葡萄早熟芽变果实发育表观遗传调控机制研究

A lot of researches have suggested that the development of fruit is regulated by epigenetic genomics. 'Fengzao' is an early ripening bud mutant from 'Kyoho'. It matured 30 d earlier than 'Kyoho', but it is unclear how it mutated. The dynamicl changes of the whole genome DNA methylation states, coding and non-coding RNAs will be analyzed during the different berry development stages of 'Kyoho' and 'Fengzao'. The main candidate differential expression genes (DEGs) will be screened out. Then, the inhibitator of DNA methylation was employed to treat the berry of 'Kyoho', the physiological indexes, methylation states and expression of the DEGs were measured in order to verify the results of epigenetic regulation. Finally, the function of the candidate DEGs was studied through the over-expression and silent-expression. The aims was to explore the regulation relationships between DNA methylation patterns and non-coding RNAs (smallRNA, lncRNA), construct the defined transcriptional regulation network, find the methylation loci which has important influence on the early ripening trait and the structure and function of the corresponding genes, explicit the regulation mechanism of DNA methylation and non-coding RNA on berry ripening and inquire the formation of grape early ripening bud mutant. It will provide the reference for controling fruit ripening through epigenetic methods and theoretic basis for the breeding and improving of grape early ripening.

已有果实成熟突变体的研究表明果实发育受表观遗传调控，‘峰早’是源于‘巨峰’的早熟芽变，成熟提早近30天，但其早熟性状形成机理未知。本项目拟先分析‘峰早’与‘巨峰’果实不同发育阶段全基因组甲基化，lncRNA和small RNA等表观基因组的动态变化，解析葡萄果实发育过程中DNA甲基化与非编码RNA的作用方式和调控网络，筛选出两者间差异表达的候选基因；然后分析DNA甲基化抑制剂（5-氮胞苷）处理‘巨峰’后，其果实发育物候期，果实发育相关生理指标，DNA甲基化状态和差异候选基因表达水平的变化，以确证表观遗传调控因子在葡萄果实发育成熟过程中的作用；并对相关候选差异基因进行瞬时过表达与沉默表达以研究其功能；以期构建完善的葡萄早熟芽变果实发育表观遗传调控网络，探索葡萄早熟芽变形成的分子机理，为通过表观遗传途径调控葡萄成熟期提供参考，为葡萄早熟品种的改良和选育提供理论依据。

峰早是比巨峰成熟早1个月的早熟芽变，但其早熟性状变异相关基因未知。本项目对两者果实发育关键时期的果实进行了全基因组甲基化分析，果实成熟相关lncRNAs和microRNAs对比分析。甲基化分析结果表明两者间甲基化模式和变化趋势不同，峰早由于CHH甲基化水平的增加，胞嘧啶甲基化水平要高于巨峰，同时果实发育早期（蚕豆大小时）的甲基化水平决定着果实成熟进程，F3H和α-葡糖苷酶等为两者间差异甲基化基因，甲基化抑制剂5-azaC处理显著促进了巨峰葡萄提前成熟。鉴定了6个关键的lncRNAs（TCONS_00221683、TCONS_00684459、TCONS_00022149、TCONS_00167247、TCONS_00258125、TCONS_00261813）在两者间差异表达，氧化还原酶活性在其靶基因中显著富集。峰早和巨峰大部分miRNAs表达模式相似，miR399b、miR399e、miR477b-3p等为两者间差异表达的miRNA，构建了调控葡萄果实早熟的miRNAs网络，聚焦在ROS水平的调控上。上述表观组学结果均显示ROS可能与葡萄的早熟有关。我们外施H2O2和核黄素确实促进了巨峰葡萄提早成熟20天。对5-azaC，H2O2和核黄素促进巨峰葡萄早熟的转录组分析均反映出HSP20，GDSL基因为差异表达基因，对HSP20，GDSL和SOD基因进行了葡萄全基因组鉴定，明确了其结构特征和表达特性，进一步筛选出了与葡萄果实早熟有关的关键基因，在巨峰和峰早中克隆了相关基因并比较了两者间的序列差异。对HSP20和GDSL基因的亚细胞定位显示其分别定位于细胞质和细胞膜上，HSP20和GDSL基因启动子上均有有MYB结合位点MRE，启动子功能分析表明HSP20和GDSL基因启动子均具活性。项目结合甲基化，lncRNA和microRNA分析结果，开发了促进葡萄提早成熟的简单有效方法，筛选出了与葡萄早熟有关的关键基因，构建了葡萄早熟基因表达网络，揭示了葡萄果实提早成熟的新机理，为调控葡萄成熟期提供了参考，为葡萄早熟品种的改良和选育提供了理论依据。