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以红肉苹果‘紫红3号’(新疆红肉苹果与‘富士’F_1代)的红色幼嫩叶片为外植体诱导的红色愈伤组织为试材,初步探讨生长素调控苹果花青苷代谢机理。根据拟南芥AtARF3蛋白序列在苹果基因组中Blast比对得到一个生长素信号相关基因(MDP0000173151),暂命名为MdARF3。克隆测序发现该基因的开放阅读框长度为2 127 bp,编码708个氨基酸。进化树分析表明,MdARF3与AtARF3在同一进化支上,推测它们具有相似的功能。愈伤组织在含有0.3 mg·L~(-1) NAA的MS培养基培养2 h之后其MdARF3上调表达,表达量极显著高于无NAA培养基(对照),而CHS、CHI、F3H、DFR、UFGT及LDOX等花青苷合成结构基因的表达量均极显著低于对照,并且MdARF3表达量与培养基中NAA浓度呈显著正相关(相关系数为0.98),与愈伤组织花青苷含量呈显著负相关(相关系数为–0.89),推测MdARF3对培养基中生长素快速做出反应调控花青苷合成;通过原核诱导获得了MdARF3的重组蛋白,为进一步研究MdARF3蛋白在花青苷代谢途径中的功能奠定了基础。
Red calluses induced by explants of red flesh of red apple “Zihong No.3” (Xinjiang red meat apple and ’Fuji’ F_1 generation) were used as materials to study the effect of auxin on apple anthocyanin metabolism mechanism. According to the Arabidopsis AtARF3 protein sequence, an auxin signal-related gene (MDP0000173151) was obtained by Blast comparison in the apple genome and named as MdARF3. The open reading frame of this gene was found to be 2 127 bp, encoding 708 amino acids. Phylogenetic tree analysis showed that MdARF3 and AtARF3 are on the same branch of evolution, suggesting that they have similar functions. MdARF3 was up-regulated in MS medium containing 0.3 mg · L -1 NAA for 2 h, and the expression level of MdARF3 was significantly higher than that in NAA-free medium (control), while the contents of CHS, CHI, F3H, DFR , UFGT and LDOX and other anthocyanin biosynthesis genes were significantly lower than the control, and the expression of MdARF3 and the medium NAA concentration was significantly correlated (correlation coefficient was 0.98), and anthocyanin content of callus (Correlation coefficient of -0.89). It was speculated that MdARF3 could rapidly respond to auxin in culture medium to regulate the synthesis of anthocyanin. MdARF3 recombinant protein was obtained through prokaryotic induction. To further study the effect of MdARF3 on anthocyanin metabolism The function of the way laid the foundation.