茉莉C病毒侵染性克隆的构建及CP基序分析

Infectious clone construction of jasmine virus C and motif analysis of CP

【目的】构建茉莉C病毒(JaVC)福建分离物基因组全长cDNA侵染性克隆,克隆9省JaVC分离物的CP基因并比较分析基序差异,调查JaVC在我国茉莉产区的分布和传播情况。【方法】提取JaVC检测呈阳性的茉莉叶片总RNA,以反转录后的cDNA为模板扩增获得JaVC基因组全长序列并构建全长cDNA克隆pXT-JaVC-FJ;同时构建了外壳蛋白(coat protein,CP)融合红色荧光蛋白mCherry的克隆(pXT-JaVC CP-mCherry)。利用农杆菌浸润法侵染本生烟,通过RT-PCR检测法和激光共聚焦扫描显微镜观察法验证JaVC侵染性。克隆其他8省JaVC分离物的3'末端包含CP的片段并测序分析CP基序差异。通过田间调查明确JaVC在茉莉上的发生情况和其传播介体。【结果】pXT-JaVC-FJ浸润本生烟可引起系统侵染,说明该克隆具有侵染活性。所有JaVC分离物的CP均编码296个氨基酸,JaVC中国台湾分离物的CP与各分离物核苷酸序列相似性为82.27%‒91.36%,与广东分离物相似性最高;氨基酸序列相似性为92.23%‒96.82%,与云南分离物相似性最高;各分离物CP的氨基酸序列在32‒35位点上差异显著,具有6种不同的氨基酸基序排列,分别为SEHA、GENA、REGT、SENA、GGDA和GGNA。田间调查显示,JaVC在中国茉莉植株上广泛分布且可在蓟马中检测到JaVC。【结论】成功构建了JaVC-FJ的侵染性克隆,这为该病毒的基因功能、致病机理等研究奠定了基础;通过我国茉莉产区JaVC的发生及变异情况分析,为JaVC引起的病毒病的防治提供了理论依据。

[Objective]The full-length cDNA infectious clone of jasmine virus C(JaVC),from Fujian Province was constructed.Coat protein(CP)genes of JavC isolates from 9 provinces/autonomous regions in China were cloned and the motif difference was analyzed.Thereby,the distribution and transmission of JaVC in jasmine producing areas in China were investigated.[Methods]Total RNA of jasmine leaves which tested positive for JaVC was extracted and then reverse-transcribed to cDNA.The cDNA was then used as template for amplification to obtain the full-length genome sequence of JaVC,followed by the construction of full-length cDNA clone pXT-JaVC-FJ and the CP-fused red fluorescent protein mCherry clone pXT-JavC CP-mCherry.Both of them were then transformed into Agrobacterium tumefaciens for Nicotiana benthamiana infiltration and verified through RT-PCR and confocal laser scanning electron microscopy.Samples collected from eight other provinces/autonomous regions of China positive for JaVC were then used for cloning and sequencing of the 3'end containing CP.Field investigation was conducted to identify the occurrence and transmission vector of JavC.[Results]pXT-JaVC-FJ induced systemic infection on N.benthamiana by agro-infiltration,which demonstrated that it was biologically active.CPs of all isolates encoded 296 amino acids.The CP of isolate from Taiwan of China shared 82.27%-91.36%nucleotide sequences and 92.23%-96.82%amino acid sequences with those of isolates from the 9 provinces/autonomous regions.It showed the highest similarity to that from Guangdong and Yunnan Province at nucleotide level and amino acid level,respectively.The CP from different isolates mainly showed difference at aa 32-35 and six motif arrangements of SEHA,GENA,REGT,SENA,GGDA,and GGNA were identified.Field investigation suggested that JavC was widely distributed on jasmine plants and could be detected in thrips.[Conclusion]An infectious clone of JaVC-FJ is developed,which lays a foundation for the study of gene function and pathogenic mechanism of JavC.The analysis of occurrence and variation of JavC in indifferent jasmine producing areas in China is helpful for the prevention and control of JavC diseases.