不同结构域MYB转录因子调控梨果实类黄酮合成的三级调控网络解析

MYB transcription factor plays an important role in regulating plant flavonoid secondary metabolism. The pre-project has screened all R2R3-MYB transcription factors from the pear genome and clarified the flavonoid biosynthesis pattern in pear fruits. In the present study, four pear varieties fruits with different flavonoid synthesis mode were used as materials. The first core step in this project is to analyze the sequence features of those MYB genes with possibility in regulating flavonoid biosynthesis and to forecast their target genes and their specific regulated flavonoid metabolism branches. Then through the correlation analysis of the related MYB gene expression patterns and flavonoids synthesis model, the candidate MYB genes involved in the regulation of flavonoid biosynthesis in pear fruits were screened. Furthermore, to verify the mechanism of flavonoid biosynthesis regulatory that MYB transcription factors with specific motifs could selective alter the transcriptional activities of specific flavonoid structural genes and change the quantity of products, the target genes will be tested by yeast one-hybrid experiment and chromatin immunoprecipitation (ChIP) experiment. As the last step of this project, this regulatory network will be finally proved by transient system and VIGS (virus induced gene silencing) system on pear fruits. Parsing the three-tier (Transcription factor-Structural gene-Metabolite) regulatory network of flavonoid biosynthesis in pear fruits could offer theoretical foundation for pear breeding, fruit nutritional quality regulation ,and plant flavonoids biosynthesis and regulation mechanism.

MYB转录因子在植物类黄酮次生代谢的调控中起着重要作用。本项目在前期从梨基因组中筛选出R2R3-MYB转录因子家族与梨果实类黄酮合成模式研究的基础上，以类黄酮合成模式具有典型差异的4个梨品种果实为材料，首先研究相关MYB基因的结构域特征，分析其靶基因与调控的类黄酮支路代谢；然后通过相关MYB基因的表达模式与类黄酮合成模式的相关性分析，筛选参与调控类黄酮合成的候选MYB基因；进一步基于酵母单杂、免疫共沉淀等实验手段验证这些转录因子的靶基因，探明梨果实中具有不同结构域特征的MYB转录因子选择性改变若干靶基因转录活性从而调控产物合成的类黄酮合成调控网络；最后利用VIGS技术与瞬时表达技术在梨果实上加以验证。解析梨果实类黄酮代谢中转录因子-结构基因-代谢产物的三级调控网络，为梨育种和果实营养品质调控研究以及植物类黄酮生物合成与调控机理的深入研究提供理论支撑。

类黄酮化合物是一类重要的植物次生代谢产物，对人体有重要的保健价值。MYB转录因子对植物体内类黄酮的合成、降解和运输途径有关键的调控作用。本研究从梨基因组中筛选出可能参与调控梨果实类黄酮合成的4类18个MYB转录因子，并从中筛选出4个表达模式与梨果实类黄酮合成模式高度相关的MYB基因，同时补充筛选出了PbMYB12b和MYBR类转录因子PbREVEILLE，利用瞬时过表达技术和沉默技术对这些基因的生物学功能在梨果实上进行了验证。. PbMYB10b可以通过提高PbDFR的表达促进原花青素和花青苷的积累，但其沉默后不影响类黄酮类次生代谢产物的积累，其功能具有一定的冗余性；PbMYB9可以通过上调PbANR和PbUFGT1的表达，促进黄酮醇类化合物、原花青素和花青苷的含量增加，而沉默PbMYB9会抑制原花青素、黄酮醇和花青苷的合成；PbMYB3可以正向调控PbFLS和PbMYB10的表达，但其对类黄酮组分含量影响不大；过表达PbMYB12b可以上调PbCHSb和PbFLS的表达，其对于槲皮素糖苷和异鼠李素为主的黄酮醇代谢有正向调控作用；而REVEILLE家族基因，特别是其中的PbRVE1b对于梨果实花青苷的昼夜节律性积累有重要的调控作用。进一步利用酵母单杂和Dual-luciferase等技术，验证了这些MYB转录因子与不同类黄酮代谢支路关键结构基因的互作关系，结果表明PbMYB10和PbMYB9可以和结构基因PbUFGT1的核心启动子区域互作；PbMYB12b对PbCHSb和PbFLS的启动子有较强的激活能力；REVEILLE家族特别是PbRVE1b可以结合并激活PbANS和PbDFR的启动子。. 本研究明确了梨自身果实中类黄酮合成模式与MYB转录因子参与的调控模式，筛选出的MYB转录因子与其调控途径是筛选高含量类黄酮梨品种的重要靶标基因与目标位点，为高含量类黄酮梨品种选育和植物类黄酮合成与调控模式研究提供了理论参考。