甘薯交替氧化酶基因家族生物信息学与表达分析

Bioinformatics and Expression Analysis of Alternative Oxidase Genes in Sweetpotato

为分离甘薯[Ipomoea batatas(L.)Lam.]交替氧化酶(AOX)基因家族成员,分析其在甘薯应答生物和非生物胁迫中的响应模式,本试验在筛选甘薯基因组的基础上,进行基因克隆和测序,最终从心香甘薯中获得3个AOX基因家族成员。基因结构分析结果表明,3个甘薯AOX基因的cDNA全长依次为963、1080和1032 bp,分别编码320、359和343个氨基酸,均含4个外显子和3个内含子,将其分别命名为IbAOX1a(GenBank登录号:MT992313)、IbAOX1b(GenBank登录号:MT992314)和IbAOX2(GenBank登录号:MG450566.1)。蛋白同源性分析结果显示,甘薯AOX蛋白序列在C端十分保守,仅在N端有较小差异,且序列中都存在特有的EXXH、FXHR和EEE-Y铁离子结合保守结构域。亚细胞定位预测结果表明该基因家族成员主要定位在线粒体。蛋白质二级结构预测表明,该基因家族成员主要以α-螺旋、β-转角、延伸链和不规则卷曲为主。实时荧光定量PCR(qRT-PCR)分析结果表明,IbAOXs基因的表达具有组织特异性,IbAOX1a在叶片中表达最高,IbAOX1b和IbAOX2在块根中高表达,IbAOX1a和IbAOX2在花蕾中几乎不表达。IbAOX1a和IbAOX1b在低温、干旱和盐胁迫的幼苗中均表达上调。IbAOX1a在低温块根中的表达高峰显著高于IbAOX1b,而在腐皮镰刀菌(Fusarium solani)侵染的块根中IbAOX1b的表达量显著高于IbAOX1a。无论在甘薯幼苗或块根中,IbAOX2对各种胁迫都没有响应,呈现组成型表达模式。本研究为甘薯AOX基因的功能挖掘及甘薯抗逆品种筛选提供了一定的理论依据。

This study was aimed to identify the members of alternative oxidase(AOX)gene family in sweetpotato[Ipomoea batatas(L.)Lam.],and characterize their expression patterns in response to biotic and abiotic stresses.Based on genome wide screening and gene cloning and sequencing,three members of AOX gene family were obtained from Xinxiang sweetpotato.The gene structure analysis showed that the cDNA of three AOX genes in sweetpotato were 963 bp,1080 bp and 1032 bp in length,encoded 320,359 and 343 amino acids,respectively.Three AOX genes all contained 4 exons and 3 introns,and were named as IbAOX1a(GenBank accession number:MT992313),IbAOX1b(GenBank accession number:MT992314)and IbAOX2(GenBank accession number:MG450566.1),respectively.Protein homology analysis showed that the protein sequences of IbAOX were very conservative at the C-terminus,only minor differences existed at the N-terminus,and there were special Fe-binding conserved domains of EXXH,FXHR and EEE-Y in the sequences.Subcellular localization prediction showed that IbAOX were mainly located in mitochondria.Protein secondary structure prediction showed that the primary secondary structure of the gene family wasα-helix,β-turn,extended chain and irregular coils.Real-time fluorescence quantitative PCR(qRT-PCR)analysis showed that the expression of IbAOXs gene was tissue-specific.The transcript level of IbAOX1a in leaves was the highest,while IbAOX1b and IbAOX2 were highly expressed in tuberous roots.The expression of IbAOX1a and IbAOX2 in buds were hardly detected.The expression of IbAOX1a and IbAOX1b in seedlings was stimulated under cold,drought and salt stresses.The transcript level of IbAOX1a was significantly higher than that of IbAOX1b in tuberous roots at 4℃,while the expression IbAOX1b in tuberous roots infected by Fusarium solani was markedly higher than that of IbAOX1a.However,the expression of IbAOX2 in sweetpotato seedlings or tuberous root was not altered under various stresses,showing a constitutive expression pattern.This study provides a theoretical basis for studying the functions of AOX genes and breeding sweetpotato varieties with stress resistances.