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水稻条纹病毒安徽分离物NS3基因的克隆 及其RNA沉默抑制子功能的初步鉴定
邓竹根,丁菲,贾琳,宋培培,江彤
0
(安徽农业大学植物保护学院,合肥 230036;德州经济开发区东艺园林有限公司,德州 253000;山东省平度市农业局,平度 266700)
摘要:
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。
关键词:  水稻条纹病毒  NS3基因  克隆  序列分析  RNA沉默抑制子
DOI:
基金项目:安徽省教育厅自然科学基金重点项目(KJ2012A113, KJ2011A120)和安徽省自然科学基金(11040606M68)共同资助。
Cloning and preliminary identification of RNA silencing suppressor of gene NS3 of Rice stripe virus Anhui isolate
DENG Zhu-gen,DING Fei,JIA Lin,SONG Pei-pei,JIANG Tong
(School of Plant Protection, Anhui Agricultural University, Hefei 230036;Dongyi Landscape Co. Ltd. of Dezhou Economic Development Zone, Dezhou 253000;Pingdu Agricultural Bureau of Shandong, Pingdu 266700)
Abstract:
Rice plants showing stripe disease were collected from Ma-Anshan, Anhui province. The total RNA was extracted from samples of rice plants by TRIzol method. A pair of specific primers was designed to amplify RNA3 partial fragment of Rice stripe virus Anhui isolate by RT-PCR, and then the fragment was cloned and sequenced. Sequence analysis results showed that the fragment was consisted of 874 nts, including 636 nts of full-length gene NS3 encoding 211 amino acids. Sequence comparison showed that gene NS3 of RSV Anhui isolate shared the high sequence similarity (97.6%-99.4%) with that of RSV isolates from East China, while had relatively lower nucleotide sequence similarity (93.4%-95.4%) to that of Yunnan isolates. A phylogenetic tree based on alignment of nucleotide sequences of genes NS3 of RSV was constructed. It was found that gene NS3 of RSV Anhui isolate and those of eight RSV isolates from East China clustered into a separate branch. The results indicated that RSV isolates from East China had the closest relationship. Gene NS3 of RSV Anhui isolate was inserted into plant expression vector pBIN438, and the recombinant plant expression vector pBIN-NS3 was constructed and transformed into Agrobacterium tumefaciens. The leaves of Nicotiana benthamiana line 16c plants were co-infiltrated with A. tumefaciens mixtures carrying pBIN-NS3 and pBIN-GFP. Intense green fluorescence could be observed in the co-infiltrated patches under the UV light 6 days later. It was illustrated that the local silencing of gene GFP was inhibited, and it could be preliminarily demonstrated that protein NS3 encoded by RSV Anhui isolate was a RNA silencing suppressor.
Key words:  Rice stripe virus  gene NS3  clone  sequence analysis  RNA silencing suppressor

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