穴蚁蛉幼虫全长均一化cDNA文库的构建

通过构建穴蚁蛉幼虫(俗称蚁狮)全长均一化cDNA文库,以期了解蚁狮的抗菌肽、毒素蛋白以及其它药用蛋白种类,为日后筛选、克隆表达和分离纯化这些药用蛋白奠定基础.用针刺诱导蚁狮产生抗茵物质后,分离纯化总RNA,结合SMART全长文库与DSN(duplex-specific nuclease)均一化技术,构建全长均一化cDNA文库,并通过平板计数和菌落PCR方法鉴定文库.随机挑选192个单克隆进行5′端测序,并与NCBI数据库进行比对分析.结果显示,构建出的文库平均插入片段长度为1 200bp,原始文库的滴度达到7.5×105 CFU/mL,而扩增文库则为1.95×1011CFU/mL.在所挑选的单克隆中,共获得190条有效序列,平均读长1 340 bp,文库重组率达到98.96%.与NCBI数据库比对后发现,这些序列可能是一些编码蚁狮免疫、消化及毒性蛋白的基因.     

烤烟品种南江3号均一化全长cDNA文库构建

目的:为了获得功能基因的信息,构建烤烟品种南江3号的均一化cDNA文库.方法:用RNeasy Plant Mini Kit提取烤烟南江3号叶片和花的RNA,用反转录酶逆转录合成第一链eDNA.以LD-PCR扩增获得的双链cDNA为模板,采用基于双链特异性核酸酶(Duplex-Specific Nuclease,DSN)的均一化cDNA文库技术,构建南江3号盛花期的均一化cDNA文库.结果:构建了南江3号盛花期的均一化cDNA文库,文库重组率大为97.47%,库容量约为1.26×10<'6>,插入片段平均长度大于1.2kb.从文库中随机48个克隆进行PCR检测,挑取20个克隆进行测序,序列通过BLAST比对结果显示文库可能包含大量基因和ESTs序列.结论:该文库为研究南江3号的基因功能和资源提供材料来源.     

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。     

莱芜猪肝脏组织全长cDNA文库的构建和鉴定

目的:构建莱芜猪肝脏组织全长cDNA文库,以便研究与莱芜猪优良性状相关的基因。方法:采用改良的异硫氰酸酸胍一步法制备总RNA;利用SMART技术,以PrimeScript反转录酶逆转录合成第一链cDNA,通过LD-PCR扩增获得cDNA双链;经蛋白酶K消化和CHROMA SPIN-400柱分级分离后,收集500 bp以上的cDNA片段,并与pMD18-T载体连接,转化大肠杆菌DH5α感受态细胞,建成原始文库;随机挑取单菌落,用HindⅢ和EcoRⅠ进行双酶切鉴定重组子插入片段大小。结果:经鉴定,原始文库的滴度为2.8×105 cfu/mL,重组率约为98%,插入片段大小为0.5～2 kb,平均插入片段长度大于1 kb。结论:建立的cDNA文库质量良好,可以用于目的基因的筛选。     

中华大蟾蜍精巢组织全长cDNA文库的构建及泛素延伸蛋白基因Bg-ubi52的获得

运用SMART技术构建了中华大蟾蜍(Bufo bufo gargarizans)精巢全长cDNA文库。提取中华大蟾蜍精巢总RNA,用Clontech公司SMARTTM cDNA文库构建试剂盒反转录合成第一链cDNA,LD-PCR扩增获得全长cDNA双链;经SfiⅠ酶切、层析柱分离后,500bp以上的片段与λTriplEx2载体连接并包装,建成原始文库。经鉴定,原始文库滴度为2.21×106pfu/ml,重组率为91%;文库扩增后的滴度为2.94×109pfu/ml,重组率为93.7%。插入片段大小分布于0.4~2.0kb之间,平均长度约为1.0kb,说明已构建文库质量较高,为进一步筛选、克隆精巢特异表达基因奠定了基础。从该文库中克隆到了泛素延伸蛋白基因,全长561bp,包含完整的5′和3′非编码区,编码128个氨基酸,即泛素的76个氨基酸后融合了52个氨基酸的核糖体L40蛋白。     

Construction and characterization of normalized cDNA library of maize inbred MO17 from multiple tissues and developmental stages

A comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10 830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarray was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using the BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate genes, and developing microarrays in maize genomics research.__________From Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 198–206.Original English Text Copyright © 2005 by Z. Zhang, F. Zhang, Tang, Pi, Zheng.This article was submitted by the authors in English.     

Enhanced error-prone RCA mutagenesis by concatemer resolution

Error-prone rolling circle amplification (RCA) is a promising alternative to error-prone PCR for random mutagenesis. The main disadvantage of error-prone RCA is the low transformation efficiency of the DNA concatemer produced in the amplification reaction. We improved the method by introducing loxP recombination site of bacteriophage P1 Cre recombinase into the target plasmid and reducing the concatemer by Cre recombinase to plasmid-sized units, increasing the number of transformants 50-fold in non-error-prone and 13-fold in error-prone conditions. The efficiency improvement was verified by obtaining 115 ± 57 ceftazidime resistant colonies per recombined RCA reaction from randomly mutated TEM-1 β-lactamase gene library whereas only 9 ± 11 colonies were gained without recombination. Supplementation of the error-prone RCA with Cre/loxP recombination is a simple and useful tool to increase the transformable library size.     

莱芜猪心脏cDNA文库的构建与鉴定

以莱芜猪心脏为供试材料,采用改进的异硫氰酸胍法提取心脏总RNA。通过SMART技术反转录合成全长cD-NA,经LD-PCR扩增后与pMD18-T载体连接并转化细菌DH5α,测定滴度和重组率,构建成莱芜猪心脏全长cDNA文库。经初步检测,原始文库的滴度为1.8×105CFU/mL,重组率约为94.4%。从原始文库随机挑取13个单菌落过夜培养,提取质粒,经HindⅢ和EcoRⅠ双酶切,凝胶电泳显示插入片段大小在0.3-2.0kb之间。     

High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe.

We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants. cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules. The transformation frequencies obtained by the method are 10(6) colonies per 10(8) cells transfected with 2 micrograms of library and 1 microgram of vector, 50-60% of which contain pcD molecules. The high-efficiency alkali cation method circumvents many of the shortcomings of the spheroplast method generally used for Schiz. pombe transfection. The vectors are maximized for the efficiency of library transduction and minimized for the rearrangements of pcD molecules during propagation in yeast. This system allows rapid screening of multi-million cDNA clone libraries for rare cDNAs in a routine scale of experiments. Using this system, various mammalian cDNAs that are extremely difficult, time-consuming, or unclonable to clone by other methods have been cloned.     

A binary vector-based large insert library for Brassica napus and identification of clones linked to a fertility restorer locus for Ogura cytoplasmic male sterility (CMS).

We constructed and characterized a large DNA insert library for Brassica napus that would facilitate genome-related research and map-based cloning efforts in Brassica species. This library, consisting of 92,160 clones arrayed in 384-well microtiter dishes, was based on a conventional plant transformation vector (binary vector), and was constructed using a single ligation with transformation efficiency of over 5000 recombinants per microliter of ligation mixture. Every clone in this library contains an insert in the size range of 30-190 kb, facilitating both chromosome walking and plant transformation. Screening this library with three DNA markers (C2, F10, and CabR) that are linked to a fertility restorer locus for Ogura cytoplasmic male sterility (CMS) identified at least 17 positive clones for each probe. Among the 17 positive clones identified by C2, nine are linked to the restorer locus. Marker F10 identified 21 clones, of which only two are linked to the restorer locus. None of 68 clones identified by CabR is linked to the restorer locus. A stability test using two clones identified by the C2 marker indicated that large DNA inserts are stable in this conventional vector in both Escherichia coli and Agrobacterium.     

甜菜夜蛾cDNA文库的构建

以甜菜夜蛾Spodoptera exigua(Hübner)幼虫为材料建立了cDNA表达文库,经检测文库的初始滴度为1.1×105pfu/mL,重组率为97%,扩增后文库的滴度为5.4×107pfu/mL。用设计的2对引物筛选该文库,得到468 bp的1个片段,分析后证实是几丁质合成酶基因I保守区域的1个片段。该cDNA文库为进一步筛选甜菜夜蛾功能基因奠定了基础。     

人胃癌组织cDNA文库的建立

本文直接从一例人低分化胃腺癌组织中提取RNA和分离poly(A)~+RNA,进而合成双链cDNA。再以λgt10作为克隆载体和大肠杆菌C600hfl作为受体菌,构建了人胃癌cDNA文库。该文库中含有1.10×10~5重组子,重组率约为68.8％,克隆效率为2.21×10~6克隆/μgcDNA。