柑橘类柠檬苦素生物合成相关基因CiOSC的转录调控机制研究

Limonoids are important secondary metabolites with various bioactive functions, such as anticancer, antimicrobial, antioxidant and insecticidal capacities. Exploitation of limonoids from citrus is of great value for medical care and increasing economic benefits for citrus processing industry. However, the genetic mechanisms involved in the biosynthesis of limonoids in citrus have not been reported. We have previously demonstrated that Citrus oxidosqualene cyclase gene-CiOSC plays a crucial role in the biosynthesis of limonoids..The citrus germplasm with high and low limonoids, the mutant with rare content and the transgenic lines of CiOSC knock-out will be used for transcriptome sequencing analysis. Co-expression network for limonoids regulation will be constructed via `Weighted gene co-expression network analysis (WGCNA). The transcription factors (TFs) were fished by the techniques of yeast one-hybrid system (Y1H). Based on the results of transcriptome and Y1H, TFs clearly related to the regulation of CiOSC gene will be selected. The candidate genes were verified by the tobacco rattle virus mediated virus-induced gene silencing (TRV-mediated VIGS), CRISPR/Cas9 editing system and gene over-expression..In order to clarify the mechanism of TFs regulating the CiOSC gene, the binding cis-element of TFs will be validated by dual-luciferase assay (LUC), electrophoretic mobility shift assay (EMSA) (in vitro), and chromatin immunoprecipitation (CHIP) (in vivo). .This research will unveil the molecular mechanism of TFs on regulating the CiOSC gene, toward further identification of the key genes on the pathway of limonoids biosynthesis. The outcome of the study will lay the foundations for understanding the genetic control of limonoids biosynthesis in citrus, and provide the information for genetic improvement of citrus with high limonoids productivity.

柑橘类柠檬苦素是一类有益人体健康的次生代谢产物，提高其产量对提升柑橘的经济效益有重大意义。目前对类柠檬苦素生物合成的遗传调控机理研究较少。本课题组在前期的研究中发现，氧化鲨烯环化酶基因-CiOSC是调控类柠檬苦素生物合成的一个重要基因。本研究利用类柠檬苦素含量差异显著的种质及CiOSC基因敲除株系进行转录组测序，分析与类柠檬苦素生物合成相关的调控网络。结合酵母单杂交鉴定出调控CiOSC基因的转录因子。通过瞬时表达、基因敲除和过表达等技术验证基因功能；利用荧光素酶系统、凝胶阻滞实验、CHIP等鉴定转录因子结合的顺式作用元件；结合对转基因株系代谢组的精准分析，研究调控CiOSC基因对类柠檬苦素生物合成的影响，阐明转录因子调控CiOSC基因的机制，并通过基因调控网络研究，鉴定与类柠檬苦素生物合成相关的重要基因，为阐明类柠檬苦素生物合成的调控机理，通过遗传改良有效控制类柠檬苦素的生物合成奠定基础。

柑橘类水果富含对人体健康有益的物质。其中，类柠檬苦素是一类具有抗癌、降低胆固醇、抗病毒和抗虫等多种生物活性的次生代谢产物，提高其产量对提升柑橘的经济效益有重大意义。目前对类柠檬苦素生物合成的遗传调控机理研究较少。本课题组在前期的研究中发现，氧化鲨烯环化酶基因-CiOSC是调控类柠檬苦素生物合成的一个重要基因。本研究利用类柠檬苦素含量差异显著的种质及CiOSC基因敲除株系进行转录组测序，通过权重基因共表达网络分析，找到与类柠檬苦素显著相关的两个模块，筛选出10个转录因子作为候选基因。基于已发表的柑橘基因组克隆到CiOSC基因启动子，利用酵母单杂交未筛选到候选转录因子，参考本课题组组装的染色体级别柑橘基因组克隆CiOSC基因启动子发现，该启动子与已发表的9个柑橘基因组的序列相似度为70%左右，显示该启动子序列在已发表的柑橘基因组中存在组装错误问题。目前正在筛选结合CiOSC基因启动子的转录因子。同时正在对10个转录因子进行柑橘遗传转化。已获得过量表达AP2转录因子的转基因株系的类柠檬苦素含量为对照的1.14~1.95倍，基因表达水平为对照的2.5~156.26倍。类柠檬苦素含量和基因表达水平之间达显著正相关，相关系数为0.972。结果表明，AP2是一个正调控类柠檬苦素的基因，正在验证其是否直接结合CiOSC基因启动子。在柑橘或者体外合成类柠檬苦素时，AP2可能是一个潜在调控类柠檬苦素含量的转录因子。