苹果MdMYB32通过自身EAR抑制序列抑制花青苷的生物合成

doi:10.3864/j.issn.0578-1752.2018.24.009

摘要/Abstract

摘要：

目的研究苹果MYB转录因子家族MdMYB32的生物学信息、表达水平及其在花青苷合成中的功能,旨在为进一步完善花青苷合成代谢机理提供参考。方法以新疆红肉苹果杂种一代优系为试材,克隆MdMYB32,分析其进化树和蛋白序列;测定其在不同果实及在不同胁迫处理下的表达水平,通过转基因验证其在花青苷合成中的功能,并通过酵母单杂交分析其互作关系。结果 qRT-PCR分析表明MdMYB32在花青苷含量高的‘红脆9号’苹果中表达水平较低,而在花青苷含量低的‘红脆6号’苹果中表达水平较高,与花青苷含量呈负相关;且盐胁迫和冷胁迫均能抑制MdMYB32的表达;在进化上,MdMYB32与AtMYB32,MdMYB16和AtMYB4在同一个进化枝上,且MdMYB32蛋白序列在C端含有一个EAR抑制序列;在红肉愈伤中过表达MdMYB32能够抑制ANS的表达水平,降低花青苷含量,而过表达切除EAR抑制序列后的LESMdMYB32后,不能明显改变ANS的表达水平和花青苷含量;酵母单杂交和Chip-PCR分析表明MdMYB32和LESMdMYB32均能够结合ANS的启动子。结论 MdMYB32能够结合ANS启动子,并通过自身EAR抑制序列抑制花青苷的生物合成。

关键词: 苹果, MYB转录因子, MdMYB32, EAR抑制序列, 酵母单杂

Abstract:

【Objective】 In order to improve the metabolic mechanism of anthocyanin synthesis, several aspects of apple MdMYB32 in MYB transcription factors were studied, including the bio-informatics, the expression level and the function in the anthocyanin synthesis.【Method】 We cloned the MdMYB32 in Malus sieversii f. neidzwetzkyana F1 population and analyzed its phylogenetic tree and protein sequence. The expression level of MdMYB32 in different apple fruits with different stress treatments was studied. We verified its function in anthocyanin biosynthesis by transgene and analyzed its interaction by yeast one-hybrid.【Result】 qRT-PCR analysis showed that the expression level of MdMYB32 in 'Hongcui No. 9' with high anthocyanin content was lower than that in 'Hongcui No. 6' with low anthocyanin content. The expression level of MdMYB32 was negatively correlated with the content of anthocyanin. Both salt stress and cold stress could inhibit the expression of MdMYB32. The phylogenetic tree indicated that MdMYB32, AtMYB32, MdMYB16 and AtMYB4 were located in the same evolutionary branch, and MdMYB32 protein contained an EAR inhibitory sequence at the C-terminus. Overexpressing MdMYB32 in red-fleshed callus could inhibit the expression of ANS and reduce the anthocyanin content. However, when we overexpressed LESMdMYB32 (knocked out the EAR sequence of MdMYB32) in red-fleshed callus, we found that it could not affect the expression of ANS and the anthocyanin content. Yeast one-hybrid and Chip-PCR analyses showed that MdMYB32 and LESMdMYB32 could bind the promoter of ANS.【Conclusion】 MdMYB32 could bind the promoter of ANS and inhibit the anthocyanin biosynthesis by its own EAR inhibitory sequence.

Key words: apple, MYB transcription factors, MdMYB32, EAR inhibitory sequence, yeast one-hybrid

许海峰,杨官显,王意程,姜生辉,王楠,陈学森. 苹果MdMYB32通过自身EAR抑制序列抑制花青苷的生物合成[J]. 中国农业科学, 2018, 51(24): 4690-4699.

XU HaiFeng,YANG GuanXian,WANG YiCheng,JIANG ShengHui,WANG Nan,CHEN XueSen. Apple MdMYB32 Inhibits the Anthocyanin Biosynthesis by Its Own EAR Inhibitory Sequence[J]. Scientia Agricultura Sinica, 2018, 51(24): 4690-4699.

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