甜叶菊UDP-糖基转移酶基因启动子克隆及功能分析

Steviol glycosides(SGs) is a kind of low-calorie and high intensity natural sweeteners, which mainly contain stevioside (St) and rebaudioside A (RA) , accounting for 60% -70% and 15% - 20% respectively. Compared with other SGs, RA is the sweetest component, and pure sweetness. Improve rebaudioside A in stevia leaves is an important direction for breeding. The project around stevioside biosynthesis of UDP-glycosyltransferase (UDP-glycosyltransferases, UGTs) gene regulation research. Promoter cloning from genomic DNA UGT76G1, UGT85G2, and its deletion fragments with different GUS gene fusion construct recombinant plasmids, then use rhizogenes-mediated into arabidopsis. To explore these promoters drive expression of the GUS reporter gene by transient and stable expression in arabidopsis leaves, and thus the promoter function and domain research, analytical its stevioside synthetic molecular mechanism , is directed to improve the foundation of stevia AR content.Optional the sequence of promoter by the UGT85C2 and UGT76G1 promoter expression patterns， then construct the new vectors by artificial promoter. To validate the feasibility of this vector for the stevia metabolic regulations，then transforming the vector into stevia. It may establish the foundation for the further research on stevia genetic engineering.

甜菊醇糖苷是天然高倍甜味剂，以甜菊糖苷（stevioside，St）和莱鲍迪A（rebaudioside A，RA）含量最高，分别占为60%-70%和15%-20%。RA比其它糖苷甜度更高、甜味纯正，提高甜叶菊叶片的RA含量是育种的重要方向。本项目围绕甜菊醇糖苷生物合成过程中的UDP-糖基转移酶（UDP-glycosyltransferases，UGTs）基因启动子开展研究。从甜叶菊克隆 UGT76G1、UGT85G2基因启动子，将全长或不同缺失片段的启动子与 GUS 基因构建成重组质粒，利用发根农杆菌介导进入拟南芥。通过拟南芥探究这些启动子表达模式，对其功能和结构进行研究，解析其在甜叶菊发育期间甜菊糖苷合成的分子调控机制，为定向提高甜叶菊RA含量打下基础。根据启动子表达模式，优化启动子序列，构建人工启动子，改造现有载体，验证该载体在甜叶菊代谢调控中的可行性，为甜叶菊基因工程研究奠定基础。

研究基于甜叶菊甜菊醇糖苷合成相关基因UGT76G1、UGT85G2启动子序列和其片段在拟南芥和甜叶菊的表达情况。其主要内容包括：克隆甜菊醇糖苷合成相关基因UGT76G1、UGT85G2启动子序列；构建含GUS表达载体，导入拟南芥，分析其在拟南芥中的表达模式；分析光照时间、激素等处理对其在转基因拟南芥表达的影响，分析启动子调控序列。构建人工启动子，将其通过质粒分别与UGT85G2、UGT76G1基因连接后转化甜叶菊，检测验证转基因材料甜叶菊叶片中甜菊醇糖苷含量、基因表达量及田间形状变化，分析优化后启动子序列是否有效。获得以下成果：克隆UGT76G1和UGT85C2启动子片段，预测其可能的调控元件，构建表达载体，从分子水平上分析其表达模式，根据结果获得人工启动子。为解析相关基因在甜叶菊叶片发育期间甜菊糖苷合成的分子调控机制，定向提高甜叶菊RA含量打下一定的基础。