悦目金蛛丝腺SMART RACE cDNA文库的构建与鉴定

运用SMART 技术构建了悦目金蛛丝腺SMART RACE cDNA文库.经检测,文库所含全长cDNA的长度主要集中在500 bp～2000 bp 之间;把双链cDNA通过T/A克隆、随机挑选阳性克隆并测序后,得到1条752 bp的全长cDNA序列.以该文库为模板,用依据这条全长cDNA序列设计的基因特异性引物与接头引物进行RACE, 3'RACE 和5'RACE的产物拼接后的全长序列与上述全长cDNA序列一致.结果表明,该文库适于用RACE方法从中分离在悦目金蛛丝腺中表达基因的全长cDNA.本文还对SMART 技术的特点和局限性进行了讨论.     

基因组研究中全长cDNA克隆的策略

全长cDNA的克隆是目前人类基因组研究中的一个重要方面,与大规模基因组测序相比较,cDNA测序克隆用相对较少费用获得更多的功能基因信息,故也是符合我国国情的基因组研究的主要方向。如何更加高效地获得新的全长cDNA,本文结合作者自己工作中的经验,对从全长文库的建立到全长cDNA的测序及克隆方法作了较为详细的介绍。     

大乳头水螅RACE cDNA文库的构建

目的:为筛选和克隆大乳头水螅发育调控相关基因的全长cDNA,构建大乳头水螅RACE cDNA文库.方法:提取大乳头水螅总RNA后从其中分离mRNA,运用SMART技术构建RACE cDNA文库.为鉴定所构建文库的质量,根据GenBank中大乳头水螅actin基因cDNA序列设计5'RACE和3'RACE的引物及用于扩增actin基因编码区全长序列的引物.结果:琼脂糖凝胶电泳结果表明,RACE cDNA文库中全长cDNA的长度集中在500-2 000bp之间.5'RACE、3'RACE PCR及扩增actin基因编码区全长序列时均以本文构建的大乳头水螅RACE cDNA文库为模板,这3个PCR反应均能扩增出产物,产物大小与目标片段预计大小相似.PCR产物分别经T/A克隆及测序后证明为大乳头水螅actin基因cDNA的相应序列.结论:RACE cDNA文库的成功构建为通过RACE方法获得大乳头水螅功能基因cDNA全长序列奠定了基础.     

利用改进的Oligo-capping法构建手掌参全长cDNA文库

Oligo-capping法是构建全长cDNA文库的重要方法之一.以名贵中药手掌参(Gymnadenia conopseaR.Br.)的幼芽组织为材料,通过减少mRNA的用量,用pUC18作载体,设计特异引物对第1链cDNA进行PCR扩增,按片段大小分级回收cDNA,形成了一种以Oligo-capping法为基础,构建珍稀材料全长cDNA文库的快速、简单的技术体系.利用该方法,首次成功地构建了手掌参的全长cDNA文库.经综合评价,文库容量达到了8×105个/μg cDNA,全长cDNA比例达到68%,重组率超过96%,说明本实验的改进非常成功.该文库的建成为手掌参的遗传资源保护和功能基因发掘等理论与应用研究奠定了基础.     

青杄均一化cDNA文库构建及EST序列分析

以青杄花粉和针叶为材料,将青杄全长cDNA与Gateway供体载体pDONR222重组,构建了其非剪切型全长cDNA原始文库,利用基因组DNA饱和杂交技术对原始cDNA文库进行均一化处理,构建青杄的均一化全长cDNA文库。文库的总库容量为1.1×106CFU/mL,平均插入片段长度大于1.0 kb,重组率大于95%。定量RT-PCR检测表明,青杄高丰度表达基因EF1-α在均一化cDNA文库中的表达量下降了约41倍。接着对文库中随机的5 144个克隆进行了测序,获得高质量的有效EST(expressedsequence tag)序列为5 144条,经拼接共获得单一基因(unigene)为2 717个,其中包括片段重叠群(contig)628个和单一EST序列(singlet)2 089个。NCBI同源比对分析表明,其中1 887个序列unigenes获得分子功能注释,这些EST涉及细胞生长、信号转导、转录、抗逆、能量代谢等功能。这些数据有助于对青杄的相关功能蛋白及分子机制开展进一步的研究。     

全长cDNA克隆的三种方法的比较研究

为了得到一段EST所在基因的全长,采用了快速扩增cDNA末端（RACE）、cDNA文库构建、λphage测序引物与特异性引物结合,非放射性探针进行噬菌体原位杂交等方法分别对该基因进行了克隆,结果得到了一致的全长cDNA,三种不同实验方法的应用,为更方便、有选择性地克隆全长cDNA基因奠定了基础。     

自噬基因APG4基因结构的生物信息学分析

利用生物信息学手段对一种与人类细胞凋亡有关的基因－自噬基因（Autophagy5,简称APG5）的基因组全序列进行拼接和基因结构分析,同时该基因的一个新的可变性剪切变异体（APG5β）被首次证实及报道,利用PCR技术从人胚脑cDNA文库和B细胞cDNA文库中调取APG5基因,装入绿色荧光蛋白pEGFP-C1表达载体,测序确定其核苷酸序列,进行数据库搜索。该基因组全序列分散在核酸序列数据库GenBank的几个没序列条目中,将相关克隆的基因组序列做相似性比对,用DOTPLOT方法确定其相互位置关系,并进行序列拼接,得到全长基因,利用GenScan,Genefinder等基因识别软件确定其内含子,外显子,转录起始位点,加尾信号等。分析其基因结构,在此基础上进一步分析其cDNA中外显子数目和排列顺序,证实了从B细胞cDNA文库中克隆所得的APG5为一种可变性剪切变异体。利用生物信息学工具,成功地接接出全长为150kb人类APG5基因基因组全序列,确定其有8个外显子。根据剪切位点的特性找出其在序列中的位置,分别计算出内含子,外显子的长度,首次证实并报道APG5β是第3外显子缺失的可变性剪切变异体,将验证确实的APG5β转染至人肝细胞株和HeLa细胞株,在共聚焦激光显微镜下可观察到其在细胞凋亡时的特异表达,在后基因组研究中生物信息学的应用有非常广阔的前景。对可变性剪切所产生的蛋白多样性分析提供了一种辅助分析手段。     

利用巢式PCR技术和人类基因组草图搜索法快速获得人类睾丸生精细胞凋亡相关基因TSARG2

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段 (BE6 44 5 42 )入手 ,设计了基因特异性引物和载体特异性引物进行巢式PCR扩增 ,结合人类基因组草图搜索法 ,从睾丸cDNA文库中快速分离出人类睾丸凋亡相关基因TSARG2的 5′末端而获得全长cDNA ,GenBank登录号为AY0 40 2 0 4(保密期为 1年 ) ,同时应用生物信息学的方法克隆了该基因在小鼠中的同源基因 ,GenBank登录号为AF395 0 83。TSARG2基因的cDNA全长为 12 33bp ,包含 6个外显子 ,基因组跨越 115kb ,编码由 30 5个氨基酸组成的、分子量为 34 75 1的蛋白质 ,与已知蛋白质无明显同源性。查询最新的人类基因组工作草图 ,该基因定位在染色体 4q33～ 34 .1。     

利用改进的Cap-trapper法构建拟斯卑尔脱山羊草(Ae.speltoides Tausch)全长cDNA文库

Captrapper法是目前全长cDNA文库构建的重要方法之一。在引进、消化、吸收的基础上,通过设计引物,同位素检测,对末端转移反应条件控制等方面做了一些切实可行的改进,形成了一套简单易行的技术体系。利用改进的Captrapper方法,成功构建了小麦B基因组的可能供体种拟斯卑尔脱山羊草(Ae.speltoides)的全长cDNA文库。经综合评价,文库的全长比例达到89.6%,重组率为99%,库容量超过3.0×106cfu。     

利用SMART方法快速克隆异黄酮代谢途径多基因的研究

采用改进的酸酚法提取高质量的大豆叶片RNA,利用SMART思想和方法构建大豆叶片全长cDNA文库,直接以一级库液稀释液为模版进行PCR,快速克隆得到异黄酮代谢途径相关的5个基因。与传统的从DNA、RNA出发克隆基因,以及构建文库再进行基因筛选的克隆方法相比,该方法得到的基因均为全长基因,适用于快速、简便的进行多基因全长克隆。     

SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

Background  

cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library.     

Identification of unique transcripts from a mouse full-length, subtracted inner ear cDNA library

A small-scale full-length library construction approach was developed to facilitate production of a mouse full-length cDNA encyclopedia representing approximately 250 enriched, normalized, and/or subtracted cDNA libraries. One library produced using this approach was a subtracted adult mouse inner ear cDNA library (sIEa). The average size of the inserts was approximately 2.5 kb, with the majority ranging from 0.5 to 7.0 kb. From this library 22,574 sequence reads were obtained from 15,958 independent clones. Sequencing and chromosomal localization established 5240 clusters, with 1302 clusters being unique and 359 representing new ESTs. Our sIEa library contributed 56.1% of the 7773 nonredundant Unigene clusters associated with the four mouse inner ear libraries in the NCBI dbEST. Based on homologous chromosomal regions between human and mouse, we identified 1018 UniGene clusters associated with the deafness locus critical regions. Of these, 59 clusters were found only in our sIEa library and represented approximately 50% of the identified critical regions.     

Construction and characterization of a normalized yeast two-hybrid library derived from a human protein-coding clone collection

The nuclear yeast two-hybrid (Y2H) system is the most widely used technology for detecting interactions between proteins. A common approach is to screen specific test proteins (baits) against large compilations of randomly cloned proteins (prey libraries). For eukaryotic organisms, libraries have traditionally been generated using messenger RNA (mRNA) extracted from various tissues and cells. Here we present a library construction strategy made possible by ongoing public efforts to establish collections of full-length protein encoding clones. Our approach generates libraries that are essentially normalized and contain both randomly fragmented as well as full-length inserts. We refer to this type of protein-coding clone-derived library as random and full-length (RAFL) Y2H library. The library described here is based on clones from the Mammalian Gene Collection, but our strategy is compatible with the use of any protein-coding clone collection from any organism in any vector and does not require inserts to be devoid of untranslated regions. We tested our prototype human RAFL library against a set of baits that had previously been searched against multiple cDNA libraries. These Y2H searches yielded a combination of novel as well as expected interactions, indicating that the RAFL library constitutes a valuable complement to Y2H cDNA libraries.     

Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments.

We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes were then captured from the solutions using the digoxigenin-antidigoxigenin paramagnetic beads followed by recovery of the enriched double-stranded cDNA expression library. We have observed a linear relation between the capture of full-length cDNAs in the library and the fold enrichment in the subtracted cDNA population.     

Generation of Arabidopsis Mutants by Heterologous Expression of a Full-Length cDNA Library from Tomato Fruits

Heterologous expression of cDNA library in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with a plant transformation-ready binary vector that contained a higher percentage of full-length cDNAs since synthesized double-stranded cDNA was size-selected using gel electrophoresis, with cDNA sizes of 2–5 kb being gel-purified for ligation onto the binary vector. Sequencing of 81 cDNA clones indicates that 75% (61) are full-length genes, which is similar to sequencing of inserted cDNA in Arabidopsis. The library was used to transform Arabidopsis plants. Among the 7,000 mutants, one was found to be a dwarf due to the expression of an ATP synthase, and another vegetative mutant did not produce flowers even after 7 months. The technique was validated by reintroducing the tomato ribosomal protein L9 gene and can be used in any other plant species as a gene discovery tool.     

玫瑰红绿海葵触手cDNA表达文库的构建和初步分析

海葵共有 10 0 0多种 ,栖息于世界各地的海洋中 ,从极地到热带、从潮间带到深海都有分布 ,我国地处温带和亚热带 ,海葵种类较多[1 ] 。海葵的单体呈圆柱状 ,柱体开口端为口盘 ,口盘周围环生众多触手 ,触手上有被称为刺丝囊的毒腺结构 ,能分泌毒液 ;柱体下端为基盘 ,海葵以其基盘吸附于甲壳、海藻等物体上 ,较少运动。海葵毒液成分主要是蛋白质和多肽 ,它们对甲壳类动物有较大毒性 ,对人或其它高等动物的毒性则相对较小。现代研究表明 ,海葵多肽毒素有多种药理效果 ,这些毒素主要作用于神经及心血管系统 ,可引起强心、降低血压、麻痹肌肉等多…     

Vector-capping: a simple method for preparing a high-quality full-length cDNA library.

Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.