甘蔗ScBAK1基因及其可变剪接体的克隆与表达分析

Cloning and expression analysis of ScBAK1 gene and its alternative spliceosome in sugarcane

可变剪接(Alternative splic ing,AS)是真核生物基因表达调控研究的热点.BAK1(Brassinosteroid insensitive1-associated receptor kinase 1)是植物中丝氨酸/苏氨酸蛋白激酶的一种,可调控植物的生长发育及先天免疫反应.为揭示BAK1基因在甘蔗应答不良外界环境方面的作用,利用电子克隆及RT-PCR方法从甘蔗品种崖城05-179叶片c DNA中克隆到1个BAK1基因及其1个可变剪接体,分别命名为Sc BAK1(Gen Bank登录号:KP032226)和Sc BAK1 S1(Gen Bank登录号:KP032227).生物信息学分析结果表明,Sc BAK1/Sc BAK1 S1基因的ORF长1 860 bp/1 770 bp,编码蛋白含有619/589个氨基酸残基,分子量(Mr)为6.928×104/6.576×104;两种编码蛋白均定位于质膜上,含有信号肽,为酸性、分泌脂溶性蛋白;它们的二级结构以α-螺旋和无规则卷曲为主,其次是延伸链,无β-螺旋;蛋白功能预测显示其主要作为细胞膜蛋白,还参与氨基酸及辅酶因子生物合成.荧光定量PCR分析结果显示,Sc BAK1 S1基因的表达在非生物(水杨酸SA、Cu Cl2、PEG、脱落酸ABA、Na Cl、茉莉酸甲酯JA)和生物胁迫(黑穗病菌)下均受到抑制,而Sc BAK1却受SA、Cu Cl2、PEG、Na Cl和黑穗病菌的诱导.结果还表明,相较于Sc BAK1在甘蔗抗黑穗病性、渗透胁迫以及细胞生长方面发挥作用来说,Sc BAK1 S1缺失的氨基酸序列或数目在Sc BAK1的抗逆性方面扮演了重要角色.Sc BAK1和Sc BAK1 S1的不同丰度表达为深入解析Sc BAK1基因在生物和非生物逆境条件下的功能奠定了基础.

Alternative splicing（AS） is an important part of regulation of eukaryotic gene expression.BAK1（Brassinosteroid insensitivel-associated receptor kinase 1） is a specific type of plant serine/threonine protein kinases,and can regulate growth and development and natural immunization.To reveal the responses of sugarcane BAK1 gene to the adverse environment,a ScBAK1 gene and its alternative spliceosome,termed ScBAK1（GenBank accession number：KP032226） and ScBAK1 S1（GenBank accession number：KP032227）,were cloned from leaf cDNA of Yacheng 05-179 utilizing the methods of electronic cloning and RT-PCR.The open reading frame（ORF） length of ScBAKl/ScBAK1 S1 gene was 1 860bp/l 770 bp,encoding619/589 amino acids residues.The predicted molecular weight of the protein was 69.28 kDa/ 65.76 kDa.Both proteins were located in plasma membrane,estimated as acid,hydrophikic and secretive proteins.Random coil and alpha helix gave priority to extended strand in their secondary structure without beta turn.The most important protein function was cell envelope,secondly biosynthesis of amino acids and cofactors.Real-time quantitative PCR analysis revealed that the expression of sugarcane ScBAK1 S1 gene exhibited the reduced expression trend under smut fungus stress and various abiotic exogenous stresses,including SA,CuCl_2,PEG,ABA,NaCl and JA,while the expression of ScBAK1 gene was induced by SA,CuCl_2,PEG,NaCl and smut fungus stresses.The phenomenon showed that the absent sequences or amounts of ScBAK1 S1 gene plays a key role in the response of ScBAK1 to the stress of sugarcane smut fungus,osmotic stress and cell growth.The differential expression of ScBAK1 and ScBAK1 S1 lays a foundation for further research on the function of ScBAK1 gene under biotic and abiotic stress.