Long85-5226 3.94H04-4396 6.76H Long90-050997.73H04-4517 4.35H Long91-113113.82H04-4526 3.05H Long91-1178 4.61H04-4568 3.82H Long91-1825 3.46H04-4703 3.54H Long94-4081 1.45H04-47477.68H Longfu970189 3.94H05-5093 2.48H Longfumai11 1.18H05-5170 3.05H Longfumai12 4.25H2006Za002 1.26H Longfumai13 5.41H2006Za055 1.73H Longfumai148.11H2006Za091 3.10H Longfumai15 3.54H Aiganzao48.00H Longfumai16 3.65H Beijing1538.509Longfumai59.83H Beijing584.50H Longfumai618.85H Beijing683.009Longfumai70.84H Beijing83793.00H Longfumai8 3.10H Chuanfu133.50H Longfumai98.00H Chuanyu633.00H Longfumai10 4.11H Dongfanghong398.00H Longjian19690.009Een136.00H Longjian4691.009 Fengkang1087.50H Longjian6484.00H Fengkang1592.00H Longmai10 1.66H Fengkang785.509Longmai1112.34H Fengkang883.009Longmai129.06H Hongmangmai87.009Longmai1310.12H Hongtuzi28.009Longmai14 1.89HJian2664.509Longmai1611.54H Jiangdongmen32.009Longmai19 4.53H Jilimai45.00H Longmai20 2.08HJimai1056.009Longmai23 2.80HJimai1776.50H Longmai26 5.60HJinan551.009Longmai2720.989Jing942885.50H Longmai2911.86Hand 18S rRNA bands in 1.2%(w/v)agarose gels.cDNA was synthesized using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Transgen)with anchored Oligo(dT)18primer accord-ing to the manufacturer’s instructions.Quantitative real-time PCR (qRT-PCR)was performed on an ABI 7300Real-Time system (Applied Biosystems).A genome-speciﬁc primer set DFR-B-RT-F/R unique to the TaDFR -B gene was designed using DNAMAN v6software and employed to investigate the expression level of each TaDFR -B allele (Table 2).Each reaction contained 12.5l l of 29TransStart Green qPCR SuperMix (TransGen),4l l of diluted cDNA sample,0.5l l of each primer,0.5l l of Passive Reference Dye and 7l l of ddH 2O in a ﬁnal volume of 25l l.PCR conditions were as follows:95°C for 30s,followed by 40cycles at 95°C for 5s,55°C for 15s and 72°C for 31s.After completion of the PCR,a melting curve was generated to analyze the speciﬁcity of the gene by increasing the temperature from 55to 95°C.Three technical replicates were performed for each cDNA sample.The wheat actin gene (Table 2)was used as an internal control.The relative expression level of each allele was calculated by the 2-DD CT method following the instruction manual provided along with the Trans-Start Green qPCR kit(TransGen).Fig.1Schematic diagrams of the locations of genome-speciﬁc primer sets used for ampliﬁcation of the TaDFR -A,TaDFR -B,and TaDFR -D genes.The locations of primer sets are represented by arrows .Promoters are represented by lines ,exons and introns are indicated by grey boxes and white boxes ,respectivelyTable 1continued Cultivar or advanced line Average GI 9100%a TaDFR -B Cultivar or advanced line Average GI 9100%TaDFR -B Jingdong 192.50H Longmai 3020.41H Jingdong 692.509Longmai 5 3.92H Jingdong 895.00H Longmai 24 1.66H Jingshuang 1695.009Ningchun 1639.50H Jinmai 2462.00H Ningmai 642.00H Jinmai 2575.00H Nongda 14689.509Jinmai 6771.509Nongda 31188.00H Jiusanhong 80.50H Nongda 45100.00H Ke 92-387 6.71H Sumai 135.50H Kefeng 1011.75H Taiyuan 56667.009Kefeng 937.02H Xiaobingmai 3348.50H Kehan 179.11H Yanan 1194.00H Kehan 18 5.22H Yanan 1593.50H Kehan 1919.61H Yangmai 15836.50H Kehan 20 1.05H Yangmai 332.50H Kenjianmai 18.54HZa 0180.61HaThe GI values were measured by Dr.Shihe Xiao in 2001and 2002(Xiao et al.2004);H indicates TaDFR -Bb allele (PCR products digested by Hin f I);9indicates TaDFR -B a or TaDFR -Bc (no PCR products digested by Hin f I)Transient expression assayA pAHC-PSK vector with the maize ubiquitin pro-moter was used as the basic plasmid for making constructs for transient assays.Both TaDFR and TaMyb10-D were ampliﬁed from genomic DNA of wheat cv.Chinese Spring(CS)using DFR-F/R primers and Myb10-D-F/R primers,respectively (Table2).The TaDFRb gene was cloned into the pAHC-PSK vector via Not I and Sac I multiple cloning sites(designated as pAHC-PSK-DFR),while the Sma I and Not I restriction sites were employed for the TaMyb10-D gene(designated as pAHC-PSK-Myb10).Both vectors,as well as the pAHC-PSK control vector,were delivered into CS coleoptiles by particle bombardment as described by Ahmed et al. (2003).Mature seeds were removed from the spikelet, surface-sterilized with70%ethanol for1min,fol-lowed by10%(v/v)Domestos(Lever)for15min, and then rinsed three times with sterilized distilled water.Seeds were then placed crease down on moist ﬁlter paper in plastic Petri dishes and incubated24h at 4°C in darkness in order to synchronize germination. The Petri dishes were put in a darkroom at23°C for germination.Coleoptiles were collected48h after germination and transferred onto MS media(Murash-ige and Skoog1962).For bombardment,the concen-tration of each vector was1l g/l l and5l l was used for coating gold particles(1.0l m).The bombardment parameters were8.5cm target distance and1,100psi acceleration pressure.After bombardment,pigmenta-tion of coleoptiles was observed under the microscope after24h of culture on MS media at25°C in darkness.ResultsWheat DFR gene ampliﬁcation and sequence analysisTen cultivars differing in PHS response were initially employed to amplify the full genomic sequences, including partial sequences of the upstream regions of TaDFR-A,TaDFR-B and TaDFR-D,using two pairs of genomic-speciﬁc primers(Fig.1;Table2)and their full-length sequences were obtained through sequence assembly.The results of multiple alignments indicated that whereas there was no polymorphism in the TaDFR-D gene among these wheat cultivars, allelic variations in TaDFR-A and TaDFR-B were identiﬁed although no association between TaDFR-A genotypes and PHS response was apparent.In the case of TaDFR-B,compared with the TaDFR-Ba allele,two allelic variants were found with one allelic variant having an8bp insertion(CTCTAGGA)at position-282to-275bp in the promoter region and a SNP at443bp in theﬁrst intron in PHS resistant varieties(Fig.2).The second allelic variant had only the same SNP in the same position.According to theTable2Primer sets used in this studyPrimer set Upstream(50–30)Downstream(50–30)Annealing temperature(°C)DFRAF1/R1CGCGCAACAACCAGCAAAAT TGGAGAAGAGAAGCCTCTCT54DFRAF2/R2CAAGGAGCGGCTGTCCATC AGTGCTGGCTTATTATGTCTC57DFRBF1/R1TGGCCATGTAAAAGAAAGG TGCCACAAGTAATATAGCAAC55DFRBF2/R2CAAGGAGCGGCTGTCCATC CTATTATGTCTCGGCACCAA56DFRDF1/R1CGCGCAACAACCAGCAAAAT CATTCTACGTACTTTCCTCGGGGT56DFRDF2/R2CAAGGAGCGGCTGTCCATC CGCAGCAGCGCTGGCTTATT55DFR-B-RT-F/R GGAAGGATATCCCAGCCATT CTCCAGCAACGGCTTGTT55DFR-B–F/R(CAPSmarker)TGCGGTCTGGCGGGGTACGT ACGTCGAGAGAGAGAGGGAGGGG60DFR-F/R ACAATGGACGGGAGCAAAG TCATACTACTACCGAGGTGGAAAA55Myb10-DF/R TGCACACACTCACACCGAGAG ATAAGAATGCGGCCGCCTAGCAA58AGCCACGCCAACTActinF/R CTCCCTCACAACAACCGC TACCAGGAACTTCCATACCAAC602005Supplement of the Wheat Gene Catalogue (McIntosh et al.2005),these two alleles were desig-nated as TaDFR -Bb and TaDFR -Bc ,respectively.TaDFR -B allelic variations and their association with PHS response in a set of red-grained Chinese wheat germplasmAs shown above,the TaDFR -A and TaDFR -B allelic variations were identiﬁed in ten wheat varieties employed to amplify the full-length sequences of the three TaDFR homologues,with TaDFR -Bb being in PHS resistant cultivars.In order to explore the association between these TaDFR allelic variations and PHS response,a set of 120red-grained Chinese cultivars and advanced lines (Table 1)was used to perform a preliminary association study.The 8bp insertion sequence and its upstream adjacent sequence in TaDFR -Bb formed a Hin f I restriction enzyme site (GANTC)(Fig.2).A CAPS marker was designed and the TaDFR -Bb genotype could be identiﬁed by digesting the ampliﬁed product of the promoterregionFig.2Sequence alignment among TaDFR -B allelesof the TaDFR -B gene with Hin f I restriction enzyme (Fig.3).Whereas no association was found between TaDFR -A allelic variations and PHS response in this set of red-grained Chinese wheats (data not shown),polymorphism of TaDFR -B was detected using this CAPS marker (Table 1).The occurrence of two PCR fragments ampliﬁed by this marker in 120red-grained cultivars was as indicated in Fig.4.Among them,71were PHS resistant cultivars,of which 70had the 8bp insertion;18were intermediate resistant with 13having the 8bp insertion;31were PHS susceptible cultivars of which 17had the 8bp insertion.The GIs of the 120genotypes were consistent over 2years (R =0.898,P \0.0001),with mean values of 39.9and 39.4in years 2001and 2002,respectively.Analysis of variance indicated a signiﬁcant GI differ-ence between the two clusters separated on the basis of the 8bp insertion,whose presence contributed to an average GI of 23.6%,compared with 69.5%in the absence of the insertion (Student’s t test,P \0.01).We concluded that this 8bp insertion was closely associated with PHS resistance in this set of red-grained Chinese wheat cultivars.Expression of TaDFR -B gene in immature wheat seedsIn order to investigate the effect of the 8bp insertion in the promoter region on the expression level of the TaDFR -B gene,quantitative real-time PCR (qRT-PCR)was conducted to assess the relative expression levels of TaDFR -B alleles in the immature seeds of seven wheat cultivars and lines at 5DPA and 10DPA,respectively (Fig.5).TaDFR -B expression was gen-erally higher in the whole seeds at 5DPA than at 10DPA,consistent with a previous report by Himi andNoda (2004).Among three PHS susceptible cultivars,TaDFR -B expression level in Jingdong 6(without the 8bp insertion)was clearly lower than that in Nongda 45and Nongda 311(with the insertion).Similarly,among three cultivars with intermediate PHS response,TaDFR -B expression in Hongtuzi (without the insertion)was lower than that in Aiganzao and Yangmai 3(with the insertion).A preliminary con-clusion was that the 8bp insertion in the promoter region of TaDFR -B enhanced its expression level.Moreover,among cultivars with the 8bp insertion,the expression of TaDFR -B was highest in the PHS resistant cultivar Long 05-5061,followed by the intermediate-tolerant cultivar Aiganzao,and lowest in PHS susceptible cultivar Nongda 311(Fig.5).This suggested a positive correlation between the expres-sion levels of TaDFR -B alleles and PHS resistance in red-grained wheatvarieties.Fig.3Electrophoresis of Hin f I digested PCR products of TaDFR -B alleles in wheat cultivars differing in PHS response.M,Marker I (bands from lower to upper are 100,200,300,400,500,600and 700bp).1.Longmai 13(TaDFR -Bb ,average GI 10.11%);2.Kenjiu 1(TaDFR -Bb ,6.77%);3.Jilimai (TaDFR -Bb ,45%);4.Taiyuan 566(TaDFR -Ba or TaDFR -Bc ,67%);5.Long 94-4081(TaDFR -Bb ,1.44%);6.Longfumai 10(TaDFR -Bb ,4.11%);7.Fengkang 8(TaDFR -Ba or TaDFR -Bc ,83%);8.Kefeng 10(TaDFR -Bb ,11.75%);9.Kefeng 9(TaDFR -Bb ,37.0233%);10.Jingshuang 16(TaDFR -Ba or TaDFR -Bc ,95%)Fig.4Association between PHS response (average GI)and number of wheat varieties with and without the TaDFR -Bb allele (with or without the 8bp insertion)Transient expression of TaDFR -BIt was reported that the Myb10transcription factor could transiently activate ﬂavonoid biosynthetic genes and induce red pigmentation in wheat coleoptiles (Himi et al.2011).DFR is a structural gene involved in an important step in the ﬂavonoid biosynthesis path-way upstream of anthocyanin,proanthocyanidin,and phlobaphene,and contributing to pigmentation of various plant tissues (Winkel-Shirley 2001;Polashock et al.2002;Himi and Noda 2004).Therefore,it was of interest to check whether transient over-expression of TaDFR -Bb could produce red pigmentation in wheat coleoptiles.Expression vectors pAHC-PSK-TaDFR harboring TaDFR-Bb and pAHC-PSK-Myb10harbor-ing the wheat Tamyb10gene were thereforeconstructed.Coleoptiles of wheat cv.Chinese Spring were bombarded with the two vectors,respectively.As shown in Fig.6,obvious red pigmentation was observed in wheat coleoptiles bombarded with pAHC-PSK-Myb10as reported by Himi et al.(2011),whereas no red spots appeared on coleoptiles bombarded with pAHC-PSK-TaDFR.This result indicated that enhanced expression of TaDFR alone could not induce the synthesis of anthocyanins or appearance of red pigmentation.DiscussionRed-grained wheat cultivars are usually more resistant to PHS than white-grained ones;however,some are still susceptible.The red grain color of resistant wheat genotypes directly alters levels of grain dormancy and resistance to PHS (Flintham 2000;Warner et al.2000;Himi et al.2002).DFR is an important structural gene for production of red pigment (anthocyanin,proanth-ocyanidin,and phlobaphene)in the ﬂavonoid synthe-sis pathway (Winkel-Shirley 2001;Polashock et al.2002).However,the mechanism underlying higher PHS resistance of red-grained wheats remains unclear.In this paper,a panel of 120red grained Chinese wheat cultivars and advanced lines was used to characterize TaDFR allelic variants and their associations with PHS response.While no polymorphism in TaDFR -D or association between TaDFR -A genotypes and PHS response were evident in this set of red-grained Chinese wheats,a novel TaDFR -B allele,TaDFR -Bb ,which was characterized by an 8bp insertion sequence (CTCTAGGA)in the promoter region,did show a close relationship with higher PHS resistance.A CAPS marker was then developed andvalidatedFig.5Relative expression levels of TaDFR -B alleles in immature seeds at 5and 10DPA of seven red-grained wheat cultivars and lines differing in PHS response.Nongda 45(TaDFR -Bb ,average GI 100%);Nongda 311(TaDFR -Bb ,88%);Jingdong 6(TaDFR -Ba or TaDFR -Bc ,92.5%);Aiganzao (TaDFR -Bb ,48%);Yangmai 3(TaDFR -Bb ,32.5%);Hongtuzi (TaDFR -Ba or TaDFR -Bc ,28%);Long 05-5061(TaDFR -Bb ,2.80%)Fig.6Transient expression of TaDFR -B gene in wheat coleoptiles.a Wheat coleoptile transformed with pAHC-PSK-DFR-B;b ,c Wheat coleoptiles transformed with pAHC-PSK-Myb10.The red arrows indicate red pigmentswith this Chinese wheat germplasm set.Preliminary association study indicated that wheat genotypes with the insertion were more resistant to PHS than those lacking it.Further study on the expression levels of TaDFR-B alleles revealed that the8bp insertion in the promoter region of TaDFR-B enhanced expression of this gene at the transcription level.Several regulatory elements have been identiﬁed in the50upstream region of TaDFRs,including two or three G-box core elements and myb-binding elements, two Dof elements,a TACpyAT element,and a core element for the SBF-1factor(Himi and Noda2004). TaDFR-B carries three combinations of a G-box element and a myb-binding element in its promoter region;whereas TaDFR-A and TaDFR-D only have two combinations of these elements.This may be responsible for the higher expression level of TaDFR-B in wheat grains(Himi and Noda2004).Although the 8bp insertion in the promoter region of TaDFR-Bb did not reside in any of the elements mentioned above, it formed a repeat sequence with an adjacent sequence (data not shown).Previous reports showed that the numbers of repeat sequences in the promoter region of a gene could affect the expression level of such genes (Espley et al.2009;Vinces et al.2009).Thus,it could be inferred that the8bp insertion might increase the expression level of TaDFR-Bb by enhancing the binding of related transcription factors through changed secondary structure of the upstream region of TaDFR-B during transcription.In addition,Tamyb10is also a strong candidate for the red grain color gene(Himi et al.2011).Tamyb10 recognizes a CCT/AACC element in the promoter and activates related genes such as CHS,CHI,F3H and DFR in theﬂavonoid synthesis pathway(Chopra et al. 1996;Grotewold et al.1998).Wheat coleoptiles transiently transformed with Tamyb10produced red pigmentation as observed by Himi et al.(2011)and in this study.However,although DFR is a very important structural gene in theﬂavonoid synthesis pathway, transient expression of TaDFR-B alone did not activate red pigmentation.The reason might be that the pigment in wheat grain is synthesized by four emzymes in theﬂavonoid synthesis pathway:viz., CHS,CHI,F3H and DFR;and DFR locates at the downstream of other three enzymes(Holton and Cornish1995;Chopra et al.1996;Mol et al.1998; Himi and Noda,2004).Therefore,transient expression of the TaDFR-B gene alone in the coleoptiles could not rescue the red pigment without increased levels of the products of the other three enzymes in theﬂavonoid synthesis pathway.The results described in this preliminary study demonstrate involvement of TaDFR alleles in PHS resistance of red-grain wheats.Furthermore,the development of a CAPS marker to detect the explored TaDFR-Bb allele will be useful in germplasm selec-tion and marker-assisted breeding for enhanced PHS resistance in red-grained wheats.Acknowledgments This work was supported in part by the China National Basic Research Program(2009CB118300). ReferencesAdkins SW,Bellairs SM,Loch DS(2002)Seed dormancy mechanisms in warm season grass species.Euphytica 126:13–20Ahmed N,Maekawa M,Utsugi S,Himi E,Ablet H,Rikiishi K, Noda K(2003)Transient expression of anthocyanin in developing wheat coleoptile by maize C1and B-peru regu-latory genes for anthocyanin synthesis.Breed Sci53:29–34 Appels R,Francki M,Chibbar R(2003)Advances in cereal functional genomics.Funct Integr Genomics3:1–24 Bewley JD(1997)Seed germination and dormancy.Plant Cell 9:1055–1066Carson CB,Hattori T,Rosenkrans L,Vasil V,Peterson PA, McCarty DR(1997)The quiescent/colorless alleles of viviparous1show that the conserved B3domain of Vp1is not essential for ABA-regulated gene expression in the seed.Plant J12:1231–1240Chopra S,Athma P,Peterson T(1996)Alleles of the maize P gene with distinct tissue speciﬁcities encode Myb-homol-ogous proteins with C-terminal replacements.Plant Cell 8:1149–1158Derera NF,Bhatt CM,McMaster GJ(1977)On the problem of pre-harvest sprouting of wheat.Euphytica26:299–308 Espley RV,Brendolise C,Chagne´D,Kutty-Amma S,Green S, Volz R,Putterill J,Schouten HJ,Gardiner SE,Hellens RP, Allan AC(2009)Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples.Plant Cell21:168–183Flintham JE(2000)Different genetic components control coat imposed and embryo-imposed dormancy in wheat.Seed Sci Res10:43–50Flintham JE,Adlam RE,Gale MD(1999)Seedcoat and embryo dormancy in wheat.In:Weipert D(ed)8th International symposium on pre-harvest sprouting in cereals.Associa-tion of Cereal Research,Detmold,pp67–76Gale MD,Lenton JR(1987)Preharvest sprouting in wheat a complex genetic and physiological problem affecting bread making quality in UK wheats.Aspects Appl.Biol 15:115–124Gale MD,Flintham JE,Arthur ED(1983)Alpha-amylase production in the late stages of grain development-an early sprouting。

对双重减量的看法英语作文

对双重减量的看法英语作文The Double Reduction Policy: An Analysis and Evaluation。

Introduction。

The Double Reduction Policy, also known as "双减政策"in Chinese, refers to the Chinese government's initiativeto reduce both excessive homework and after-school tutoring burdens on students. This policy has been widely discussed and debated as it aims to alleviate the stress and pressure faced by students in China's highly competitive education system. In this essay, we will analyze and evaluate the Double Reduction Policy, taking into consideration its benefits, drawbacks, and potential long-term implications.Benefits of the Double Reduction Policy。

One of the key benefits of the Double Reduction Policyis the reduction of excessive academic workload on students. Many students in China face immense pressure to excelacademically, often leading to long hours of homework and additional tutoring after school. By reducing excessive homework assignments, students can have more time for extracurricular activities, hobbies, and rest, which are crucial for their overall well-being and personal development.Another advantage of the Double Reduction Policy is the potential to narrow the education gap between urban and rural areas. In China, there is a significant disparity in the quality of education between urban and rural schools. Students in urban areas often have access to better resources and educational opportunities, while those in rural areas struggle to keep up. By reducing the burden of excessive homework and after-school tutoring, students in rural areas can have more time to focus on self-study and catch up with their urban counterparts.Moreover, the Double Reduction Policy can promote a healthier parent-child relationship. In many Chinese families, parents invest a significant amount of time and money in their child's education, often sacrificing theirown well-being. With the reduction in after-school tutoring, parents can spend more quality time with their children, engaging in activities that foster emotional bonding and support their overall development.Drawbacks and Challenges。

混合料双面击实成型英文

混合料双面击实成型英文Mixing Material Double-Sided Consolidation and FormingIn recent years, the field of material engineering has witnessed significant advancements in the development of new techniques for processing and shaping materials. One such technique that has gained considerable attention is the Mixing Material Double-Sided Consolidation and Forming method. This cutting-edge approach allows for the creation of highly durable and versatile materials, which have a wide range of applications in various industries. In this article, we will explore the process and benefits of this innovative technique.1. IntroductionThe Mixing Material Double-Sided Consolidation and Forming method, also known as MMDF, is a technique that enables the blending of different materials to form a composite product. Unlike traditional manufacturing methods, MMDF not only combines different materials but also enhances their properties through a double-sided consolidation process. This method involves the application of heat and pressure to the mixture, resulting in a tightly bonded composite material.2. ProcessThe MMDF process can be broken down into several distinct steps. Firstly, the desired materials are selected based on their compatibility and desired properties of the final product. These materials are then thoroughly mixed together using specialized equipment, ensuring a uniform distribution. The mixed material is then placed between two heated molds, which aresubjected to high pressure. This pressure helps in consolidating the mixture and driving out any air or voids present. The molds maintain their high pressure and temperature for a specific period, allowing the materials to bond and form a solid, dense composite. Finally, the molds are cooled down, and the composite is removed, ready for further processing or utilization.3. BenefitsThe MMDF technique offers several advantages over traditional material processing methods. One of the key benefits is the ability to create composite materials with tailored properties. By selecting different materials and adjusting the mixing ratio, manufacturers can produce composites with specific characteristics such as improved strength, durability, flexibility, or conductivity. Moreover, since the MMDF process eliminates the need for adhesives or mechanical fasteners, the resulting materials have enhanced structural integrity and longevity. The tight bond achieved through consolidation also ensures a high level of dimensional accuracy, allowing for precision manufacturing.4. ApplicationsThe versatility of MMDF makes it suitable for a wide range of applications across various industries. In the automotive sector, this technique can be utilized to develop lightweight yet sturdy components, resulting in improved fuel efficiency. In the construction industry, the MMDF method can be employed to fabricate composite building materials with enhanced insulation properties. Additionally, the electronics industry can benefit from the ability to produce components with superior electrical conductivity and thermal dissipation. Furthermore, the medical field canleverage MMDF to create biocompatible materials for implants or prosthetics, promoting better patient outcomes.5. Future DevelopmentsMoving forward, the MMDF technique holds immense potential for further advancements and innovations. Researchers are actively exploring the integration of nanomaterials into the mixtures, aiming to enhance the composites' properties at the atomic level. Additionally, advancements in automation and robotics can streamline the production process, reducing costs and increasing efficiency. The ongoing research into the optimization of material compositions and processing conditions will continue to broaden the range of applications for MMDF.In conclusion, the Mixing Material Double-Sided Consolidation and Forming technique is a revolutionary method for creating composite materials with exceptional properties. Through its unique blending and consolidation process, MMDF enables the development of materials that possess enhanced strength, durability, and flexibility. With its wide range of applications across industries, this technique is poised to transform the field of material engineering. As research and development continue to progress, we can expect even more exciting possibilities for the future of MMDF.。

关于双减的英语作文题

关于双减的英语作文题Double reduction is a growing phenomenon in today's society. People are constantly striving to simplify their lives and reduce unnecessary stress. This can be seen in various aspects of life, such as work, relationships, and personal belongings.In the workplace, double reduction is evident in the rise of remote work and flexible schedules. People are no longer tied to a traditional 9-to-5 office job. They can now work from the comfort of their own homes or choose when and where they want to work. This allows for a better work-life balance and reduces the stress of commuting and strict office hours.In relationships, double reduction can be seen in the rise of online dating and casual relationships. People are no longer interested in the traditional dating scene and the pressure of finding "the one." Instead, they prefer to keep things casual and have multiple partners or engage inshort-term relationships. This reduces the emotional stress and commitment that comes with long-term relationships.When it comes to personal belongings, minimalism is becoming increasingly popular. People are realizing that they don't need to own a lot of things to be happy. They are decluttering their homes and getting rid of unnecessary possessions. This not only reduces physical clutter but also mental clutter, allowing for a more peaceful and stress-free living environment.In conclusion, double reduction is a trend that is becoming more prevalent in today's society. People are simplifying their lives and reducing unnecessary stress in various aspects such as work, relationships, and personal belongings. This allows for a better work-life balance, less emotional stress in relationships, and a more peaceful living environment.。

进出口货物名称中英对照(103)

进出口货物名称中英对照(103)进出口货物名称中英对照(103)进出口货物名称中英对照(103)double-crank ward cot 双摇柄病床double-cross leno 全绞式纱罗double-current generator 双流发电机double-current key 双流电键double-current relay 双流式继电器double-cut file 双纹锉double-cut plough 双层犁double-cut shears 双刃剪切机double-deck bus 两层公共汽车double-deck exhaust box 双层排气箱double-deck machine 两层拔丝机double-deck pallet 双层货盘double-deck plough 双层犁double-deck stock wagon 双层家畜车double-deck twister 双层捻线机double-deck vibrating sieve 双层振动筛double-depth plough 双层犁double-detection receiver 两次检波接收机double-dial timer 双标度计时器double-dial 双标度盘double-die-turning finishing hydraulic press 双模具翻转抛光液压机double-diffused device 双扩散器件double-diffused integrated circuit 双扩散集成电路double-diffused phototransistor 双扩散型光电晶体管double-diffused transistor 双扩散型晶体管double-discharge gear pump 双出口齿轮泵double-discharge pump 双排量泵double-discharge spiral water turbine 双排量涡轮式水轮机double-door and dual-temperature refrigerator 双门双温电冰箱double-door household refrigeration 双门家用冰箱double-door refrigerator 双门冰箱double-dragon lampstand 双龙灯座double-drum boiler 双汽包锅炉double-drum drier 双滚筒干燥机double-drum electric block 双滚筒电葫芦double-drum tilt rig 双滚筒倾斜钻机double-drum winch 双滚筒绞车double-duty lube truck 两用润滑油运输车double-duty regulator 双作用调节器double-ear fancy colour vase with lotus design 斗彩勾莲双耳樽double-ear mortar-pan 双耳泥箕double-eccentric fiber-optic connector 双偏芯光纤连接器double-edge blade 双面刀片double-edge chisel 双刃凿double-edge grinder 双边研磨机double-edge hacksaw blade 双面钢锯条double-edge milling cutter 二面刃铣刀double-edge prunning saw 双边齿修树锯double-edge safety razor blade 双面安全刀片double-effect lithiumbromide absorption refrigerating machine 两效型溴化锂吸收式制冷机double-emitter chopper transistor 双发射极斩波晶体管double-end fired furnace 两头烧火加热炉double-end hexagon ring wrench 双头六角扳手double-end hexagon straight wrench 双头六角直口扳手double-end mill 双端铣刀double-end plug gauge 双端塞规double-end punch 双头冲double-end scraper 双头刮刀double-end set screw wrench 双头紧定螺钉扳手double-end slotting cutter 双头键槽铣刀double-end socket wrench 双套筒扳手double-end spark plug wrench 双头火花塞扳手double-end tungsten iodide lamp 双头碘钨灯double-end wrench 双口扳手double-ended boiler 双面燃烧式锅炉double-ended clamp 双头线夹double-ended cord 双头塞绳double-ended gall stone scoop 双头胆石匙double-ended magnetron 双端出线磁控管double-ended open jawed spanner 双头开口爪形扳手double-ended probe 双头探针double-ended spanner 双头扳手double-ended wrench 双头扳手double-ends fabric 双经布double-entry compressor 双进口压缩机double-entry pump 双进口泵double-extraction condensing turbine 双抽气凝气式汽轮机double-extraction non-condensing turbine 双抽气背压式汽轮机double-face blanket 鸳鸯毡double-face carborundum disk 双面金刚砂片double-face corrugated board 双面瓦楞纸板double-faced adhesive tape 双面胶粘带double-faced blacksmith's hammer 八角锤double-faced embroidery with varied colours and patterns 双面异色异样绣double-faced embroidery with varied colours 双面异色绣double-faced embroidery 双面绣double-faced flocking 双面植绒double-faced grinding and polishing machine 双面研磨抛光机double-faced hand saw 双刃手锯double-faced in head claw hammer 头部八角羊角锤double-faced in neck claw hammer 颈部八角羊角锤double-faced pile 双面绒double-faced satin 双面缎double-flow reaction turbine 双流反动式涡轮机double-flow reheat-condensing steam turbine 双流再热冷凝式汽轮机double-flow type boiler 双流式锅炉double-flue boiler 双炉胆锅炉double-frequency generating set 双频发电机组double-frequency generator 双频发电机double-fried pork 回锅肉double-furnace boiler 双炉膛锅炉double-gate field effect transistor 双栅场效应晶体管double-gear block 双联齿轮double-gear cone transmission 复式锥轮传动装置double-gear sliding block 两联滑动齿轮double-geared headstock 双套齿轮床头double-geared roller crusher 双齿辊破碎机double-glazed unit 双层玻璃窗double-glazing unit 双层玻璃窗double-grid tube 双栅管double-grid valve 双栅阀double-head boxing machine 双头箱式缝纫机double-head endmill 双头立铣刀double-head flat scraper 双头平刮刀double-head milling machine 双轴铣床double-head rail 双头轨double-head spring needle knitting machine 汗绒布二用针织机double-head turning tool welded with carbide tip 硬质合金双头车刀double-head tyre 双缘轮胎double-head wire stitching machine 双头铁丝订书机double-head wrench 双头扳手double-headed bolt tension meter 双头螺栓张力计double-headed camera 双头摄影机double-headed spray gun 双口喷枪double-headed tube bender 双头弯管机double-header boiler 双筒式水管锅炉double-heterostructure junction laser 双异质结激光器double-high rolling mill 双辊式轧机double-hook oil ring 双刃刮油环double-horizontal-shaft forced type concrete mixer 强制式双卧轴混凝土搅拌机double-hung window 双挂拉窗double-image range-finder 双像测距仪double-image telemeter 双像测距仪double-impeller impact breaker 双叶轮冲击破碎机double-impeller pump 双叶离心泵double-input mixer 双输入混频器double-intergrating accelerometer 二重积分加速度计double-j bolt 双钩穿钉double-knitted plaited fabric 双面编结添纱针织物double-loop towel 双面毛圈毛巾double-m-derived filter 双m推演式滤波器double-mica spacer 双层云母垫片double-mode cyclotron 双重加速法回旋加速器double-optical projection stereoplotter 双镜光学投影立体绘图仪double-pass spectrometer 双光道分光计double-pass wired glass machine 双加料夹丝玻璃成形机double-pass-out turbine 双抽气式汽轮机double-pipe exchanger 双套管换热器double-pipe heat exchanger 双套管换热器double-piston explosive press 双活塞爆炸压力机double-piston 双活塞double-plate clutch 双片式离合器double-plunger plastic injection moulding machine 双柱塞塑料注模机double-plunger pump 双柱塞泵double-plunger ultramicro pump 双柱塞超微量泵double-ply bag 双层袋double-ply hosiery 双层袜double-point breaker 复式断电器double-point crank press 双点曲轴压力机double-point double-throw switch 双刀双掷开关double-point gear 多点啮合齿轮double-point scriber 两端划针double-point tool 双刃刀具double-pointed nail 双头钉double-pole cutout 双极断路器double-pole double-throw switch 双刀双掷开关double-pole fuse 双极熔断器double-pole switch 双极开关double-poppet throttle valve 双座节流阀double-projection direct-viewing stereo-plotter 双投影直接观测立体测图仪double-projection plotter 双投影测图仪double-projection stereoscope plotting instrument 双投影立体测图仪double-purchase winch 双辘绞车double-purpose interchangeable tip square blade screwdriver 可换头方杆两用螺丝刀double-range instrument 双量程仪表double-range recording flowmeter 双量程记录式流量表double-reheat stream turbine 二次再热式汽轮机double-resonator klystron 双腔速调管double-resonator 双腔谐振器double-return valve 双回流阀double-roll crusher 双辊破碎机double-roller mill 双辊研磨机double-roller stretching and letting-out machine 双辊伸展拉软机double-rolling pipe-reducing machine 双辊缩管机double-row self-aligning ball bearing 双列自调式球轴承double-row spherical ball bearing 双列球面球轴承double-screw continuous mixer 双螺杆连续混合机double-screw conveyor 双螺杆输送机double-screw extruder 双螺杆挤出机double-screw mixer 双螺杆混合器double-screw open and close machine 双螺杆式启闭机double-screw plastic extruder 双螺杆塑料挤压机double-screw plodder 双螺杆压条机double-screw pump 双螺杆泵double-screw reactor 双螺杆反应器double-screw tuner 双螺旋调谐器double-seat stop valve 双座截止阀double-seat valve 双座阀double-section filter 两节滤波器double-shared double wheeled plow 双轮双铧犁double-shed jacquard 双梭口提花机double-side carbon paper 双面复写纸double-side cementing machine 双面粘合机double-side coated art printing paper 双面铜版纸double-side hacksaw blade 双面刃钢锯条double-side milling cutter 两面铣刀double-side planer 双面刨床double-side saw 双面锯double-side sealing machine 双边封口机double-side terry fabric knitting machine 双面毛圈织物针织机double-side tipping wagon 双边倾卸车double-side wood planer 双面木工刨床double-sideband signal generator 双边带信号发生器double-sideband suppressed carrier modulator 双边带抑制载波调制器double-sideband transmitter 双边带发射机double-sided automatic blanking and forming press 双边自动冲压和成形压机double-sided board 双面板double-sided compressor 双面进气压气机double-sided diskette 双面软盘double-sided drawing press 双边拉压机double-sided floppy 双面软磁盘double-sided four-point press 双边四点压力机double-sided high speed press 双柱高速压力机double-sided khaki 双面卡其布double-sided loop pile fabric 双面毛圈起绒织物double-sided needling loom 双面针刺机double-sided plate trimming shears 双边修边剪切机double-sided plush 双面绒double-sided printed circuit board 双面印刷电路板double-sided socket 双向插座double-sided spinning machine 双面纺丝机double-sided transfer presses 双点传输压力机double-size wash tub 双料大型洗衣盆double-skip hoist 双翻斗卷扬机double-speed electric hoist 双速电升降机double-speed manual hydraulic press 双速手动液压机double-speed synchronous motor 双速同步电动机double-spindle contour copying milling machine 双轴平面仿形铣床double-spindle hammer crusher 双轴锤式轧碎机double-stage air pump 双级空气泵double-stage ammonia refrigerating machine set 双级氨制冷机组double-stage compressor 双级压缩机double-stage turbine 双级涡轮机double-stage vortex pump 双级旋涡泵double-step vortex dust remover 双级涡旋除尘器double-strand mill 双线轧机double-strand pig machine 双链式铸铁机double-strand winder 双束绕丝机double-stroke solid-die automatic cold header 双击整模自动冷镦机double-stub tuner 双短线调谐器double-suction centrifugal pump 双吸离心泵double-suction pump 双吸泵double-suction ventilator 双吸口通风机double-surface transistor 双面晶体管double-surface wood planer 双面木工刨床double-swiveling uncoiler and recoiler 并列回转式开卷-卷取机double-table transfer printing press 双面转移印花压烫机double-tariff system meter 双价制计double-thickness sheet glass 双厚窗玻璃double-thread hob 双头滚刀double-throw crank-shaft 双弯曲轴double-throw disconnecting switch 双掷隔离开关double-throw switch 双掷开关double-tip baler 双嘴打包机double-tool cross-slide 双刀横向滑板double-tool holder 双刀刀架double-trace scope 双迹示波器double-track skip hoist 双料斗卷扬机double-transit oscillator 双渡越振荡器double-tread tyre 双胎面轮胎double-trough type mixer 双槽式拌和机double-tube biologic microscope 双筒生物显微镜double-tube bracket light support 双筒壁灯架double-tube converter 双套管转换器double-tube core barrel 双筒取心筒double-tube heat exchanger 套管热交换器double-tube injector 双管喷射器double-tube pressure gauge 双管压力表double-tube reactor 双套管反应器double-tube washing machine 双桶洗衣机double-tuned amplifier 双调谐放大器double-tuned detector 双调谐检波器double-tuned filter 双调谐滤波器double-tuned intermediate frequency amplifier 双调谐中频放大器double-twist twisting machine 倍捻捻线机double-twisting machine 倍捻加捻机double-unit motor 双电枢电动机double-voke snowsuit 双v形肩轭式雪衫裤double-wall corrugated paper 双壁瓦楞纸double-wall corrugated pipe 双壁波纹管double-walled cryogenic storage tank 双壁深冷储罐double-walled steel tank 双壁钢罐double-width press 双全张印报机double-wing electric web for trapping insect 双翼灭虫电网double-wire machine 并纱机double-worm mixer 双螺旋混合机double-wound transformer 双绕变压器double-woven blanket 双层织造毯子doubled yarn 合股纱线doubledeck bed 双层床doubler twister 并捻机doubler winder 并纱机doubler 倍频器doublerie 印花帆布doubles 双股棉线doublet cord 双绞线doublet magnifier 二重放大镜doublet oscillator 赫兹振荡器doublet 紧身上衣doublete 双色花塔夫绸doubling and lapping machine 对折卷板机doubling and rolling machine 对折卷布机doubling and twisting machine 并捻机doubling machine 并丝机doubling register 加倍寄存器doubling roller 贴合辊doubling thread 纺线doubling winder 并丝机doublure 漂白军服粗呢doubly clad optical fiber 双包层光纤doubly resonant oscillator 双谐振振荡器doubly-coated fiber 双涂层纤维doubly-fed polyphase shunt commutator motor 双馈多相并励换向器电动机doubly-fed repulsion motor 双馈推斥电动机douche can 冲洗桶douche pipe 冲洗管douche 冲洗器doudynatron 双负阻管dough batch 打面机dough brake 碾面机dough kneading machine 和面机dough mill 调面机dough mixer 和面机dough mixing machine 和面机dough-making machine 和面机doughing machine 揉面机doughnut antenna 绕杆式天线进出口货物名称中英对照(103) 相关内容:。

反转录PCR原理步骤小结

Human Tissue
Protocol of Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
In the one-step protocol, the components of RT and PCR are mixed in a single tube at the same time. The oneபைடு நூலகம்step protocol generally works well for amplifying targets that are reasonably abundant. Alternatively, RT-PCR can be done in two steps, first with the reverse transcription and then the PCR. The two-step protocol is usually more sensitive than the one-step method; yields of rare targets may be improved by using the two-step procedure.
Note to wear gloves and mask at all times to avoid RNase contamination.
Polymerase Chain Reaction (PCR)
Principle of PCR
The first step of the PCR reaction separates the double strands into single strands (strands 1 and 2) by heat denaturation. The second step is a process called primer annealing, i.e., the formation of partially double-stranded regions by interaction with the primer binding sites on each strand with the corresponding primer. DNA polymerase is then used to extend the primer region and to synthesize full-length complementary strands (light and dark blue lines) of strands 1 and 2, yielding two new double strands 1-3 and 2-4. This reaction requires the presence of deoxynucleoside triphosphates, which serve as substrates for the polymerase.

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J OURNAL OF B ACTERIOLOGY,Dec.2002,p.7042–7046Vol.184,No.24 0021-9193/02/$04.00ϩ0DOI:10.1128/JB.184.24.7042–7046.2002Copyright©2002,American Society for Microbiology.All Rights Reserved.NOTESAnalysis of Infection Thread Development Using Gfp-andDsRed-Expressing Sinorhizobium melilotiDaniel J.Gage*Department of Molecular and Cell Biology,University of Connecticut,Storrs,Connecticut06269Received2May2002/Accepted28August2002Sinorhizobium meliloti growth inside infection threads was monitored after inoculation of alfalfa with red-orgreen-tagged bacteria.Most threads were populated with single bacterial types.Mixed infections were presentbut gave mixed nodules less often than expected.These patterns are explained by a model describing bacterialgrowth during infection.Sinorhizobium meliloti cells grow as free-living organisms but also as nitrogen-ﬁxing symbionts inside root nodule cells of alfalfa and related species(2,4,10–13).Bacteria must get from the root surface to inner root tissue,where they will populate nodule cells.Thus,they grow and divide inside a plant-derived tubule,called an infection thread(7–9).Infection threads grow down host root hairs,and S.meliloti grows and divides inside the thread,keeping itﬁlled.The thread branches during growth through root tissue,and bacteria eventually exit the resulting network and enter the cytoplasm of nodule cells. Studies of mixed infections during symbiosis can lead to insights into the infection and invasion process because they allow investigators to discern spatially distinct subpopulations of bacteria,and thus,something about growth and behavior of the whole population can be inferred.Earlier studies showed that mixingﬂuorescent and nonﬂuorescent S.meliloti gave infection threads that contained both types of bacteria.Both strains entered the infection thread but did not randomly mix. Instead,the growing bacteria formed sectors that apparently originated from the clonal expansion of a few founder cells(8). In this study,I inoculated alfalfa with mixtures of S.meliloti tagged with red or greenﬂuorescent proteins.This allowed observation of all bacteria inside infection threads and showed that infection was more complicated that previously thought. Alfalfa seedlings placed on microscope slides covered with BNM agar(8)were inoculated with a1:1mixture of two S. meliloti strains.Strain L5-30/pDG71(p trp-Gfpmut3)constitu-tively produced Gfp(mut3)under the control of a Salmonella enterica serovar Typhimurium trp promoter(3,6),and strain L5-30/pDG77(p trp-DsRed)constitutively produced DsRed. Five days after inoculation,green infection threads were seen along the length of the root,but red threads were seen only near the top of the root.This was likely because DsRed ma-tures slowly compared to Gfp(mut3)(1).After7to10days,red and green infection threads were visible all along the root(Fig.1A).Sectored infection threads containing red and green bac-teria were readily observed(Fig.1B).In addition,other types of mixed infection threads were seen.Threads with a jumbled population of red and green bacteria and root hairs containing dual infection threads where one was red and one was green were observed(Fig.1C and D). Even this classiﬁcation is a simpliﬁcation because jumbled threads often gave rise to threads with sectors(Fig.1E).Root hairs sometimes contained dual threads,each of which was sectored(Fig.1F).Dual threads containing bacteria of a single type could fuse and then form threads that contained red and green bacteria jumbled together(Fig.1D).Not all infection threads invade nodules;most terminate, and accompanying nodules never develop(14).An unan-swered question is whether mixed infection threads that do deliver bacteria to nodules give rise to nodules that contain mixed populations of bacteria.If so,then the portion of threads that contain mixed populations should equal the por-tion of nodules that contain mixed populations.To test this, alfalfa seedlings were inoculated with a1:1mixture of L5-30/ pDG71(p trp-Gfpmut3)and L5-30/pDG77(p trp-DsRed)at densities of105,106,107,and108cells per slide.Slides were observed7to10days later,and threads were classiﬁed as red, green,or mixed.Nodules were allowed to develop for a total of 20to30days.Selected nodules were surface sterilized, crushed,and plated on selective agar.Colonies were observed to determine if the population inside each nodule was mixed.I excluded lobed nodules and nodules from tight clusters from analysis because these may have arisen from closely spaced infection threads of different colors.Figure2shows results from experiments in which2,546 infection threads were classiﬁed.At the time I had not yet classiﬁed dual threads as a type of mixed infection;therefore, the mixed category included only sectored and jumbled types. Infection threads per plant increased with increasing inoculum size,except on plants inoculated with the highest number of bacteria.Note that these experiments revealed only mixed infections that started when red and green cells populated an infection thread.Cases where two independent green,or red, cells populated a thread could not be discerned and counted as*Mailing address:University of Connecticut,Department of Mo-lecular and Cell Biology,75N.Eagleville Rd.,U-44,Storrs,CT06269. Phone:(860)486-3092.Fax:(860)486-1784.E-mail:gage@uconnvm.7042FIG.1.Examples of mixed infection threads following coinoculation of alfalfa with red and green ﬂuorescent bacteria.(A)Infection threads containing only Gfp-expressing or DsRed-expressing S.meliloti .(B)Sectored infection thread in which the mixed population gave rise to a series of sectors which increase in length along the thread.The tipmost sector advanced into the epidermal cell body,branched (top arrow),and penetrated the underlying cell (bottom arrow),leaving the other sectors behind.(C)Jumbled type of mixed infection thread.Gfp-expressing and DsRed-expressing cells appear randomly mixed throughout the infection thread.(D)Dual-type mixed infection thread.The infected root hair contained two infection threads,one ﬁlled with Gfp-expressing bacteria and the other ﬁlled with DsRed-expressing bacteria (top arrow and left inset).These two infection threads fused together at the base of the root hair (yellow sphere),and new infection threads grew from the fusion point.These new threads contained green and red bacteria mixed together in a jumbled fashion (bottom arrow and right inset).(E)Dual infection threads inside a root hair.The threads began as jumbled-type threads but later gave rise to green sectors at their tips (arrows show the extent of the sectors in each thread).(F)Root hair infected with dual infection threads.Each thread contains sectors of Gfp-expressing and DsRed-expressing bacteria.(G)Curled root hair with red and green microcolonies of S.meliloti in the interior bend of the curl.The red microcolony has given rise to an infection thread containing only DsRed-expressing bacteria.V OL .184,2002NOTES 7043mixed.In another set of experiments,1,109threads were ob-served and classi ﬁed according to type —unmixed,dually in-fected,jumbled,or sectored.These experiments showed that sectored infection threads were the most prevalent type of mixed thread and accounted for slightly less than half of all mixed threads (Table 1).Of 170nodules,146contained bacteria that grew on selec-tive plates;10of the 146contained both red and green strains.Thus,the portion of nodules that contained mixed populations (7.4%)was less than the portion of threads that contained mixed infections (15.2%)(Table 1).This indicated that at most,half of mixed infection threads gave rise to nodules populated with both types of bacteria.Half is a maximum because some mixed nodules may have arisen from closely spaced infection threads containing bacteria of different colors.A model describing the dynamics of bacterial growth inside of infection threads was developed.In the model,bacterial growth rate,growth zone size,number of founder cells,and the ratio of marked strains can be speci ﬁed.Sizes and placement of sectors are calculated and presented numerically and graph-ically.The modeling program is at /ϳgage/Pages/research.html.For Fig.3A,the model was started with a small thread containing six alternating equal-sized sectors.The growth zone was set at 60␮m;bacteria which were that distance,or less,from the tip of the thread grew and divided with a doubling time of 4h.Modeling indi-cates that the size of the penultimate sector should be equal to the size of the growth zone divided by ln2.Based on the observed sizes of these sectors,the 60-␮m growth zone is likely typical of real growth zone sizes.The model predicts that sector size will increase along the length of the thread.This occurs because sectors at the rear stop growing earlier than those near the advancing tip.Such increases in sector length are commonly seen in sectored threads (Fig.1B and F)(8).It also predicts that sectored threads would result in nodules populated with only one of the two strains.This occurs because the only subpopulation that continues to multiply inde ﬁnitely are descendants of the bac-teria which were at the tip of the thread when it initiated.All founder cells which were not at the very tip will eventually fall behind the growth zone and cease to increase in number.A concrete example of this is shown in Fig.1B.The quantitative data presented above also supporttheFIG.2.Quanti ﬁcation of infection thread (IT)types following coinoculation of alfalfa seedlings with Gfp-expressing and DsRed-expressing S.meliloti .(A)Plant roots were examined by epi ﬂuorescence 7to 10days after inoculation with the indicated number of bacteria,and the number of red,green,and mixed (sectored and jumbled)infection threads per plant was determined.(B)Data for mixed infection threads replotted as the percentage of total infection threads per plant at each inoculation density.TABLE 1.Infection thread types following coinoculation withred-and green-tagged S.meliloti aThread typeNo.(%)of typeTotal........................................................................................1,109Mixed b .....................................................................................169(15.2)Dual.........................................................................................34(3.1)Jumbled..................................................................................57(5.1)Sectored..................................................................................78(7.0)aResults of two experiments in which the three types of mixed infection threads were categorized.bTotal of dual,jumbled,and sectored types.7044NOTES J.B ACTERIOL .model.The percentage of nodules containing both red-and green-tagged bacteria (7.4%)was about half of what was ex-pected based on the percentage of mixed threads (15.2%).Therefore,at most,half the mixed infections gave rise to nod-ules populated with both bacterial types.In addition,the per-centage of mixed but nonsectored threads was 8.2%.This agrees well with the percentage of nodules containing red and green bacteria.Thus,the data are consistent with the idea that jumbled and dual-type threads are responsible for populating the mixed nodules.The random distribution of bacteria in jumbled-type infec-tion threads appears to con ﬂict with the growth model.How-ever,the fact that jumbled threads often become sectored suggests that the growth patterns of these types are nottooFIG.3.Models of infection thread growth.(A).A computer model was used to simulate sectored infection thread growth.For the case shown,it was assumed that the thread started with six alternating bright and dark sectors of equal size.The doubling time for the growing bacteria (those 60␮m or less from the growing tip of the thread)was 4h.The black brackets indicate the 60-␮m growth zone.Each row is a representation of the length of the thread and its six sectors at each time point.Growth of the thread is exponential until it reaches 60␮m in length (dashed line)and becomes linear thereafter,when only cells in the tipmost 60␮m contribute to subsequent growth.Once the tipmost sector is more than 60␮m in length,it will be the only sector which continues to increase in size (see the last column for an example).(B)Micrograph of Gfp-labeled S.meliloti inside an infection thread.Note the braided appearance of the population,which is typical for growing infection threads.Threads that have terminated growth often lose this braided appearance.(C)Diagram of a small,two-column infection thread giving rise to a sectored infection thread.(D)Diagram of a small,two-column infection thread giving rise to a jumbled infection thread and later conversion into a sectored thread.The braiding of columns populated with single bacterial clones of different colors (Fig.2D)would give rise to the appearance of a random arrangement of bacteria,especially in cases where three or more columns are involved.Lowercase letters indicate the original cells;lowercase letters with primes indicate ﬁrst-generation daughter cells.V OL .184,2002NOTES 7045different.An addition to the model can explain jumbled growth (Fig.3).Actively growing threads contain two or three braided columns of bacteria(8)(Fig.3B).Sectored growth should occur when bacteria of a single type load into the thread side by side(Fig.3C).When similar types of bacteria initially load end to end,threads which appear jumbled should result(Fig. 3D).If,through faster growth,or random movement,the tip-most bacteria of a jumbled thread become a single type,then continued growth should eventually result in the appearance of a large terminal sector(Fig.3D and1E).Theoretical work suggests that the frequency at which mixed nodules occur has important implications for the evolution of rhizobium-host symbioses(5).My work suggests that bacterial growth patterns inside infection threads can inﬂuence whether nodules contain mixed or single strains of bacteria.Thus,such growth patterns may ultimately inﬂuence the ecology and evo-lution of this rhizobium-plant symbiosis.This work was supported by 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