“草原海棠”MiGR15启动子片段插入参与调控果实绿熟的分子机制研究

The traits evaluation and utilization of wild apple germplasm resources is significance for apple genetic improvement. Our preliminary results showed that the green-ripe fruit of "Malus ioensis Britton" was closely associated with the fragment insertion in MiGR15 promoter region. On this basis, the chlorophyll content and expression of ethylene response genes of apple peels transient overexpressing or silencing MiGR15 will be measured to identify the role of MiGR15 in regulating the green-ripe fruit. Next, GUS activity assay of apple fruit overexpressing Pinsert::GUS or Ploss::GUS will be determined to identify the effect of insertion squence in promoter region on gene expression. At the same time, the chlorophyll content and gene expression will be detected in the fruits of 35S::MiGR15, Pinsert::MiGR15 or Pdel::MiGR15 transgenic tomato to characterize the effect of insertion squence in promoter region on expression and function of MiGR15. Finally, transgenic calluses overexpressing or scilencing MiGR15 and MiGR15-interacting ethylene receptor together or each alone, are used to clarify the signal transduction mechanism of MiGR15. The project implementation will be of great significance for clarifing the molecular mechanism of green-ripe fruit of "Malus ioensis Britton" and the new variety breeding of green-ripe apple.

野生苹果资源的性状评价和利用对于苹果品种改良具有重要意义。项目组前期研究结果表明，“草原海棠”果实绿熟表型与MiGR15基因启动子片段插入密切相关。在此基础上，本项目拟通过比较过量和沉默MiGR15的病毒载体注射的苹果果皮中叶绿素含量及基因表达，鉴定该基因在调控绿熟中的作用；通过比较P插入::GUS及P缺失::GUS注射的苹果果实GUS染色及酶活性测定，揭示启动子片段插入对基因表达的影响。同时，拟通过比较35S::MiGR15、P插入::MiGR15及P缺失::MiGR15转基因番茄的果实表型、叶绿素含量及基因表达等，鉴定启动子片段插入对MiGR15基因表达及功能的影响；通过单独及共转化MiGR15及与之互作的乙烯受体的转基因愈伤组织的鉴定，明确其信号转导机制。通过本项目的实施，对于解析“草原海棠”果实绿熟性状形成的分子机理及未来绿熟苹果新品种的培育均具有重要意义。

野生苹果资源的性状评价与利用对于苹果品种改良具有重要意义。本项目利用资源圃中果实深绿的“草原海棠”为研究对象，通过比较35S::MdGR15、P插入::MdGR15以及P缺失::MdGR15三种表达载体的转基因番茄植株的表型，对MdGR15的功能进行了鉴定；并通过将其与栽培品种“金冠”进行基因组及转录组比较，成功鉴定了对苹果果实叶绿素积累具有正调控作用的转录因子MdGLK1，该基因过量表达获得了深绿色的番茄果实；利用DAP-seq技术以及转基因苹果愈伤组织的转录组分析对其调控的靶基因进行了鉴定；同时，还发现负调控叶绿素积累的关键酶基因MdSGR1也与“草原海棠”和“金冠”果实色泽的差异密切相关，通过转化番茄进行功能互补对该基因的功能进行了验证；更为重要的是，该基因的启动子区域在两份资源间存在一段AG重复的In/del序列，采用瞬时表达技术证实该In/del序列在调控基因表达中具有重要的调控作用。通过本项目的实施，对于解析“草原海棠”果实绿熟性状形成的分子机理及未来绿熟苹果新品种的培育均具有重要意义。