云南省昆明地区番茄斑萎病毒核衣壳蛋白和运动蛋白变异分析

为了明确对番茄斑萎病毒(tomato spotted wilt virus,TSWV)免疫的番茄YNAU335自交系表现出TSWV感病症状(抗性被打破)的原因,在排除YNAU335自交系不纯和或混杂其它感病番茄材料的因素外,选取96172I(感病)和YNAU335(抗性被打破)自交系感病植株,进行TSWV核衣壳蛋白(nucleocapsid protein,NP)和运动蛋白(movement protein,MP)基因的RT-PCR检测,并对阳性克隆进行基因测序分析。结果表明:(1)2个自交系中均克隆出TSWV的NP和MP基因,共获得5条NP(登录号:MK628735～MK628739)和3条MP(登录号:MK883723、MK883724和MK887284)多态性基因序列。(2)96172I感病材料中克隆出以上所有基因序列,YNAU335感病材料中克隆出其中的3条NP和1条MP基因序列。(3)对YNAU335中TSWV特有的NP和MP氨基酸突变位点进行分析,结果发现4个特异突变位点可能与打破YNAU335抗性有关,4个突变位点分别为:NP 18位氨基酸G突变为V,36位T突变为I,39位L突变为R,MP 274位E突变为K。(4)系统发育分析显示,5条NP和3条MP与云南省已登记的NP和MP聚类在不同的分支,表明云南省昆明地区TSWV存在丰富的遗传多样性。

Variation Analysis of Nucleocapsid Protein and Movement Protein of Tomato Spotted Wilt Virus around Kunming Area, Yunnan Province

The tomato inbred line YNAU335, previously behaved immune to tomato spotted wilf virus (TSWV), exhibited with TSWV symptoms which indicated that the resistance of YNAU335 to TSWV had been overcome. To clarify the resistance breaking determinant, we firstly identified both the infected plants of susceptible 96172I and resistance breaking YNAU335 inbred lines with RT PCR method to clone the nucleocapsid protein (NP) and movement protein (MP) genes, and the target genes of positive clones were further analyzed by sequencing. The results were shown as follows. (1) The NP and MP genes of TSWV existed in the two inbred lines, and five NP (Accession No. MK628735 MK628739) and three MP (Accession No. MK883723, MK883724 and MK887284) polymorphic gene sequences were also gained. (2) All the gene sequences mentioned were cloned from 96172I infected material, whereas, YNAU335 infected material possessed the three NP and one MP of the above gene sequences. (3) Amino acid mutations in the NP and MP of YNAU335 inbred line were more analyzed. Four specific mutations might be related to the resistance breaking event of YNAU335 inbred line: substitution G to V at position 18(G18V), T36I and L39R in the NP, and E274K in the MP. (4) Phylogenetic analysis revealed that the five NP and three MP, and the registered NP and MP from Yunnan province were clustered in different branches, which indicated the presence of abundant genetic diversity of TSWV in Yunnan province.