橡胶树胶乳cDNA文库的构建及其EST序列的功能分析  

Construction and EST Analysis of cDNA Library from Latex in Hevea brasiliensis

作　　者：段翠芳[1] 曾日中[1] 聂智毅[1] 黎瑜[1] 魏小弟[1] 

机构地区：[1]中国热带农业科学院橡胶研究所,农业部热带作物生物学重点开放实验室,海南儋州571737

出　　处：《热带作物学报》2007年第3期46-51,共6页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金资助项目(No.30360051);中国热带农业科学院科技基金(2005-2006);农业部热带作物生物学重点开放实验室重大研究项目(No.KLOF0601;2006-2007);基本科研经费专项(2007)资助

摘　　要：为了分离和克隆橡胶树胶乳中与胶乳代谢、橡胶生物合成和茉莉酸生物合成等途径相关的功能基因,以橡胶树无性系热研7-33-97品系新开割树的胶乳为材料,用SDS裂解法提取胶乳总RNA,磁珠法纯化mRNA,逆转录合成第1链cDNA,并合成双链cDNA,经电泳检测,双链cDNA的大小主要在1.0～2.0kb之间,收集大于500bp的cDNA片段,以DH10B为宿主菌,pBluescriptII为载体,构建未经扩增的橡胶树胶乳cDNA初级文库。该文库的库容大于5.0×105Pfu/mL,重组率大于90%。随机对文库的3200个克隆进行了EST测序,用所测序列与GenBank非冗余蛋白质数据库进行BlastX比对,并将比对上的基因进行生物学功能注释和分类。研究结果表明,所构建的胶乳cDNA初级文库质量较高,适合包括低丰度表达基因在内的橡胶树胶乳功能基因的筛选,是研究橡胶树胶乳中重要功能基因的最主要资源和开展橡胶树胶乳代谢与橡胶生物合成的分子机制研究的基础。In order to isolate and clone the functional genes involved in the latex metabolism,rubber biosynthesis pathway,and jasmonic acid biosynthesis pathway in Hevea brasilensis,a cDNA library was constructed from the la-tex in this research.The latex was collected from the newly tapped clone 7-33-97,RNA was extracted through SDS method,and then to purify by magnetic beads.The primary library had a total of 5×105 phages for the latex,in which above 90 % of the phages were recombinants.Insert sizes mainly ranging from1 000 bp to 2 000 bp were ob-tained.About 3 200 clones were randomly sequenced from the library;the Blast X analysis was carried out with the non-redundant protein database,and afterwards,the genes were made in classification according their functions.The cDNA library was in high quality,which was suitable for the detection and identification of functional genes in-cluding the lowabundance gene in latex.This library would provide a good foundation for investigating the molecu-lar mechanism of rubber biosynthesis and latex metabolism,and it was also an important resource for cloning the im-portant functional genes in latex.

关 键 词：橡胶树 胶乳 CDNA 文库 EST 

分 类 号：S794.104[农业科学—林木遗传育种]