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

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。

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

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.