Acta Entomologica Sinica ›› 2022, Vol. 65 ›› Issue (12): 1598-1605.doi: 10.16380/j.kcxb.2022.12.005

• RESEARCH PAPERS • Previous Articles     Next Articles

Functional analysis of pax3 gene in Bombyx mori by the binary transgenic CRISPR/Cas9 system

PU Shang-Kun1, WANG Lei1, TAN An-Jiang2,3, WEI Guo-Qing1,*   

  1.  (1. School of Life Sciences, Anhui Agricultural University, Hefei 230036, China; 2. School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, China; 3. Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu 212100, China)
  • Online:2022-12-20 Published:2023-01-19

Abstract:  【Aim】 This study aims to investigate the biological functions of pax3 gene in Lepidoptera insects using the silkworm, Bombyx mori, as a model. 【Methods】 The exon sequence of Bmpax3 from B. mori was verified by PCR amplification. The expression profile of Bmpax3 in the head, cuticle, fat body, midgut, Malpighian tubules, anterior silk gland, middle silk gland, posterior silk gland and gonads (including testis and ovary) of the day-3 5th instar larvae of B. mori was detected by qRT-PCR. The Bmpax3 knockout mutants were constructed using the binary transgenic CRISPR/cas9 system to analyze the effects of Bmpax3 mutation on the larval survival, somite differentiation and gender difference of B. mori. 【Results】 Bmpax3 was expressed in the head, midgut and silk gland of the day-3 5th instar larvae of B. mori, with its highest expression level in the anterior silk gland. The egg hatching rate of the Bmpax3 mutants was about 90%, but about 80% of mutants died in the 1st instar larval stage, and only about 10% of the mutated individuals survived to the adult stage. And there was gender difference in the number of surviving adults, in which the number of male survivors was significantly higher than that of female survivors. About half of the surviving adults showed abnormal differentiation in distal abdominal somites, disorder of cuticle stripes, deficiency of partial abdominal sternum and developmental defects in reproductive organs and other auxiliary organs around them. 【Conclusion】 Bmpax3 mutation has a great impact on the survival and morphological development of B. mori, suggesting that Bmpax3 may participate in the growth and development processes in B. mori.

Key words: Bombyx mori; Bmpax3, CRISPR-Cas9, survival rate, somite differentiation