家蚕BmVNCP基因表达与BmNPV感染增殖相关性的鉴定分析  

Identification and Analysis of the Correlation Between the Expression of BmVNCP Gene and Infection and Proliferation of BmNPV in Silkworm, Bombyx mori

作　　者：邵露璐 何小柏 高娟[1] 宋秋云 张业顺 方瑷 武康晖[1,2] 王学杨 张国政[1,2] Shao Lulu;He Xiaobai;Gao Juan;Song Quyun;Zhang Yeshun;Fang Ai;Wu Kanghui;Wang Xueyang;Zhang Guozheng(School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212100,China;Key Laboratory of Silkworm and Mulberry Genetic Improvement,Ministry of Agriculture and Rural Affairs,Sericultural Research Insttute,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212100,China)

机构地区：[1]江苏科技大学生物技术学院,江苏镇江212100 [2]中国农业科学院蚕业研究所,农业农村部蚕桑遗传改良重点实验室,江苏镇江212100

出　　处：《蚕业科学》2022年第4期293-301,共9页ACTA SERICOLOGICA SINICA

基　　金：财政部与农业农村部国家现代农业产业技术体系项目。

摘　　要：为解析家蚕AN品系高抗BmNPV的分子机制,采用家蚕抗性品系AN与易感品种C108杂交及多代回交,结合累代BmNPV攻毒选择,构建了高抗BmNPV的近等基因系C108_AN;在5龄起蚕经口添食BmNPV多角体1×10^(8) mL^(-1)悬浊液浸渍桑叶对C108及C108_AN攻毒,对感染后12、24、36、48、60 h的中肠组织进行转录组测序发现,C108_AN样本中检出BmNPV基因个数与表达量均大幅减少,病毒虽然能够侵染但不能完成复制;转录组中编码230 aa的家蚕病毒核衣壳蛋白基因(命名为BmVNCP)在C108_AN样本中显著高表达,表明与BmNPV抗性可能具有关联性。在BmN细胞中过表达BmVNCP基因可以抑制BmNPV的增殖,在BmN细胞中进行靶向BmVNCP的CRISPR/Cas9基因编辑,未能观察到BmNPV增殖和vp39基因表达的稳定增加;BmN细胞内过表达BmVNCP基因可以明显提高宿主IMD信号通路中Dredd基因的表达水平,对FADD基因转录水平无影响,PGRP-LB和PGRP-LF基因表达量极低以致qRT-PCR无法检出。综合分析上述结果认为,家蚕细胞或组织中BmVNCP基因高水平表达不能够阻止BmNPV侵染,但能够降低BmNPV侵染后病毒基因的表达以及病毒增殖,BmVNCP基因产物可能是通过IMD信号通路触发免疫机制。In order to analyze the molecular mechanism of B. mori nuclear polyhedrosis virus(BmNPV) resistance of Bombyx mori AN strain, the susceptible strain C108 was crossed with the resistant strain AN and then repeatedly backcrossed C108, and each generation was screened for BmNPV resistance. A near-isogenic line C108_AN with high resistance to BmNPV was constructed. The C108 and C108_AN silkworms in the fifth instar were tested by feeding mulberry leaves impregnated with BmNPV polyhedra of 1×10^(8) mL^(-1) suspension. Transcriptome sequencing was performed on midgut tissue at 12, 24, 36, 48, 60 h after infection, and it was found that the number of BmNPV genes and gene expression levels in C108_AN midgut tissue samples were both significantly reduced. Although the virus could infect, it was not able to complete replication and proliferation. It was found that the viral nucleocapsid protein gene(named BmVNCP) encoding 230 aa was significantly expressed in the C108_AN sample, indicating a possible correlation with BmNPV resistance. Overexpression of BmVNCP gene in BmN cells could significantly reduce BmNPV proliferation, but the BmVNCP-targeting CRISPR/Cas9 gene editing in BmN cells showed no BmNPV proliferation or stable increase of vp39 gene expression. Overexpression of BmVNCP gene in BmN cells could significantly increase the expression level of Dredd gene in IMD signaling pathway, but had no effect on the transcription level of FADD gene in the pathway. At the same time, the expression levels of PGRP-LB and PGRP-LF genes in the IMD pathway were too low to be detected by qRT-PCR. Comprehensive analysis of the above results showed that high-level expression of BmVNCP gene in silkworm cells or tissues could not prevent BmNPV infection, but could reduce the viral gene expression and virus proliferation after BmNPV infection. The product of BmVNCP gene might trigger the immune mechanism through IMD signaling pathway.

关 键 词：家蚕 家蚕核型多角体病毒 家蚕病毒核衣壳蛋白基因 基因过表达 病毒感染 病毒增殖 

分 类 号：S884.51[农业科学—特种经济动物饲养]