番茄SYTA的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2017.15.008

摘要/Abstract

摘要： 【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（qRT-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pCV-SYTA-mGFP，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。

关键词: 番茄, S.l SYTA, 克隆, 表达分析, TMV-GFP

Abstract: 【Objective】 The objective of this study is to clone Solanumlycopersicum SYTA (S.l SYTA), to analyze its bioinformatic characteristics, tissue expression and subcellular localization, the effect of green fluorescence protein (GFP) labeled Tobacco mosaic virus (TMV) on the expression level of S.l SYTA, its effect on the infection and movement of TMV and to provide a theoretical basis for further investigation of the S.l SYTA influence on the process of the infection and movement of the plant viruses. 【Method】The primer pairs of S.l SYTA were designed using Primer Premier 5.0 software based on the sequence of homologous SYTA in the genome ofthe S. lycopersicum. A complete coding sequence of S.l SYTAwas isolated and obtained by RT-PCR method. The sequence characteristics ofS.l SYTA was analyzed by bioinformatics tools. Multiple sequence alignments between S.l SYTAand its homologous ones from other species and a phylogenetic tree of homologous species were made by MEGA 7.0. The subcellular localization of S.l SYTA was analyzed through fusing with GFP protein. The real-time fluorescent quantitative PCR (qRT-PCR) was used to analyze the expression of S.l SYTAin different tissues and the change of its expression level in the TMV-GFP infected S. lycopersicum. The S.l SYTA was transiently expressed in the Nicotiana benthamiana by agroinfiltration, and then ELISA was used to detect the accumulation and movement of TMV-GFP. 【Result】A full-length SYTA of 1 620 bp was cloned from S. lycopersicum. Sequence alignment and bioinformatics analysis revealed that the deduced amino acids ofS.l SYTA had common characteristics of SYTs family with an N-terminal transmembrane region, a linker of variable size, and two C-terminal C2 domains. Furthermore, SYTA has the closest phylogenetic relationship to that of the N. sylvestris and N. tomentosiformis in amino acids sequences, and distanced from that of Cucumis sativus. Subcellular localization results showed that S.l SYTA was distributed at plasma membrane. The expression level of S.l SYTAin different tissues of S. lycopersicum was root＞leaf＞stem. The expression of S.l SYTAin S. lycopersicum obviously increased on the 1st day and decreased to the normal level on the 7th day after inoculation of TMV-GFP.Following the transient expression of S.l SYTA in N. benthamiana, TMV-GFP was inoculated. After 5 days, TMV-GFP had moved to the new leaf of the N. benthamiana. But no TMV-GFP was found in the new leaf of the control N. benthamiana in which the empty vectors were transiently expressed. ELISA assay results showed that the accumulation of the TMV-GFP in the samples under treatment was obviously higher than that in the control group after 5 days of TMV-GFP inoculation.【Conclusion】S.l SYTAhas the typicalcharacteristics of the SYTs family. S.l SYTAlocated on the plasma membrane of epidermal cell of N. benthamiana. The S.l SYTA expressed highest in root of S. lycopersicum. Under the stress of TMV-GFP, the expression of S.l SYTA in S. lycopersicum obviously increased firstly and then decreased to the normal level. In the S.l SYTA transient expression of N. benthamiana, the S.l SYTA could promote the initial accumulation and movement of TMV-GFP.

Key words: Solanum lycopersicum, S.l SYTA, gene cloning, expression and analysis, TMV-GFP

潘琪，刘旭旭，彭浩然，蒲运丹，张永至，叶思涵，吴根土，青玲，孙现超. 番茄SYTA的克隆及表达分析[J]. 中国农业科学, 2017, 50(15): 2936-2945.

PAN Qi, LIU XuXu, PENG HaoRan, PU YunDan, ZHANG YongZhi, YE SiHan, WU GenTu, QING Ling, SUN XianChao. Cloning, Expression Analysis of Solanum lycopersicum SYTA[J]. Scientia Agricultura Sinica, 2017, 50(15): 2936-2945.

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