芸薹属物种(B.napus,B.oleracea,B.rapa)MAPK1家族的克隆、进化和表达特征

Cloning, Evolution and Expression Features of MAPK1 Gene Family from Brassica Species (B. napus, B. oleracea, B. rapa)

【目的】克隆甘蓝型油菜、甘蓝和白菜MAPK1家族,分析3个物种MAPK1转录表达器官特异性。【方法】在电子克隆的基础上,利用RACE(rapid amplification of cDNA ends)技术获得3个物种MAPK1全长cDNA序列和gDNA序列。利用实时荧光定量PCR(qRT-PCR)分析3个物种MAPK1家族的器官特异性表达特征。【结果】从甘蓝型油菜、甘蓝和白菜中克隆了MAPK1家族共4个成员基因BnMAPK1-1、BnMAPK1-2、BoMAPK1和BrMAPK1,它们的gDNA长度为2 512—2 525 bp,均有3个外显子和2个内含子,最长标准mRNA为1 599—1 620 bp,ORF均为1 100 bp。系统进化分析表明,甘蓝型油菜MAPK1家族来自于甘蓝和白菜MAPK1之和,其中,BnMAPK1-1对应于BoMAPK1,BnMAPK1-2则对应于BrMAPK1。qRT-PCR结果显示3个物种MAPK1在所有检测器官中均有表达,但器官特异性在种间差异显著,暗示快速进化。【结论】克隆了甘蓝型油菜、甘蓝和白菜MAPK1的全长cDNA序列和gDNA序列,支持甘蓝和白菜通过天然种间杂交形成异源四倍体物种甘蓝型油菜的假说,芸薹属MAPK1基因和蛋白在序列和结构上非常保守,但转录表达的器官特异性上进化极快,近缘种间差异显著。

Objective】The objective of this study is to isolate MAPK1 gene family from Brassica napus,Brassica oleracea and Brassica rapa and analyze the transcriptional expression profiles of MAPK1 in various organs of these three species【.Method】On the basis of in silico cloning,the full-length cDNA and gDNA of MAPK1 genes were isolated from the three species using RACE（Rapid Amplification of cDNA Ends） technology.Organ specificity of MAPK1 in various organs of the three species was detected by quantitative real-time PCR（qRT-PCR）.【Result】In this study,four MAPK1 genes including BnMAPK1-1,BnMAPK1-2,BoMAPK1 and BrMAPK1 were isolated from B.napus,B.oleracea and B.rapa.Their gDNA sequences were 2 512-2 525 bp,containing three exons and two introns.Standard mRNA lengths of these genes were 1 599-1 620 bp,all with an ORF of 1 100 bp.Phylogenetic tree showed that BnMAPK1 gene family was originated from the assemblage of B.oleracea and B.rapa MAPK1 genes,BnMAPK1-1 and BnMAPK1-2 corresponding to BoMAPK1 and BrMAPK1,respectively.qRT-PCR showed that MAPK1 genes expressed in all tested organs,but organ specificity showed significant differences among species implying quick evolution【.Conclusion】 In this study,the full-length cDNA and gDNA of MAPK1 genes were isolated from B.napus,B.oleracea and B.rapa.It supports the assumption that B.napus is a heterotetraploid species of B.oleracea and B.rapa by natural interspecific hybridization.Brassica MAPK1 genes show much conservation in gene and protein sequences and structures,but the transcription organ specificity evolves very fast and show significant differences among closely related species.