南方根结线虫诱导辣椒基因CaRKNIF2的克隆与分析  

Cloning and Analysis of Gene CaRKNIF2 Isolated from Capsicum annuum Induced by Meloigogyne incognita

作　　者：郑井元[1,2] 邹学校[1] 茆振川[2] 陈国华[2] 谢丙炎[2] 

机构地区：[1]中南大学研究生院隆平分院,长沙410125 [2]中国农业科学院蔬菜花卉研究所,北京100081

出　　处：《中国农业科学》2010年第24期5046-5054,共9页Scientia Agricultura Sinica

基　　金：国家重点基础研究发展计划(2009CB119000);国家自然科学基金项目(30671412);农业公益性行业科研专项(nyhyzx07-050)

摘　　要：【目的】明确辣椒(Capsicum annuum L.HDA149)中与南方根结线虫(Meloidogyne incognita)不亲和互作过程中抗性相关WRKY转录因子的基因结构及其表达模式。【方法】采用RACE结合RT-PCR方法克隆cDNA序列,并根据cDNA序列设计特异引物扩增DNA序列,Southern杂交确定基因拷贝数,实时荧光定量PCR方法分析该基因在线虫侵染后辣椒的不同组织和不同时间点的表达。【结果】从辣椒中克隆到一个WRKY转录因子基因CaRKNIF2(GenBank登录号:GQ253367),该基因编码区全长1662bp,编码553个推定的氨基酸,为单拷贝基因。CaRKNIF2基因编码区全长DNA序列2530bp,包含5个内含子和6个外显子。在线虫接种诱导处理时,该基因表达具有组织特异性,根尖组织中的表达量最高;接种3h后在根尖组织中的表达量开始升高,12h最高,此时相对表达量约为3.1倍。【结论】CaRKNIF2在线虫接种处理后的表达上调,表明该基因参与了抗性基因Me3介导的辣椒与根结线虫的不亲和互作,可能在此过程中具有重要功能。【Objective】 The aim of the study was to clarify the characterization and expression of one WRKY transcription factor in pepper （Capsicum annuum L.HDA149） during the incompatible interaction between HDA149 and Meloidogyne incognita.【Method】The combination of RACE and RT-PCR was used to clone the cDNA of a WRKY transcription factor named CaRKNIF2 from pepper inoculated with nematode,and the coding sequences of DNA were amplified with specific primers designed according to the cDNA sequence,copy number of the gene was identified by Southern blot,and the gene expression in various tissue and in different time points after inoculation with nematode were analysed by real-time RT-PCR.【Result】 The cDNA of CaRKNIF2 （GenBank Accession No： GQ253367） containing an open reading frame of 1 662 bp and encoding 553 amino residues was cloned from pepper.The genome DNA length of CaRKNIF2 was 2 530 bp,and contained 5 introns and 6 exons.CaRKNIF2 was a single copy gene in genome identified by Southern blot.The real-time RT-PCR revealed that the gene expressed tissue-specific with the expression level was the highest in tissue of root tips after inoculation with M.incognita.The expression level of gene CaRKNIF2 in root tips of pepper was increased gradually from 3 to 12 h after inoculation with M.incognita and reached a peak at 12 h.The relative gene expression level at 12 h after nematode inoculation was 3.1 times higher than that of the control.【Conclusion】 The expression of CaRNIF2 was up-regulated by the nematode inoculation,which implied that the gene might play an important role in the process of incompatible interaction between M.incognita and C.annuum variant HDA149 mediated by resistant gene Me3.

关 键 词：WRKY转录因子 CaRKNIF2 CDNA SOUTHERN杂交 基因表达 

分 类 号：S436.418[农业科学—农业昆虫与害虫防治]