家蚕BmRpt4基因在翅膀发育中的功能研究

Functional analysis of the BmRpt4 gene during wing development in the silkworm, Bombyx mori

【目的】 研究分析编码蛋白酶体调控复合物亚基基因BmRpt4(Regulatory particle triple-A ATPase4,BGIBMGA010794)在家蚕翅膀发育过程中的功能。【方法】 利用CRISPR/Cas9基因组编辑技术,将BmRpt4的gRNAs和Cas9 mRNA直接注射到家蚕胚胎中,观察G0代家蚕个体雏翅率,并分析雏翅个体中BmRpt4基因突变情况。【结果】 经统计,对照组G0代家蚕个体均表现为正常翅,而实验组注射BmRpt4的gRNAs和Cas9 mRNA后,G0代约66.7%家蚕个体表现出小翅表型。在分子水平上对小翅表型个体进行检测,发现BmRpt4基因在两个gRNA及其之间位置均有不同长度片段的缺失或者插入,说明在这些突变个体中BmRpt4基因被不同程度的敲除,从而其功能缺失,导致出现小翅表型。【结论】 该研究结果表明编码蛋白酶体调控亚基的BmRpt4基因在家蚕翅膀发育中具有重要作用,为蛋白酶体在生物组织器官发育中调控作用研究提供重要实验依据。

[Objectives] To study and analyze the function of the BmRpt4 gene (Regulatory particle triple-A ATPase 4, BGIBMGA010794) during wing development in the silkworm Bombyx mori. [Methods] BmRpt4 gRNAs and Cas9 mRNA were directly injected into silkworm embryos using the CRISPR/Cas9 genome editing technique. Phenotypes of G0 adults were observed and the proportion of adults with minute wings was statistically analyzed. Mutation of BmRpt4 was detected in adults with minute wings. [Results] All G0 adults in the control group had normal wings whereas 66.67% of G0 adults in the experimental group had minute wings. The BmRpt4 gene was deleted, or inserted, in different length fragments at the two gRNAs locii in G0 individuals with minute wings. These results indicate that individuals developed minute wings due to knock-down of the BmRpt4 gene. [Conclusion] The BmRpt4 gene plays an important role in wing development in the silkworm, and in regulating proteasome in the development of biological tissues and organs.