利用CRISPR/Cas9双元转基因体系探究家蚕配子生成素结合蛋白基因Bmggnbp2的功能

【目的】配子生成素结合蛋白2(gametogenetin binding protein 2,ggnbp2)在哺乳动物配子发生等生殖相关过程中发挥重要作用。本研究以家蚕Bombyx mori为模型,探究驯化基因ggnbp2的生物学功能,为鳞翅目昆虫中该基因的深入研究提供线索。【方法】在NCBI GenBank数据库通过序列比对检索到翅目昆虫ggnbp2基因的序列;通过贝叶斯法构建系统发育树进行ggnbp2基因的系统发育分析。根据我们前期研究建立的家蚕-野桑蚕Bombyx mandarina群体基因组多态性数据,对家蚕中Bmggnbp2进行选择信号分析;利用已发表的基因芯片数据分析Bmggnbp2在家蚕5龄第3天幼虫中的组织表达特征。利用CRISPR/Cas9双元转基因体系构建Bmggnbp2敲除突变体,测定分析突变体与对照品系Nos-Cas9在繁殖性状(单雌产卵量和卵孵化率)以及与对照品系U6-Bmggnbp2 sgRNA和Nos-Cas9经济性状(全茧重、蛹重和茧壳重)的差异。【结果】ggnbp2在鳞翅目昆虫中广泛存在,且高度保守,并且在家蚕中具有强烈的人工选择信号。Bmggnbp2在家蚕5龄第3天幼虫的脑、卵巢、精巢和丝腺中均高度表达。利用CRISPR/Cas9双元转基因体系成功获取家蚕Bmggnbp2基因的嵌合体敲除突变体△Bmggnbp 2。与对照Nos-Cas9精巢的典型肾形不同,突变体△Bmggnbp 2的精巢呈椭圆形,并且表面出现一层较厚的黄色覆盖物,但突变体△Bmggnbp 2单雌产卵量和卵孵化率与对照品系Nos-Cas9以及全茧重、蛹重和茧壳重与对照U6-Bmggnbp2 sgRNA和Nos-Cas9均没有显著差异。【结论】GGNBP2在鳞翅目昆虫中对繁殖的影响可能并没有在哺乳动物中那么至关重要,其具体的作用机制仍需要进一步探讨。

Exploration of the function of the gametogenetin binding protein gene Bmggnbp2 in Bombyx mori by CRISPR/Cas9 binary transgenic system

【Aim】Gametogenetin binding protein 2(ggnbp2)plays an important role in mammalian gametogenesis.In this study,we used Bombyx mori as a model to explore the biological function of the domesticated gene ggnbp2,so as to provide clues for the further study of this gene in lepidopteran insects.【Methods】The sequences of ggnbp2 genes in lepidopteran insects were retrieved from the NCBI GenBank by blast,and the phylogenetic analysis of the ggnbp2 genes was made through constructing the phylogenetic tree by Bayesian method.Based on the genomic polymorphism data of B.mori and B.mandarina populations established in our previous study,the selection signal of Bmggnbp2 in B.mori was analyzed,and the tissue expression pattern of Bmggnbp2 in the day-35th instar larvae of B.mori was analyzed based on the published gene chip data.The Bmggnbp2 knockout mutants were constructed in the binary transgenic system CRISPR/Cas9,and the differences in the reproductive traits(number of eggs laid per female and egg hatching rate)between the mutant and the control line Nos-Cas9 and the economic traits(cocoon weight,pupal weight and cocoon shell weight)between the mutants and the control lines U6-Bmggnbp2 sgRNA and Nos-Cas9 were assayed and analyzed.【Results】ggnbp2 is widely distributed in lepidopteran insects and highly conserved,and has strong artificial selection signal in B.mori.Bmggnbp2 was highly expressed in the brain,ovary,testis and silk gland of the day-35th instar larvae of B.mori.The chimeric knockout mutant△Bmggnbp 2 of Bmggnbp2 gene was successfully obtained by binary transgenic system CRISPR/Cas9.The testes of the control line Nos-Cas9 are typical reniform,while those of the mutant△Bmggnbp2 are oval in shape and have a thick yellow covering.The number of eggs laid per female and egg hatching rate between the mutant△Bmggnbp2 and the control line Nos-Cas9 and the cocoon weight,pupal weight and cocoon shell weight between the mutant△Bmggnbp 2 and the control lines U6-Bmggnbp2 sgRNA and Nos-Cas9 showed no significant difference.【Conclusion】The effect of GGNBP2 on reproduction in lepidopteran insects may not be as important as that in mammals,and its action mechanism still needs to be further explored.