Construction of a Full-Length cDNA Library of Gossypium hirsutum L. and Identification of Two MA
夏黑葡萄花及果实全长cDNA文库的构建
（ 南京农业大学 园艺学院，江苏 南京 210095）
摘要： 构建葡萄花果全长 cDNA 是开展葡萄花及果实发育的分子机理研究的重要工作基础，有助于重要相关基因 的克隆、功能分析、调 控 及 其 利 用。以 生 产 上 广 泛 栽 培、性 状 优 良 且 极 具 代 表 性 的 夏 黑 葡 萄 为 试 材，应 用 优 化 的 Creator SMART cDNA Construction Kit 技术，fu / mL，库容为 6 鉴定的结果显示： 插入片段大小为 1． 0 ～ 3． 0 kb，重组率为 99% 良 基 因 提 供 了 方 便，已 成 为 发 现 新基因、进行基因克 隆 与 功 能 基 因 组 学 研 究 的 重 要 工具［3，4］，目前已发现的基因 来，为 方 便 基 因 克 隆 以及 EST 的大量 测 序，众 多 研些 重 要 的 模 式 生 物［5，6］和重要 农 作 物［7］ 全 长 cDNA 文 库，获 得 了 大 量 的 重 要 数 据 ，极 大 地 促 进 了 功 能 基 因 完整阅读框 3'和 5'端的非编码区的全长基 因序 列，并 能 够 有 效 地 克 隆 低 丰 度 的、稀 有 mRNA
constructed with SMART
1． 4． 2 cDNA 第 1 链的合成 在预冷的 0． 5 mL 无 菌离心管中混合如下试剂： 1 μL 总 RNA 样品、1 μL SMART Ⅳ Oligo 引物、1 μL CDS 3 mol / L 接头、2 μL 双 蒸 水 ，混 匀 混 合 物 ，在 微 量 离 心 机 中 稍 离 心 使 混 合 物都集中管底； 72℃ ，2 min 后 冰 上 冷 却 2 min，再 稍 离心使内容物都集 中 到 管 底，并 向 每 一 个 管 中 先 后 加入 2． 0 μL 5 × First-Strand 缓 冲 液，1． 0 μL DDT （ 20 mmol / L） ，1． 0 μL dNTPs （ 10 mmol / L） ，1． 0 μL PowerScriptTM 反 转 录 酶，反 应 总 体 积 为 10 μL，混 合 均 匀，稍 离 心； 在 PCR 仪 上 42℃ 温 育 1 h，70℃ 15 min 终止第 1 链的合成，置于 － 20℃ 保存。 1． 4． 3 cDNA 第 2 链 的 合 成 在 获 得 第 1 链 DNA 的基础上，采 用 LD PCR （ Long distance PCR ） 扩 增， 获得双链 cDNA。在 0． 5 mL 反 应 管 中 加 入 如 下 试
cdna文库的构建和测序方法
cdna文库的构建和测序方法**CDNA Library Construction and Sequencing Methods**The construction of a cDNA library is a fundamental technique in molecular biology, enabling researchers to isolate, clone, and study specific genes. The process begins with the isolation of mRNA from a target cell or tissue. This mRNA is then reverse-transcribed into cDNA using a reverse transcriptase enzyme. The resulting cDNA fragments are subsequently cloned into a suitable vector, such as a plasmid or bacteriophage, to form a recombinant DNA molecule. These recombinant molecules are then introduced into host cells, typically bacteria, for amplification and storage.cDNA文库的构建是分子生物学中的一项基础技术，它使研究者能够分离、克隆和研究特定的基因。
该过程始于从目标细胞或组织中分离出mRNA。
随后，使用逆转录酶将这种mRNA逆转录成cDNA。
接着，将得到的cDNA片段克隆到合适的载体中，如质粒或噬菌体，形成重组DNA分子。
然后，这些重组分子被引入宿主细胞，通常是细菌，进行扩增和保存。
**Sequencing of cDNA Library**Once the cDNA library is constructed, sequencing becomes the next crucial step. Sequencing technologies have evolved significantly in recent years, with next-generation sequencing (NGS) platforms becoming increasingly popular. NGS allows for high-throughput sequencing, generating millions of reads in a short time. Thesereads are then aligned to a reference genome or transcriptome, enabling the identification and analysis of individual genes and gene expression patterns.cDNA文库构建完成后，测序成为下一个关键步骤。
核酸技术问题解答
核酸技术问题解答问题1：我想克隆一个基因家族中的一个基因，这个基因家族的很多基因全长cdna已经被克隆，鸡中的该基因还未克隆，我本来打算根据该基因家族的保守区设计简并引物从鸡中扩增出一段，然后利用race得到全长cdna，可是作了很久的简并引物pcr都没得到什么结果，我想在genbank中查一下有没有该基因的est，不知道该怎么查啊？还请熟悉这方面工作的战友多加指教。
答：请你按照如下步骤：二，步入界面后你将看见存有一个“search”的大空地儿，在此黏上你的基因序列（随便就是全长还是片段，全长不好一些，所贴的基因序列和你必须克隆的物种亲缘关系越近越好），然后找出“choosedatabase”的附加栏，挑选里面的est栏或者estothers （因为你必须克隆鸡子的），页面\按钮；三，在出现的界面里点击“format!”,等待新弹出的窗口完全打开后，里面会出现n 多est序列，找到鸡子的对应的序列；四，页面对应的序列号可以发生代莱窗口，在这个窗口的最后面就是你必须的est序列，后面的就不必我再罗索啦。
问题2：现在手头上有两个老鼠的est克隆，都是1kb左右，但是已知人的cdna有6kb，老板要我拿到全长的老鼠的cdna，对这方面的工作一点经验都没有，不知哪位大侠可以给一点意见，由est去做或者是自己做rt？但是有6kb，rt不知道能行吗？答：几条途径：1，利用生物信息学资源展开电子克隆，进而赢得全长基因；2，利用race技术赢得全长基因；3，利用est得控针筛全长cdna库，进而获得全长基因；问题3:est就是由cdna文库的随机测序赢得，cdna文库源自细胞的mrna,那么这些mrna与否经过剪辑加工为明朗的mrna,还是pre-mrna？如果是由成熟的mrna经反转录为cdna的话，那么est就是没有内含子的，如果有些cdna来自pre-mrna,那么est就可能有内含子？我没搞过cdna文库，所以有些稀奇古怪。
Construction of a Full-Length cDNA Infectious Clone of a European-Like Type 1 PRRSV Isolated in the
CONSTRUCTION OF A FULL-LENGTH cDNA INFECTIOUS CLONE OF A EUROPEAN-LIKE TYPE 1 PRRSV ISOLATED IN THE U.S. Ying Fang, Kay. S. Faaberg, Raymond R. R. Rowland, J. Christopher-Hennings, Asit K. Pattnaik, Fernando Osorio, and Eric A. Nelson 1. INTRODUCTION The recent emergence of a unique group of European-like Type 1 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in the U.S. presents new diagnostic and disease control problems for a swine industry that has already been seriously impacted by the traditional North American Type 2 PRRSV. Genetic and antigenic analysis from our laboratories demonstrated that this group of U.S. Type 1 PRRSV has features that distinguish it from typical European Type 1 PRRSV.1, 2 In order to further characterize this group of U.S. Type 1 PRRSV and provide an essential tool for the future construction of a new generation of genetically engineered PRRSV vaccines for both Type 1 and Type 2 PRRSV, we constructed a full-length cDNA infectious clone of a U.S. Type 1 PRRSV. This is the first Type 1 infectious clone shown to replicate well in MARC-145 cells and represents the second infectious clone of Type 1 PRRSV. In addition, this infectious clone represents a recent member of this genotype, differentiating itself from the Lelystad infectious clone 3 derived from a 15-year-old strain of PRRSV. 2. MATERIALS AND METHODS A European-like Type 1 PRRSV isolate, SD 01-08 (P34) was used for construction of a full-length infectious clone. SD 01-08 was isolated in 2001 from a group of 8-week-old pigs showing no clinical signs. BHK-21 cells were used for initial transfection, and MARC-145 cells were used for virus rescue and subsequent experiments. To construct the full-length cDNA clone, seven overlapping fragments (except the 5’ and 3’ ends) flanked by unique restriction enzyme sites were amplified by RT-PCR and Y. Fang, J. Christopher-Hennings, E. A. Nelson, South Dakota State University, Brookings, South Dakota. K. S. Faaberg, University of Minnesota, St. Paul, Minnesota. R. R. R. Rowland, Kansas State University, Manhattan, Kansas. A. K. Pattnaik, F. Osorio, University of Nebraska, Lincoln, Nebraska.**605Y. FANG ET AL.cloned into the pCR-Blunt II-Topo vector. These fragments were assembled into the low copy number plasmid, pACYC177, by restriction enzyme digestion, ligation, and transformation. The 5’ and 3’ ends of the genome were determined using a GeneRacer kit (Ambion) and assembled into pACYC177 vector. To rescue infectious virus, capped RNA was transcribed in vitro from the pACYC177 clone and transfected into BHK-21C cells using DMRIE-C (Invitrogen). Cell culture supernatant obtained 48 hours post- transfection was serially passaged on MARC-145 cells. Rescue of infectious virus was N specific monoclonal antibodies (MAbs). For discrimination between the cloned virus ORF7 region of the cloned virus using site-directed mutagenesis. Growth kinetics was examined by infecting MARC-145 cells with cloned virus and parental virus at a MOI of 0.1. Infected cells were collected at various times post- infection, and the virus titers were determined by IFA on MARC-145 cells and expressed as fluorescent focus units per ml (FFU/ml). Plaque morphology between the cloned virus and parental virus was compared by plaque assay on MARC-145 cells.3. RESULTS AND DISCUSSIONA full-length genomic cDNA clone of a European-like (U.S. Type 1) PRRSV, strain SD 01-08 was constructed. This construct contains a bacteriophage T7 RNA polymerase promoter at the 5’ terminus of the viral genome, an additional guanosine residue introduced between the T7 promoter and the first nucleotide of the viral genome, 15047 nucleotides full-length genome of SD 01-08 and a poly (A) tail of 41 residues incorporated at the 3’ end of the genome. The in vitro transcribed capped RNA was transfected into BHK-21 cells. Forty-eight hours post-transfection, cells were examined by IFA using nucleocapsid (N) protein specific MAb SDOW17 (Fig. 1A). Results showed that about 5% of cells transfected with pSD 01-08 RNA expressed the N protein. Supernatants from the transfected cells were passaged to naïve MARC-145 cells. After 48 hours postinfection, MARC-145 cells were tested using Type 1 PRRSV specific, anti-Nsp2 MAb ES2 36-19 (Fig. 1B), and a MAb recognizing both genotypes, SDOW 17 (Fig. 1C). A Type 2 PRRSV specific, anti-N MAb MR39 (Fig. 1D) was used as a negative control. The results showed that both Nsp2 and N proteins were detected in MARC-145 cells inoculated with supernatant from transfected BHK-21 cells. Upon further passage in MARC-145 cells (passage 2 on MARC-145 cells), cytopathic effects (CPE) were observed within 48 to 72 hours post- infection. These results indicate that viable and infectious PRRSV was rescued from the cells transfected with in vitro transcribed RNA. The cloned virus from the second MARC-145 cell passage was also passaged on porcine alveolar macrophages (PAM). IFA results confirmed the presence of virus replication in PAM (Fig. 1E and 1F), which indicates that cloned virus possessed the ability, as its parental virus, to replicate not only in MARC-145 cells but also in PAM. The growth properties of the cloned virus were compared with that of parental virus. Results showed that there were no significant differences in growth kinetics and plaque morphology between cloned virus and its parental virus (data not shown).606 confirmed by immunofluorescent assay (IFA) using Type 1 and Type 2 PRRSV Nsp2 and and parental SD 01-08 virus, a ScaI restriction enzyme site was engineered into theCONSTRUCTION OF A cDNA INFECTIOUS CLONE OF TYPE 1 PRRSV Figure 1. Rescue and passage of cloned U.S. Type 1 virus, SD 01-08. Picture A, BHK-21C cells transfected with in vitro transcribed RNA from the full-length cDNA clone. Pictures B, C, and D, MARC-145 cells were infected with cloned virus rescued from BHK cells. Cells were fixed and stained with PRRSV specific monoclonal antibodies (MAbs) at 48 hours post-transfection (or infection). A. Anti-N MAb SDOW17; B. Anti-Nsp2 MAb ES2 36-19 (Type 1 PRRSV specific); C. Anti-N MAb SDOW17; D. Anti-N MAb MR40 (Type 2 PRRSV specific). Pictures E and F, porcine alveolar macrophages were infected with parental virus (E) and cloned virus (F), IFA stained with anti-N MAb SDOW17.In conclusion, we successfully constructed a full-length cDNA infectious clone of a U.S. Type 1 PRRSV. The cloned virus maintained similar in vitro growth properties as that of parental virus. The availability of this U.S. Type 1 infectious clone provides an important research tool to study the virulence factors and pathogenic mechanisms of PRRSV. In conjunction with the traditional North American Type 2 infectious clones,4-7 a new generation of genetically engineered chimeric PRRSV vaccines can be constructed.4. REFERENCES1. Y. Fang, D.-Y. Kim, S. Ropp, P. Steen, J. Christopher-Hennings, E. A. Nelson, and R. R. R. Rowland, Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States, Virus Res.100, 229-235 (2004).2. S. L. Ropp, C. E. Mahlum Wees, Y. Fang, E. A. Nelson, K. D. Rossow, M. Bien, B. Arndt, S. Preszler, P. Steen, J. Christopher-Hennings, J. E. Collins, D. A. Benfield, and K. S. Faaberg, Characterization of emerging European-like PRRSV isolates in the United States, J. Virol .78, 3684-3703 (2004).3. J. J.Meulenberg, J. N. Bos-de Ruijter, R. Van de Graaf, G. Wensvoort, and M. Moormann, Infectious transcripts from cloned genomic-length cDNA of porcine reproductive and respiratory syndrome virus, J. Virol .72, 380-387 (1998).4. H. S. Nielsen, G.-P. Liu, J. Nielsen, M. B. Oleksiewicz, A. Bøtner, T. Storgaard, and K. S. Faaberg, Generation of an infectious clone of VR-2332, a highly virulent North American-type isolate of porcine reproductive and respiratory syndrome virus, J. Virol .77, 3702-3711 (2002).5. J. G. Calvert, M. G. Sheppard, S.-K. W. Welch, Infectious cDNA clone of North American porcine reproductive and respiratory syndrome (PRRS) virus and uses thereof, US Patent 6,500,662 (2002).607 To differentiate cloned virus from the parental virus, we engineered a ScaI restriction enzyme site at nucleotide 42 of ORF7. A 1057- bp RT-PCR fragment derived from the cloned virus was cleaved by ScaI . In contrast, the RT-PCR fragment derived from the parental isolate was not cleaved by ScaI.Y. FANG ET AL. 6086. J. G. Calvert, M. G. Sheppard, S.-K. W. Welch, Infectious cDNA clone of North American porcine reproductive and respiratory syndrome (PRRS) virus and uses thereof, US Patent Application 20030157689 (2003).7. H. M.Truong, Z. Lu, G. Kutish, J. Galeota, F. A. Osorio, and A. K. Pattnaik, A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo markers of virulence and transmissibility characteristics of the parental strain, Virology325, 308-319 (2004).。
分子生物学10-gene libraries
Genomic libraries: made from genomic DNA cDNA libraries: made from cDNA （DNA complementary to mRNA, copy of mRNA）
Genomic Library
A genomic library contains clones of all the genes from a species genome Restriction fragments of a genome can be packaged into phage using about 16 – 20 kb per fragment This fragment size will include the entirety of most eukaryotic genes Once a library is established, it can be used to search for any gene of interest
Low enzyme concentration short digestion time
Sau 3A Sau 3A Sau 3A Sau 3A
粗毛栓菌cDNA文库的构建和漆酶基因的表达分析
ISS N 100727626C N 1123870ΠQ中国生物化学与分子生物学报Chinese Journal of Biochemistry and M olecular Biology2009年6月25(6):528～533粗毛栓菌cD NA 文库的构建和漆酶基因的表达分析张希根, 何 川, 张义正3(四川大学生命科学学院,四川省分子生物学与生物技术重点实验室,成都 610064)摘要 粗毛栓菌(Trametes gallic a )能够分泌多种胞外氧化酶并且快速降解木质纤维素.为了快速高效分离鉴定粗毛栓菌木质纤维素降解酶相关基因,用T rizol 试剂提取不同培养条件下粗毛栓菌总RNA ,用CreatorT MS MART T M cDNA Library C onstruction K it 和Advantage○R 2PCR K it 成功构建了该菌全长cDNA 文库.原始文库滴度为115×105cfu ,重组率达99%,插入片段在017～210kb 之间,平均大小约1kb.随机取16个重组子进行测序,全长cDNA 序列完整性率为8517%;并筛选到1个漆酶基因,编码区长1551bp ,预测的蛋白质由517个氨基酸残基组成,分子量为55141kD ,等电点为4176.用半定量RT 2PCR 法分析了该漆酶基因在不同培养条件下的表达水平.结果显示,高浓度的碳源,氮源,Cu 2+均能诱导此基因的表达,该结果为漆酶基因的表达调控机制的深入研究奠定了基础.关键词 粗毛栓菌;cDNA 文库;漆酶;基因表达;反转录PCR 分析中图分类号 Q78Cloning of Laccase G ene from a Constructed cD NALibrary of Trametes gallicaZH ANG X i 2G en ,HE Chuan ,ZH ANG Y i 2Zheng3(College o f Life Sciences ,Sichuan Univer sity ,K ey Laboratory o f Molecular Biology andBiotechnology o f Sichuan Province ,Chengdu 610064,China )Abstract Tramets gallica secrete a series of extracellular oxidases to rapidly degrade lignocellulose.T o studythe relevant lignocellulolytic enzymes under different cultural conditions ,a cDNA library was constructed byusing the S MART (S witching Mechanism At 5′end of RNA T ranscript ,S MART T M )K it and the Advantage ○R 2PCR K it using the total RNA was extracted from T .gallica .Over 99%of 115×105independent clones in the cDNA library contained insertions of 017～210kb.16clones were selected randomly for sequencing and 14of which contained full 2length cDNA sequences ,including a laccase that encodes a protein of 517amino acid residues with m olecular weight 55141kD and pI 4176.The expression levels of the laccase in different culture conditions was analyzed by semi 2quantitative RT 2PCR.The result dem onstrated that the gene expression wasinduced by high concentrations of carbon ,nitrogen s ource and Cu 2+.This laccase 2expressing construct appeared to be useful for further studies of the enzyme expression regulations.K ey w ords Trametes gallica ;cDNA library ;laccase ;gene expression ;RT 2PCR analysis收稿日期:2009201210;接受日期:2009203218国家自然科学基金资助项目(N o.30470984)3联系人 T el :028*********;E 2mail :yizzhang @Received :January 10,2009;Accepted :M arch 18,2009Supported by National Natural Science F oundation of China (N o.30470984)3C orres ponding auth or T el :028*********;E 2m ail :yizzhang @ 栓菌(Trametes )是一类能有效地降解木质纤维素及多种难降解的苯酚类有机物的丝状真菌,它能用于研究植物生物质利用和自然界有机污染物的生物治理[1～4].粗毛栓菌HT C (T .gallica HT C )是一株可分泌20余种漆酶的白腐丝状真菌,比其它许多真菌,如香菇(Lentinula )、平革菌(Phanerochaete )、侧耳菌(Pleurotus )等能更有效地降解木质素,并具有很高的木质纤维素降解酶活性,可作为生物制浆、纸浆漂白、环境保护等方面的工业生产理想候选菌[5～7].虽然目前对T .gallica HT C 的基因工程研究取得了一些进展,但多限于对木质纤维素降解酶基因的克隆等,其研究水平与其他真菌相比,还存在着很大的差距[8～10].构建在不同培养条件下生长的T .gallica HT C 全长cDNA 文库是克隆相关基因并研究其生物学特性的基础.因此,本室用在不同生长条件下的T.gallica HT C制备RNA,用S MART技术,构建了全长cDNA文库,为分离新的全长序列的功能基因,对T.gallica HT C木质纤维素降解酶基因的特异表达及其应用等研究奠定基础.漆酶(laccase,EC11101312,Lac)是一种具有多个铜原子的含糖氧化酶,广泛分布于动植物和微生物中,其中真菌Lac被认为参与了腐木中木质素的降解22自然界碳循环的重要步骤.Lac被广泛用于生物制浆、漂白或用于降解污水中的酚类有毒物以及化合物的改造;利用木质素生产有用化合物,制造生物电极等,因而具有极大的工业应用价值[11～14].真菌Lac多为同工酶家族,通常由不同的基因家族所编码,而编码各种Lac同工酶的基因的表达调控又受到其菌株培养条件的影响[15～17].本实验室曾从T.gallica HT C中分离纯化出多种Lac同工酶,但对其表达调控机制仍知之甚少.本实验从T.gallica HT C的cDNA文库中成功地筛选到1个新Lac基因,通过半定量RT2PCR方法对该基因的表达模式进行了初步分析,为进一步筛选其它Lac同工酶基因以及对此类基因家族的表达分析提供了重要依据.1 材料和方法111 材料粗毛栓菌HT C(T.gallica HT C)由山东菏泽师范学院孙迅教授提供,4℃保存于PDA培养基中.该系列培养基由K irk基本培养基[18]改进而来,每种培养基(L)都含有2g K H2PO4,015g MgS O4・7H2O,011 g CaCl2和100ml微量元素溶液.微量元素溶液(L): 3g MgS O4・7H2O,015g MnS O4,1g NaCl,011g FeS O4・7H2O,011g C oCl2,011g ZnS O4・7H2O,011g CuS O4・5H2O,0101g K Al(S O4)2・12H2O,0101g H3BO3,0101g Na2M oO4・2H2O,115g氮三乙酸(NT A).在上述营养盐液中设计高碳高氮、高碳低氮、低碳高氮、低碳低氮4种营养组合条件:其中氮营养(酒石酸铵)浓度为214mm olΠL,定为低氮;碳营养(葡萄糖)浓度为1010gΠL,定为高碳,在此基础上分别设置了高氮,其浓度为24mm olΠL;低碳,葡萄糖浓度110gΠL;其中CuS O4浓度为200μm olΠL.另外,设置6种不同铜离子含量的培养基:用24mm olΠL酒石酸铵作为氮源,1%葡萄糖作为碳源,CuS O4浓度分别为:0、015、5、50、100、200μm olΠL.总RNA抽提T rizol试剂、M2M LV Reverse T ranscriptase购自Invitrogen公司,文库构建试剂盒Creator T M S MART T M cDNA Library C onstruction K it与Advantage○R2PCR K it购自Clontech公司Taq聚合酶购自T aK aRa公司,其余试剂均为分析纯或化学纯. 112 T.gallica HTC培养将T.gallica HT C接种于PDA培养基斜面, 28℃下活化培养;然后转接在PDA平板培养5～7 d,待菌丝长满整个平板后,用打孔器制成直径为12 mm的菌塞,接种于改进的K irk基本培养基中,250 ml三角瓶,每瓶25ml培养基液体,pH510,定量接种1个菌塞,振荡(120rΠmin,28℃)培养12～14d.113 总RNA提取收集T.gallica HT C菌体,立即在液氮中速冻,再放置-80℃保存.将011g样品放入研钵中,加液氮速冻研磨成粉末状后,加入装有1ml T rizol试剂的离心管里,按T rizol试剂说明书进行操作抽提RNA.使用紫外分析仪检测其浓度,琼脂糖凝胶电泳检测其完整性.114 双链cDNA的合成按Creator T M S MART T M cDNA Library C onstruction K it说明书中严格进行操作.在5μl反应体系中依次加入110μg poly(A)+RNA、10μm olΠL S MRART I V Olig o(5′2AAG CAG TGGT AT CAACG CAG A GTGG CC2 ATT ACGG CCGGG23′)1μl、C DSⅢΠ3′PCR引物5′2AT2 TCT AG AGG CC G AGG CG G CCG AC ATG2d(T)30N-1N2 3′(N=A,G,C or T;N-1=A,G,or C)1μl,用无RNase水补到5μl.72℃2min后向反应液中加入5×第一链反应缓冲液2μl及20mm olΠL DTT、10mm olΠL dNTP混合液1μl、P owerScript逆转录酶各1μl,于42℃孵育60 min,置于冰浴中终止反应,于-20℃保存或直接进行PCR扩增.引物为5′PCR引物(5′2AAG CAGTGG2 T ATCAACG CAG AGT23′)、C DSⅢΠ3PCR引物.循环条件为:首先把PCR仪预热到95℃,再95℃1min, 25个循环:95℃15s,68℃6min.115 cDNA分级分离纯化及文库构建合成的双链cDNA经S fiⅠ酶切后再经CHROMA SPI N2400柱层析分级分离,共收集14管,每管约40μl,然后分别从每管中取4μl经琼脂糖凝胶电泳检测,收集大于015kb的cDNA片段,沉淀后重悬在7μl去离子水中,在T4DNA连接酶作用下,与质粒载体pDNR2LI B(经SfiⅠ酶处理过)于16℃下连接16 h.连接产物转化感受态细胞大肠杆菌JM109.925第6期张希根等:粗毛栓菌cDNA文库的构建和漆酶基因的表达分析 116 文库质量鉴定和扩增取适量原始文库菌液,稀释100倍、1000倍后取10μl均匀地涂在含30μgΠml氯霉素的LB平板上,每个倍数设3个重复.文库滴度公式pfuΠml=菌落数×稀释倍数×103Π稀释的菌体铺板μl数,文库总库容量=文库滴度×总体积数(ml).随机挑取17个克隆,提取质粒经SfiⅠ酶切后电泳检测,可测定其重组率及重组片段的大小.取适量原始文库(115×105重组子)均匀地涂在10个含30μgΠml氯霉素的LB平板上(150mm),37℃倒置培养16～18h,用5 ml含有25%甘油LB培养基洗脱每个平板所有的菌落,将每板的菌液混合即得到了扩增文库,检测文库滴度,分装,放置-80℃保存.117 DNA测序和序列分析随机选取含重组克隆的菌落经过夜培养后,送北京三博远志生物技术有限责任公司测序,在NC BI 上网站中的G enBank内对测得的DNA序列进行比对,并分析其结果.118 Lac基因的表达分析用半定量RT2PCR分析T.gallica HT C中Lac基因在不同培养条件下的相对表达水平.设计一对特异引物:FP:5′2ATGG CCAGGTTCCAGTCTCTCC23′, RP:5′2CTGGTCGTCAGG CG AG AG CG23′,分别以不同cDNA作为模板,目的产物长度为1551bp.PCR反应程序:在25μl反应体系中依次加入模板DNA约10 ng,10×T aq缓冲液215μl,215mm olΠL dNTP2μl,10 mm olΠL上下游引物各1μl,Taq DNA聚合酶1U.在BI O2RAD公司生产的扩增仪上进行PCR扩增.反应条件为94℃预变性4min,每次循环94℃变性30s, 62℃退火30s,72℃延伸2min,共30次循环,最后72℃再延伸10min.以T.gallica HT C中组成型表达的三磷酸甘油醛脱氢酶基因(Gpd)为内参,设计扩增Gpd基因的特异引物:FP:5′2CGT ATCGTCCTC C2 GT AATG C23′,RP:5′2ACTCGTTGTCGT ACCAGG AG2 3′,扩增产物长度为907bp,PCR反应程序同上,但将循环数减少到20.将PCR产物电泳后拍照,在AlphaImager软件上进行光密度扫描分析.2 结果211 双链cDNA的合成与分级利用T rizol法提取的T.gallica HT C菌丝体总RNA经紫外分光光度计测定,A260ΠA280值为1193,所得总RNA纯度较高,符合建库要求.合成的双链cDNA产物经110%琼脂糖凝胶电泳结果显示,不同大小的cDNA成一弥散带,最大片段超过310kb,主要集中在015～210kb(Fig.1),表明合成的cDNA是比较完整的.合成产物用SfiⅠ完全酶切后过CHROMA SPI N2400柱分级分离,根据111%琼脂糖凝胶电泳检测结果,合并合适大小片段的收集管样品(5～7管),乙醇沉淀,重悬于ddH2O中.Fig.1 E lectrophoresis analysis of synthesized ds2cDNA of Trametes gallica HTC M:D L15000DNA marker;1: Agarose gel electrophoresis analysis of the synthesized ds2cDNA synthesized by LD2PCR,appeared mainly as the size of015～210kb212 cDNA文库的构建文库构建过程中cDNA和pDNR2LI B载体的连接比例决定了连接效率,设计3种cDNA片段与质粒体积比为015∶1、1∶1、115∶1.结果表明,比例为115∶1时,产生重组体的效率最高.将所有的cDNA 与载体连接后电击转化大肠杆菌JM109感受态细胞,电击条件是215kV,25μF,300Ω.将所有转化细胞合并,涂平板检测,该文库滴度为115×105cfu.随机挑取17个克隆,提取质粒经SfiⅠ酶切后电泳.结果表明,每个克隆均有插入片段,重组率>99%,插入片段大小大多在017～210kb间,平均大小1kb 左右(Fig.2).扩增文库的总容量达到312×109,适合长期保存.213 漆酶cDNA序列分析从T.gallica HT C的cDNA文库中随机挑选16个阳性克隆测序,同时对序列进行全长分析.有14条序列具有相关同源信息,除T g02,T g09两序列外,其余12条均为完整编码cDNA序列,长度为016～1165kb,完整性比率为8517%.这些同源基因包括酶类、结构蛋白、信号传导因子等(T able1).其中T g13序列与Lac基因具有非常高的同源性,命名为Tg Lac1.序列分析表明,ORF编码1个由517个氨基酸组成的蛋白,预测分子量为55141kD,等电点为035中国生物化学与分子生物学报25卷Fig.2 E lectrophoresis analysis of cDNA clones digested with SfiⅠ M:λEco T14DNA marker;1～17:Sam ples of recombinant clones.All of17recombinant clongs were digested by SfiⅠfrom recombinant clones picked at random and analyzed by agarose gel electrophoresis analysis.The average size of inserted fragments was approximately110kb.The m ost inserts were over015kb4176,已登录G enBank(登录号F J598130).将此蛋白序列与其它真菌中已报道的Lac进行多序列比对,再利用MEG A410软件采用邻接法构建系统进化树(Fig.3).结果表明,Tg Lac1与T.sp.C230,T.trogii 产生的Lac同源性达95%,与G.tusgae产生的Lac 同源性较低,为69%.T able1 List of some cDNA fragments comp ared with sequence from G enB ankClone number Putative function LengthΠbp C om pared species Identity(%) T g01Ribos omal protein759Laccaria bicolor S238N2H8265T g02Argininosuccinate synthetase813Coprinopsis cinerea okayama7#13078T g03Alpha2tubulin1254Coprinopsis cinerea okayama7#13080T g04Phosphatidylinositol32kinase1271Coprinopsis cinerea okayama7#13072T g05Hypothetical protein591Laccaria bicolor S238N2H8271T g06G lycoside hydrolase family16protein1560Laccaria bicolor S238N2H8280T g07M itochondrial ribos omal protein894Laccaria bicolor S238N2H8267T g08ATP synthase A chain subunit9760Pleurotus ostreatus95T g09G lycoside hydrolase1620Legionella pneumophila str.Lens24T g10Heat shock protein603Laccaria bicolor S238N2H8253T g11Cytochrome P450oxidoreductase848Trametes ver sicolor87T g12Hypothetical protein732Cryptococcus neo formans var.neo formans J EC2155T g13Laccase1554Trametes sp.C3095T g14Hydrophobin2603Lentinula edodes41Fig.3 Phylogenetic relationship betw een laccase amino acid sequences of different fungi Analysis of hom ology tree from several kinds of laccase amino acid sequences was per formed by using MEG A410.Bootstrap percentage values were shown at nodes,G enBank register numbers were in the right.The Tg Lac was highly conserved to T.sp.C230and T.trogii(95%indentity as a whole),but only69%indentity to G.tsugac 135第6期张希根等:粗毛栓菌cDNA文库的构建和漆酶基因的表达分析 214 Lac 基因的表达分析半定量RT 2PCR 分析T .gallica 在不同培养条件下Tg Lac 1的转录水平.结果(Fig.4)表明,在碳氮组合培养条件时,当碳源、氮源在较高浓度培养条件下,Tg Lac 1转录水平明显增加;当氮浓度不变时,高碳比低碳条件下的Tg Lac 1转录水平增加不到2倍,而当碳浓度不变时,高氮比低氮条件下的Tg Lac 1转录水平却增加了3～5倍.在不同Cu 2+培养条件时,当不加CuS O 4时,Tg Lac 1的转录水平几乎为零;但随着CuS O 4浓度的提高,伴随着Tg Lac 1转录水平相应的提高,在Cu 2+浓度为200μm ol ΠL 时,Tg Lac 1的转录水平比Cu 2+浓度为015μm ol ΠL 时增加了4倍.Fig.4 Expression of TgLac 1under different concentrations of carbon 2nitrogen (A)and Cu 2+(B) The higher m olecular weight band in each case represented the level of Tg Lac 1transcripts present.The lower m olecular weight band represented the TgGpd fragment am plified as an internal PCR control.T ΠC :Expression ratio of Tg Lac 1to TgGpd control.1:Nitrogen 2and carbon 2limited media ;2:Nitrogen 2su fficient and carbon 2limited media ;3:Nitrogen 2limited and carbon 2su fficient media ;4:Nitrogen 2and carbon 2su fficient media3 讨论311 高质量cDNA 文库的构建策略及评价发现和确证新基因的功能的关键在于获取尽可能多的全长cDNA.构建T .gallica HT C 的全长cDNA 文库,极大地方便了T .gallica HT C 新功能基因的筛选工作,是进一步进行T .gallica 木质纤维素降解酶相关基因克隆和鉴定的重要基础.完成高质量cDNA 文库的构建,首先需要分离到高质量的总RNA ,并且mRNA 种类越多,cDNA 文库就越完整[19].由于存在一些不同生长发育时期和不同生长条件下特异表达的重要基因,本室选取了不同培养条件下的T .gallica HT C ,经分别对木质纤维素酶酶活的测定分析,在12～14d 正是mRNA 转录和表达最为旺盛的时期.为了重点研究木质纤维素酶的生产条件以及表达调控机制,分别选取了不同生长状况下的T .gallica HT C 菌丝体,用T rizol 试剂提取法抽提总RNA ,直接混合建库,这样避免了在mRNA 分离和纯化中所造成的RNA 丢失.为了高效和大规模地获得全长基因序列,并实现对低丰度mRNA 基因的克隆,本室采用了S MART 技术[20]来构建cDNA 文库.此方法保证PCR 扩增出来的为全长cDNA.同时将cDNA 片段进行分级分离,除去其中的小片段(小于500bp ),增加了大片段插入到载体中的几率,提高文库的质量.评价一个cDNA 文库的质量,主要是看该cDNA 文库的库容量和插入的cDNA 片段长度.通过以上措施使本室所构建的文库具较好的代表性和完整性.经鉴定,cDNA 克隆重组率为99%,文库库容量为312×109,而插入片段长度多分布于700～2000bp 之间.测序验证部分插入cDNA 全序列,经生物信息学分析,序列完整性比例为8517%.已测的基因包含了多种参与生理过程的重要功能基因,在T .gallica HT C 中均为首次报道,更值得一提的是,它们中有许多具有完整的开放阅读框,跟其他物种的序列同源性很低或无同源性,提示它们极有可能是T .gallica HT C 中的特有基因,对这些基因的深入研究,可为将来揭示T .gallica HT C 生长过程的分子机制和发育进化中的特殊地位提供物质基础.T .gallica HT C 全长cDNA 文库的成功构建,为后续的235中国生物化学与分子生物学报25卷大规模测序,以及全长新基因的克隆和功能研究奠定了坚实的基础.312 漆酶基因的表达由于T.gallica HT C中降解木质纤维素酶系统的复杂性以及编码它们的基因的复杂性和它们在染色体上的连锁关系,木质纤维素降解酶基因的转录表达以及它们在木质纤维素降解中的作用方式并不清晰.T.gallica HT C作为高产Lac菌株,有必要开展此类基因在不同的培养条件下的转录应答分析,以确定它对不同的营养因子的转录应答和它们在降解天然木质纤维素的作用模式.本实验从cDNA文库中成功地筛选到1个Lac基因,与对应的Tg Lac1在T.gallica HT C中的转录水平相对较高.用半定量RT2PCR方法初步分析了T.gallica HT C在4种碳源与氮源营养组合以及不同Cu2+浓度培养基培养12～14d时Tg Lac1的表达,确定不同营养因子对其表达的影响.结果显示酒石酸铵、葡萄糖、Cu2+浓度对Tg Lac1表达影响十分显著.当浓度提高时,Tg Lac1的转录水平相应提高,这表明该基因在转录水平上受到碳源、氮源、Cu2+的诱导表达调控;对Tg Lac1而言,氮源、Cu2+浓度对其表达调控的影响比碳源要更大.由此可见,Lac的表达与菌株的培养条件有很重要的关系,同时为进一步分析T.gallica HT C中Lac 同工酶基因与其它木质纤维素酶基因特异表达及其表达调控机制等提供了重要依据.参考文献(R eferences)[1] Raghukumar C,D’S ouza2T iclo D,Verma A K.T reatment of coloredeffluents with lignin2degrading enzymes:an emerging role of marine2derived fungi[J].Crit Rev M icrobiol,2008,34(324):1892206 [2] Naik N M,Jagadeesh K S,Alagawadi A R.M icrobial decolorizationof spentwash:a review[J].Indian J M icrobiol,2008,48(1):41248 [3] Parawira W.The status and trends in food,industrial andenvironmental biotechnology research in Z imbabwe[J].A frican JBiotechnol,2008,7(10):137721384[4] Ciullini I,T illi S,Scozzafava A,et al.Fungal laccase,cellobiosedehydrogenase,and chem ical mediators:C ombined actions for thedecolorization of different classes of textile dyes[J].Biores ourtechnol,2008,99(15):700327010[5] D ong J L,Zhang Y W,Zhang R H,et al.In fluence of cultureconditions on laccase production and is ozymes patterns in the white2rotfungus T ramets gallica[J].J Basic M icrobiol,2005,45(3):1902198[6] Sun X,Zhang R,Zhang Y.Production of ling ocellulolytic enzymesby T rametes gallica and detection of polysaccharide hydrolase andlaccase activities in polyacrylam ide gels[J].J Basic M icrobiol,2004,44(3):2202231[7] 谢君,孙迅,任路,等.粗毛栓菌产生木质纤维素酶及其降解植物生物质的研究[J].高技术通讯(X ie Jun,Sun Xun,RenLu,et al.Production of lignocellulolytic enzymes and wheat strawdegradation of Trametes gallic[J].High T echnol Lett),2001,8(8):11216[8] K ersten P,Cullen D.Extracellular oxidative systems of the lignin2degrading Basidiomycete Phanerochaete chrysosporium[J].FungalG enet Biol,2007,44(2):77287[9] 胡敏珊,张义正,曾凡亚.粗毛栓菌(Trametes gallic)基因启动子的分离与鉴定[J].四川大学学报(Hu M in2Shan,Zhang Y i2Zheng,Z eng Fan2Y a.Is olation and characterization of prom oters fromTrametes gallic[J].J S ichuan Univ),2002,39(2):3402344 [10] 孙迅,江明锋,李校,等.用cDNA微阵列技术快速筛选粗毛栓菌的表达基因[J].生物化学与生物物理进展(Sun X,Jiang M F,Li X,et al.Rapid screening of expressed genes of Trametesgallica by cDNA m icroarray[J].Prog Biochem Biophys),2004,31(4):3562360[11] R odríguez C outo S,T oca Herrera J L.Industrial and biotechnologicalapplications of laccases:A review[J].Biotechnol Adv,2006,24(5):5002513[12] Riva ccases:blue enzymes for green chem istry[J].T rendsBiotechnol,2006,24(5):2192226[13] C outo S R,T oca2Herrera J ccase production at reactor scale byfilamentous fungi[J].Biotechnol Adv,2007,25(6):5582569 [14] Pant D,Adhlleya A.Biological approaches for treatment of distillerywastewater:A review[J].Biores our T echnol,2007,98(12):232122334[15] C ollins P J,D obs on A.Regulation of laccase gene transcription inTrametes ver sicolor[J].Appl Environ M icrobiol,1997,63(9):344423450[16] M ansur M,Suarez T,G onzalez A E.Differential gene expression inthe laccase gene fam ily from basidiomycete I262(CECT20197)[J].Appl Environ M icrobiol,1998,64(2):7712774[17] S oden D M,D obs on A D.Differential regulation of laccase geneexpression in Pleurotus sajor2caju[J].M icrobiology,2001,147(7):175521763[18] K irk T K,Farrell R L.Enzym ic”combustion”:the m icrobialdegradation of lignin[J].Annu Rev M icrobiol,1987,41:4652505 [19] 张晖,张静,田杰,等.婴幼儿小肠组织全长cDNA文库的构建及鉴定[J].中国实用儿科杂志(Zhang Hui,Zhang Jing,T ianJie,et al.C onstruction and identification of a cDNA library ofin fant’s small intestine[J].Chin J Pract Pediatr),2008,23(2):1332135[20] Zhu Y Y,M achleder E M,Chenchik A,et al.Reverse transcriptasetem plate switching:a S M ART approach for full2length cDNA libraryconstruction[J].Biotechniques,2001,30(4):8922897335第6期张希根等:粗毛栓菌cDNA文库的构建和漆酶基因的表达分析 。
利用DSN和SMARTTM技术构建绿僵菌产孢时期均一化全长cDNA文库
农业生物技术学报 Journal of Agricultural Biotechnology 2007,15(5):884~887*基金项目： 国家自然科学基金 （No.30170630） 和重庆市重点自然科学基金(No.8564)资助。
**通讯作者。
Author for correspondence.教授， 主要从事病原真菌与寄主昆虫之间相互作用的分子机理研究以及杀虫真菌农药研制。
EMail:<Yuxianxia@>. 收稿日期：20070109 接受日期： 20070315 ·研究论文· 利用 DSN 和 SMART TM技术构建金龟子绿僵菌产孢时期均一化全长cDNA 文库 *张石柱，王中康，彭国雄， 曹月青，殷幼平，谢 磊，刘 静，夏玉先 **(重庆大学生物工程学院基因工程研究中心，重庆市杀虫真菌农药工程技术研究中心， 功能基因及调控技术重庆市重点实验室，重庆 400030) 摘要： 为高效获得与绿僵菌产孢相关的全长 cDNA ， 采用 DSN （duplexspecific nuclease ） 均一化技术与 SMARTTM(switching mechanism at 5忆 end of RNA transcript) 建库技术相结合的方法，构建了杀虫真菌金龟子绿僵菌（ var.)菌株 CQMa102产孢时期的均一化全长 cDNA 文库。
经检测， 原始文库滴度为2.1伊 10 6 cfu/mL ， 库容量超过6伊 10 6。
随机挑取 100个克隆，PCR 方法测得文库重组率大于95％,插入片段平均长度1500bp 。
小规模测序分析表明， 全长基因的比例 超过60％。
对组成性表达基因 3磷酸甘油醛脱氢酶 和微管蛋白 均一化前后丰度检测表明， 其丰度均有大幅度的下降，基本满足节约筛库的要求。
一种构建全长cDNA文库的方法
自 ２０ 世 纪 ７０ 年 代 中 期 首 例 ｃＤＮＡ 克 隆 问 世 以 来 ， 已 经 发 展 了 许多 旨 在 提 高 ｃＤＮＡ 合 成 效 率 和 长 度 的 方 法 ， 并 对载管该系统在载体的设计方面 非常成熟， 然而， 该系统构建过 价 值 。 传 统 ｃＤＮＡ 文 库 构 建 方 法 由 于 反 转 录 能 力 差 ， ｃＤＮＡ 内切酶位点保护不彻经不能适 应 目 前 大 规 模 、高 通 量 、高 效 的 功 能 基 因 组 研 究 需 要 。因 此 ， 寻找一种此， 笔者以人胎盘组织为材 料， 研究了一种以 ＬＤ!ＰＣＲ 为基础的简人胎盘组织， ＪＧ４５ 质粒和 Ｅ．ｃｏｌｉ ＪＭ１０９ 菌株， 由 西北农林科技大学动物科技学院实验室保存； Ｍ!ＭＬＶ 反转 录酶， ＳｆｉⅠ限制性内切酶， Ｐｙｒｏｂｅｓｔ ＤＮＡ 聚合酶， Ｔ４ ｌｉｇａｓｅ 及 ＤＥＰＣ， 购自大连宝生物公司； 质粒小量快速提取试剂盒， ＰＣＲ 产 物 纯 化 试 剂 盒 ， 购 自 Ｂｉｏ!Ｄｅｖ 公 司 ； Ｔｒｉｚｏｌ， 购 自 Ｉｎｖｉｔｒｏｇｅｎ 公 司 ； 引物 合 成 和 测 序 ， 由 上 海 ｉｎｖｉｔｒｏｇｅｎ 生 物 公 司公司完成； 其余试剂均为国产分析纯。引物序列为：
Ｓｔｕｄｙ ｏｎ Ｆｕｌｌ Ｌｅｎｇｔｈ ｃＤＮＡ Ｌｉｂｒ ａｒ ｙ Ｃｏｎｓｔｒ ｕｃｔｉｏｎ ＬＩＵ Ｈｏｎｇ!ｊｕｎ ｅｔ ａｌ （Ｃｏｌｌｅｇｅ ｏｆ Ａｎｉｍａｌ Ｓｃｉｅｎｃｅ ａｎｄ Ｔｅｃｈｎｏｌｏｇｙ， Ｎｏｒｔｈｗｅｓｔ Ａ＆．Ｆ Ｕｎｉｖｅｒｓｉｔｙ， Ｙａｎｇｌｉｎｇ， Ｓｈａａｎｘｉ ７１２１００） Ａｂｓｔｒ ａｃｔ Ａ ｓｉｍｐｌｅ ｍｅｔｈｏｄ ｏｆ ｆｕｌｌ ｌｅｎｇｔｈ ｃＤＮＡ ｌｉｂｒａｒｙ ｃｏｎｓｔｒｕｃｔｉｏｎ ｂａｓｅｄ ｏｎ ｌｏｎｇ!ｄｉｓｔａｎｃｅ ＰＣＲ ｗａｓ ｅｓｔａｂｌｉｓｈｅｄ． Ｔｏｔａｌ ＲＮＡ ｆｒｏｍ ｔｈｅ ｈｕｍａｎ ｐｌａｃｅｎｔａ ｔｉｓｓｕｅｓ ｗｅｒｅ ｉｓｏｌａｔｅｄ， ａｎｄ ｔｈｅ ｆｉｒｓｔ ｃＤＮＡ ｗａｓ ｓｙｎｔｈｅｓｉｚｅｄ ｔｈｒｏｕｇｈ ＲＴ!ＰＣＲ ｗｉｔｈ ｔｈｅ ｈｅｌｐ ｏｆ Ｍ!ＭＬＶ ｒｅｖｅｒｓｅ ｅｎｚｙｍｅ． Ｔｈｅｎ ｄｏｕｂｌｅ ｓｔｒａｎｄ ｃＤＮＡ （ｄｓ!ｃＤＮＡ） ｗｉｔｈ ｔｈｅ ｒｅｓｔｒｉｃｔｉｏｎ ｓｉｔｅｓ ｏｆ ＳｆｉⅠ ｗａｓ ｓｙｎｔｈｅｓｉｚｅｄ ｔｈｒｏｕｇｈ ＬＤ!ＰＣＲ （Ｌｏｎｇ!ｄｉｓｔａｎｃｅ!ＰＣＲ） ａｎｄ ｌｉｇａｔｅｄ ｉｎｔｏ ｔｈｅ ＪＧ４５ ｖｅｃｔｏｒ ｗｉｔｈ ｔｈｅ ｒｅｓｔｒｉｃｔｉｏｎ ｓｉｔｅｓ ｏｆ Ｓｆｉ Ｉ． Ｒｅｃｏｍｂｉｎａｎｔ ｖｅｃｔｏｒｓ ｗｅｒｅ ｔｒａｎｓｆｏｒｍｅｄ ｉｎｔｏ Ｅ．ｃｏｌｉ ＪＭ１０９ ａｎｄ ｔｈｅｎ ａｍｐｌｉｆｉｅｄ． Ｔｈｅｎ ｑｕａｌｉｔｉｅｓ ｏｆ ｔｈｅ ｃＤＮＡ ｌｉｂｒａｒｙ ｗｅｒｅ ａｎａｌｙｚｅｄ． Ｔｈｅ ａｖｅｒａｇｅ ｃａｐａｃｉｔｉｅｓ ｏｆ ｔｈｅ ｃＤＮＡ ｌｉｂｒａｒｙ ｗａｓ ａｂｏｕｔ ７．０１×１０５ ｃｌｏｎｅｓ ｐｅｒ μｇ ｄｓ!ｃＤＮＡ ｗｉｔｈ ｒｅｃｏｍｂｉｎａｎｔ ｒａｔｅ ｏｆ ９６％． Ａｍｏｎｇ ｔｈｅ ８０ ｄｅｔｅｃｔｅｄ ｒａｎｄｏｍｌｙ ｓｅｌｅｃｔｅｄ ｃＤＮＡ ｃｌｏｎｅｓ， ｎｏ ｒｅｐｅｔｉｔｉｖｅ ｓｅｑｕｅｎｃｅ ｗａｓ ｆｏｕｎｄ． ｃＤＮＡ ｌｉｂｒａｒｙ ｃｏｎｓｔｒｕｃｔｅｄ ｗｉｔｈ ｔｈｉｓ ｍｅｔｈｏｄ ｗａｓ ｓｕｉｔａｂｌｅ ｆｏｒ ｆｕｎｃｔｉｏｎａｌ ｇｅｎｅ ａｎａｌｙｓｉｓ． Ｋｅｙ ｗｏｒ ｄｓ Ｆｕｌｌ ｌｅｎｇｔｈ ｃＤＮＡ； Ｌｏｎｇｄｉｓｔａｎｃｅ!ＰＣＲ； Ｍｅｔｈｏｄ ｏｆ ｃＤＮＡ ｌｉｂｒａｒｙ ｃｏｎｓｔｒｕ分别为 和 ５．７３×１０μｇ ｄｓ&# ＤＮＡ， 通过酶切鉴定 ｃＤ ｋｂ， 在 １００ 个随机提取的质粒 中 仅图见图 ３。
利用PLDMV
热带作物学报2022, 43(4): 684 692 Chinese Journal of Tropical Crops收稿日期 2021-12-20；修回日期 2021-12-27基金项目 海南省自然科学基金高层次人才项目（No. 320RC717）；国家自然科学基金项目（No. 32072390）。
作者简介 杨秀坤（1996—），女，硕士研究生，研究方向：农艺与种业（园艺）。
*通信作者（Corresponding author ）：朱国鹏（ZHU Guopeng ），E-mail ：******************；周 鹏（ZHOU Peng ），E-mail ：*****************.cn 。
利用PLDMV/Twin-Strep 侵染性克隆纯化HC-Pro 病毒蛋白杨秀坤1,2，沈文涛2，庹德财2，王 赫1,2，言 普2，黎小瑛2，朱国鹏1*，周 鹏1,2*1. 海南大学园艺学院/海南省热带园艺作物品质调控重点实验室，海南海口 570228；2. 中国热带农业科学院热带生物技术研究所，海南海口 571101摘 要：番木瓜畸形花叶病毒（Papaya leaf distortion mosaic virus , PLDMV ）是一种新的潜在威胁番木瓜种植业的病毒，其辅助成分蛋白酶（helper component- proteinase, HC-Pro ）是PLDMV 编码参与病毒复制、运动、寄主植物症状表现的多功能蛋白，因此纯化获得具有功能活性的HC-Pro 蛋白，研究其多功能性具有重要意义。
本研究利用In-Fusion 拼接策略和E.coli Cell-Free 快速构建植物病毒侵染性克隆法，一步快速地将28个氨基酸组成的蛋白标签Twin-Strep 插入到PLDMV HC-Pro 氨基端，成功获得了基于农杆菌的携带Twin-Strep 标签的PLDMV 侵染性克隆pPLDMV-Strep 。
千里光S-腺苷甲硫氨酸合成酶(SAMS)的结构域与功能位点分析3
S -(SAMS)a ,a ,b ,c*a.b.c.,563003cDNA S-S-adenosylmethionine synthase,SAMS 3(GenBank ID:KC149908.1)3945.4843.40kD 3-D -helix/-strand SPOUT SAMS S -SAMS :Q949.783.5:A1007-7847(2015)03-0203-07Functional Role Determined by Structural Domains ofS -adenosylmethionine Synthase (SAMS)in Senecio scandensBuch.-Ham.ex D.DonTAN Hao a ,WEN Chun-ju a ,QIAN Qian b ,QIAN Gang c*(a .Department of Medical Cosmetology;b .Department of Clinic Medicine;c .Department of Cell Biology and Genetics,ZunyiMedical College,Zunyi 563003,Guizhou,China )Abstract:Three highly conserved motifs of S -adenosylmethionine synthase(SAMS)were selected to observe the relationship between structural domains and their functional sites based on our previous construction on a high-quality of full-length-enriched cDNA library in Senecio scandens Buch.-Ham.ex D.Don.Here,S -adenosylmethionine synthase gene (SA MS )was isolated depending on analysis of its open readingframe (ORF).As shown in our results,cDNA clone (GenBank ID:KC149908.1)encodes a protein composed of 394amino acid residues with the theoretical isoelectric point of 5.48and the predicted molecular weightof 43.40kD.And then,3-D model shape alignments indicate that a genuine hydrophobic core composed of SPOUT-domain and a relaxed -helix/-strand complexity is a key methyl group donor for the methyltrans ferase reactions involving DNA,RNA,proteins,and phospholipids.This work hereby elucidates that SAMSprotein binding sites are attributed to the structural properties determined by the highly conserved motifs.2014-09-022014-10-17([2013]6501)(201410661001)2008-611994-E-mail:tanhao0219@*1969-E-mail:pengjiaqiong@ Received data:2014-09-02Accepted date:2014-10-17Foundation item:The initial work project of undergraduate in Zunyi Medical College ([2013]6501),The innovation project of undergraduate in Guizhou province (201410661001)The special foundation administered by supervisor in Guizhou province of China (QZH-2008-61)Biographies:TAN Hao (1994-),female,Dianjiang county of Chongqing city,undergraduate of Zunyi Medical College,E-mail:tanhao0219@.;*Corresponding author:QIAN Gang (1969-),male,Dianjiang county of Chongqing city,professor of Zunyi Medical College,PhD,E-mail:pengjiaqiong@.19393156Jun.15151IntroductionRegulation of the tetrapyrrole biosynthesis pathway is complex and involves several regulatory systems.Protein function can be thought of on dif ferent interdependent levels and may be divided in to three major categories:molecular function,bio logical process and cellular component[1].S-adenos-ylmethionine synthase(SAMS),the second most prevalent enzyme substrate in cells after ATP,is the major methyl donor for essential methylation re actions and serves as a substrate in polyamine biosynthesis[2].In addition to its role in radical SAMS enzymes,it transfers one electron from an iron-sul fur cluster to the SAMS cofactor,which is then cleaved into methionine and a highly oxidizing radi cal[3].As the biological function of protein molecule is accurately described by its three-dimensional st-ructure,protein-fold structural domains and inter acting components in whole metabolic networks[4,5], ones of the most common motivations for predicting the protein structure,are used to gain insight into the protein's biological function.It is nevertheless an efficient process in the biosynthesis reaction and RNA transcription termination in vitro[6];despite the heavy metabolic demands,which vary according to the changeable gene sites and affect functional roles of this pathway,no direct evidence is available to clarify SAMS functional assay from relationship be tween the key residues of highly conserved motifs and the protein-fold structural domains.Senecio scandens Buch.-Ham.ex D.Don,pre dominantly selfing annual,plays an important role in anti-microorganism involved in Chinese tradi tional medicinal plant and has a widespread distri bution in a few ecological habitats of China[7].Owing to its important antibacterial source in Chinese tra ditional medicine,the biological features should be distinguished at the molecular level to facilitate breeding,gene discovery or industrial applications. As a general trend,the biological usefulness of the predicted protein models relies on the accuracy of the structure prediction[8],although a structural ins-ight into the arrangement of the components in such complexes is still limited[9].Recent advances in co-mputer algorithms for predicting protein structure and function have alleviated this problem and pro vide biologists with valuable information about their proteins of interest[10].Therefore,we here focus on:1) clarifying SAMS functional sites determined by its highly conserved motifs;2)presenting3-D model alignments for a better understanding of the struc tural correlations to functional roles;3)elucidating the phylogenetic relationship of the S-Adenosylme thionine in the high plants.2Materials and methods2.1Plant materialsThe experimental materials Senecio scandens were harvested from the diverse eco-geographic re gions of Yunnan-Guizhou plateau.In this study,the elite antibacterial sample(SC-36)with the high quality of antibacterial feature was selected to con struct cDNA library according to the methods of Shapiro and Baneyx[11],using a series of standard ization bacteria involving Staphylococcus aureus, Pseudomonas ae-ruginosa,Escherichia coli, Salmonella paratyphi,Shigella flexneri,Aeromonas sobria,and Edwardsiella tarda.2.2Construction of full-length cDNA library and sequence data trimmingLeaf tissue of the experimental seedlings was harvested for RNA extraction,using TRIzol-RNA Total RNA Isolation Kit(Invitrogen,China). SMART cDNA library construction kit was applied to generate a full-length cDNA library according to the manufacture's suggestions.The ligation product (5L)of the resultant double cDNA and the vectorKey words:structural domain;three-dimension model alignment;S-adenosylmethionine synthase(SAMS); Senecio scandens Buch.-Ham.ex D.DonCLC number:Q949.783.5Document code:A Article ID:1007-7847(2015)03-0203-07Life Science Research2015193203209 2043pDNR-LIB was transferred to electrocompetent cell XL1-Blue(25L).The plasmid DNA of each clone was directly prepared from bacterial cultures of a glycerol stock plate by the RCA method using a TempliPhi HT DNA amplification kit(GE Health care,UK).End sequencing of10000clones was carried out with iCycler iQ SYBR Green PCR (BIO-RAD Co.,LTD.,USA)using M13sense and antisense primer.Raw sequence data(chro matograms)were base-called using the Phred pro gram and vector sequences were then detected by using cross-match.The low quality region(Phred quality score<20,and more than>20bases re peated)was discarded.We trimmed off the vector sequences of both ends of each read using the sim4 program[7].Sequences data of lengths shorter than100 bases after the trimming process were also omitted for further analysis.2.3Sequence alignment and phylogenetic an-alysisThe cDNA sequence prediction was conducted with GenScan software(/GEN SCAN.html).Sequence similarity analysis in Gen Bank was performed using the Blast2.1search tool (/blast/).ClustalW soft ware(/clustalw/)was used for al-ignment of multiple sequences.Identified ORFs of one transcript(SAMS)was translated into amino acid sequences,and multiple alignments of deduced amino acid sequences were performed using ClustalW with default options[12].Nucleotides and amino acid sequence analyses were performed with DNA MAN program.Phylogenetic trees and molecular evolutionary analyses were constructed based on the bootstrap Neighbor-joining(NJ)method with a Jukes-Cantor model for DNA sequences and Pois son correction model for amino-acid sequences by MEGA v4.0[13].The stability of internal nodes was as-sessed by bootstrap analysis with1000replicates.2.4Prediction and functional assays of protein moleculeSAMS was selected to perform further bioinfor matics analysis according to the methods of Umeza wa et al[14].Signature amino acid patterns for SAM synthetases were retrieved from the PROSITE database of protein families and domains.The se quences excluded in all searches were submitted to the InterPro version4.2with DBrelease12.1to iden tify their functional domains.To predict the bio physics characteristics of the putative protein of SAMS,software on the ExPASy Proteomics Server (/)was used.SignalP-4.0soft ware was applied to analyze the protein signal pep tide(http://www.cbs.dtu.dk/services/SignalP/).The prediction and analysis for the protein structural domain and functional site were finished using PROSITE software(/prosite/). Th e3-D shape of the putative protein conservative domain was performed with the3-D Conservative Domain Architecture Retrieval Tool of Blast(http:// /),and its alignment model was obtained from Database of VAST model(http://www. /blast/).3Results3.1Sequence characteristics and molecular evolutionary of SAMSHere,the isolation of a cDNA encoding SAMS is obtained from a full-length cDNA library in Senecio scandens Buch.-Ham.ex D.Don.The pre sent gene(GenBank ID:KC149908.1)encodes a protein composed of394amino acid residues,with the theoretical isoelectric point of5.48and the predicted molecular weight of43.40kD.As shown in Fig.1,47different genera are accepted to present the phylogenetic tree depending on these selections of the highest scores of E-values in the same species.Based on the deduced amino acid sequence of SAM,a combined phylogenetic tree reveals that the present accession(Senecio scandens Buch.-Ham.ex D.Don)has the closest genetic relation to Populus trichocarpa among the selected species. 3.2Determination on conserved sequences As a result of sequence alignments by running a BlastN search against the GenBank"nr/nt" databases,a complete coding sequence of SAM gene is selected to perform with sequencing analy sis.Seven representative accessions are further apS-(SAMS)20515Fig.1Phylogenetic tree of SAMS in 47representa tive speciesSenecio scandens Buch.-Ham.ex D.Don (KC149908.1)Ricinus communis (XP_002512570.1)Ipomoea batatas (ABP35525.1)Brassica rapa subsp.pekinensis (Q5DN-B1.1)Sorghum bicolor (XP_002457705.1)Triticum aes tivum (B0LXM0.1)Cajanus cajan(AEY85025.1).Fig.2Sequence alignments of the amino acids of SAMS in 7accessions involving the most diverse genetic distanceSenecio scandens Buch.-Ham.ex D.Don (KC149908.1)Ricinus communis (XP_002512570.1),Ipomoea batatas (ABP35525.1)Brassica rapa subsp.pekinensis (Q5DNB1.1)Sorghum bicolor (XP_002457705.1)Triticum aes tivum (B0LXM0.1)Cajanus cajan (AEY85025.1).XP_002512570.1SC-36AEY85025.1Q5DNB1.1XP_002457705.1B0LXM0.1ABP35525.1SC-36AEY85025.1XP_002512570.1Q5DNB1.1XP_002457705.1B0LXM0.1ABP35525.1SC-36AEY85025.1XP_002512570.1Q5DNB1.1XP_002457705.1B0LXM0.1ABP35525.1SC-36AEY85025.1XP_002512570.1Q5DNB1.1XP_002457705.1B0LXM0.1ABP35525.1SC-36AEY85025.1XP_002512570.1Q5DNB1.1XP_002457705.1B0LXM0.1ABP35525.172748272757572154156164154157157154236238246236239239236318319328318321321318394394403393396396393plied to determine the conservative motif sequences depending on the most diverse genetic distance.As shown in Fig.2,the amino acid sequence compari-sons validate the correctness of the current classification of the SAMS domain protein,sharing 92.71%homology with these of the high plant counterparts.Accordingly,three conservative motifs are further used to observe the relationship between structuraldomains and functional sites,included in N-terminal conserved region,M-conserved region and C -terminal conserved region.3.3Structurally similar alignments of conservative domain in SAMSNext,the 3-D conformation of the putative domain of SAMS protein is applied to determine thefunctional site linkage to structural domain of those conservative residues,using the DALI -server.Just as demonstrated by the alignments of 3-D shape,the highly consistent structural distributions are2063foundbetween SAMS of Ricinus communis (XP_002512570.1)and the present protein,involved of N-terminus (Fig.3A),M-conserved region (Fig.3B)and C-terminus (Fig.3C).3.4The topology prediction on SAMSThe topology prediction also observes that theminimal conservative core contains the so -calledSPOUT-domain (related to the SpoU-and TrmD-methyltransferases)in Senecio scandens Buch.-Ham.ex D.Don (SC-36).The result shows that theSAMS topology structure is comprised of six com pactly folded domains of -helix/-strand sheets and a long protruding globular loop (Fig.4A).In this case,a relaxed -helix/-strand subunit is inserted into the core fold of the SPOUT -domain (Fig.4B).As a result of this observation,a deep trefoil knot is involved in both the formation of the co -factor -binding site and part of the dimer interface,whichFig.3Structure-based domain alignments of 3-D shape of SAMS in Senecio scandens Buch.-Ham.ex D.Don andRicinus communis(A)N-terminal conserved domain;(B)N-terminal conserved domain;(C)M-conserved domain.The same configuration (yellow marked);Senecio scandens Buch.-Ham.ex D.Don (SC -36);Ricinus communis (XP_002512570.1).(C)(B)(A)Fig.4Overall 3-D conformations of SAMS protein from Senecio scandens Buch.-Ham.ex D.Don (A)-helix;(B)SPOUT-class.(B)(A)is the other defining feature of the SPOUT-domain fold.Moreover,the knot region is stabilized by an extended network of hydrophobic interactions thatform a genuine hydrophobic core.4DiscussionWhen a gene has been identified,there maybe more than one polymorphism within these ortho logues from different species.A mutation causing a radical change in the amino acid is more likely to affect the properties of the protein than a conserva tive amino acid substitution [15].Some of the most suc-cessful approaches use a phylogenetic tree to rank the residues by evolutionary importance and thenS-(SAMS)20715map this ranking onto a structure if one is available[16]. Here,our results indicate that a stable conformation from the key residues of highly conserved motifs is likely to be needed for protein function across species.Bioinformatics analysis shows that these re gions in three conserved units of SAMS protein are highly identical sequences among the different species(Fig.1and Fig.2),based upon the target-template sequence alignment of those accessions. Thus,the greatest degree of consensus residues has confidently been used to clarify the structural corre lations to some potential functional sites,because of their similar sequences and secondary structures. As proteins from different evolutionary origins may have similar structure,threading methods are designed to match the query sequence directly onto the3-D structures of other solved proteins,with the goal of recognizing folds similar to the query even when there is no evolutionary relationship between the query and the template protein[17].Structural data can be used to detect proteins with similar function whose sequences have diverged beyond a level sim ilarity that can reliably detected using sequence comparison methods[1].Therefore,our target-to-te-mplate model alignment(Fig.3)will also provide an accurate structural comparison on3-D model tem plate from the most contrasting diverse species, even if there is no evolutionary relationship between our present target sequence and the template pro tein.After the major model alignments have been made,the level of conservative sequence observed for the residues strongly suggests a functional im portance for these amino acids in structural do mains.As expected for template-based homology models,structure-based domain alignments of SAMS proteins indicate that the crucial functional sites are dominated by three conserved motifs and none of the mutated amino acid positions is in the vicinity of the SAMS-binding sites. Interestingly,3-D alignments of protein-fold domains in this study show that the highly struc tural similarity results likely in the same and/or similar function in proteins.Just as shown in Fig.4, a genuine hydrophobic core from the core fold of the SPOUT-domain may be the key methyl group donor for the methyltransferase reactions involving DNA,RNA,proteins,and phospholipids.As shown in Fig.3and Fig.4,the other positively charged area involves residues of the irregularly structured sur face loop which is close in space to the cluster of conservative Arg(from R278to I303).The irregularly structured surface loop is poorly conservative,and its utilization would be in agreement with the obser vation that in many other SPOUT-methyltransferas es insertion or extension elements are exploited as auxiliary RNA-binding elements[18].Taylor et al.[19],as parallel with our results,also found that the preva lence of backbone-mediated interactions with the ligand correlates with the lack of sequence conser vation for amino acids in the SAMS-binding pock ets between Nep1and other members of the SPOUT-class of methyltransferases.The topology and the location of the co-factor-binding site are exactly conservative in SPOUT-domain even if the cofactor adopts an extended conformation in the classical Rossman-fold methyltransferases[20].In conclusion,phylogenetic tree and sequence alignment are applied to detect highly conserved motifs of SAMS protein for observing the relation ship between structural domains and functional sites.Both3-D model and structural domain align ments indicate that a genuine hydrophobic core from SPOUT-class may perform with the methyl transferase reactions,which composed of the key residues of highly conserved motifs of SAMS pro tein.This work sheds light on the functional sites are attributed to the structural domains in S-adeno sylmethionine synthase.References:[1]LEE D,REDFERN O,ORENGO C.Predicting protein function from sequence and structure[J].Nature Review,2007,8(12): 995-1005.[2]FONTECAVE M,ATTA M MULLIEZ E.S-adenosylmethionine:nothing goes to waste[J].Trends in Biochemical Sciences, 2004,29(5),243-249.[3]HUNTER C N,DALDAL F,THURNAUER M,et al.The Purple Phototrophic Bacteria[M].New York:Springer-Verlag,2008.81-95.[4]GELLY J C,LIN H Y,DE BREVERN A G,et al.Selectiveconstraint on human pre-mRNA splicing by protein structural properties[J].Genome Biology and Evolution,2012,49:966-975.2083[5]ARNOLD K,BORDOLI L,KOPP J,et al.The SWISS-MODELworkspace:a web-based environment for protein structure homology modelling[J].Bioinformatics,2006,22(2):195-201.[6]SABATY M,ADRYANCZYK G,ROUSTAN C.et al.Coproporphyrin excretion and low thiol levels caused by point muta tion in the rhodobacter sphaeroides S-adenosylmethionine syn thetase gene[J].Journal of Bacteriology,2010,192(5):1238-1248.[7]QIAN G,PING J,LU J,et al.Construction of full-length cDNAlibrary and development of EST-derived simple sequence re peat(EST-SSR)markers in Senecio scandens[J].Biochemical Genetics,2014,52(8):494-508.[8]ROY A,KUCUKURAL A,ZHANG Y.I-TASSER:a unifiedplatform for automated protein structure and function predic tion[J].Nat Protocols,2010,5(4):725-738.[9]MAYNE SLN,PATTERTON H G.Bioinformatics tools for thestructural elucidation of multi-subunit protein complexes by mass spectrometric analysis of protein-protein cross-links[J].Briefings Bioinformatics,2011,12(6):660-671. [10]BROEKER N K,GOHLKE U,MULLER J J,et al.Singleamino acid exchange in bacteriophage HK620tailspike protein results in thousand-fold increase of its oligosaccharide affinity[J].Glycobiology,2013,23(1):59-68.[11]SHAPIRO E,BANEYX F.Stress-based identification andclassification of antibacterial agents:second-generation Escherichia coli reporter strains and optimization of detection[J].Antimicrobial Agents and Chemotherapy,2002,46(8):2490-2497.[12]THOMPSON J D,HIGGINS D G,GIBSON T J.CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice[J].Nucleic Acids Research, 1994,22(22):4673-4680.[13]TAMURA K,DUDLEY J,NEI M,et al.MEGA4:molecularevolutionary genetics analysis(MEGA).Software Version4.0[J].Molecular Biology and Evolution,2007,24(8):1596-1599.[14]UMEZAWA T,SAKURAI T,TOTOKI Y,et al.Sequencingand analysis of approximately40000soybean cDNA clones from a full-length-enriched cDNA library[J].DNA Research, 2008,15(6):333-346.[15]URRUTIA A O,HURST L D.Codon usage bias covaries withexpression breadth and the rate of synonymous evolution in humans,but this is not evidence for selection[J].Genetics,2001, 159(3):1191-1199.[16]YAO H,KRISTENSEN D M,MIHALEK I,et al.An accurate,sensitive,and scalable method to identify functional sites in protein structures[J].Journal of Molecular Biology,2003,326(1):255-261.[17]BOWIE J U,LUTHY R,EISENBERG D.A method to identifyprotein sequences that fold into a known three-dimensional s-tructure[J].Science,1991,253(5016):164-170. [18]MOSBACHER T G,BECHTHOLD A,SCHULZ G E.Structureand function of the antibiotic resistance-mediating methyltransferase AviRb from streptomyces viridochromogenes[J].Jour-nal of Molecular Biology,2005,345(3):535-545. [19]TAYLOR A B,MEYER B,LEAL B Z,et al.The crystal structure of Nep1reveals an extended SPOUT-class methyltrans ferase fold and a pre-organized SAMS-binding site[J].Nucleic Acids Research,2008,36(5):1542-1554.[20]TKACZUK K L,DUNIN-HORKAWICZ S,PURTA E,et al.Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases[J].BMC Bioinformatics,2007,(8):73.S-(SAMS)209。
干旱胁迫下耐旱稻中旱3号全长cDNA文库的构建
分子植物育种，2007，第5卷，第3期，第367-370页Molecular Plant Breeding, 2007, Vol.5, No.3, 367-370研究报告Research Report干旱胁迫下耐旱稻中旱3号全长cDNA文库的构建刘运华1,2刘灶长1*周立国1,2余舜武1刘鸿艳1罗利军1*1 上海市农业生物基因中心, 上海, 201106;2 华中农业大学植物科学院, 武汉, 430070* 通讯作者, lzc@; lijun@摘要干旱胁迫导致很多基因的表达发生变化。
以干旱胁迫下的耐旱稻品种中旱3号为材料，我们用SMART技术(RNA转录5'末端模板转换技术)成功构建一个高质量的干旱胁迫诱导的节水抗旱稻中旱3号全长cDNA文库。
体外包装后感染大肠杆菌XL1-Blue宿主菌，未扩展文库的滴度为1.94×106 pfu/mL，重组率为85.15%。
扩展后文库的滴度为 1.45×1011 pfu/mL。
将λ噬菌体臂转入pTriplEx2质粒，从中随机挑选100个克隆，PCR扩增检测插入片段长度，结果显示文库插入片段在500~2 000 bp之间。
关键词水稻, SMART技术, 干旱胁迫, 全长cDNA文库, 抗旱相关基因Construction of Full Length cDNA Library from Drought-tolerant Rice cv. Zhonghan No. 3 Cultivated under Drought StressLiu Yunhua1,2Liu Zaochang1*Zhou Liguo1,2Yu Sunwu1 Liu Hongyan1Luo Lijun1*1 Shanghai Agrobiological Gene Center, Shanghai, 201106;2 College of Plant Science, Huazhong Agricultural University, Wuhan, 430070* Corresponding authors, lzc@; lijun@Abstract Drought stress affects plant physiological process by causing altered gene expression. A high quality full-length cDNA library of drought-stressed rice cv. Zhonghan 3 was constructed with SMART (switching mecha-nism at 5' end of the RNA transcript) technique. The λ phage packaging reaction and library amplifi cation in E.coli strain XL1-Blue were performed. The titer of unamplifi ed library was 1.94×106 pfu/mL and the recombinant rate was 85.15%. The amplifi ed library obtained a 1.45×1011 pfu/mL recombinants. Finally the λTriplEx2 clone was transformed to pTriplEx2 plasmid and 100 clones were picked randomly from the library for screening size of cDNA inserts through PCR. Results indicated that most of the inserts were between 500 and 2 000 bp. Keywords Rice (Oryza sativa L.), SMART technique, Drought stress, Full-length cDNA library, Drought-toler-ance related genes农业生产的发展往往同城市发展共同竞争着优良而且有限的土地资源。
类NADC30猪繁殖与呼吸综合征病毒FJZ03株感染性克隆的构建及鉴定
中国预防兽医学报Chinese Journal of Preventive Veterinary Medicine第42卷第11期2020年11月V ol.42No.11Nov.2020doi ：10.3969/j.issn.1008-0589.201911018类NADC30猪繁殖与呼吸综合征病毒FJZ03株感染性克隆的构建及鉴定勾明郗1,2，林志锋3，邓小莺1,2，黄晓紫1,2，毛婉1,2，徐叶3，李娜1,2，杨小燕1,2，魏春华1,2*，刘建奎1,2*（1.龙岩学院生命科学学院，福建龙岩364000；2.福建省家畜传染病防治与生物技术重点实验室，福建龙岩364000；3.福建农林大学动物科技学院，福建福州350002）摘要：为构建类NADC30猪繁殖与呼吸综合征病毒（PRRSV ）FJZ03株的感染性克隆，本研究将该株病毒全基因组分成6段，通过同义突变将其中的A 14378G ，引入了Mlu I 酶切位点为分子标记。
经RT-PCR 分别扩增后，克隆至改造后的载体pSK 中构建中间质粒。
经测序鉴定各质粒正确后，分段连接至pSK 中构建全长cDNA 克隆质粒pSK-FJZ03。
将鉴定正确的pSK-FJZ03转染Marc-145细胞并传代，收集出现CPE 后的病毒液，经RT-PCR 扩增后的产物经Mlu I 酶切和测序鉴定；同时采用激光共聚焦试验鉴定，获得的拯救病毒命名为RvFJZ03。
分别测定RvFJZ03在Marc-145细胞与潴肺泡巨噬细胞（PAMs ）中的生长曲线，比较拯救病毒与亲本病毒的生长特性。
将获得的拯救病毒RvFJZ03在Marc-145细胞中连续传代，对不同代次的重组病毒分别测定病毒效价，并经RT-PCR 扩增后通过Mlu I 酶切和测序鉴定。
测序鉴定结果表明，正确构建了感染性克隆质粒pSK-FJZ03；将其转染Marc-145细胞后并传至第3代出现典型CPE ，经激光共聚焦试验、RT-PCR 及测序鉴定表明拯救了重组病毒RvFJZ03。
全长cDNA主要构建方法的比较
全长cDNA主要构建方法的比较摘要全长cDNA文库的构建是进行功能基因组研究的一种经济、快速、有效的途径,克服了传统cDNA 文库的缺点,本文主要介绍了两种较为实用的方法，分别是SMART法和Cap trapper法。
关键词：全长cDNA构建SMART法Cap trapper法随着测序技术和计算机科学的不断发展，大部分生物和人类的基因组全序列测定高速完成。
cDNA作为基因克隆的一种重要工具，在帮助人们更好的发现新基因和研究基因功能上发挥了巨大的作用。
但是，由于传统的cDNA由于反转录能力差，cDNA酶切位点保护不彻底和非cDNA片段插入导致克隆片段短、无效克隆多和全长率低等缺点,因而无法满足大规模、高通量、高效的功能基因组研究需要。
而全长cDNA序列大多数拥有5’和3’端非编码区序列,因而弥补了传统cDNA文库构建方法的缺陷,成为目前基因克隆的一种重要方法。
目前主要有CAPture法，Oligo capping法，SMART法，Cap jumping法以及Cap trapper 法。
本文主要介绍优点突出的两个方法，SMART法和Cap trapper法。
SMART 方法SMART 方法是Chenchik 等1996 年提出的[3]，该方法利用PowerscriptTMRT 反转录酶的反转录、末端转移活性和内切酶sfiⅠ的特性，快速、简单地构建全长cDNA 文库。
PowerscriptTMRT 反转录酶是M-MLVR点突变而来的，丧失了RnaseH 的活性，但保留着野生型聚合酶转移酶的活性，能够长距离的反转录，又可识别mR-NA5’帽子结构。
原始一链合成中，转移酶的活性低，延伸效率低。
用于反转录的5’端poly(A)引物和延伸模板分别含有sfiI（A）、sfiI（B）识别位点的寡聚核苷酸序列。
而截短的一链cDNA，反转录酶没有识别到mRNA5’帽子结构，一链cDNA3/ 端不能被延伸、合成和扩增二链。
full-length 基因
full-length 基因摘要：1.基因的概念与作用2.全长基因的特点3.全长基因在生物体内的功能与应用4.全长基因研究的发展前景正文：基因是生物体内负责遗传信息传递和生物性状表现的遗传物质。
在过去的几十年里，基因研究取得了举世瞩目的成果，尤其是全长基因的探索，为我们揭示了生命世界的奥秘。
全长基因是指基因编码区内完整的DNA序列，包括了编码蛋白质所需的所有信息。
相较于传统的基因研究，全长基因研究更能全面地揭示基因的功能和作用。
全长基因的研究方法主要有基因组测序、基因编辑等技术。
这些技术使得科学家们能够快速地获取生物体的基因信息，进而研究基因在生物体内的作用机制。
全长基因在生物体内的功能与应用十分广泛。
首先，全长基因是生物体构建生命体系的基础。
基因编码蛋白质，而蛋白质是生物体细胞功能的主要承担者。
通过研究全长基因，我们可以更深入地了解生物体的生长发育、生理功能等各个方面。
其次，全长基因与疾病的发生和发展密切相关。
许多疾病都是由基因突变导致的，通过研究全长基因，我们可以找到潜在的治病基因，为疾病的诊断和治疗提供新思路。
此外，全长基因研究还为农业、畜牧业等领域带来了革命性的变革。
通过改良全长基因，我们可以培育出抗病、抗旱、高产等优良品种，以满足人类社会的需求。
在全长基因研究的发展前景方面，随着科学技术的不断进步，全长基因研究将更加深入。
在未来，我们可以期待以下几个方面的突破：一是基因编辑技术的优化，使得全长基因的编辑和调控更加精确和高效；二是全长基因疗法的应用，通过修复或替换异常基因，治疗遗传性疾病；三是基因驱动技术的出现，有望解决生物入侵和疾病传播等问题。
总之，全长基因研究为我们揭示了生物体的奥秘，为人类社会带来了巨大的利益。