受青枯菌诱导表达的马铃薯转录因子类StWRKY8的克隆与表达分析  

Cloning and Expression Analysis of the Potato Transcription Factor St WRKY8 Like Gene Induced by Ralstonia solanacearum

作　　者：薛珍[1] 李卉[1] 孔超越 段婷婷[1] 郜刚[1] 

机构地区：[1]山西师范大学生命科学学院,山西临汾041004

出　　处：《中国农业科学》2015年第21期4219-4226,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31271774);山西省自然科学基金(2012011032-2)

摘　　要：[目的]从接种青枯菌(Ralstonia solanacearum)的马铃薯中薯3号(Solanum tuberosum)中克隆类St WRKY8部分序列,分析其序列结构特征,研究该基因在感、抗病基因型马铃薯中受诱导的差异表达模式及其不同的组织表达定位。[方法]分别培养抗病基因型马铃薯ED13、感病基因型中薯3号至9—10叶期,以伤根浸菌法接种青枯菌菌株PO41。提取叶片总RNA,反转录为cDNA,使用PCR筛选cDNA差减试剂盒构建消减文库,筛选出384个阳性克隆作为原始SMART cDNA文库,采用5'-RACE法根据SMART-RACE cDNA扩增试剂盒说明克隆全长类St WRKY8。并分别应用生物信息学软件Bio Edit、BLAST、MEGA 5.0分析类St WRKY8的基因序列、序列相似性比对以及建立系统进化树。利用Prot Param、Prot Scale、SWISS-MODEL、Net Phos2.0 Server、WOLF PSORT、Target P1.1Server等在线服务器预测类St WRKY8的理化特性、三级结构、磷酸化位点以及亚细胞定位。提取马铃薯ED13、中薯3号接种青枯菌后的叶片总RNA,采样时间点分别为处理后6 h、12 h、1 d、2 d、3 d、4 d和6 d,运用反转录PCR和荧光定量PCR检测类St WRKY8的表达情况。以类St WRKY特异PCR产物制备地高辛标记探针,取材料茎、叶组织制作石蜡切片,并与标记探针进行原位杂交,定位其组织表达。[结果]获得了类St WRKY8 cDNA部分序列,长563 bp,包括一个完整开放阅读框258 bp,编码85个氨基酸。类St WRKY8属于第二类WRKY,锌指结构类型为C2H2,具有一个典型的WRKY保守结构域。其氨基酸序列与茄科(Solanaceae)其他成员具有高度同源性,与马铃薯转录因子St WRKY8相似性高达98%。预测类St WRKY8等电点约为9.1,半衰期大于5.5 h,为水溶性蛋白,其三维结构为非球状分子,含有3个磷酸化位点,亚细胞定位于细胞内。类St WRKY8受青枯菌诱导后表达上调,且在感、抗病基因型马铃薯中表达有差异。类St WRKY8在感病基因型马铃薯接种青枯菌6 h内表达量较低,[Objective]Clone the partial cDNA of the St WRKY8-like gene from a cultivated Solanum tuberosum Zhongshu 3 after inoculation with Ralstonia solanacearum.Analyze the St WRKY8-like gene coding sequence,and study the differential expression pattern of St WRKY8-like between susceptible and resistant S.tuberosum toward R.solanacearum and the tissue-specific expression of St WRKY8-like.[Method]R.solanacearum-resistant ED13 and R.solanacearum-susceptible Zhongshu 3 S.tuberosum were root-inoculated with R.solanacearum strain PO41.RNA were extracted from the leaves and reserve-transcribed into cDNA which was subjected for the construction of subtractive cDNA bank with the PCR Select cDNA Subtraction Kit.384 positive clones were obtained as SMART cDNA bank and used as a template for 5′-RACE with SMART-RACE cDNA Amplification Kit to PCR amplify the St WRKY8-like gene.The St WRKY8-like gene sequence and DNA sequence similarity were analyzed by Bio Edit and BLAST tools.Phylogenetic trees were established by MEGA 5.0.The biochemical feature,tertiary structure,phosphorylation sites and sub-cellular location of the St WRKY8-like protein were predicted by Prot Param/Prot Scale,SWISS-MODEL,Net Phos2.0 Server,WOLF PSORT/Target P1.1 Server,respectively.The RNA were extracted from the leaves of ED13 and Zhongshu 3 S.tuberosum at 6 h,12 h,1 d,2 d,3 d,4 d,and 6 d post inoculation of the R.solanacearum strain PO41 for RT-PCR and real-time PCR analysis of the St WRKY8-like gene expression.Digoxin-labeled St WRKY8-like specific probes were in situ hybridized with S.tuberosum stem and leaf sections to determine its expression in different tissues.[Result] The partial cDNA of the St WRKY8-like gene（563 bp） was obtained,which contained a 258 bp open reading frame coding a peptide with 85 amino acids.The St WRKY8-like protein has a classical conserved WRKY domain with a zinc finger motif of C2H2 and belongs to the subgroup II of WRKY family.The amino acid sequence of St WRKY8-like protein was highly close to the other WRKY members of So

关 键 词：马铃薯 青枯菌 转录因子 类StWRKY8 基因表达 组织定位 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]