野葛异黄酮2-hydroxyisoflavanone糖碳苷合酶基因的分离与功能分析

The C-glycosides of isoflavonoids show various biological functions including an anti-tumor activity. A significant example of an isoflavonoid C-glycoside is puerarin, a compound that contributes to the major medicinal effects of Pueraria lobata. The synthesis of those C-glucosides by a chemical approach costs very much but with an extremely low yield. On the other hand, synthetic biology technique will serve as an alternative way to produce those compounds. However, the prerequisite for this new technology is to understand how the C-glycosyl bond is formed. To date, the gene encoding 2-hydroxyisoflavanone C-glucosyltransferase for the biogenesis of puerarin is not isolated yet. This project will aim to clone the gene coding for the 2-hydroxyisoflavanone C-glucosyltransferase from Pueraria lobata using comparative transcriptomics. For determining the glycosyltransferase candidate genes from the transcriptome data sets, the relevance of the glycosyltransferase gene transcript abundance to puerarin accumulation will be studied. After the selection of the glycosyltransferase candidates, their roles in puerarin biosynthesis will be investigated by silencing their expressions in Pueraria lobata hairy roots or overexpressing them in soybean hairy roots. Once the role of the candidate genes in puerarin biosynthesis is confirmed, the biochemical function of the target glycosyltransferase will be investigated by performing an in vitro enzyme assay with the substrate 2-hydroxyisoflavanone. The successful lunch of this project will not only form a basis for the elucidation of the C-glycosyl bond formation during puerarin biosynthesis but also will provide key genes for producing isoflavone C-glycosides via the synthetic biology technique.

异黄酮糖碳苷具有抗肿瘤等多种生物活性，化学合成糖碳苷成本高、产率低，近期合成生物学技术的发展为异黄酮糖碳苷的制备提供了崭新思路，但该项技术的前提是其糖碳苷合成机制的解析。以异黄酮葛根素为例，其合成路径中的2-hydroxyisoflavanone糖碳苷合酶基因至今未被分离。本项目拟以野葛植物为研究材料，挖掘葛根素合成路径中的2-hydroxyisoflavanone糖碳苷合酶基因；利用反向遗传学与生物化学技术，分别在植物体内与体外对所分离的目标糖碳苷合酶基因的功能展开分析，并拟进一步解析目标糖碳苷合酶调控葛根素合成的分子机制；本项目的成功开展在理论上将为解析异黄酮糖碳苷合成机制提供分子证据、在应用层面上将为运用合成生物学技术制备异黄酮糖碳苷提供关键基因元件。

异黄酮糖碳苷具有抗肿瘤等多种生物活性，人们对于其糖碳苷形成机制的理解较为有限，例如催化异黄酮葛根素合成的主要糖碳苷合酶基因至今仍未得到分离。本项目旨在利用各种技术手段，从葛种植物中大量分离葛根糖基转移酶基因，并对它们的功能进行鉴定，取得了以下研究结果：.1、以实验室前期建立的野葛植物根与叶的比较转录组学文库，筛选出了11个葛根特异性表达的糖基转移酶基因，利用常规RT-PCR技术成功分离了其中7个糖基转移酶基因；.2、利用原核蛋白表达体系，成功获得上述7个糖基转移酶纯化蛋白，并通过体外酶促反应，发现其中3个糖基转移酶(分别被命名为PlUGT4、PLUGT15、PLUGT57)均能催化异黄酮7-O-糖苷键的形成，而其余4个糖基转移酶在体外条件下未能检测到生物活性。基因表达与代谢物积累相关性分析显示PlUGT4、PLUGT15、PLUGT57均与异黄酮糖氧苷合成密切相关。相关科研成果在国际SCI杂志Frontiers in Plant Science上进行了公开发表(Wang et al., Frontiers in Plant Science, 2019, 10:28)；.3、以广西地区野葛与粉葛为研究材料，植物化学分析发现其茎可能为葛根素的主要合成场所，然后由茎运输至根中进行储存。构建野葛与粉葛植物根与茎的比较转录组学文库，从中发现共计276个糖基转移酶基因，通过加权相关性分析了这276个糖基转移酶基因的表达与葛根素积累的相关性，发现了控制葛根素合成的糖基转移酶模块，结合进化树分析，最终筛选出了3个与葛根素生物合成相关性最高的糖基转移酶(分别被命名为PlUGT82、PlUGT83、PLUGT86)。相关科研成果已在SCI杂志Biomolecules上公开发表(Xi et al., Biomolecules, 2023,13(1):170);.4、为进一步探索葛糖基转移酶基因，与广西农科院合作完成了广西粉葛植物的全基因组测序工作，并优化了野葛植物发根遗传转化体系以便在植物体内开展基因功能分析研究。相关成果已在中科院1区杂志Horticulture Research杂志进行了公开发表(Shang et al., Horticulture Research, 2022, 9: uhab035)。