摘要
谷胱甘肽转移酶(glutathione transferases,GSTs)在植物抵御逆境胁迫中具有非常重要的作用。从高羊茅叶片中克隆获得FaGST基因,对其进行酶切位点分析,设计酶切引物,通过BamHI和PstI双酶切将目的基因片段连接在真核表达载体pCAMBIA 1300-35 S上,成功构建pCAMBIA 1300-35 S-FaGST过表达载体。将过表达载体转化感受态(DH 5α)细胞,利用农杆菌介导法遗传转化菊苣,通过潮霉素(Hyp)进行筛选后获得阳性植株。经PCR鉴定呈阳性,结果表明,pCAMBIA 1300-35 S-FaGST过表达载体已成功导入菊苣中,获得转基因菊苣阳性苗9株,利用实时荧光定量PCR对其表达量进行检测,发现该基因表达量明显提高。这将为下一步分析该基因的抗逆性研究奠定基础。
Glutathione transferases(GSTs)play an important role in response to adversity stress in plants.In this study,the FaGST gene was cloned from Festuca arundinacea leaves,and restriction site of the gene was analyzed and the restriction primer was also designed.Meanwhile,the target gene fragment was connected to the eukaryotic expression vector pCAMBIA 1300-35 S by Bam HI and Pst I double restriction,and the pCAMBIA 1300-35 S-FaGST over-expression vector was successfully constructed.The overexpressing vector was transformed into Escherichia coli competent cells(DH 5α),and Agrobacterium-mediated genetic transformation was used to obtain positive plants by hygromycin(Hyp)-resistance screening.It was identified as positive by PCR and the result showed that pCAMBIA 1300-35 S-FaGST overexpressing vector had been successfully introduced into chicory,and 9 transgenic chicory positive seedlings were obtained.The expression level of FaGST was detected by real-time quantitative PCR and it was found that the standard level of FaGST gene was significantly increased.This would lay a foundation for further analysis of the stress resistance of FaGST gene.
作者
陈锡
陈莹
刘晓霞
刘重阳
吴溪
姚文琴
赵德刚
王小利
CHEN Xi;CHEN Ying;LIU Xiaoxia;LIU Chongyang;WU Xi;YAO Wenqin;ZHAO Degang;WANG Xiaoli(Guizhou Institute of Prataculture,Guizhou Academy of Agricultural Sciences,Guiyang 550006,China;Guizhou University,College of life sciences,the Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region,Ministry of Education,the National-Local Joint Engineering Research Center of Karst Region Plant Resources Utilization&Breeding,Guiyang 550025,China)
出处
《种子》
北大核心
2022年第3期32-36,共5页
Seed
基金
贵州省科技计划项目([2019]1302)
贵州省高层次创新人才培养项目([2016]4003)
国家自然科学基金项目(31860674)
黔科合平台人才([2018]5634-2)。
关键词
菊苣
FaGST
遗传转化
表达分析
chicory
FaGST
genetic transformation
expression analysis
