毛竹PheERF5基因克隆及生物信息学分析

Cloning and bioinformatics analysis of PheERF5 gene from moso bamboo(Phyllostachys edulis)

【目的】探究毛竹ERF转录因子基因的结构与功能,为揭示毛竹ERF亚家族成员的抗逆境胁迫机制提供参考。【方法】利用RNA-Seq和RT-PCR技术从毛竹幼苗叶片中克隆PheERF5基因的全长cDNA,利用SMART:Main page、Prot-Param、ProtScale、NetPhos 3.1、TMHMM Server v.2.0、Softberry等在线分析软件对PheERF5蛋白进行生物信息学分析,采用DNAMAN7.0软件对PheERF5蛋白氨基酸序列进行比对分析,运用MEGA 6.0软件对PheERF5蛋白和水稻Oryza sativa AP2/ERF家族成员蛋白进行系统发育分析。【结果】从毛竹幼苗叶片中克隆获得PheERF5基因的全长cDNA片段,其开放阅读框序列全长为1017 bp,共编码339个氨基酸残基,编码蛋白相对分子质量为35.96 kD,等电点为4.58,其基因ID号为PH01001057G0620。PheERF5蛋白具有1个AP2保守结构域,属于AP2/ERF转录因子家族,为带负电荷的不稳定亲水性蛋白,其无信号肽,无跨膜结构,定位于细胞核。该蛋白存在48个磷酸化位点,以丝氨酸残基位点为主。PheERF5蛋白氨基酸序列与小米Setaria italica XP_004967470.1、水稻XP_015629650.1、二岁短柄草Brachypodium distachyon XP_003565510.1等禾本科植物的ERF氨基酸序列同源性高达84%以上,且与这些物种的AP2保守结构域同源性较高。PheERF5蛋白与124个AP2/ERF家族转录因子成员的系统发育进化分析结果显示,PheERF5与Os12g41030的亲缘关系最近,与Os12g41030、Os05g25260、Os03g60120、Os06g06540、Os01g12440和Os01g46570同属于ERF亚家族B-5子组转录因子成员,该组成员具有植物抗逆性生物学功能,主要参与植物抗病原菌侵染。【结论】PheERF5属于典型的植物AP2/ERF家族转录因子,为ERF亚家族B-5子组转录因子,可能在毛竹抗生物或非生物胁迫过程中发挥重要作用。

【Objective】The exploration of the structure and function of the ERF transcription factor gene in moso bamboo provide a theoretical foundation for improving the stress resistance of moso bamboo.【Method】Using RNASeq and RT-PCR methods to clone the cDNA fragment of PheERF5 gene from the leaves of moso bamboo seedlings,using SMART:Main page,Prot-Param,ProtScale,NetPhos 3.1,TMHMM Server v.2.0,Softberry etc.online analysis software to analyze bioinformatics of the PheERF5 protein,using DNAMAN7.0 software to compare and analyze the amino acid sequence of PheERF5 protein,and using MEGA 6.0 software to analyze phylogenetic of PheERF5 protein and AP2/ERF family member proteins from rice Oryza sativa.【Result】The cDNA fragment of PheERF5 gene was cloned from the leaves of moso bamboo seedlings,the gene ID No.was PH01001057G0620.The open reading frame(ORF)of PheERF5 gene was 1017 bp in length,encoding 339 amino acids.The putative protein PheERF5 was 35.96 kD in relative molecular mass with a theoretical isoelectric point of 4.58.This protein had a AP2 conserved domain belonging to the AP2/ERF transcription factor family.It was a negatively charged and unstable hydrophilic protein,which had no signal peptide,no transmembrane structure and localized to the nucleus.The PheERF5 protein had 48 phosphorylation sites,mainly serine residue sites.The amino acid sequence of PheERF5 protein had more than 84%homology with the ERF amino acid sequence of Gramineae plants such as Setaria italica(XP_004967470.1),Oryza sativa(XP_015629650.1),Brachypodium distachyon(XP_003565510.1)and had high homology with the AP2 conserved domain of these species.The phylogenetic analysis of PheERF5 protein and 124 AP2/ERF family transcription factor members showed that PheERF5 had the closest relationship with Os12g41030,and was clustered into ERF subfamily B-5 group together with Os12g41030,Os05g25260,Os03g60120,Os06g06540,Os01g12440 and Os01g46570.The B-5 group transcription factors play an important role in plant stress resistance,especially defense pathogen attack.【Conclusion】The PheERF5 belongs to a typical plant AP2/ERF family transcription factor,which is a B-5 subgroup of ERF subfamily transcription factor,and may be play an important role in the resistance of biotic or abiotic stress for moso bamboo.