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

孙 霞；张 静；印 锦；秦 笙；张国政；李木旺

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DOI：10.7679/j.issn.2095-1353.2019.115

作者单位：1. 江苏科技大学生物技术学院，镇江 212018；2. 中国农业科学院蚕业研究所，镇江 212018

中文关键词：雏翅；蛋白酶体；CRISPR/Cas9技术；基因组编辑；家蚕

英文关键词：minute wing, proteasome, CRISPR/Cas9, genomic editing, silkworm

中文摘要：【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。

英文摘要：[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.

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