Acta Entomologica Sinica ›› 2021, Vol. 64 ›› Issue (3): 309-317.doi: 10.16380/j.kcxb.2021.03.002

• RESEARCH PAPERS • Previous Articles     Next Articles

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

LIU Hong-E1, WANG Li-Zhi1, LIU Zu-Lian2, YI Mei-Yan1, FENG Qi-Li1, HUANG Yong-Ping2, XIANG Hui1,*   

  1.  (1. Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China; 2. Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China)
  • Online:2021-03-20 Published:2021-04-20

Abstract: 【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-3 5th 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-Bmggnbp2sgRNA 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-3 5th instar larvae of B. mori. The chimeric knockout mutant △Bmggnbp2 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 Bmggnbp2 and the control lines U6-Bmggnbp2sgRNA 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.

Key words: Lepidoptera; Bombyx mori, gametogenetin binding protein 2, transgenic CRISPR, testis, egg hatching rate