木薯ERF转录因子基因MeERF5克隆及表达分析

Cloning and expression analysis of ERF transcription factor gene MeERF5 in cassava

  • 摘要: 【目的】克隆木薯ERF转录因子基因MeERF5,并分析其在多种逆境胁迫下的表达模式,为深入研究MeERF5基因在木薯逆境胁迫应答中的调控机制提供参考。【方法】PCR扩增木薯品种华南8号的MeERF5基因编码区(CDS)全长序列,对其进行生物信息学分析,并利用根癌农杆菌介导法进行蛋白亚细胞定位。通过实时荧光定量PCR(qRT-PCR)检测MeERF5基因在木薯不同组织及多种逆境胁迫处理下的相对表达量。【结果】克隆获得MeERF5基因CDS序列为948 bp,与Phytozome数据库中的参考序列(登录号:Manes.01G085200.1)的核苷酸序列相似性为100.00%,其编码315个氨基酸残基,蛋白分子量为77.84 kD,理论等电点(pI)为5.08,属于酸性蛋白,其二级结构中α-螺旋占16.51%,β-转角占3.81%,无规则卷曲占62.22%,延伸链占17.46%,亚细胞定位于细胞质。通过多序列比对及系统进化分析发现,MeERF5蛋白含有1个AP2/ERF保守结构域,与同属大戟科的橡胶树ERF蛋白氨基酸序列相似性最高(76.8%),亲缘关系最近。MeERF5基因在木薯不同组织中均有表达,其中,在茎中的相对表达量最高,在腋芽中的相对表达量最低。MeERF5基因能快速响应低温胁迫、干旱胁迫、氧化胁迫、盐胁迫及ABA处理,均出现诱导表达上调的现象,其中,在氧化胁迫下,MeERF5基因的相对表达量持续上升;在干旱胁迫、盐胁迫及ABA处理下,MeERF5基因的表达量先上升后降低;在低温胁迫下,MeERF5基因在处理5 h时的相对表达量达到最大值,之后有所下降,但在处理48 h时再度上升。【结论】克隆获得的MeERF5基因属于AP2/ERF类转录因子,参与木薯多种非生物胁迫应答过程。

     

    Abstract: 【Objective】To clone ERF transcription factor gene MeERF5 and analyze expression pattern of the gene under stress conditions,so as to provide reference for studying regulation mechanism of MeERF5 gene in cassava.【Method】The full length of coding sequence(CDS)of MeERF5 gene in cassava variety SC8 was amplified by PCR and bioinformatics analysis was conducted. Subcellular localization was performed through Agrobacterium tumefaciens mediation method. Then relative expression of MeERF5 gene in different tissues and organs under different abiotic stress was identified by quantitative real-time PCR(qRT-PCR).【Result】CDS sequence of MeERF5 gene obtained through cloning was 948bp,which was 100.00% similar with a nucleotide sequence of reference sequence(accessing number:Manes.01G085200.1)in the Phytozome database,encoding 315 amino acid residues. MeERF5 protein had a theoretical molecular weight of77.84 kD and theoretical isoelectric point(p I)of 5.08,indicating it was an acidic protein. In its secondary structure,α-helix accounted for 16.51%,β-turn 3.81%,and random coil 62.22%,extended strand 17.46%. Subcellular localization was in the cytoplasm. Multiple sequence alignment and phylogenetic analysis showed that MeERF5 protein contained a conserved AP2/ERF domain,sharing a highest sequence similarity(76.8%)and close genetic relationship with ERF proteins in Hevea brasiliensis. MeERF5 gene was expressed in various tissues and organs of cassava,and its highest expression showed in stem,while the expression was the lowest in lateral bud. MeERF5 gene could quickly response to low temperature stress,drought stress,oxidation stress,salt stress and ABA treatment,thus showing up-regulated of induction expression. Under oxidation stress,relative expression of MeERF5 gene was up-regulated. Under drought stress,salt stress and ABA treatment,the expression of MeERF5 gene was increased firstly and then decreased. Under low temperature stress,the expression of MeERF5 gene peaked at 5 h and then decreased,but increased again at 48 h.【Conclusion】MeERF5 gene obtained through cloning is AP2/ERF transcription factor participates in abiotic stress responses in cassava.

     

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