蒜芥茄SsFLS2基因的克隆及其表达分析  

Cloning and expression analysis of SsFLS2 gene in Solanum sisymbriifolium Lam

作　　者：孙茂 吴丽艳[1] 龚亚菊[1] 鲍锐[1] 桂敏[1] 黎志彬[1] 杜光辉 SUN Mao;WU Li-yan;GONG Ya-ju;BAO Rui;GUI Min;LI Zhi-bin;DU Guang-hui(Horticultural Research Institute,Yunnan Academy of Agricultural Sciences,Kunming 650205,China;School of Agriculture,Yunnan U-niversity,Kunming 650500,China)

机构地区：[1]云南省农业科学院园艺作物研究所,昆明650205 [2]云南大学农学院,昆明650500

出　　处：《西南农业学报》2024年第8期1669-1676,共8页Southwest China Journal of Agricultural Sciences

基　　金：云南省重大科技计划专项(202302AE090006);云南省农业基础研究联合专项面上项目(202301BD070001-052);国家自然科学基金项目(31960594);云南大学研究生科研创新基金项目(ZC-23234029)。

摘　　要：【目的】克隆云南野生蒜芥茄(Solanum sisymbriifolium Lam.)中的FLS2基因,并对其编码蛋白的理化性质、亚细胞定位、系统进化以及表达情况予以分析,初步探究野茄FLS2基因在黄萎病胁迫下的生物学功能。【方法】基于前期所测转录组数据(蒜芥茄接种黄萎病病原菌),克隆获取蒜芥茄FLS2基因,命名为SsFLS2;利用生物信息学分析软件对SsFLS2基因的理化性质进行分析,并通过实时荧光定量PCR(Real-time fluorescence quantitative PCR,RT-qPCR)检测其在蒜芥茄根、茎、叶的表达以及在接种黄萎病病原菌后不同时间的表达情况。【结果】蒜芥茄SsFLS2基因全长3655 bp,具有完整的ORF框,编码1126个氨基酸。其编码蛋白的分子式为C_(5596)H_(8814)N_(1494)O_(1627)S_(4),理论分子量为124.34 kD,理论等电点(pI)为7.33,总平均亲水性系数为0.081。该蛋白二级结构主要由42.81%的无规则卷曲、40.23%的α-螺旋、13.23%的延伸链以及3.73%的β-折叠组成,且存在跨膜结构,定位于细胞膜上,其中可被磷酸化且超过阈值线的位点,共计151个。SsFLS2蛋白的氨基酸序列与马铃薯(Solanum tuberosum)同源蛋白(XP 006358149.2)的关系最近。RT-qPCR检测发现,在蒜芥茄根、茎、叶中均有SsFLS2基因的表达,且根、叶中的相对表达量极显著高于茎部;接种黄萎病病原菌后72 h内,处理组和对照组均在24 h时,SsFLS2基因的相对表达量大幅度增加且极显著高于其他时间点。【结论】本研究成功克隆蒜芥茄的SsFLS2基因,并对其编码蛋白的理化性质及基因表达情况等进行分析。结果表明,SsFLS2是蒜芥茄响应黄萎病胁迫的重要基因,结果可为进一步研究该基因在蒜芥茄黄萎病抗性中的功能奠定基础。【Objective】The study aimed to clone the FLS2 gene from wild eggplant species(Solanum sisymbriifolium Lam.)in Yunnan prov⁃ince,and to analyze the physicochemical properties,subcellular localization and phylogenetic evolution of its encoded protein,as well as the expression,in order to preliminarily explore the biological functions of the wild eggplant FLS2 gene under the stress of verticillium wilt.【Method】In the study,the FLS2 gene,named SsFLS2,was cloned based on the transcriptome data(S.sisymbriifolium inoculation with verti⁃cillium pathogens).The physical and chemical properties of SsFLS2 gene were analyzed by using bioinformatic analysis software,and real⁃time fluorescence quantitative PCR(RT⁃qPCR)was used to detect the expression of S.sisymbriifolium roots,stems and leaves,as well as the expression at different times after inoculation of verticillium pathogens.【Result】The SsFLS2 gene was 3655 bp in length,with a complete open reading frame(ORF),and encoded 1126 amino acids.The molecular formula of the encoded protein was C5596 H8814 N1494 O1627 S4,the theoretical molecular weight was 124.34 kD,the theoretical isoelectric point(pI)was 7.33,and the total mean hydrophilicity coefficient was 0.081.The secondary structure of SsFLS2 was mainly composed ofα⁃helix(40.23%),random coil(42.81%),β⁃folding(3.73%),and extended chain(13.23%),and there was a transmembrane structure,localized on the cell membrane,in which the sites could be phosphoryl⁃ated and exceeded the threshold line,a total of 151.The results of homologous sequence alignment and phylogenetic evolutionary analysis showed that the amino acid sequence of SsFLS2 protein was most closely related to the potato(Solanum tuberosum)homologous protein(XP 006358149.2).RT⁃qPCR assay indicated that SsFLS2 gene was expressed in roots,stems,and leaves of S.sisymbriifolium,and the relative expression level of SsFLS2 gene was highly significant higher in roots and leaves than in stems.The relative expression level of SsFLS2 gene was substant

关 键 词：蒜芥茄 Flagellin sensing 2(FLS2) 基因克隆 生物信息学 基因表达 

分 类 号：S641.1[农业科学—蔬菜学]