甘蔗黄叶病毒P0蛋白分子特性及其抑制RNA沉默活性  

Molecular Characterization and Analysis of Suppressing RNA Silence of P0 Protein Encoded by Sugarcane yellow leaf virus

作　　者：林艺华[1] 肖胜华[1] 刘营航 陈建生[1] 傅华英[1] 陈如凯[1] 高三基[1,2] 

机构地区：[1]福建农林大学农业部福建甘蔗生物学与遗传育种重点实验室,福州350002 [2]广西大学广西蔗糖产业协同创新中心,南宁530004

出　　处：《中国农业科学》2014年第23期4627-4636,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31170345);国家现代农业产业技术体系(CARS-20-2-4)

摘　　要：【目的】甘蔗黄叶病(yellow leaf,YL)是一种主要的甘蔗病毒病害,在全球多数甘蔗种植国家或地区普遍发生,已对甘蔗生产造成严重威胁。其病原为甘蔗黄叶病毒(Sugarcane yellow leaf virus,SCYLV),属于黄症病毒科马铃薯卷叶病毒属成员。研究SCYLV编码的沉默抑制子P0蛋白的分子特征、保守结构域及其在自然寄主甘蔗上抑制RNA沉默的功能,为下一步从RNA沉默水平解析SCYLV致病分子机理奠定基础。【方法】利用RT-PCR克隆技术获得SCYLV中国分离物CHN-FJ4编码的RNA沉默抑制子基因P0及其两个缺失突变体P0Δ2-15(N端缺失15个氨基酸)和P0Δ155-256(C端缺失102个氨基酸),然后定向克隆到单子叶植物表达载体p Ubi-nos(空载体)上,获得重组质粒。利用甘蔗嫩叶组织瞬时表达系统鉴定病毒RNA沉默抑制子技术,结合Image J软件定量分析P0及其两个缺失突变体抑制外源基因EYFP瞬时表达活性的变化。番茄丛矮病毒(Tomato bushy stunt virus,TBSV)编码的P19作为沉默抑制子阳性对照。【结果】P0核苷酸序列的遗传进化分析结果显示,所获得的SCYLV病毒分离物CHN-FJ4为BRA基因型,与其他BRA基因型分离物氨基酸序列一致性为96.1%—98.4%。利用MEME在线软件预测P0蛋白的保守结构域,结果表明P0蛋白含有3个显著的保守区。分别位于第1—60、76—125和161—210氨基酸残基。通过Datamonkey在线服务器(http://www.datamonkey.org)提供的5种运算方法分析P0蛋白氨基酸位点的选择压力,结果显示P0蛋白具有8个氨基酸正向选择位点。将含有P0及其缺失突变体的重组质粒连同含EYFP报告基因的p TEM12质粒采用基因枪轰击法分别导入甘蔗嫩叶组织细胞,轰击后48—120 h,P0和P19逐渐地提高EYFP荧光表达点数和荧光表达水平;在120 h,与p Ubi-nos空载体(不含沉默抑制子)对照组相比,P0和P19均显著地提高甘蔗嫩叶组织EYFP荧光表达点数和荧光表达水平,分别达1.6倍和4.0【Objective】Yellow leaf（YL） disease of sugarcane is one of viral diseases in major sugarcane-producing regions inChina and worldwide, causing a serious threat to sugarcane industry. Sugarcane yellow leaf virus（SCYLV）, a member of the genus Polerovirus and family Luteoviridae, is the causal agent of YL. The aim of this study is to characterize the conserved regions of P0 protein encoded by SCYLV, and to test its functionality as a RNA silencing suppressor in natural host sugarcane（Saccharum spp.hybrids）. The findings will contribute to further investigate the molecular mechanism of SCYLV pathogenesis at RNA silencing level.【Method】P0 and its two deletion mutants, P0Δ2-15（15-aa deletion in N terminal） and P0Δ155-256（102-aa deletion in C terminal） from SCYLV CHN-FJ4 isolate were obtained by RT-PCR and cloned into an expression vector under control of the maize ubiquitin 1promoter and Agrobacterium nopaline synthase terminator. Each of P0 or its deletion mutant constructs were co-bombarded with the reporter gene coding for enhance yellow fluorescent protein（EYFP） into sugarcane young leaf segments, and transient EYFP expression was quantified using Image J software. The Tomato bushy stunt virus-encoded P19 was included as a model suppressor.【 Result 】 Phylogenetic analysis revealed that the CHN-FJ4 isolate clustered with isolates of the BRA genotype and shared96.1%-98.4% aa identified with them. MEME software online revealed that the P0 proteins contained three significant conserved regions positioned at 1-60, 76-125, and 161-210 aa residues. Besides, eight positive sites were detected using five different approaches under the datamonkey web-server. P0, P0Δ2-15 and P0Δ155-256 were co-bombarded with EYFP into sugarcane young leaf segments, respectively. In young leaf segments co-expressing a suppressor P0 or P19, EYFP foci account and EYFP expression level were increased at 48-120 h post-DNA introduction compared with that in the absence of a suppressor. At 120 h post-bombardment

关 键 词：甘蔗黄叶病毒 P0蛋白 RNA沉默抑制子 黄色荧光蛋白 

分 类 号：S435.661[农业科学—农业昆虫与害虫防治]