粉葛PtERF98基因克隆及表达分析

Cloning and Expression Analysis of PtERF98 Gene from Pueraria thomsonii Benth.

乙烯响应因子(ethylene responsive factors,ERF)是植物特有的一类转录因子,在乙烯信号转导通路、植物生长发育和逆境胁迫响应中起重要的调控作用。为探明PtERF98转录因子在粉葛(Pueraria thomsonii Benth.)块根采后生理性变质(post-harvest physiological deterioration,PPD)过程中的表达调控机制,本研究采用RT-PCR技术从火山粉葛叶组织中克隆PtERF98基因,并对其进行生物信息学、组织表达特异性分析和在PPD过程中的表达分析。结果显示,PtERF98基因CDS序列长450 bp,编码149个氨基酸残基;PtERF98蛋白理论分子量为16.93 kDa,理论等电点为8.06,为亲水性不稳定蛋白。蛋白结构分析显示,PtERF98具有典型的AP2保守结构域,属于ERF转录因子家族成员,定位于细胞核,无跨膜螺旋结构,含有1个信号肽和16个磷酸化位点。氨基酸序列同源性和系统进化关系分析表明,PtERF98与大豆(Glycine max)GmERF98同源性最高且亲缘关系最近。组织表达分析显示,PtERF98基因在火山粉葛的根、茎、叶中均有表达,且在不同组织中的相对表达量存在差异,叶中表达量最高。此外,PtERF98的表达受到PPD的显著诱导,表达水平随着PPD时间延长呈先升高后下降趋势,并在采后48 h达到最高,推测该基因可能参与粉葛块根的PPD过程。本研究可为深入了解PtERF98基因的功能及其在粉葛PPD过程中的表达调控作用奠定研究基础。

The ethylene responsive factors(ERF)are plant-specific transcription factors which play cru-cial regulatory roles in ethylene signal transduction pathways,plant growth and development,as well as stress responses.In order to investigate the expression and regulation mechanism of the PtERF98 transcription factor during the post-harvest physiological deterioration(PPD)process in roots of Pueraria thomsonii Benth.,the PtERF98 gene was cloned from the leaf tissue of P.thomsonii Volcano cultivar using RT-PCR technology.The bioinformatics,tissue expression specificity and expression characteristics during the PPD process of PtERF98 were analyzed.The results showed that the PtERF98 gene had a CDS sequence of 450 bp,encoding 149 amino acid residues.The PtERF98 protein had theoretical molecular weight and isoelectric point of 16.93 kDa and 8.06,respectively,and was a hydrophilic unstable protein.Protein structure analysis indicated that PtERF98 contained a typical AP2 conserved domain,so it was categorized as a member of the ERF transcription factor family;it was localized in the nucleus without transmembrane helix structure,but contained one signal peptide and 16 phosphorylation sites.Amino acid sequence homology and the phylogenetic analysis indicated that PtERF98 had the highest homology and the closest phylogenetic relationship with GmERF98 of soybean(Gly-cine max).Tissue expression analysis demonstrated that PtERF98 gene was expressed in the roots,stems and leaves of Volcano cultivar.There were differences in expression level among different tissues,and the highest expression level exhibited in leaves.Moreover,PtERF98 expression was significantly induced by PPD,and its expression level first increased and then decreased over time,reaching the peak at 48 hours post-harvest,which suggested that this gene may be involved in the PPD process of P.thomsonii tuberous roots.This study could lay the groundwork for further understanding the function of PtERF98 gene and its regulatory roles in the PPD process of P.thomsonii.