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

doi:10.3864/j.issn.0578-1752.2015.21.003

摘要/Abstract

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StWRKY8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StWRKY8。并分别应用生物信息学软件BioEdit、BLAST、MEGA 5.0分析类StWRKY8的基因序列、序列相似性比对以及建立系统进化树。利用ProtParam、ProtScale、SWISS-MODEL、NetPhos2.0 Server、WOLF PSORT、TargetP1.1 Server等在线服务器预测类StWRKY8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StWRKY8的表达情况。以类StWRKY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StWRKY8 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StWRKY8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StWRKY8相似性高达98%。预测类StWRKY8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StWRKY8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StWRKY8在感病基因型马铃薯接种青枯菌6 h内表达量较低，而在抗病基因型马铃薯接种青枯菌6 h内高强度表达。该基因主要在茎叶维管束系统的韧皮部表达，具有组织特异性。【结论】类StWRKY8具有转录因子的一般结构特征，受青枯菌等病菌的诱导表达，同时受寄主植物固有抗性的影响，在感、抗病基因型中表达模式不同。推测其在植物防御反应中发挥作用并参与机体调控。

关键词: 马铃薯, 青枯菌, 转录因子, 类StWRKY8, 基因表达, 组织定位

Abstract: 【Objective】Clone the partial cDNA of the StWRKY8-like gene from a cultivated Solanum tuberosum Zhongshu 3 after inoculation with Ralstonia solanacearum. Analyze the StWRKY8-like gene coding sequence, and study the differential expression pattern of StWRKY8-like between susceptible and resistant S. tuberosum toward R. solanacearum and the tissue-specific expression of StWRKY8-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 StWRKY8-like gene. The StWRKY8-like gene sequence and DNA sequence similarity were analyzed by BioEdit and BLAST tools. Phylogenetic trees were established by MEGA 5.0. The biochemical feature, tertiary structure, phosphorylation sites and sub-cellular location of the StWRKY8-like protein were predicted by ProtParam/ProtScale, SWISS-MODEL, NetPhos2.0 Server, WOLF PSORT/TargetP1.1 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 StWRKY8-like gene expression. Digoxin-labeled StWRKY8-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 StWRKY8-like gene (563 bp) was obtained, which contained a 258 bp open reading frame coding a peptide with 85 amino acids. The StWRKY8-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 StWRKY8-like protein was highly close to the other WRKY members of Solanaceous plants and in 98% similarity with StWRKY8 in R. solanacearum. The StWRKY8-like protein is predicted as a hydrosoluble protein with an isoelectric point of 9.1 and half-life period of 5.5 h. It is a non-spherical protein, contains 3 phosphorylation sites and locates in the cytoplasm. The StWRKY8-like gene was upregulated after the R.solanacearum infection, and differentially expressed in susceptible and resistant S. tubosum. The expression of StWRKY8-like in resistant S. tubosum was significantly higher than in susceptible S. tubosum at 6 h post infection. It was mainly expressed in the vascular system of the stem and leaves. 【Conclusion】 StWRKY8-like was a putative transcription factor gene, upregulated at R. solanacearum infection and differentially expressed in susceptible and resistant S. tubosum. It may be involved in the S. tubosum defense against R. solanacearum.

Key words: Solanum tuberosum, Ralstonia solanacearum, transcription factor, StWRKY8-like, gene expression, tissue-specific expression

薛珍，李卉，孔超越，段婷婷，郜刚. 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[J]. 中国农业科学, 2015, 48(21): 4219-4226.

XUE Zhen, LI Hui, KONG Chao-yue, DUAN Ting-ting, GAO Gang. Cloning and Expression Analysis of the Potato Transcription Factor StWRKY8 Like Gene Induced by Ralstonia solanacearum[J]. Scientia Agricultura Sinica, 2015, 48(21): 4219-4226.

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