芒果AP2/ERF转录因子MiERF2基因的克隆及表达分析

Cloning and Expression Analysis of MiERF2,an AP2/ERF Transcrip-tion Factor in Mango

芒果(Mangifera indica L.)是热带地区主要的经济作物之一,也是典型的呼吸跃变型水果,其成熟衰老与乙烯有关。AP2/ERF转录因子在植物中广泛存在,研究发现这类转录因子参与果实内源乙烯合成的信号转导,是乙烯信号通路中的关键基因,在植物生长发育、逆境胁迫响应及贮藏运输过程中发挥重要作用。为了探究AP2/ERF转录因子对采后芒果成熟衰老的影响,本研究以贵妃芒果品种为研究对象,通过PCR技术克隆得到了1个长度为915 bp,编码305个氨基酸的MiERF2基因。通过生物信息学分析得知:MiERF2蛋白分子式为C_(1449)H_(2259)N_(433)O_(470)S_(11),分子量为33618.16 Da,总原子数为4622,理论等电点为7.66,带30个正电残基,带29个负电残基,蛋白具有亲水性,结构不稳定;MiERF2蛋白不含信号肽和跨膜区域,磷酸化修饰以苏氨酸为主,包含1个AP2保守结构域;二级结构以66.12%的无规则卷曲为主,还含有24.67%的α-螺旋、1.32%的β-折叠、7.89%的延长链,其三维结构模型包含有3个β-折叠和1个α-螺旋,符合AP2结构域的特征。motif分析和多序列比对结果表明,MiERF2蛋白属于ERF亚家族成员。系统进化分析表明,与MiERF2蛋白亲缘关系最近的是同为漆树科植物的阿月浑子PvERF109-like。洋葱亚细胞定位结果显示,MiERF2蛋白定位于细胞核。实时荧光定量分析表明,MiERF2基因的表达量在不同采收成熟度下通过400μg/mL乙烯利处理后表达情况不同:在6成熟芒果中,处理组MiERF2基因的表达量在9 d达到峰值,之后显著低于对照组;在8成熟芒果中,除18 d以外,对照组中MiERF2基因的表达量在贮藏过程中均显著高于处理组,推测该基因在乙烯调控果实成熟过程中起负调控作用。本研究为揭示ERF转录因子在芒果采后成熟衰老过程中的调控作用提供理论依据。

Mango(Mangifera indica L.)is one of the major cash crops in the tropics and a typical respiratory leap fruit,whose ripening senescence is associated with ethylenee.AP2/ERF transcription factors are widely present in plants,and it is found that the transcription factors are involved in the signal transduction of endogenous ethylene synthesis in fruits,and are key genes in the ethylene signaling pathway,playing an important role in plant growth and development,re-sponse to adversity stress and storage and transportation.To investigate the effect of AP2/ERF transcription factor on post-harvest mango ripening and senescence,a MiERF2 gene of 915 bp in length,encoding 305 amino acids,was cloned by PCR using Guifei mango as the target of this study.The molecular formula of MiERF2 protein was C_(1449)H_(2259)N_(433)O_(470)S_(11).The molecular weight was 33618.16 Da,the total atomic number was 4622.The theoretical isoelectric point was 7.66,with 30 positively charged residues and 29 negatively charged residues.The protein was hy-drophilic and structurally unstable.MiERF2 didn’t contain a signal peptide,didn’t contain a transmembrane region,and phosphorylation modification was mainly threonine.MiERF2 contained a conserved structural domain of AP2,the sec-ondary structure was dominated by 66.12%of random curl,also contained 24.67%ofα-helix,1.32%ofβ-fold,7.89%of the extended chain.Its three-dimensional structure model contained threeβ-fold and oneα-helix,consistent with the characteristics of the AP2 structural domain.The results of motif analysis and multiple sequence matching indicated that MiERF2 protein belonged to the ERF subfamily.Phylogenetic analysis showed that the closest relative to MiERF2 was the pistachio PvERF109-like,which is also a member of the Lacertidae family.Onion subcellular localization results showed that MiERF2 was localized in the nucleus.Quantitative real-time fluorescence analysis showed that the expres-sion of MiERF2 differed after 400μg/mL ethephon treatment at different harvest maturity.In 60%maturity mango,the expression of MiERF2 in the treatment group peaked at 9 d and then was significantly lower than that in the control group.In 80%maturity mango,the expression of MiERF2 in the control group was significantly higher than that in the treatment group during storage,except for 18 d.It is suspected that this gene would play a negative regulatory role in ethylene regulation of fruit ripening.This study would provide a theoretical basis for revealing the regulatory role of ERF transcription factors in postharvest ripening and senescence of mango.