利用简并引物克隆天山雪莲sikPIP基因片段及RNAi载体的构建  

Cloning of sikPIP Gene cDNA Fragment of S.involucrata Kar.et Kir.by Degenerate Primers and Construction of RNAi Vector

作　　者：孙辉[1] 祝建波[1] 

机构地区：[1]石河子大学生命科学学院农业生物技术重点实验室,石河子832003

出　　处：《生物技术通报》2010年第6期107-112,共6页Biotechnology Bulletin

基　　金：国家"863"计划项目(2007AA021401);国家转基因重大专项(2008ZX005-004)

摘　　要：利用NCBI、Bloekmaker、CODEHOP、Primer Premier5.0等数据库和生物软件设计简并引物并利用简并引物RT-PCR,首次从天山雪莲中克隆到474bp的PIP基因cDNA片段,命名为sikPIP,其与GenBank中非洲杂交菊PIP基因氨基酸序列的同源性高达93.0%。从上述序列中选取200bp的序列,利用2对同尾酶将正义片段和反义片段插入到植物表达载体CAM2300-35S-Ocs中,成功构建天山雪莲RNAi载体CAM2300-35S-Ocs-sikPIP。为接下来的遗传转化,进一步探明sikPIP基因在天山雪莲中的功能奠定了基础。By using some internet databases and biological softwares such as NCBI,Blockmaker,CodeHop and Primer Premier 5.0 to design degenerate primers and degenerate primers RT-PCR,a 474 bp fragment of PIP gene cDNA was acquired from S.involucrata Kar.et Kir.for the first time and was named sikPIP.It presented a high homology of 93.0%with PIP gene amino acid sequence of Gerbera hybrid cultivar registered in GenBank.A 200 bp sequence was selected from the sequence and inserted its sense fragment and anti-sense fragment to plant expression vector CAM2300-35S-Ocs,using isocaudarner,to construct a RNAi plant expression vector CAM2300-35S-Ocs-sikPIP.The experiment makes preparation for the genetic transforming and understanging the function of sikPIP in S.involucrata Kar.et Kir.ulteriorly.

关 键 词：天山雪莲 sikPIP 简并引物 RNAI 

分 类 号：S567.239[农业科学—中草药栽培]