家兔Zfx基因shRNA干扰载体构建及验证

The shRNA interference vector construction and validation from Lepus Zfx gene

研究主要是为了构建出针对家兔Zfx基因的shRNA重组载体及效果验证。根据家兔Zfx基因mRNA序列特征,设计出3条shRNA片段,再与线性化的pLL 3.7载体进行连接重组,最后经过qRT-PCR检测Zfx基因mRNA表达量筛选出有效重组表达载体并通过体内试验进行效果验证。结果表明:试验组shRNA-c和shRNA-f干扰效率分别为74%和67%,与空白对照组相比差异极显著(P<0.01);试验组shRNA-e干扰效果为17%,与空白对照组相比差异不显著(P>0.05)。用shRNA-c和shRNA-f进行体内试验,结果显示,两试验组雄性率分别为44.92%(P>0.05)和33.16%(P<0.01)。本试验成功构建针对家兔Zfx基因的重组载体,体内试验显示子一代出现性别偏移,说明Zfx基因对动物的性别决定有一定作用,关于导致体内验证出现反转的原因仍需进一步验证。

This study was mainly to construct a shRNA recombinant vector for rabbit Zfx gene and to verify its effect. Three shRNA fragments were designed according to the sequence characteristics of Zfx gene in rabbits, and then linked to the linearized pLL 3.7 vector for recombination. Finally, the effective recombinant expression vector was screened by qRT-PCR to detect the expression level of Zfx gene. The results showed that the interference rates of shRNA-c and shRNAf in the experimental group were 74% and 67%, respectively, which were significantly different from the blank control group(P<0.01), and the interference effect of shRNA-e in the experimental group was 17%, which was not significantly different from the blank control group(P>0.05). In vivo experiments with shRNA-c and shRNA-f showed that the male rates in the two experimental groups were 44.92%(P>0.05) and 33.16%(P<0.01), respectively. This experiment successfully constructed a recombinant vector for rabbit Zfx gene. In vivo test showed that the offspring had sex deviation, which indicated that Zfx gene played a certain role in animal sex determination. The reason for the inversion of the in vivo verification still needs further verification.