介导蓝莓果实色泽发育调控MIR156/SPLs的鉴定及其作用机理研究

Color development is a significant determinant of fruit quality in blueberry. However, the regulatory mechanisms underlying fruit color development remain largely unknown. MIR156s and their targets, SPL genes, act as regulatory module that plays important roles during plant development and growth. Nevertheless, little is known about their functions in fruit color development, particularly fruit degreening. We propose to systematically identify the MIR156/SPLs modules from the MIR156 and SPL families in blueberry, which are involved in the regulation of fruit coloration and degreening. Subsequently, we will uncover the target genes of the identified MIR156/SPL modules using ChIP-Seq and other molecular and genetic techniques, and further investigate if and how the target genes affect fruit color development. Meanwhile, using approaches combing yeast two-hybrid and other molecular and genetic techniques, the interactors of the identified SPL proteins will be identified, and their roles in regulating fruit color development will be examined. Further, we will investigate how the SPLs and their interactors control the downstream target genes. Last but not least, the upstream regulators of the SPL genes will be explored in response to the regulation of fruit color development. Thus, the outcome of the proposed work is expected to reveal the mechanisms whereby miRNA156/SPLs regulate fruit color development in blueberry. We believe that this study will not only provide novel targets for the “precise improvement” of the coloration trait and quality of blueberry fruits, but may also present a reference framework and testable models for studying fruit color development in other plant species.

果实色泽发育是蓝莓果实品质形成的一个重要环节，然而目前关于蓝莓果实色泽发育调控机理研究还鲜有报道。MIR156及其靶标基因SPL在植物生长发育中发挥着重要的作用，然而关于MIR156/SPL调控果实色泽发育研究还少有报道，尚未见到其在果实褪绿调控方面的报道。本项目将从果实褪绿与着色的角度在蓝莓MIR156和SPL家族中鉴定介导果实色泽发育调控的MIR156/SPLs；然后利用ChIP-Seq与分子遗传等手段鉴定MIR156/SPL的靶标基因并阐释其在果实色泽发育中的功能，同时利用酵母双杂等技术鉴定与SPL互作的色泽发育调控因子并阐释二者对SPL靶标基因协同或拮抗作用机制；最后阐释调控蓝莓SPL响应果实色泽发育的上游因子，从而纵/横向揭示MIR156/SPL对蓝莓果实色泽发育的调控机理。该项目的实施将为针对蓝莓色泽及品质性状进行精准改良提供新思路，同时为在其它物种中展开相关研究提供理论参考。

果实色泽发育是蓝莓果实品质形成的一个重要环节，然而目前关于蓝莓果实色泽发育调控机理研究还鲜有报道。MIR156及其靶标基因SPL在植物生长发育中发挥着重要的作用，然而关于MIR156/SPL调控果实色泽发育研究还少有报道，尚未见到其在果实褪绿调控方面的报道。本项目首先从基因组水平上鉴定了4组介导果实色泽发育调控的MIR156/SPLs作用模块 。随后，利用分子遗传、生物化学、细胞生物学、转录组学、代谢组学等技术手段纵向阐释了多个VcMIR156/VcSPLs调控蓝莓果实色泽发育的作用机理：利用模式植物拟南芥和番茄揭示了VcMIR156a/VcSPL12能够通过调控花青苷和叶绿素积累相关基因的表达，从而影响蓝莓色泽发育（褪绿和着色）的作用模式；利用蓝莓遗传转化体系创制了VcMIR156a和多个VcSPLs的过表达突变体，同时结合蓝莓果实瞬时基因表达和抑制等技术手段，进一步阐释了VcMIR156a/VcSPL12通过直接靶定乙烯合成基因ACO6和ACS1的启动子调控其表达，从而影响乙烯的产生，进而调控蓝莓果实的色泽发育；同时发现VcSPL12可以直接结合到蓝莓叶绿素合成基因VcDVR的启动子、VcSPL13a可以结合叶绿素结合蛋白基因VcLHCB的启动子，从而激活这2个基因的表达，进而促进蓝莓果实叶绿素的积累。最后，利用生物信息学、蛋白-蛋白互作、分子遗传等技术手段鉴定了VcMYBPA1等与VcSPL12互作且参与色泽发育的调控因子，探索了其上游调控因子情况。本项目准确挖掘了蓝莓色泽发育的调控因子从而为针对蓝莓色泽及品质性状进行精准改良提供了新思路，同时为在其它物种中展开果实色泽发育机理研究提供理论参考。目前在本项目的资助下已在国际期刊上发表论文7篇，其中中科院分区1区论文3篇，后期研究成果将陆续发表；培养研究生5名；进行线上线下相结合的国内国际交流，完成了本项目的预期目标。