木糖异构酶基因xylA的克隆及其表达载体的构建  

Cloning of xylA Gene and Construction of Its Plant Expression Vector

作　　者：孙磊[1] 张国军[1] 闫爱玲[1] 徐海英[1] 

机构地区：[1]北京市农林科学院林业果树研究所,北京100093

出　　处：《生物技术通报》2011年第7期167-170,共4页Biotechnology Bulletin

基　　金：现代农业产业技术体系建设专项资金项目(nycytx-30);北京市优秀人才培养资助项目(20104D002020000007)

摘　　要：木糖异构酶基因xylA是一种正向选择标记基因,在植物基因工程中使用该标记可以获得安全的转基因植物。构建了以xylA基因为选择标记的植物表达载体。从大肠杆菌Top10中扩增出xylA基因,插入到质粒pCAMBIA2301的XhoI位点,通过酶切和PCR检测插入片段的正确性,得到载体pCAMBIA2301-xylA,将pBI121载体上的‘35S-GUS-Nos’表达框插入到pCAMBIA2301-xylA的EcoR I和Hind III位点。得到中间载体pCAMBIA2301-xylA-GUS,用SacI和SmaI切下克隆载体上的CBF1基因替代pCAMBIA2301-xylA-GUS中的GUS片段,用电转化法将获得的表达载体转化到农杆菌中,为将来获得安全的转基因抗寒植株奠定基础。Xylose isomerase gene was the positive selective marker which can be used to obtain safe transgenic plants in the genetic engineering.In this research,the XylA gene was amplified from the genomic DNA of Escherichia coli ＇Top10＇ by using specific primers containing Xho I enzyme site.XylA gene fragment which was inserted into the plasmid pCAMBIA2301 to substitute the nptII gene,was identified by enzyme digestion and PCR,therefore,the pCAMBIA2301-xylA was obtained.＇35S-GUS-Nos＇ fragment was cut from the vector pBI121 and linked to the EcoR I/Hind III sites of pCAMBIA2301-xylA to get pCAMBIA2301-xylA-GUS.CBF1 was then cut by Sma I/Sac I form the cloning vector and cloned into pCAMBIA2301-xylA-GUS.Finally,the recombinant vector pCAMBIA2301-xylA-CBF1 was successfully constructed and transformed into Agrobacterium tumefaciens ＇EHA105＇ by electroporation.The following research of genetic transformation could be done to obtain the cold tolerance transgenic plants with safe marker gene.

关 键 词：木糖异构酶基因 标记基因 植物表达载体 克隆 

分 类 号：Q943.2[生物学—植物学]