苹果MdAMR1/2在Vc合成中的调控功能及其分子机理研究

Vc is not only an important component of fruit quality, and affects the plant growth and resistance. The understanding on regulatory mechanism of Vc synthesis regulation is the basis of Vc genetic improvement for crop plants, e.g. apple fruits. Based on the early screening of the gene, to know the molecular mechanism and pathway regulating Vc synthesis of MdAMR1/2, as candidate genes which were negatively related with Vc content in apple, in this project, the following work will be done. (1) to further investigate the relationship between the MdAMR1/2 expression and Vc synthesis and stress, and obtain transgenic apple lines via overexpressing and silencing MdAMR1/2 into ‘GL3’ apple. Then we will detect the influence of the expression level of AMR1/2 on Vc synthesis and resistance of stresses (e.g. high light) in apple transgenic lines. (2) the technologies(e.g. Co-IP, Y2H, BiFC, Pull down) will be used to screen and verify the interaction proteins with MdAMR1/2. The candidate proteins will be transformed into apple to confirm the relationship to MdAMR1/2 and the Vc synthesis, and know molecular network on the regulatory Vc synthesis of MdAMR1/2. (3) To ascertain the transcriptional regulatory pathway of MdAMR1/2, the promoters will be separated, and yeast one hybrid and ChIP-PCR will be used to screen and identify their binding transcription factors. The aim of this project is to ascertain molecular mechanism and regulatory pathway in regulating Vc biosynthesis of AMR1/2 in apple, and lay the foundation for understanding the regulation mechanism of Vc synthesis and fruit breeding for Vc content.

Vc不仅是果实品质的重要组成，而且影响着植物的生长发育和抗逆性。探明植物Vc合成调控机制是Vc遗传改良的基础。项目组前期基因筛选和苹果转基因研究发现，两个SCF泛素连接酶家族成员MdAMR1/2与苹果Vc含量呈显著负相关，本项目将进一步分析MdAMR1/2表达特性与Vc合成及逆境胁迫的关系，在苹果中过量和沉默MdAMR1/2的表达，获得转基因株系，分析MdAMR1/2表达改变对苹果Vc合成和光等逆境抗性的影响；利用Co-IP、酵母双杂等技术分析其互做蛋白，筛选验证其互做蛋白并转化苹果愈伤组织分析其与Vc合成的关系，研究MdAMR1/2调控Vc合成的分子网络；同时，分离MdAMR1/2启动子，利用酵母单杂技术筛选其结合的转录因子，以探索MdAMR1/2的转录调控途径。通过研究，将探明MdAMR1/2与苹果Vc合成的关系及其调控途径，为认识Vc合成的调控机制和果实Vc含量改良奠定基础。

水果是人类主要的天然Asc（即Vc）来源，与猕猴桃等果实相比，苹果果实低Asc的原因主要是果实的合成能力弱。本项目研究发现，在基因表达调控上，苹果Asc的合成存在明显的转录后/翻译后调控特性。苹果中拟南芥AtAMR1同源基因MdAMR1L1和MdAMR1L2均含有F-box结构域，在果实中高度表达，与Asc含量呈负相关，且其受光的抑制。苹果果实中VIGS实验结果表明，MdAMR1L1/2的沉默能显著增加果实中Asc含量。在MdAMR1L1/2过量和沉默转基因苹果中，MdAMR1L1过表达显著降低了叶片中的Asc含量，干扰增加Asc含量，但MdAMR1L2过表达对Asc含量无明显影响，这表明MdAMR1L1对苹果Asc含量具有负调控功能。然而，MdAMR1L1过表达并没有降低Asc合成相关基因的mRNA表达，反而增加了MdGMP1、MdGME1等的表达水平，这一结果进一步被RNA-seq数据证实，且Asc正调控相关转录因子MdERF98表达量也显著上调。有趣的是，我们对MdGMP1蛋白表达研究发现，在MdAMR1L1转基因材料中 MdGMP1蛋白表达水平降低，且在MdAMR1L1过表达转基因苹果中GMP活性及其催化产物GDP-Man含量降低。通过CHIP-seq，酵母双杂、双分子荧光互补、免疫共沉淀证实，MdAMR1L1能与MdGMP1互做，且泛素化抑制剂MG132能明显抑制MdAMR1L1过表达转基因苹果中MdGMP1蛋白表达及Asc含量的降低。项目同时证实，MdERF98作为转录因子能正调控MdGMP1 的转录，其表达受到苹果体内Asc含量的反馈调控。过表达MdAMR1L1引起的Asc含量降低上调了MdERF98表达，进而促进MdGMP1基因的转录，而Asc水平的增加又会抑制MdERF98和MdGMP1基因的转录水平。项目研究证实，MdAMR1L1 可通过泛素化翻译后调控MdGMP1蛋白水平，负调控苹果Asc的合成，且MdERF98可响应体内Asc含量的降低，调控着MdGMP1等基因的转录水平，参与了Asc信号的反馈调控。通过项目研究，我们探明苹果AMR1L1/2调控苹果Asc合成的机制及其分子调控途径，深化了人们对植物及果实Asc合成调控机制的认知，获得了新的基因资源和提高Asc含量的新途径，为培育高Asc含量的果实提供了理论方法和资源。