加工番茄SlAGO4A基因过表达及干扰载体构建与遗传转化  

Overexpression of tomato SlAGO4A gene and construction of interfering vector and its genetic transformation

作　　者：许鹏程[1] 路遥[1] 周东[1] 刘丽[1] 刘丽娜[1] 郑银英[1] 

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

出　　处：《生物技术》2017年第6期511-516,共6页Biotechnology

基　　金：国家自然科学基金项目("番茄OPA3-like基因的功能研究";No.31460466)

摘　　要：[目的]构建加工番茄SlAGO4A基因过表达及干扰载体,获得相应的转基因番茄植株。[方法]利用PCR技术从番茄c DNA文库中获得2 727 bp的加工番茄SlAGO4A基因。构建SlAGO4A基因的过量表达载体35S:SlAGO4A。以获得的SlAGO4A基因为模板,获得了SlAGO4A PIWI的220 bp的片段,构建SlAGO4A基因的RNAi干扰载体RNAi-PIWI,通过农杆菌介导的遗传转化,获得SlAGO4A过表达及干扰转基因番茄阳性植株,并利用实时荧光定量PCR(QRTPCR)技术检测过表达番茄阳性植株中的SlAGO4A基因的表达水平。[结果]经PCR鉴定获得5株独立转化的里格87-5干扰转基因加工番茄株系,获得6株独立转化的里格87-5过量表达转基因加工番茄株系,其6株SlAGO4A基因过表达的阳性植株的表达量均有不同程度的上调。[结论]为阐明SlAGO4A基因在加工番茄抗病毒信号通路中的功能奠定基础。[Objective]Constructing tomato SlAGO4A gene overexpression and interference vector,to obtain the corresponding transgenic tomato plants.[Methods]The 2 727 bp tomato SlAGO4A gene was obtained from the tomato c DNA library by PCR.SlAGO4A gene overexpressing vector 35 S： SlAGO4A was constructed.The SlAGO4A gene as template,a 220 bp fragment of SlAGO4A PIWI was obtained,then RNAi-PIWI of SlAGO4A gene was constructed.Agrobacterium tumefaciens-mediated genetic transformation was used to obtain SlAGO4A gene overexpression and interfere ransgenic positive plants.Fluorescence quantitative PCR（QRT-PCR） technique was used to detection the expression level of SlAGO4A gene in overexpressing plants.[Results]Five different Rieg 87-5 transgenic tomato lines were obtained and detected by PCR,and six different Rieg 87-5 transgenic tomato lines were obtained,and the expression of SlAGO4A gene in the positive plants was increased in varying degrees.[Conclusion]This study lays the foundation for clarifying the function of SlAGO4A gene in processing tomato antiviral signaling pathway.

关 键 词：加工番茄 SlAGO4A 遗传转化 PCR 

分 类 号：Q78[生物学—分子生物学]