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【目的】克隆获得番茄 SYTA (Solanum lycopersicum SYTA，S.l SYTA),分析其基因序列生物信息学特征和预测蛋白的结构特征,明确S.l SYTA亚细胞定位和组织表达,并分析其在绿色荧光蛋白(green fluorescence protein,GFP)标记的烟草花叶病毒(Tobacco mosaic virus,TMV)侵染下的表达变化及其对TMV移动的影响,为明确S.l SYTA在植物病毒侵染致病过程中的作用提供理论依据。【方法】根据番茄基因组含有的SYTA同源基因序列,利用Primer Premier 5.0软件设计克隆引物,采用RT-PCR技术克隆S.l SYTA全长序列;应用生物信息学方法分析该基因的序列特征;使用MEGA 7.0对S.l SYTA蛋白序列及其同源序列进行多序列比对,并构建同源物种间系统进化树;通过与GFP蛋白融合进行亚细胞定位;利用实时荧光定量PCR(q RT-PCR)检测番茄各部位S.l SYTA的表达量以及在TMV胁迫的番茄中S.l SYTA的表达变化;构建植物瞬时表达载体p CV-SYTA-m GFP,通过农杆菌介导在本氏烟草中瞬时表达,TMV-GFP攻毒,利用酶联免疫吸附实验(ELISA)检测在本氏烟中瞬时表达S.l SYTA时TMV-GFP的积累和移动情况。【结果】克隆得到1 620 bp的S.l SYTA基因开放阅读框全长,序列比对及生物信息学分析表明,其编码的氨基酸序列具有SYTs家族的典型特征,含有N端的跨膜区、胞间连接区和C端的两个C2结构域;多序列对比及系统进化树分析发现,与茄科林生烟草、绒毛状烟草等植物亲缘关系较近,与黄瓜较远;亚细胞定位显示S.l SYTA定位于细胞质膜。在番茄的根、茎和叶中S.l SYTA的表达量从高到低依次为根>叶>茎;TMV-GFP侵染番茄导致其S.l SYTA表达量在接种后第1天显著上调,在第7天降至正常水平。在S.l SYTA瞬时表达的本氏烟叶片部位接种TMV-GFP,TMV-GFP在接种第5天时已经到达新叶,而接种部位仅表达空载体对照的本氏烟新叶中未观察到TMV-GFP,且接种第5天时TMV-GFP在接种叶和新叶中的积累量均明显高于其在空载体对照处理的叶片。【结论】获得的S.l SYTA具有SYTs家族的典型特征。S.l SYTA定位于细胞质膜,S.l SYTA在番茄根中表达量最高。在TMV-GFP胁迫下,番茄中S.l SYTA表达呈现先上升后下降至正常水平。在本氏烟中瞬时表达S.l SYTA有利于TMV-GFP侵染初期的积累和移动。
【Objective】 The objective of this study was to clone and identify the bioinformatics features of SYTA and SY SYSC in tomato. The structural and teature of S1 SYTA was identified, and its expression in green fluorescence protein (GFP) -induced tobacco mosaic virus (TMV) infection and its effect on the migration of TMV in order to provide a theoretical basis for clarifying the role of Sl-SYTA in the pathogenesis of plant virus infection . 【Method】 According to the sequence of SYTA homologous genes contained in tomato genome, the cloned primers were designed by Primer Premier 5.0 software and the full-length sequence of Sl SYTA was cloned by RT-PCR. The sequence characteristics of the gene were analyzed by bioinformatics method. Multiple sequence alignment of Sl SYTA protein sequence and its homology sequence was carried out to construct phylogenetic tree between homologous species. Subcellular localization was performed by fusion with GFP protein. Each tomato was detected by q RT-PCR The expression level of Sl SYTA and the expression of Sl SYTA in tomato under TMV stress. The plant transient expression vector p CV-SYTA-m GFP was constructed and transiently expressed in N. benthamiana via Agrobacterium tumefaciens, TMV-GFP challenge , The accumulation and migration of TMV-GFP were detected by enzyme-linked immunosorbent assay (ELISA) when transiently expressing Sl SYTA in N. benthamiana. 【Result】 The full-length open reading frame (ORF) of 1 620 bp Sl SYTA gene was cloned. Sequence alignment and bioinformatics analysis showed that the amino acid sequence encoded by SYTS gene was typical of the SYTs family. It contained N-terminal transmembrane region, And C-terminal two C2 domains. Multiple sequence alignment and phylogenetic tree analysis showed that the phylogenetic tree was more closely related to plants such as Lycopersiconidae and Nicotiana tabacum than cucumber, and the subcellular localization of Sl SYTA Plasma membrane. The expression level of Sl SYTA in roots, stems and leaves of tomato was root> leaf> stem from highest to lowest. Infection of tomato with TMV-GFP resulted in a significant up-regulation of S1 SYTA expression on the first day after inoculation, Days dropped to normal levels. TMV-GFP was inoculated on N. benthamiana leaf spot transiently expressed in Sl SYTA, TMV-GFP reached the new leaf on the 5th day after inoculation while no TMV-GFP was observed in the new leaf of N. benthamiana at the inoculation site only expressing the empty vector control , And the accumulated amount of TMV-GFP in inoculated leaves and new leaves on the 5th day of inoculation was significantly higher than that of the control on empty vector. 【Conclusion】 The obtained S.l SYTA has typical characteristics of SYTs family. S.l SYTA localized in the plasma membrane, S.l SYTA expression in tomato roots highest. Under TMV-GFP stress, the expression of S.l SYTA in tomato increased first and then decreased to normal level. Transient expression of S.l SYTA in N. benthamiana facilitates the accumulation and mobilization of TMV-GFP in the early stages of infection.