摘要： WRKY转录因子参与调控与植物免疫反应相关的转录重编程，对植物抗病性发挥调控作用。为了解析WRKY在野生角瓜对根结线虫（RKN）抗性中的作用，通过RT-PCR并结合RACE技术从角瓜根系中克隆CmWRKY20全长cDNA序列，采用DNAStar和DNAMAN进行氨基酸序列与系统进化分析，通过qRT-PCR进行基因表达分析，利用农杆菌介导的烟草叶片细胞转化法进行编码蛋白的亚细胞定位。结果表明，CmWRKY20转录因子cDNA序列全长1 603 bp，5'非翻译区646 bp，3'非翻译区320 bp，ORF长度1 017 bp，编码338个氨基酸，GenBank登录号MN365875；CmWRKY20具有1个核定位信号和1个WRKY保守结构域，锌指基序为C₂H₂，归类于WRKY第Ⅱ组，与黄瓜、甜瓜等作物WRKY的氨基酸序列同源性较高，亲缘关系较近；CmWRKY20表达表现为抗病材料高于感病材料，根、茎高于叶片，CmWRKY20受根结线虫快速诱导，水杨酸（SA）和茉莉酸甲酯（MeJA）对CmWRKY20表达既有协同效应又有拮抗作用，CmWRKY20蛋白被定位于细胞膜上。CmWRKY20可能通过SA/JA信号传导参与对根结线虫防卫反应的调控。

关键词: 角瓜, 根结线虫, CmWRKY20, 克隆, 基因表达, 亚细胞定位

Abstract: WRKY transcription factors are involved in the regulation of transcriptional reprogramming associated with plant immune responses,and play a regulatory role in plant disease resistance. To identify the role of WRKY transcription factor in resistance of Cucumis metuliferus against root-knot nematode (RKN),full-length cDNA sequence of CmWRKY20 was cloned from the roots of C. metuliferus by RT-PCR combined with RACE tecnology. Two softwares of DNAStar and DNAMAN were used for amino acid sequence and phylogenetic evolutionary analyses. Tissue-specific expression characteristics and induced expression profiles of the gene were determined through qRT-PCR,and Agrobacterium-mediated transformation of tobacco leaf cells was used to investigate subcellular localization of its coding proteins. The results showed that full cDNA sequence of CmWRKY20 was 1 603 bp in length,including 5' untranslated part of 646 bp,3' untranslated part of 320 bp,an open reading frame of 1 017 bp encoding a protein of 338 amino acids consisting of a conserved WRKY domain with a nuclear localization signal and a zinc finger motif of C₂H₂,belonging to the WRKY subgroup Ⅱ. CmWRKY20 was deposited in GenBank with Accession No. MN365875,and the amino acid sequence of CmWRKY20 was highly homologous and closely related to cucumber and melon. CmWRKY20 showed higher relative expression in the resistant than the susceptible response,and higher in roots and stems than in leaves. CmWRKY20 was rapidly induced by root-knot nematode infection,salicylic acid (SA) and methyl jasmonate (MeJA) had synergistic or antagonistic effects on the expression of CmWRKY20. CmWRKY20 protein was localized on the cell membrane. CmWRKY20 may be involved in the regulation of defense response to root-knot nematode through SA/JA signal transduction.

Key words: Cucumis metuliferus, Root-knot nematodes, CmWRKY20, Cloning, Gene expression, Subcellular localization

中图分类号: 

• Q78

• S436.421