iMyAP基因过表达载体转化甘蓝型油菜

Transformation of iMyAP Gene Over-expression Vector into Brassica napus

本研究以甘蓝型油菜湘油15号叶片cDNA为模版,克隆油菜黑芥子酶协助蛋白iMyAP基因全长CDS,目的片段序列和GenBank上报道的甘蓝型油菜序列iMyAP12-Y10156同源性达到100%。将得到的iMyAP基因片段与pMD19-T载体连接,命名为T-1152。用BamHⅠ和XbaⅠ双酶切T-1152和pHB载体,回收片段经T4DNA连接酶连接得到iMyAP基因过表达载体pHB-1152。通过农杆菌介导将pHB-1152转化至甘蓝型油菜湘油15号,PCR鉴定显示成功获得2株转基因植株。本实验为进一步研究iMyAP基因的功能奠定了良好的基础。

Taking the cDNA from Brassica napus Xiangyou 15 leaves as template,full-length CDS of induced Myrosinase-associated proteins（iMyAP） gene was cloned in this study.BLAST analysis shows that the homology between the cloned sequence and a iMyAP sequence from Brassica napus（iMyAP12-Y10156） in GenBank is up to 100%.The cloned sequence was ligated into pMD19-T vector to obtain T-1152,Then T-1152 and pHB were dou ble digested by BamHⅠ and XbaⅠ.The restriction fragments were recycled and ligated by T4 DNA Ligase to obtain iMyAP gene over-expression vector pHB-1152.Then pHB-1152 was transformed into Brassica napus Xiangyou 15 mediated by Agrobacterium tumefaciens and two transgenic plants was detected by PCR identification.This research would lay a foundation for further investigating the function of iMyAPgene.