番木瓜环斑病毒西瓜株系弱毒突变体的筛选与应用

Screening and application of attenuated mutants of Papaya ringspot virus-watermelon strain

为防治番木瓜环斑病毒(Papaya ringspot virus,PRSV)在我国葫芦科作物上引起的病毒病害,以PRSV西瓜株系山东分离物(PRSV-SD)侵染性cDNA克隆为基础,采用定点突变方法将辅助成分-蛋白酶保守氨基酸137位和346位的天冬酰胺(N)和417位的缬氨酸(V)突变为丙氨酸(A),应用农杆菌浸润法接种西葫芦叶片并分析突变对PRSV-SD致病力的影响,筛选弱毒突变体,进而评价其交叉保护效果。结果表明,与野生型PRSV-SD相比,获得的3个突变体N137A、N346A和V417A,接种后在西葫芦植株上的症状明显减轻,衣壳蛋白在叶片中的积累水平分别为野生型PRSV-SD的24.0%、13.0%和4.0%,均为弱毒突变体。当保护间隔期为10 d时,弱毒突变体N137A具有完全的交叉保护效果,N346A可延迟发病15 d,而V417A无交叉保护效果。当间隔保护期为15 d时,弱毒突变体N137A和N346A的保护效率分别为100.0%和26.7%,而V417A无交叉保护效果。

To control the diseases caused by Papaya ringspot virus(PRSV)in cucurbit crops,the asparagine(N)at positions 137 and 346 and valine(V)at position 417 of the helper component-protease(HC-Pro)of a PRSV isolate of watermelon strain from Shandong Province(PRSV-SD)were mutated to alanine(A)in an infectious cDNA clone via site-directed mutagenesis.The virulence of the resultant mutants on Cucurbita pepo plants were analyzed by agroinfiltration,and the cross protection efficiency of attenuated mutants were evaluated.The results indicated that,compared with those of the wild type PRSV-SD,the symptoms of mutants N137 A,N346 A and V417 A on C.pepo plants were significantly attenuated,and their accumulative levels were significantly reduced to 24.0%,13.0%and 4.0%of the wild type virus,indicating that all of them were attenuated mutants of PRSV-SD.When the protective interval was ten days,the attenuated mutant N137 A provided complete cross protection effect,and N346 A delayed the appearance of viral symptom by 15 days,while V417 A showed no cross-protective effect.When the protective interval was 15 days,the protective efficiencies of attenuated mutants N137 A and N346 A on C.pepo plants were 100.0%and 26.7%,respectively,while V417 A had no cross-protective effect.