花生中促丝裂原蛋白活化激酶6a(AhMAPK6a)的克隆与表达分析

Cloning and Expression Analysis of a Mitogen-activated Protein Kinase 6a Gene in Peanut

促丝裂原蛋白活化激酶(MAPK)是一类保守的丝氨酸/苏氨酸(Ser/Thr)蛋白激酶,是一类植物对逆境胁迫反应途径中的关键家族基因。本研究利用已经构建的花生种子全长cDNA文库,以拟南芥MAPK序列为模板,结合大规模EST测序和RACE克隆等方法,从花生中克隆得到了一个MAPK基因,命名为AhMAPK6a,并在GenBank上注册,注册号为KP329549。其cDNA序列全长1492bp,开放阅读框(ORF)含有1191bp,编码一条含有397个氨基酸的蛋白序列。不同物种中MAPK基因的氨基酸序列比对发现,该基因含有典型的Ser/Thr保守基序,与拟南芥、大豆、本生烟的同源性分别为86%、94%和90%。不同组织部位的荧光定量(qPCR)分析表明,该基因在叶片中的表达量最高,其次在根中,在茎、花、子叶和下胚轴中的表达量极低,表明该基因可能在花生的根和叶的生长发育中发挥较大作用。在脱落酸(ABA)和聚乙二醇(PEG)胁迫下,表达量发生明显变化,可以推测该基因在干旱胁迫反应中发挥重要作用。

Mitogen-activated protein kinases （MAPKKs） are encoded by a large family of serine/ threonine/tyrosine protein kinases. MAPK cascades are key family which involved in plant stress re- sponse. Using the constructed full-length cDNA library of peanut （Arachis hypogaea L. ） seed and u- sing the AtMAPK as template, a new MAPK gene, designated AhMAPK6a was isolated from peanut via a large number of sequences of expressed sequence tag （EST） and RACE clone. The sequence of the gene was submitted to GenBank and its GenBank ID was KP329549. The sequence of AhMAPK6a cDNA were 1492 bp, and ORF was 1191 bp. The number of deduced amino acid was 397. Alignment results of the amino acid sequences of MAPK gene in different species indicated that the gene contains typical Ser/Thr conserved motifs, and its homology with Arabidopsis, soybean, and Nicotiana bentharniana is 86%, 94~1] 90~, respectively. Fluorescence quantitative PCR （qPCR） results for tissue expression analysis indicated that the expression of AhMAPK6a was the highest in peanut leav- es, and next in the roots. The expression level is very low in the stem, flower, cotyledon and hypo- cotyls. These results showed that the gene may play important role in the roots and leaves growth of the peanut. The expression significantly changed under ABA and PEG stress, so that it can be hy- pothesized that the gene plays an important role in peanut drought stress regulation.