影响茉莉H病毒沉默抑制子功能关键氨基酸的鉴定  

Identification of key residues affecting RNA silencing suppressor activity of Jasmine virus H

作　　者：朱丽娟[1,2] 张崇涛 王耀伟 江朝杨 解晓盈 白雅妮 韩艳红 ZHU Lijuan;ZHANG Chongtao;WANG Yaowei;JIANG Chaoyang;XIE Xiaoying;BAI Yani;HAN Yanhong(College of Plant Protection,Fujian Agriculture and Forestry University;Vector-borne Virus Research Center,Fujian Agriculture and Forestry University;College of Life Sciences,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)

机构地区：[1]福建农林大学植物保护学院 [2]福建农林大学媒介病毒研究中心 [3]福建农林大学生命科学学院,福建福州350002

出　　处：《福建农林大学学报（自然科学版）》2022年第1期27-32,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建省自然科学基金(2017J01600);国家自然科学基金(31900153).

摘　　要：茉莉H病毒(JaVH)是一种天竺葵黄斑病毒属病毒,可引起严重的茉莉病毒病害.该病毒基因组含有5个开放阅读框,分别编码2个复制相关蛋白、2个运动蛋白和1个外壳蛋白(CP),其中,CP可能是一个RNA沉默抑制子,并且与致病性密切相关.构建JaVH CP瞬时表达重组质粒pXT-CP,将其与pGDG混合浸润本生烟,浸润第5天利用紫外灯照射,观察到浸润区域仍具强烈荧光,表明CP是一个较强的沉默抑制子.为进一步鉴定CP的关键功能位点,将JaVH外壳蛋白与同属其他病毒进行比对后构建了18个pXT-CP突变体,利用农杆菌共浸润瞬时表达系统和Western blot明确了13个影响JaVH的CP功能的关键氨基酸位点,包括Trp 28、Arg 40、Arg 58、Gly 75、Ile 83、Thr 84、Val 108、Phe 111、Ser 115、Lys 123、Tyr 124、Arg132和Lys 200.Jasmine virus H(JaVH)is a member of Pelarspovirus,which can cause severe damage to jasmine plants.The genomic RNA of JaVH encodes 5 open reading frames,including 2 replication proteins,2 movement proteins and 1 coat protein(CP).To fully understand the role of the CP as RNA silencing suppressor which can be critical for the pathogenicity of JaVH,the CP of JaVH was cloned onto the pXT vector to construct the recombinant express vector pXT-CP which was subsequently co-infiltrated with pGDG onto Nicotiana benthamiana.Obvious green fluorescence patterns were observed in the co-infiltrated areas under long wave ultraviolet light 5 d post inoculation,which suggested that the JaVH CP was a strong RNA silencing suppressor.To further identify the key residues affecting RNA silencing suppressor activity of CP,18 mutants were constructed after aligning the CPs of all known Pelarspovirus for Agrobacterium-mediated transient expression and Western blotting.The results showed that 13 residues were critical to the suppressive activity,including Trp 28,Arg 40,Arg 58,Gly 75,Ile 83,Thr 84,Val 108,Phe 111,Ser 115,Lys 123,Tyr 124,Arg 132,Lys 200.

关 键 词：茉莉H病毒 外壳蛋白 RNA沉默抑制子 

分 类 号：S432.41[农业科学—植物病理学]