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采集安徽马鞍山水稻条纹叶枯病感病稻株,TRIzol法提取样本总RNA,设计特异性引物进行RT-PCR扩增,获得水稻条纹病毒(RSV)安徽分离物的RNA3部分片段,克隆并测序。序列分析表明,该片段全长874 nts,含有完整的NS3基因,NS3基因序列长度为636 nts,编码211个氨基酸。序列比对发现,RSV安徽分离物NS3基因与来源于华东各省市RSV分离物NS3基因间的序列相似性高达97.6%～99.4%,而与RSV云南分离物NS3基因间的序列相似性相对较低,为93.4%～95.4%。构建RSV NS3基因系统关系树,发现RSV安徽分离物NS3基因与来源于华东各省市8个RSV分离物NS3基因聚成一个单独的分支,说明华东各省市各个RSV分离物亲缘关系最近。将RSV安徽分离物NS3基因插入植物表达载体pBIN438,构建重组植物表达载体pBIN-NS3并转化农杆菌。将含pBIN-NS3的农杆菌和含pBIN-GFP的农杆菌共浸润16c本氏烟叶片,6天后紫外灯下观察发现,共浸润区表现出强烈的绿色荧光,说明16c本氏烟GFP基因局部沉默被抑制,可以初步证明RSV安徽分离物编码的NS3蛋白是一个RNA沉默抑制子。
Total RNA was extracted by TRIzol method. Specific primers were designed and amplified by RT-PCR. The RNA3 fragment of Anhui isolates from rice stripe virus (RSV) was obtained, cloned and sequenced. Sequence analysis showed that the fragment was 874 nts in length and contained the complete NS3 gene. The length of the NS3 gene was 636 nts, encoding 211 amino acids. Sequence alignment revealed that the sequence similarity between NS3 gene of RSV Anhui isolate and NS3 gene from RSV isolates in East China was as high as 97.6% -99.4%, while the sequence similarity with RSV Yunnan isolate NS3 gene was relatively low , 93.4% ~ 95.4%. The phylogenetic tree of RSV NS3 gene was constructed. It was found that the RSV Anhui isolate NS3 gene was clustered into a single branch with 8 NSV isolates from all provinces and municipalities in East China, indicating that the RSV isolates from provinces and municipalities in East China had the closest genetic relationship. The RSV Anhui isolate NS3 gene was inserted into the plant expression vector pBIN438, and the recombinant plant expression vector pBIN-NS3 was constructed and transformed into Agrobacterium. The Agrobacterium containing pBIN-NS3 and Agrobacterium containing pBIN-GFP were co-infiltrated with 16c N. benthamiana leaves. After six days, the co-infiltration area showed strong green fluorescence, indicating that the local cv. G gene of 16c N. benthamiana Silencing was inhibited, and it was tentatively demonstrated that NS3 protein encoded by RSV Anhui isolate is an RNA silencing inhibitor.