三叶木通促分裂原活化蛋白激酶基因mapk3的克隆及表达分析

植物MAPK信号途径在植物生长发育以及多种逆境胁迫响应和激素调控过程中发挥着至关重要的作用。本文利用RACE-PCR技术克隆三叶木通促分裂原活化蛋白激酶基因mapk3的全长c DNA序列,并对其进行生物信息学分析和时空表达分析。克隆所得的Aktmapk3基因的ORF全长序列为1 164 bp,编码387个氨基酸,其编码的蛋白具有ATP结合位点,MAPK激酶保守结构和丝氨酸/苏氨酸蛋白激酶激活位点,推测其可能通过被磷酸化而激活以及通过磷酸化下游蛋白而执行生理功能。实时荧光定量PCR结果显示,Aktmapk3基因在三叶木通各组织器官均有表达,在芽成熟叶片以及花中表达量比较高,在茎、幼叶和果肉中的表达量最低,暗示该基因可能参与了三叶木通芽的形成和花的发育。

Cloning and Expression Analysis of Mitogen-Activated Protein Kinases Gene of mapk3 of Akebia trifoliata

Plant MAPK (mitogen-activated protein kinases) cascades play a crucial role in the process of hormone signal transduction,various stresses responses and plant growth and development.The full-length cDNA sequence of Akebia trifoliata mapk3 gene was amplified by using the technology of rapid amplification of cDNA ends (RACE) method.Subsequently,characterization of Aktmapk3,including gene alignment,phylogenetic tree,conserved domain and protein structure were analyzed by bioinformatics,while the expression level of mapk3 in different tissues of Akebia trifoliata were analyzed by real-time quantitative PCR method.Result showed that the Aktmapk3 gene contained an 1 164 bp open reading frame (ORF) which encodes 387 amino acids,and the coding protein contained an ATP binding site (IPR017441),MAPK conserved site (IPR003527),and Serine/threonine-protein kinase active site (IPR008271) suggesting that this protein might be activated by phosphorylation,and then phosphorylate downstream protein to executive physiologic function.The result of Real-time PCR exhibited that Aktmapk3 was the highest abundance in bud,mature leaf and flower,suggested that Aktmapk3 might be involved in the development of bud and flower.