木尔坦棉花曲叶病毒“C4 ORF”编码蛋白对病毒致病性的影响  

Effects of Proteins Encoded by“C4 ORFs”of Cotton Leaf Curl Multan Virus on Viral Pathogenicity

作　　者：郑信诗 尚鹏祥 李景远 丁新伦[1] 吴祖建[1] 张洁[1] ZHENG XinShi;SHANG PengXiang;LI JingYuan;DING XinLun;WU ZuJian;ZHANG Jie(Institute of Plant Virology,College of Plant Protection,Fujian Agriculture and Forestry University/State Key Laboratory for Ecological Pest Control of Fujian and Taiwan Crops/Key Laboratory of Plant Virology of Fujian Province,Fuzhou 350002)

机构地区：[1]福建农林大学植物保护学院植物病毒研究所/闽台作物有害生物生态防控国家重点实验室/福建省植物病毒学重点实验室,福州350002

出　　处：《中国农业科学》2021年第10期2095-2104,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金面上项目(31672005);福建省自然科学基金面上项目(2019J01656);福建农林大学科技创新专项(CXZX2017324,CXZX2016132)。

摘　　要：【目的】通过分析发现木尔坦棉花曲叶病毒(cotton leaf curl Multan virus,CLCuMuV)C4开放阅读框(open reading frame,ORF)附近可编码3个不同大小的蛋白,而NCBI数据库登录的CLCuMuV各个分离物编码的C4蛋白大小也不尽相同,本研究试图通过分析该3个假定的“C4 ORF”编码蛋白对病毒致病性的影响,以明确CLCuMuV C4蛋白ORF的确切定位。【方法】根据双生病毒编码蛋白的保守性,在CLCuMuV C4 ORF附近查找到3个大小不等的ORF,分别标记为C4-L(567 nt)、C4-M(546 nt)和C4-S(303 nt)。利用同源重组的方法将该3个ORF分别构建到PVX异源表达载体,通过农杆菌介导的植物接种法,分析PVX介导的3个蛋白在本氏烟表达对本氏烟症状的影响。利用同源重组的方法构建CLCuMuV野生型及C4-L或C4-S缺失突变型的侵染性克隆,同样通过农杆菌介导的植物接种法,分别与其伴随DNA-β分子的侵染性克隆接种本氏烟,分析C4-L或C4-S缺失对CLCuMuV致病性的影响,并利用Southern blot和Western blot分别对接种烟草的病毒基因组的积累量和病毒C4蛋白的表达量进行分析。同时利用Gateway系统载体对C4-L和C4-S蛋白在本氏烟叶片表皮细胞中的亚细胞定位进行分析。【结果】PVX异源表达载体接种本氏烟,结果发现PVX-C4-L和PVX-C4-S接种的本氏烟叶片卷曲、叶柄伸长,并且PVX-C4-S症状更重,而PVX-C4-M接种的本氏烟症状较轻或基本无症状,Western blot试验也在PVX-C4-L和PVX-C4-S接种组检测到了C4蛋白的表达,说明C4-S ORF编码的蛋白对PVX异源病毒的致病性影响最大。侵染性克隆接种本氏烟,结果发现野生型(CLCuMuV)和C4-L突变型(CLCuMuV-ΔL)接种的本氏烟叶片皱缩增生、叶柄和茎秆扭曲,而C4-S突变型(CLCuMuV-ΔS)和对照组(Mock)接种的本氏烟未显示任何症状,并且前两者的植株高度明显矮于后两者,同时Southern blot试验在CLCuMuV和CLCuMuV-ΔL接种的本氏烟中均检测到大量病毒基因组的积累,【Objective】Cotton leaf curl Multan virus(CLCuMuV)was found to encode three proteins with different sizes near the open reading frame(ORF)of C4,and the size of C4 protein encoded by each isolate of CLCuMuV registered in NCBI database was not the same.The objective of this study is to analyze the effect of proteins encoded by three hypothesized“C4 ORFs”on viral pathogenicity,and to determine the exact location of C4 ORF of CLCuMuV.【Method】According to the conserved protein of geminiviruses,three ORFs with different sizes were found near C4 ORF of CLCuMuV,which were labeled as C4-L(567 nt),C4-M(546 nt)and C4-S(303 nt).The three ORFs were constructed into PVX heterologous expression vector by homologous recombination,respectively.Using an agroinoculation method,the effect of PVX mediated expression of the three proteins on the symptoms of Nicotiana benthamiana were analyzed.Using a homologous recombination method,the infectious clones of CLCuMuV wild type and C4-L or C4-S deletion mutant were constructed,and then together with infectious clone of the associated DNA-β,they were inoculated into N.benthamiana respectively by agroinoculation.The effect of C4-L or C4-S deletion on CLCuMuV pathogenicity was analyzed.Meanwhile,Southern blot and Western blot were used to analyze the accumulation of viral genome and the expression level of viral C4 protein.At the same time,the subcellular localization of C4-L and C4-S proteins in the epidermal cells of N.benthamiana leaves was analyzed by a Gateway system vector.【Result】In the PVX heterologous expression assay,the results showed that the leaf curling and petiole elongation were observed in PVX-C4-L and PVX-C4-S inoculated N.benthamiana plants,and the symptoms of PVX-C4-S inoculated plants were more severe,while those of PVX-C4-M inoculated plants were mild or almost asymptomatic.The expression of C4 protein was also detected by Western blot in PVX-C4-L and PVX-C4-S inoculated plants.The results revealed that the protein encoded by C4-S ORF had the greatest ef

关 键 词：木尔坦棉花曲叶病毒 C4开放阅读框 致病性 

分 类 号：S432.41[农业科学—植物病理学]