番茄黄化曲叶病毒北京分离物侵染性克隆的构建与其致病性

Construction and pathogenicity tests of an infectious clone of Beijing isolate of tomato yellow leaf curl virus

为明确番茄黄化曲叶病毒北京分离物(Beijing isolate of tomato yellow leaf curl virus,TYLCV-BJ)致病性的强弱,以感染TYLCV-BJ的番茄叶片DNA为模板PCR扩增获得该分离物基因组全长序列,并构建该分离物的侵染性克隆,将其分别接种到番茄、烟草和拟南芥植株上,比较该分离物和TYLCV上海分离物2(TYLCV-Shanghai 2,TYLCV-SH2)致病性的差异。结果显示,该分离物基因组全长序列同TYLCV-SH2的相似度为99.03%,在番茄和烟草植株上TYLCV-BJ比TYLCV-SH2发病更早,症状更重,TYLCV DNA和外壳蛋白积累量更高。TYLCV-BJ可以通过农杆菌Agrobacterium tumefaciens注射法在拟南芥中复制和系统侵染,而TYLCV-SH2不能有效侵染拟南芥。表明TYLCV-BJ的致病性强于TYLCV-SH2,所建立的侵染性克隆有广泛的研究和应用价值。

In order to determine the pathogenicity of Beijing isolate of tomato yellow leaf curl virus TYLCV-BJ,the whole genome sequence of TYLCV-BJ was obtained by sequencing the PCR products amplified from TYLCV-BJ-infecting tomato leaves.The genomic DNA of TYLCV-BJ was used as a template to construct its infectious clone.The pathogenicity of TYLCV-BJ isolate was compared with TYLCV-Shanghai 2 isolate(TYLCV-SH2)by inoculating the two infectious clones on tomato,Nicotiana benthamiana and Arabidopsis thaliana plants.The results showed that TYLCV-BJ isolate shared 99.03%nucleotide sequence similarity to TYLCV-SH2 isolate.Plants inoculated with TYLCV-BJ developed much earlier and more severe viral symptoms,and accumulated higher levels of viral DNA and coat protein than plants infected by TYLCV-SH2 on tomato and N.benthamiana plants.Furthermore,TYLCV-BJ could replicate and infect A.thaliana plants systematically by Agrobacterium-mediated infiltration,while TYLCV-SH2 fails to infect A.thaliana plants.Therefore,TYLCV-BJ was more virulent than TYLCV-SH2,thus can be used in more valuable and extensive study.