苹果乙烯响应因子MdERF1B-like的克隆与功能鉴定

Cloning and Functional Characterization of an Ethylene Response Factor MdERF1B-like in Apple

以‘泰山早霞’苹果发育期中未着色和着色的果实为试材,利用q RT-PCR分析花青苷和乙烯通路相关基因的表达。分离出1个乙烯响应因子,暂命名为MdERF1B-like(MDP0000167207),其开放阅读框为690bp,编码229个氨基酸。在‘泰山早霞’苹果着色与未着色果实中,MdERF1B-like表达量变化与花青苷合成基因MdDFR、MdANS和调控基因MdMYB9的表达趋势一致。系统进化树分析表明,MdERF1B-like位于第Ⅸ组,与PyERF1B-like亲缘关系最近。在‘王林’苹果愈伤组织中过表达MdERF1B-like,其花青苷含量显著高于对照。分析MdMYB9启动子序列,其长度为746 bp,序列中含有1个ERF潜在结合元件RAA(TGTTG),酵母单杂表明MdERF1B-like与MdMYB9启动子结合。进一步通过荧光素酶报告试验验证MdERF1B-like促进MdMYB9启动子的转录活性。因此,MdERF1B-like可能通过对MdMYB9的调控来促进苹果花青苷积累。

The expression levels of genes involved in anthocyanin and ethylene biosythesis were analyzed via qRT-PCR using uncolored and colored fruits of‘Taishan Zaoxia’apple at different developmental stages.An ethylene response factor was isolated and tentatively named as MdERF1 B-like(MDP0000167207),with an open reading frame of 690 bp encoding a protein of 229 amino acid residues.The expression level of MdERF1 B-like was consistent with the expression trends of anthocyanin synthesis related genes MdDFR,MdANS and the regulatory gene MdMYB9 in the colored and uncolored‘TaishanZaoxia’fruit.Phylogenic analysis indicated that MdERF1 B-like was located in group IX and was closely related to PyERF1 B-like.Overexpression of MdERF1 B-like in‘Orin’calli led to the significantly higher anthocyanin accumulation than in the control.The MdMYB9 promoter sequence of 746 bp was analyzed,which contained an ERF binding RAA motif in its promoter,and yeast one-hybrid assay showed that MdERF1 B-like bound with MdMYB9 promoter.Furthermore,luciferase reporter assay was used to verify that MdERF1 B-like can promote the transcriptional activity of MdMYB9 promoter.Therefore,MdERF1 B-like may promote the accumulation of anthocyanin through regulating the expression of MdMYB9.