甘蓝型油菜BnMAPK2基因的克隆及功能分析

Cloning and functional analysis of BnMAPK2 gene in Brassica napus

从甘蓝型油菜中分离克隆了BnMAPK2(BnaA01g21880D)基因,cDNA及其编码序列长度分别为1516 bp、1113 bp,编码370个氨基酸。生物信息学分析表明,BnMAPK2蛋白分子量为42,497.0 kD,等电点为6.36,蛋白不稳定系数38.74,为疏水性蛋白,具有MAPKs蛋白特有的STKc_TEY_MAPK_plant(cd07858)保守结构域;蛋白二级结构中α螺旋所占比例最大,为44.05%,无信号肽;与拟南芥C族AtMAPK2的亲缘关系更近。核心元件预测结果显示,BnMAPK2-P含有响应水杨酸激素、热胁迫和光照等相关顺式作用元件,包括TCA-element、HSE、AAAC-motif和MYB结合位点等。实时荧光定量PCR(qRT-PCR)结果表明,BnMAPK2在甘蓝型油菜中的各个组织器官中均有表达,受到茉莉酸甲酯、水杨酸、H_(2)O_(2)、损伤、高温和核盘菌的诱导。转基因异源表达BnMAPK2拟南芥株系的表型数据发现,与野生型相比,超量表达BnMAPK2使拟南芥植株的抽薹期提前,株高、主花序有效长度和角果数显著增加,由此推测BnMAPK2基因参与调节植物生长发育过程。本研究为深入探究BnMAPK2调控甘蓝型油菜生长发育过程的分子机制提供了参考资料和数据支撑。

The mitogen-activated protein kinase(MAPK)cascade is involved in plant growth and development and it is in response to a variety of biotic and abiotic stresses.In this study,a BnMAPK2(BnaC01g28210D)gene was isolated and cloned from Brassica napus.The cDNA and its coding sequence were 1516 bp and 1113 bp in length,respectively,encoding 371 amino acids.Bioinformatics analysis revealed that the molecular weight of BnMAPK2 protein was 42,497.0 kD,the isoelectric point was 6.36,protein instability coefficient was 38.74,it was a hydrophobic protein,and it had STKc_TEY_MAPK_plant(cd07858)conserved structure domain unique to MAPKs protein,protein secondary level.The alpha helix accounted for the largest proportion of 44.05%in the secondary structure of protein,and there was no signal peptide,which was more closely related to the C group At-MAPK2 of Arabidopsis.The core element prediction indicated that BnMAPK2-P contained related cis-acting elements in response to salicylic acid hormone,heat stress,and light,including TCA-element,HSE,AAAC-motif,and MYB binding sites.Real-time quantitative PCR(qRT-PCR)demonstrated that BnMAPK2 was expressed in various tissues and organs in Brassica napus,which was induced by methyl jasmonate,salicylic acid,H_(2)O_(2),injury,high temperature,and Sclerotinia sclerotiorum.The phenotypic data of transgenic Arabidopsis lines expressing BnMAPK2 heterologously showed that compared with the wild type,the overexpression of BnMAPK2 made the bolting period of Arabidopsis plants earlier,and significantly increased plant height,the effective length of main inflorescence,and the number of siliques.We speculated that BnMAPK2 gene was involved in the regulation of plant growth and development.This study provides reference materials and data support for in-depth exploration of the molecular mechanism of BnMAPK2 regulating the growth and development in Brassica napus.