不同转座子介导的基因捕获效率比较  

Comparison of Transposon-mediated Promoter Gene Trap Efficiency

作　　者：李庆平[1] 薛松磊[1] 史云强[1] 沈丹[1] 王赛赛[1] 陈才[1] 钱跃[1] 高波[1] 宋成义[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《农业生物技术学报》2014年第6期771-778,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31200920);江苏省产学研联合创新资金--前瞻性联合研究项目(No.SBY201320109)

摘　　要：为了比较SB(Sleeping Beauty)、PB(PiggyBac)和Tol2 3种转座子介导的基因捕获效率,本研究构建了含有3种转座子的启动子捕获载体pT3-PST-Pro-Trap。将此载体分别和SB、PB和Tol2 3种转座酶mRNA按照质量比1.0∶1.5的比例注射斑马鱼(Danio rerio)一细胞胚胎,通过检测绿色荧光蛋白报告基因(GFP)的表达水平比较不同转座子的捕获效率。结果显示,3种转座子介导的启动子捕获载体能够高效率捕获内源基因启动子,进而驱动GFP的表达;注射后培养36 h时的SB、PB和Tol2捕获效率分别为24.32%、30.70%和18.87%,PB捕获效率显著高于SB和Tol2(P<0.05);3种转座子在60 h时的捕获效率均高于36 h的,其中SB和Tol2组差异达显著水平(P<0.05),而PB组差异不显著(P>0.05)。本研究结果表明,SB、PB和Tol2转座子介导的基因捕获技术可以用于脊椎动物高通量功能基因筛选,为功能基因研究提供有效新方法。To compare the efficiency of transposon-mediated gene trap among SB（Sleeping Beauty）, PB （PiggyBac） and To12, a promoter trap vector based on those transposons named pT3-PST-Pro-Trap wasconstructed. Then the vector plasmid DNA and transposase mRNA of SB, PB and To12 according to 1.0：1.5 ratio were mixed, respectively, and injected into zebrafish（Danio rerio） one-cell embryos. The gene trappingefficiency could be determined by the expression level of green fluorescent protein report gene （GFP）. The results showed that promoter trap vector mediated by SB, PB, and To12 transposon could capture theendogenous gene promoter with high efficiency of 24.32%, 30.7% and 18.87% 36 h after injected, respectively, and GFP expression were observed. PB-mediated gene trap efficiency was significantly higherthan that of SB and To12 （P〈0.05）. Three kinds of transposon capture efficiency in 60 h period were higher than that of 36 h period, SB and To12 group were significant differences （P〈0.05）, while PB group was nosignificant difference （P〉0.05）. The results showed that PB, SB and To12 transposon mediated gene capture technology can be used to vertebrate high-throughput screening functional genes, and provide effective newmethod for the research of functional genes.

关 键 词：基因捕获 SB PB Tol2 斑马鱼胚胎 

分 类 号：S511.03[农业科学—作物学]