木榄幼苗叶片cDNA文库的构建及其表达序列标签分析

应用SMART技术构建了25‰盐度下生长4个月的木榄叶片的cDNA文库,文库滴度为10~6 cfu mL~(-1),重组率为94.4%,插入片断长度为1～2 kb.从cDNA文库中随机挑选了96个重组克隆进行序列分析,共获得94个表达序列标签(ESTs),经质量控制和聚类拼接后得到81个unigenes,包括5个片断聚合群和76个单一序列.Blastx分析结果表明这些unigencs与GenBank的Nr数据库中已报道的基因具有较高的同源性(E<10~(-5)),它们参与呼吸代谢、光合作用、糖代谢、氨基酸代谢、脂肪酸代谢以及不饱和脂肪酸生物合成等重要的生理过程,并与机体的损伤修复、胞吞作用以及PPAR信号途径等相关.     

橡胶树甲基化过滤文库的构建

为高通量富集橡胶树基因编码序列,文章首次利用大肠杆菌McrBC限制修饰系统构建了巴西橡胶树基因组甲基化过滤文库.该文库未扩增和扩增后的滴度分别为2.6×106pfu/mL和9×109pfu/mL,阳性克隆率为86.4%,插入片段大小为1～2.5 kb,平均长度为1.2 kb.随机挑选100个克隆进行测序,共拼接出81个非冗余序列,其中包括6个重叠群(Contigs)和75个单一序列(Singlets),冗余度为17.35%.Blast分析发现,39.5%非冗余序列与Nr库中功能基因同源,14.81%与EST数据库中序列同源,32.1%序列为未知序列,且部分序列为开花调控、抗虫和抗病等功能相关的同源基因.橡胶树甲基化文库的建立为橡胶树重要功能基因发现和克隆提供一条重要途径.     

溶藻弧菌毒力菌株SSH文库的构建

[目的]通过构建基因文库为溶藻弧菌毒力基因的鉴定、致病机理的阐明奠定基础。[方法]应用抑制性差减杂交技术(suppression subtrative hybridization,SSH),以毒力菌株HN08155为测试子(Tester),构建了溶藻弧菌毒力菌株HN08155的SSH差减文库。[结果]文库共含有2 873个克隆;通过菌落PCR鉴定,2 786个克隆能扩增出250~1 000bp大小的目标片段;通过Southern斑点杂交,确定毒力菌株特异基因的阳性克隆347个,经测序和同源性比对分析,其中135个克隆的序列含有已知的77个基因,其中46个基因主要为代谢途径的多种酶类的编码基因,23个基因为其它基因,8个为毒力相关功能基因。[结论]这些毒力相关功能基因包括丝氨酸蛋白酶基因prk A、热休克蛋白基因dna J、趋化因子基因che W、甲基受体趋化蛋白(methyl-accepting chemotaxis protein)基因、双组分感应子(two-component sensor)基因、Ⅳ型菌毛(typeⅣpilin Pil A)基因、生物膜相关的表面蛋白(biofilm-associated surface protein)基因和外膜蛋白(outer membrane protein)基因;该文库中还含有166个未知新基因,占文库中总基因数的68.44%,溶藻弧菌的毒力基因很可能存在于这个文库中的未知新基因中。     

新疆细毛羊2倍基因组BAC文库PCR筛选系统的构建与使用

本研究所构建的BAC文库覆盖了8倍新疆细毛羊的基因组,平均插入片段的大小为133kb,同时文库92．5%的克隆插入片段大于100kb,而且有部分克隆甚至大于300kb,假定绵羊的基因组含有3×10~6kb,根据文库的平均插入片段大小为133kb,从文库筛选到目的片段的概率为98．208%。为了验证文库有较好的覆盖率,构建了2倍基因组文库PCR筛选系统,并对位于新疆细毛羊20号染色体MHC基因邻近区段的DMB_EX2、MCMA36、CP73和BM1258 4个分子标记进行了筛选,得到的平均阳性克隆数为1．5个,从筛选结果来看,这与文库插入片段估计的8倍基因组覆盖率相当接近并且没有偏向,这使得本文库成为研究绵羊的功能基因、位置克隆和完善基因组物理图谱的极为有用的资源。     

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。     

疫霉侵染下辣椒幼苗消减文库的构建与初步分析

为了分离和鉴定辣椒中疫霉诱导基因,以高抗疫霉病辣椒品种L11为材料,以接种辣椒疫霉菌的幼嫩叶片为处理（tester）,以未接种自然生长的幼嫩叶片为对照(driver),利用抑制性消减杂交技术(suppression subtractive hybridization,SSH)构建了疫霉侵染下辣椒幼苗的消减文库。从消减文库中随机挑取30个阳性克隆,提取质粒进行PCR鉴定,显示插入片段大小大部分集中在200～1 000 bp之间,文库质量良好。随机挑取40个克隆进行测序,共获得35个有效EST序列。经Blastx分析表明：有30个EST与GenBank中其他序列有同源性,5个EST为未知功能序列。已知功能的EST序列分别编码NAC转录因子、丝氨酸/苏氨酸蛋白激酶、P450单加氧酶、叶绿素a/b结合蛋白、谷胱甘肽转移酶、几丁质酶等,这些蛋白涉及抗病信号传递、抗氧化作用、转录调控及光合作用等多种生理过程。本研究为抗病基因克隆和系统研究疫霉侵染下辣椒基因的表达奠定了重要的理论基础。     

红树林土壤微生物宏基因组文库的构建及两种新颖的β-葡萄糖苷酶基因的鉴定

宏基因组技术是挖掘微生物新酶的重要途径。通过构建红树林土壤微生物Fosmid文库,共获得了约100 000个阳性克隆,该文库外源插入片段平均长度约为30 kb,文库容量达3 Gb。通过对文库中10 000个克隆进行功能筛选,获得了17个具有β-葡萄糖苷酶活性的克隆。对其中2个表达β-葡萄糖苷酶的克隆进行亚克隆,获得2个新颖的β-葡萄糖苷酶的基因,分别命名MhGH3和bgl66。生物信息学分析表明,MhGH3基因由1 992个碱基对组成,bgl66基因由2 025个碱基对组成。在氨基酸水平上,MhGH3和bgl66编码的蛋白质与GenBank数据库中已知β-葡萄糖苷酶的一致性分别为64%和55%。     

星星草cDNA文库构建和金属硫蛋白(MT-1)基因的克隆

以受NaHCO3胁迫后的星星草叶片组织为材料构建了cDNA文库,文库的初始滴度为2.0×106pfu,重组率为95%,平均插入片段长度为0.8kb,扩增后文库的滴度为4.0×109pfu.mL-1.文库克隆随机测序获得了星星草的金属硫蛋白(MT-1)基因的全长cDNA序列,显示文库中含有一定量的全长基因.MT-1基因全长622bp,其中5'非翻译区57 bp,3'非翻译区343 bp,开放读码框长222 bp,编码73个氨基酸,氨基酸序列中具有植物MT-1蛋白特有的金属响应元件(MRE)序列,MT-1蛋白的分子量为7.814 kD,理论等电点为4.72.     

黄瓜白色果皮基因遗传规律及定位研究

以黄瓜嫩果深绿色果皮自交系1507(P1)和白色果皮自交系1508(P2)为亲本,构建6世代遗传群体(P1、P2、F1、F2、BC1P1、BC1P2),对黄瓜嫩果白色果皮基因(w)进行遗传规律分析和基因定位研究。结果表明,黄瓜白色果皮性状由隐性单基因(w)控制,深绿色对白色为显性。利用F2群体,结合分离群体分组分析法筛选得到了14个与w基因相关的SSR标记,构建了该基因的SSR连锁群,将其定位到黄瓜3号染色体上,两侧的标记为SSR23517和SSR23141,遗传距离分别为4.9cM和1.9cM。侧翼标记之间的物理距离为1 150kb,在该区域中共预测了500个候选基因。该研究对w基因的初步定位,为该基因精细定位及分子标记辅助选择育种奠定了良好的基础。     

盐胁迫下西伯利亚蓼6个时期混合叶cDNA文库的构建和初步分析

以3％NaHCO3胁迫处理西伯利亚蓼2h、6h、12h、1d、2d和3d共6个时期混合叶为材料,提取总RNA。根据Stratagene公司cDNA建库试剂盒构建了西伯利亚蓼混合叶cDNA文库。原始文库滴度为2．0&#215;10^6pfu&#183;mL^-1,扩增后文库滴度为4．5&#215;10^9pfu&#183;mL^-1,重组率为95．9％,插入片段大小在0．3—1．5kb之间,平均长度为0．45kb左右。经测序获得2575个EST序列,拼接出1977个假定独立转录本（TUTs）,其中文库中含有大量低丰度表达基因,约占TUTs总数的89．58％。BlastX分析表明,1977个TUTs中共有720个TUTs有明确功能注释,其中参与胁迫过程中代谢、能量、光合作用和蛋白合成基因表达量较高,而涉及转录、信号转导、细胞防御和救援基因表达量较低。     

鸡下丘脑cDNA文库的构建及部分克隆ESTs序列初步分析

以鸡下丘脑为实验材料,以λgt10为载体,构建了鸡下丘脑cDNA文库。结果表明,文库的滴度为3.8×10     

Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×10(6) pfu/ml and 1.62×10(9) pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142 bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers.     

用suc2信号肽捕获系统筛选小鼠胚胎cDNA文库基因

PCR扩增 1 1d小鼠胚胎cDNA文库插入片段 ,将 0 .5～ 2 0kb的扩增产物插入筛选载体的多克隆位点 ,转化suc2基因缺陷酵母宿主菌 .然后将约 1 0 5个酵母菌落接种于选择性平板上进行筛选 ,得到了 1 82个可在选择性培养基上生长的菌落 .PCR扩增显示 ,插入片段大小分布于 0 1～ 1 5kb之间 .对其中 1 4个阳性菌落的重组子进行序列测定 ,分别代表 6种不同的基因序列 ,与报告基因都有正确的读框内融合 .其中两种基因序列反复被筛到 ,分别命名为spt1、spt2 .spt1 [gi:2 772 876 6 ],可能以非编码RNA的身份参与蛋白质向细胞外分泌的过程 ,而spt2编码多个连续的赖氨酸 ,可能通过非经典途径介导蛋白质的分泌     

Characterization of expressed sequence tags generated from skin cDNA clones of Equus caballus by single pass sequencing

A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10(5) plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-redundant protein database and 206 ESTs were putatively identified. Twenty six percent of the identified ESTs were redundant. The ESTs were categorized by function. The most frequently identified functional class was translational proteins.     

山葡萄成熟果皮cDNA文库的构建及ESTs初步分析

以山葡萄成熟果皮为材料,采用Creator~（TM） SMART~（TM） cDNALibraryConstruction技术构建了cDNA文库。经鉴定,文库的库容量为1.001×10~6,重组率为94.1 1%,插入片段在0.25～1.9 kb。经随机测序,获得935个有效ESTs,聚类拼接后得到636条单基因簇（unigenes）,包括107个重叠群（contigs）和529个单拷贝（singlets）。这些数据为今后山葡萄功能基因分离克隆和功能基因组学研究奠定了基础。     

Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages

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 microarry 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 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 gene, and developing microarrays in maize genomics research.     

CHARACTERIZATION OF EXPRESSED SEQUENCE TAGS GENERATED FROM SKIN CDNA CLONES OF Equus caballus BY SINGLE PASS SEQUENCING

A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10⁵ plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-redundant protein database and 206 ESTs were putatively identified. Twenty six percent of the identified ESTs were redundant. The ESTs were categorized by function. The most frequently identified functional class was translational proteins.