甘蔗MAP激酶基因SoMAPK4克隆与生物信息学分析

目的]克隆甘蔗MAP激酶家族新基因的全长序列,为了解甘蔗抗逆胁迫机制提供依据.方法]以新台糖22为材料,提取甘蔗幼叶总RNA并反转录为cDNA;利用已知物种MAP激酶基因核苷酸序列保守区设计引物,采用5′和3′端RACE技术克隆新基因全长,并进行生物信息学分析.结果]克隆获得的甘蔗新基因与玉米ZmMAPK4同源性很高,达92.6%,将该基因命名为SoMAPK4(登录号JQ062930),基因全长1499 bp,其中开放阅读框(ORF)为1128 bp,5′非翻译区(UTR)为218 bp,3′非翻译区(UTR)为213 bp.生物信息学分析结果表明,SoMAPK4基因编码一个含376个氨基酸的蛋白质,分子量约43.5 kDa,等电点为5.51,含有11个保守的蛋白激酶亚区和MAP激酶的磷酸化位点TEY基序.结论]克隆获得甘蔗MAP激酶新基因SoMAPK4,该基因可能参与多种胁迫反应的信号传递,是研究甘蔗非生物胁迫和生物胁迫过程中信号传递的一个关键因素.

Cloning and bioinformatic analysis of Mitogen-activated protein kinase gene SoMAPK4 in sugarcane

【Objective】The present experiment was conducted to clone the full-length sequence of mitogen-activated protein kinase （MAPK） in sugarcane to explore mechanism of stress resistance in sugarcane. 【Method】The total RNA was extracted from leaves of sugarcane variety ROC22 and the cDNA was synthesized by gene racer kit. Based on the cDNA sequences of the conserved regions of plant MAPK genes， a pair of primers was designed and synthesized for cloning the full-length sequence of a novel member of MAPK gene family by RACE reaction. The bioinformatics of the cloned MAPK gene was also analyzed. 【Result】The cloned mitogen-activated protein kinase gene from sugarcane showed 92.6% homology with ZmMAPK4， and it was named as SoMAPK4 （accession No. JQ062930）， which cDNA full-length was 1499 bp with a polyA tail of 23 bp， containing a 5'-untranslated region （UTR） of 218 bp and a 3'-untranslated region （UTR） of 213 bp. The cDNA contained an ORF of 1128 bp encoding a protein of 376 amino acids with a calculated molecular weight of about 43.5 kDa and a pI of 5.51. The SoMAPK4 protein contained 11 sub-domains of protein kinase with serin/threonine specificity and phosphorylation TEY motif of MAP kinase. 【Conclusion】A novel MAPK gene SoMAPK4 was cloned. The SoMAPK4 may be involved in many signal transduction activities for various stress reactions， and is the key factor for studying signal transduction during sugarcane biotic and abiotic stresses.