甘薯IbERF071基因的克隆及表达特性分析

Cloning and expression pattern analysis of IbERF071 gene from sweet potato

为探究甘薯(Ipomoea batatas(L.)Lam.)IbERF071基因在抗蔓割病中的调控作用,根据甘薯转录组数据,从甘薯高抗蔓割病品种‘鄂薯11’中克隆获得1个编码乙烯应答的基因,命名为IbERF071,并对其进行生物信息学分析,以及侵染蔓割病病原菌、外施茉莉酸甲酯和乙烯利的基因表达模式分析。结果表明,IbERF071基因的CDS序列大小为864bp,共编码287个氨基酸;在其编码的氨基酸序列的第79~142位包含1个AP2保守结构域,在第205~254位包含1个卷曲螺旋;IbERF071氨基酸序列与牵牛InERF氨基酸序列的相似度高达89%以上,具有高度同源性,且在系统发育树上也聚为同一支;IbERF071蛋白不稳定指数、脂肪系数和总亲水性平均系数分别为48.26、47.35和-1.003,分子质量为32215.31ku,等电点为5.12,表明该蛋白是一种不稳定的亲水性蛋白质;IbERF071蛋白二级结构中,随机卷曲、α螺旋、延伸链和β转角所占比例分别为61.67%、26.83%、8.01%和3.48%,三级结构中,22%的氨基酸残基结构置信度达到64.06%,IbERF071蛋白定位于细胞核上,没有跨膜蛋白且不存在跨膜螺旋区;IbERF071基因启动子区包含真核生物启动子基本元件胁迫响应元件、茉莉酸甲酯响应元件、低温响应元件MYC、干旱诱导元件ERE、脱落酸效应元件、植物防御响应元件、水杨酸响应元件TCA等30种顺式作用元件;IbERF071基因在根、茎、叶中均有表达,且在根中表达量显著高于茎、叶,经蔓割病原菌侵染后,IbERF071基因的相对表达量显著升高,并在侵染2h时表达量达到峰值,茉莉酸甲酯处理72h和乙烯利处理48h后,其表达量显著提高。综合来看,甘薯IbERF071蛋白属于AP2/ERF超级家族,具有较高的保守性,IbERF071在调控甘薯抗蔓割病菌中的功能以及JA和ET介导的激素信号转导途径的过程中起重要作用。

In order to study the regulatory role of sweet potato IbERF071 gene in resistance to Fusarium oxysporum f.batatasinfection,based on the transcriptome data of sweet potato,a gene encoding an ethylene response was cloned from sweet potato variety‘Eshu 11’with high resistance to Fusarium oxysporum f.batatas,named as IbERF071.Bioinformatics analysis was carried out,and the gene expression patterns of fusarium wilt pathogen,methyl jasmonate and ethephon were analyzed.The results show that the CDS sequence of the IbERF071 gene is 864bp in size,encoding a total of 287 amino acids.IbERF071 contains an AP2 conserved domain at positions 79 to 142 of its encoded amino acid sequence,and a coiled spiral at positions 205 to 254.The amino acidsequences of IbERF071 and InERF share more than 89%similarity,and they exhibit a high degree of homology and are clustered together on the phylogenetic tree.The instability index,fat coefficient and average coefficient of total hydrophilicity of IbERF071 protein are respectively 48.26,47.35 and-1.003,and its relative molecular mass is 32215.31ku and its isoelectric point is 5.12,indicating that the protein is an unstable hydrophilic protein.In the secondary structure of IbERF071 protein,the proportions ofrandom coil,α-helix,extended strand andβ-turn are 61.67%,26.83%,8.01%and 3.48%,respectively.In the tertiary structure of the protein,22%of the amino acid residues have structural confidence of 64.06%.The IbERF071 protein is localized in the nucleus and it possesses no transmembrane protein and no transmembrane helical region.The IbERF071 gene promoter region contains 30 kinds of cis-acting elements including stress response element,methyl jasmonate response element,low temperature response element MYC,drought inducing element ERE,abscisic acid response element,plant defense response element and salicylic acid responseelement TCA.The IbERF071 gene is expressed in roots,stems and leaves,and the expression level in roots is significantly higher than that in stems and leaves.The relative expression level of IbERF071 gene is significantly increased after Fusarium oxysporum f.batatas infection,and reaches the peak at 2h after infection.The expression level is significantly increased after treatment withMeJA for 72h and ETH for 48h.Overall,the sweet potato IbERF071 protein belongs to AP2/ERF superfamily with a high degree ofsequence conservation,and might play an important role in regulating sweet potato resistance to Fusarium oxysporum f.batatas and the process of JA and ET-mediated hormone signal transduction pathway.