基因文库

The mRNA of pituitary glands from seven important economic Pleuronectiformes fishes was extracted,the cDNA sequences of GH gene were then isolated from Cleisthenes herzensteini,Limanda yokohamae,Pleuronichthys Cornutus,Cynoglossus robustus,Platichthys bicoloratus, Zebrias zebra and Paralichthys dentatus with RT-PCR method by using designed specific primers based on the reported GH cDNA sequences of Pleuronectiformes. The results of sequencing showed that lengths of the above cDNAs are as follows: 479 bp,564 bp,519 bp,440 bp,564 bp,440 bp and 522 bp.The GH cDNA sequences encode mature polypeptide of about 140 to 170 amino acid.The full-length growth hormone cDNAs of Paralichthys lethostigma and Scophthalmus maximus were cloned by Switching Mechanism At 5\' end of the RNA Transcript RACE technology.

从7种重要鲽形目经济鱼类——高眼鲽、黄盖鲽、木叶鲽、宽体舌鳎、石鲽和条鳎、夏鲆的脑垂体中提取mRNA，并采用RT-PCR方法，根据已克隆得到的鲆鲽鱼类生长激素基因的保守性序列设计特异性引物，克隆了含开放阅读框的生长激素(Growth Hormone，GH)cDNA序列，测序结果显示，高眼鲽、黄盖鲽、木叶鲽、宽体舌鳎、石鲽、条鳎和夏鲆的GH cDNA长度依次为479 bp、564 bp、519 bp、440 bp、564 bp、440 bp和522 bp，编码140-170个氨基酸的成熟GH多肽片段；从漠斑牙鲆和大菱鲆脑垂体中提取mRNA，利用SMART-RACE技术建立脑垂体cDNA文库，并从该文库中克隆出大菱鲆和漠斑牙鲆生长激素基因cDNA全长序列。

It has been widely used in analysis of gene expression map, analysis, discovey of new gene ,gene mutation and polymorphism analysis, genom library map, gene sequence, disease diagnosis and prevention, drug selection, tumor biology and environmental toxicology.

基因芯片技术目前已广泛应用于基因表达谱分析、新基因发现、基因突变及多态性分析、基因组文库作图、基因测序、疾病诊断和预防、药物筛选、药物开发和研究、肿瘤生物学、环境毒理学等研究领域。

After thecomplete genome extraction of the strain was performed, the genomic DNA was partiallydigested by restriction enzyme Sau3AⅠ, the DNA fragments from 1 to 5Kb was clonedinto prokaryote expression vector pET-28a-c, and transformed host bacteria. The resultsshowed that we succeeded in constructing the gene expression library of haemophilusparasuis serovar 5, which is fundamental for the study of advanced gene screening. Inaddition, primer design was performed based on haemophilus influenzae in this study. In addition, PCR was performed by using genomic DNA of haemophilus parasuisserovar 5 as the template. The results demonstrated that we obtained two neo-gene:23SrRNA gene(conserved gene belonging to the large-subunit of ribosome) and adenylatecyclase gene(encodes adenylate cyclase and participates in converting adenyl nucleosidetriphosphate to cyclic adenosine3",5"-monophosphate). Furthermore, the phylogeneticanalyses between the species was performed, and neighbor-joining tree was constructedbased on comparison of 23S rRNA gene sequences, so it was illuminated betweenHaemophilus parasuis and other species in molecular evolution relationship.

选择我国流行优势菌株副猪嗜血杆菌血清5型地方株为研究对象，提取细菌基因组DNA，用限制性内切酶Sau3AⅠ对基因组DNA进行部分酶切，回收大小为1～5Kb的DNA片段，将其连接入原核表达载体pET-28a-c，最后转化宿主菌，结果成功地构建了基因表达文库，为后续的基因筛选工作奠定基础；另外，本研究选择嗜血杆菌属的流感嗜血杆菌为参考对象进行引物的设计，以副猪嗜血杆菌血清5型地方菌株的基因组DNA为模板，进行PCR扩增反应，结果表明成功地获得两个新基因：23S rRNA基因(存在于核糖体大亚基中的保守性基因)和腺苷酸环化酶基因(负责将腺嘌呤核苷三磷酸转变为环腺苷酸)，并进一步做了不同物种之间的分子系统发育分析，构建了基于23S rRNA基因的邻接法系统发育树，阐明了副猪嗜血杆菌与其它菌种的分子进化关系。

In the research, one kind of guzmania species named ostara was employed to be as material, and the full-length cDNA plasmid library of floral organ was constructed successfully by using SMART technology. The library had 3×10^6 original titer and more than 1 kb insert fragments. After 5'EST sequencing from 2004 positive clone chosen at random and clustering analyse, 1 758 high-quality sequences and 1 365 unigenes including 175 contigs and 1 195 singlets were obtained. These unigenes had 1 283 valid ORFs. COG analysis showed that those proteins coded by EST sequences were divided into 22 classes. Through blast analysis, full length or part cDNA of some genes controlling flower color, development of floral organs, florescence regulation and other breeding value genes were obtained.

本研究以擎天凤梨属品种Ostara为材料，采用SMART技术成功构建了擎天凤梨花器官的质粒型全长cDNA文库，初始文库滴度为3×10^6，插入片段平均长度大于1kb；随机挑取2004个阳性克隆进行5'EST测序，获得高质量序列1758条，经拼接获得1365条单基因簇，其中跨叠群175个和单条序列1195个；经ORF寻找共获得有效ORF1283条；经COG分析EST序列编码的蛋白质被分为22类；经Blast分析后，获得一批花色、花器官发育、花期调控以及其它育种价值基因（包括cDNA全长或片段）。

The resulting strain, designated YES2MTM1, was transformed with a yeast genomic library. Transformants lost the plasmid overexpressing MTM1 after 5-FOA treatment. Yeast strains able to grow on nonfermentable carbon source with MTM1 deletion and overexpression of some DNA fragments were picked up and candidate suppressor genes were identified. Overexpression of five genes were identified to be able to rescue the growth defect on nonfermentable carbon source. The study will provide reference for MTM1 gene function and screening for suppressor of genes whose deletion result in irreversible damage.

为了避免MTM1缺失造成的不可逆损伤，在野生型酵母中先转入带有MTM1 基因的质粒，再敲除染色体上的MTM1 基因，随后转入基因组文库，再利用药物5-氟乳清酸(5-FOA)迫使细胞丢失表达MTM1基因的外源质粒，再筛选能在非发酵培养基上生长的转化子，通过这种方法筛选发现，POR2等5个基因的过表达可以挽救MTM1 基因缺失造成的非发酵培养基上的生长缺陷，为深入了解MTM1基因的功能提供了线索，对筛选其他造成不可逆损伤的突变基因的抑制基因提供了一条可行的研究思路。

To obtain more information of magnesium homeostasis in mitochondria, mTn-lacZ/LEU2 transposon library was transformed into mrs2 deletion mutant to screen for suppressor genes of MRS2. YMR166C, a member of mitochondrial carrier family, was identified as a suppressor gene of MRS2. Deletion of YMR166C gene can rescue the defects of mrs2 deletion mutant such as the decrease in mitochondrial magnesium concentration, Group II RNA splicing defect and growth defect on nonfermentable carbon source. For the first time we demonstrated YMR166C is involved in mitochondrial magnesium homeostasis.

为了增进对线粒体镁离子代谢调控基因的了解，利用酿酒酵母mTn-lacZ/LEU2转座子文库筛选MRS2的抑制基因，发现线粒体载体家族成员YMR166C基因的缺失可以挽救MRS2基因缺失的突变体的生长缺陷、II型内含子剪接缺陷，并可以调节线粒体镁离子浓度，首次发现YMR166C是线粒体镁代谢相关基因。

The pHybLex/Zeo-Idl plasmid and the cDNA library plasmid were sequentially transformed into the yeast swains and screened to obtain Leu2^＋ and Leu2^＋LacZ^＋ clones, with the false positive clones excluded using positive and negative controls, and the plasmid of the true positive clone was sequenced and blasted for homological analysis.

方法PCR方法扩增Id1的编码序列并定向克隆入诱饵质粒pHybLex/Zeo，构建重组诱饵质粒pHybLex/Zeo-Id1，酶切鉴定后转化入酵母株EGY48/pSH18-34，检测重组诱饵质粒有无非特异激活报告基因Leu2、LacZ作用；扩增并提取成人肺组织文库质粒，将文库质粒及重组诱饵质粒转化入酵母细胞，依次筛选Leu2^＋，Leu2^＋LacZ^＋克隆；设置阴性及阳性对照，重复筛选Leu2＋LacZ＋克隆并排除假阳性克隆，获取真阳性文库质粒，进行测序和同源性比对。

Among 33 genes in this study,25 were selected from a cotton somatic embryogenesis SSH array, considered differential expressed at different development stages such as the formation of embryogenic callus and somatic embryogenesis,the others including 5 SERK genes and 3 CKⅡgenes were acquired from the Genebank.

在所研究的33个基因中，有25个来自一个棉花的体细胞胚胎发生的SSH文库，这些基因在棉花胚性愈伤和各时期胚状体中有差异表达，另外5个SERK基因和3个CKⅡ基因来源于相关Genebank库。

The capacity of the primary library was 5.0×105 PFU/mL and the capacity of the amplified library was 8×109 PFU/mL. The cDNA library was immunoscreened by the rabbit serum against H.qinghaiensis larval tick, the positive pluques were subcloned, and their plasmids extracted and then transformed into Escherichia coli JM109. Eventually, 11 genes were obtained and designated HqL1,HqL2,HqL3,HqL4,HqL5,HqL6,HqL7,HqL8,HqL9,HqL10 and HqL11,respectively.

采用常规方法构建了青海血蜱幼蜱cDNA表达文库，初级文库的库容量为5.0×105PFU/mL，扩增后的库容量为8×109PFU/mL；用兔抗青海血蜱幼蜱阳性血清对该文库进行了免疫学筛选，对阳性噬菌体进行了亚克隆，提取质粒后转化宿主菌JM109，最终共得到幼蜱基因11个，分别命名为HqL1、HqL2、HqL3、HqL4、HqL5、HqL6、HqL7、HqL8、HqL9、HqL10、HqL11。

The result of bioinformatics analysis showed that it had no remarkable homology with the known genes in GenBank. And the RSG6 gene is localized on the chromosome 8 and 10 of Oryza sativa L. with one copy for each; its homologous fragments exist also on the chromosome 4 and 12 of rice.

精细胞与二细胞花粉的差减文库中获得的在精细胞中优势表达的克隆作为探针，筛选水稻精细胞cDNA文库而得到的基因，它与已知基因没有明显的同源性，为新发现基因。

This one mode pays close attention to network credence foundation of the businessman very much.

这一模式非常关注商人的网络信用基础。

Cell morphology of bacterial ghost of Pasteurella multocida was observed by scanning electron microscopy and inactivation ratio was estimated by CFU analysi.

扫描电镜观察多杀性巴氏杆菌细菌幽灵和菌落形成单位评价遗传灭活率。