罗汉果甜苷V生物合成关键酶-葡萄糖基转移酶的功能分析

Siraitia grosvenorii is a medicinal plant native to southern China. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 425 times sweeter than sucrose. So it depends totally on cultivation, narrow distribution, serious continuous cropping obstacle, and low content, which lead to high extraction costs. The traditional breeding method is too time-consuming and difficulty to improve the mogroside V content. Therefore, it has a great significance that exploring the biosynthetic pathway and regulatory mechanism and improving the productive efficiency of mogrosides from the gene level. On the basis of preliminary study of our research group, we plan to analysis the data involved in transcriptome and digital gene expression profiles (DGE) of S. grosvenorii by Solexa sequencing. The combination analysis of RNA-seq and DGE using are performed on three important stages of fruit development, and based on their expression pattern, five UDP-glucosyltransferases (UDPG) were selected as the candidates most likely to be involved in mogrosides biosynthesis. In this study, we attempt to explore new ways to improve the content of mogrosides by verifying and regulating of key enzymes involved in its biosynthetic pathway using in vitro and in vivo methods. We verify the function of five UDPGs by prokaryotic expression and yeast eukaryotic expression in vitro, and find out the specific UDPG enzyme involved in mogroside V biosynthesis. Then, we verify the function of the specific UDPG gene by over-expression and RNAi-intefere using transgenic system in vivo. Simultaneously, we clone the promoters and enhancers of UDPG and analysis the accurate sub-cellular localization. This study will not only demonstrate the biosynthetic mechanism of UDPGs for mogroside V, but also provide a theoretical basis and target genes for S. grosvenorii in transgenic breeding and genetic engineering.

罗汉果是我国特有的药用和甜料植物，具有重要的研究价值。但罗汉果完全依赖于栽培，适生区狭窄，连作障碍严重，加之含量低导致甜苷V生产成本居高不下。传统育种周期长、提高含量潜力有限。从分子水平阐明甜苷V生物合成途径，实现皂苷合成关键酶的基因调控对于提高甜苷V含量、降低生产成本具有重大意义。本项目以甜苷V合成最后一步关键酶-葡萄糖基转移酶（UDPG）为研究对象，通过关联分析找到5条候选的UDPG，利用反向遗传学手段通过体外（In vitro）和体内（In vivo）两种方法对其进行功能验证。体外通过原核表达和酵母真核表达验证基因功能，确定特异催化甜苷V合成的UDPG。在此基础上，通过体内转基因对UDPG进行过表达和基因沉默，同时结合启动子、增强子克隆及UDPG基因的亚细胞精细定位，最终阐明UDPG基因在甜苷V生物合成中的分子催化机制，为培育高甜苷V含量的罗汉果新品系和实现甜苷V的基因调控奠定基础。

罗汉果是我国独有的药用和甜料植物，其有效成分甜苷V具有很好的药理药效，应用前景广阔。但甜苷V含量低、生产成本高的问题严重制约着产业的发展，提高甜苷V含量的研究已成为热点。通过传统栽培和育种，周期长、效率低，从分子水平阐明甜苷V生物合成途径，找到控制甜苷V合成的关键酶基因-葡萄糖基转移酶，利用基因工程或发酵工程实现转基因超表达或高效生物转化，是提高甜苷V含量、降低生产成本的重要途径。本项目前期完成了罗汉果转录组及表达谱高通量测序，通过“RNA-Seq+DGE+含量”共表达模式筛选出5条候选UDPG基因开展功能验证。通过研究，采用RACE技术克隆了5条基因全长：SgUDPG1、SgUDPG2、SgUDPG4、SgUDPG6、SgUDPG7；分析了6条看家基因在罗汉果不同发育期果实、不同组织部位和不同倍性果实中表达稳定性，筛选出18S为表达稳定的看家基因；以表达稳定的看家基因18S为对照，采用Real-time PCR分析了5条葡萄糖基转移酶在根、茎、叶、花和10 d、20 d、30 d、40 d、50 d、60 d果实中表达规律，初步确定SgUDPG1和SgUDPG4可能参与甜苷V的生物合成；构建了SgUDPG1、SgUDPG2、SgUDPG4、SgUDPG6、SgUDPG7等 5个原核表达载体，转化到大肠杆菌中，经IPTG诱导后，经SDS-PAGE鉴定和Western blotting分析表明获得了目的可溶性基因重组蛋白；构建了5条UDPG的酵母表达载体，以UDP-葡萄糖为糖基供体，罗汉果醇、罗汉果苷IIE、罗汉果苷III和黄酮苷元等为糖基受体，进行了基因功能鉴定的体外催化实验，经LC-MS分析功能催化产物，发现SgUDPG1能够催化罗汉果醇合成罗汉果苷IE，同时也能催化槲皮素加上葡萄糖基，因此SgUDPG1为广泛底物性的罗汉果葡萄糖基转移酶。此外，SgUDPG2和SgUDPG7也为黄酮类糖基转移酶。对SgUDPG1进行了亚细胞定位，该基因定位于细胞质和细胞核中。最后，优化了罗汉果遗传转化体系，构建了SgUDPG1基因的过表达和RNAi干扰载体，通过转化到罗汉果中，最终获得3株转SgUDPG1基因过表达阳性植株。这些研究结果为加快罗汉果分子育种进程、实现甜苷V基因调控和推动罗汉果产业的发展奠定了优良的基础。