The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5＇ end of RNA transcript （SMART） approach. This library had a plaque titer of 1 x 109 pfu mL-1 and a 96...The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5＇ end of RNA transcript （SMART） approach. This library had a plaque titer of 1 x 109 pfu mL-1 and a 96% recombinant ratio of which the fragment length of inserted average cDNA sequences was 1.0 kb. Based on bioinformatics analysis of the sequences, we obtained 338 expressed sequence tags （ESTs） from 380 cDNA clones which indicated 191 contigs. These contigs consist of 89 unmatched ESTs, 9 homologous known genes in sheep, and 93 homologous sequences in species of mouse, bovine, and human beings, including 19 sequences expressed in the ovary or follicle and 14 unknown sequences. Several candidate genes associated with sheep reproduction trait such as epidermal growth factor （EGF）, estrogen receptor （ESR）, Inhibin, follicle stimulating hormone receptor （FSHR）, prostaglandin （PG）, and transforming growth factor-β （TGF-β） were identified and the homologous were cloned from this library, which will contribute to compile expression profiles and find the major genes of prolificacy of Small Tail Han sheep.展开更多
目的构建红曲霉cDNA文库，为红曲霉功能基因的研究以及筛选、克隆红曲霉次生代谢产物合成途径相关基因奠定基础。方法采用SMART（ switching mechanism at 5 ＇end of RNA transcript）技术构建红曲霉全长cDNA文库，经涂平板和酶切反应...目的构建红曲霉cDNA文库，为红曲霉功能基因的研究以及筛选、克隆红曲霉次生代谢产物合成途径相关基因奠定基础。方法采用SMART（ switching mechanism at 5 ＇end of RNA transcript）技术构建红曲霉全长cDNA文库，经涂平板和酶切反应测定文库的克隆数、重组率，PCR测定插入片断的大小。结果原始文库的库容为2．03×10^5，重组率达94％。插入片段大小多在0．7—2．0kb，平均大小在1kb左右。结论所构建文库的代表性和重组片段的序列完整性达到了用于目的基因的分离筛选和克隆表达的建库要求。展开更多
以珙桐叶片为研究材料，用Rneasy Plant Mini Kit提取珙桐叶片组织总RNA，反转录成单链cDNA，长距离PCR方法合成双链cDNA；PCR产物经蛋白酶K消化、SfiI酶切后，采用CHROMA SPIN-400柱分级分离，回收0．5kb以上的cDNA组分，以λTriplEx2...以珙桐叶片为研究材料，用Rneasy Plant Mini Kit提取珙桐叶片组织总RNA，反转录成单链cDNA，长距离PCR方法合成双链cDNA；PCR产物经蛋白酶K消化、SfiI酶切后，采用CHROMA SPIN-400柱分级分离，回收0．5kb以上的cDNA组分，以λTriplEx2载体连接并进行体外包装，构建了珙桐叶全长cDNA文库．结果表明：以XL1-Blue、BM25．8为受体菌测定的原始文库滴度为8．3×10^6pfu／mL，扩增后文库总滴度为4．16×10^8pfu／mL，重组率为97．3％；对随机挑取的噬菌斑进行PCR鉴定，表明插入片段大多分布在0．5～2．0kb之间，文库质最较高．这为进一步开展珙桐EST测序分析、目的基因克隆及功能基因组学研究奠定了基础．展开更多
文摘The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5＇ end of RNA transcript （SMART） approach. This library had a plaque titer of 1 x 109 pfu mL-1 and a 96% recombinant ratio of which the fragment length of inserted average cDNA sequences was 1.0 kb. Based on bioinformatics analysis of the sequences, we obtained 338 expressed sequence tags （ESTs） from 380 cDNA clones which indicated 191 contigs. These contigs consist of 89 unmatched ESTs, 9 homologous known genes in sheep, and 93 homologous sequences in species of mouse, bovine, and human beings, including 19 sequences expressed in the ovary or follicle and 14 unknown sequences. Several candidate genes associated with sheep reproduction trait such as epidermal growth factor （EGF）, estrogen receptor （ESR）, Inhibin, follicle stimulating hormone receptor （FSHR）, prostaglandin （PG）, and transforming growth factor-β （TGF-β） were identified and the homologous were cloned from this library, which will contribute to compile expression profiles and find the major genes of prolificacy of Small Tail Han sheep.
文摘目的构建红曲霉cDNA文库，为红曲霉功能基因的研究以及筛选、克隆红曲霉次生代谢产物合成途径相关基因奠定基础。方法采用SMART（ switching mechanism at 5 ＇end of RNA transcript）技术构建红曲霉全长cDNA文库，经涂平板和酶切反应测定文库的克隆数、重组率，PCR测定插入片断的大小。结果原始文库的库容为2．03×10^5，重组率达94％。插入片段大小多在0．7—2．0kb，平均大小在1kb左右。结论所构建文库的代表性和重组片段的序列完整性达到了用于目的基因的分离筛选和克隆表达的建库要求。
文摘以珙桐叶片为研究材料，用Rneasy Plant Mini Kit提取珙桐叶片组织总RNA，反转录成单链cDNA，长距离PCR方法合成双链cDNA；PCR产物经蛋白酶K消化、SfiI酶切后，采用CHROMA SPIN-400柱分级分离，回收0．5kb以上的cDNA组分，以λTriplEx2载体连接并进行体外包装，构建了珙桐叶全长cDNA文库．结果表明：以XL1-Blue、BM25．8为受体菌测定的原始文库滴度为8．3×10^6pfu／mL，扩增后文库总滴度为4．16×10^8pfu／mL，重组率为97．3％；对随机挑取的噬菌斑进行PCR鉴定，表明插入片段大多分布在0．5～2．0kb之间，文库质最较高．这为进一步开展珙桐EST测序分析、目的基因克隆及功能基因组学研究奠定了基础．
