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本研究对江苏、浙江、湖南和北京等四个地区的葫芦、西瓜、黄瓜、西葫芦和甜瓜上的黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)129kD和57kD蛋白复制酶基因分别进行扩增、测序和序列分析。结果显示,供试CGMMV分离物CGMMV-No.1、CGMMV-No.2、CGMMV-No.3、CGMMV-No.4和CGMMVNo.5的129kD和57kD蛋白复制酶基因序列相似性分别为99.64%和99.74%,其中CGMMV-No.1、CGMMVNo.3和CGMMV-No.4的相似性较高,三者的129kD和57kD蛋白复制酶基因序列相似性分别为99.95%和99.94%,而CGMMV-No.2的129kD和57kD蛋白复制酶基因序列与其余四个参比序列的相似性相对较低,分别为99.16%~99.27%和99.04%~99.18%;供试样品在基于129kD和57kD蛋白复制酶基因序列构建的NJ系统发育树中均被分为6个类群,CGMMV-No.1、CGMMV-No.3、CGMMV-No.4与GenBank上已报道的中国山东分离物(Accession No.KJ754195)聚为一支,CGMMV-No.5与中国辽宁分离物(Accession No.EF611826)聚为一支,而浙江的CGMMV-No.2西瓜分离物在129kD蛋白复制酶基因系统发育树中与韩国西瓜分离物(Accession No.AF417242)聚为一支,其在57kD蛋白复制酶基因系统发育树中独立成群。本研究中供试CGMMV分离物的129kD和57kD蛋白均无显著疏水性,也无高度卷曲螺旋部位,ProtParam预测显示仅CGMMV-No.4的129kD蛋白为稳定蛋白,其余均为不稳定蛋白,CGMMV-No.1、CGMMV-No.2、CGMMV-No.3和CGMMV-No.5的129kD蛋白分别有6个、6个、2个和4个可能的跨膜结构区域,CGMMV-No.2、CGMMV-No.4和CGMMV-No.5的57kD蛋白分别有13个、13个和5个可能的跨膜结构域,供试样品129kD蛋白的糖基化位点分别有2个、4个、4个、4个和4个,57kD蛋白的糖基化位点分别有2个、5个、2个、5个和2个,其129kD和57kD蛋白的紊乱区、球蛋白区、磷酸化位点以及B细胞抗原表位位点均存在差异。综合分析认为,供试CGMMV分离物复制酶基因序列相似性较高,种内稳定且保守,种间差异明显;CGMMVNo.1、CGMMV-No.3、CGMMV-No.4和CGMMV-No.5与中国山东、辽宁分离物的亲缘关系较近,可能具有相同的侵染来源,而CGMMV-No.2与韩国分离物的亲缘关系较近,序列相似性高的供试样品在系统发育树中聚为一类;供试CGMMV分离物129kD和57kD蛋白生物信息学分析结果不具规律性。
In this study, the cucumber green mottle mosaic virus (CGMMV) 129 kD and 57 kD protein replicase genes were isolated from cucurbits, watermelons, cucumbers, zucchini and melons in four areas of Jiangsu, Zhejiang, Hunan and Beijing Amplification, sequencing and sequence analysis. The results showed that the similarities of 129kD and 57kD protein replicase gene sequences of CGMMV-No.1, CGMMV-No.2, CGMMV-No.3, CGMMV-No.4 and CGMMVNo.5 were 99.64% And 99.74% respectively. The similarity between CGMMV-No.1, CGMMVNo.3 and CGMMV-No.4 was high. The similarity of the 129kD and 57kD protein replicase gene sequences of the three genes were 99.95% and 99.94% The similarity of the 129kD and 57kD protein replicase genes of No.2 to the other four reference sequences was relatively low, ranging from 99.16% to 99.27% and 99.04% to 99.18%, respectively. The test samples were duplicated based on the 129kD and 57kD protein copies The NJ phylogenetic tree constructed by the sequence of the gene was divided into 6 groups. CGMMV-No.1, CGMMV-No.3 and CGMMV-No.4 shared the same sequence with the reported Shandong isolate (Accession No. KJ754195 ), CGMMV-No.5 was clustered with Accession No. EF611826, whereas CGMMV-No.2 watermelon isolate from Zhejiang Province was found in the phylogenetic tree of 129kD protein replicase gene in China Watermelon isolate (Accession No. AF417242) is clustered together in a phylogenetic tree independently of the 57 kD protein replicase gene. The 129kD and 57kD proteins of CGMMV isolates tested in this study showed no significant hydrophobicity nor highly coiled-coil sites. ProtParam predicts that only the 129kD protein of CGMMV-No.4 is a stable protein and the rest are unstable proteins. CGMMV There are six, six, two and four possible transmembrane structural regions of CGMMV-No.2, CGMMV-No.2, CGMMV-No.3 and CGMMV-No.5 129kD protein respectively There were 13, 13 and 5 possible transmembrane domains in CGMMV-No.4 and CGMMV-No.5, respectively. There were 2 glycosylation sites for 129kD protein in the sample, 4 , 4, 4 and 4, 57kD protein glycosylation sites were 2, 5, 2, 5 and 2, the 129kD and 57kD protein disorders, globulin, phosphorylation Site and B cell epitope epitopes are different. According to the comprehensive analysis, the CGMMV isolates showed high similarity in replicase gene sequences, which was stable and conservative within the species, with significant differences among species. CGMMVNo.1, CGMMV-No.3, CGMMV-No.4 and CGMMV-No.5 And China, Shandong, Liaoning isolates closer genetic relationship may have the same source of infection, and CGMMV-No.2 kinship closer to the South Korea isolates, the sequence similarity of the test samples in the phylogenetic tree The results of bioinformatics analysis of 129kD and 57kD CGMMV isolates showed irregularity.