倍半萜药用化合物苍耳素生物合成关键P450基因的分离与功能分析

As a sesquiterpene,xanthatin has been reported to show great anti-tumor activities.The α-methylene-γ-lactone ring of xanthatin was identified as the essential moiety for the anticancer activity. Until now, the gene encoding the key P450 involved in the formation of the lactone ring of xanthatin has not been isolated. In this study, we will undertake the molecular cloning and functional analysis of the key P450 genes in xanthatin biosynthesis from the isolated glandular trichomes of Xanthium sibiricum. In the preliminary experiments of this project, the P450 genes, Xs8H1, Xs8H2, and Xs8H3, have been isolated from the isolated glandular trichomes of Xanthium sibiricum. Based on the biosynthesis of other sesquiterpenoids, we suggested that Xs8H genes might be responsible for the formation of the lactone ring of xanthatin. The correlation of the transcript level patterns of Xs8H1, Xs8H2, and Xs8H3 to the biosynthesis of xanthatin will be analyzed at the different developmental stages of Xanthium sibiricum using qRT-PCRs and HPLC analysis. RACE-PCR (Rapid Amplificatioin of cDNA Ends) technique will be applied to isolate the full-length cDNAs of Xs8H1, Xs8H2, and Xs8H3. The biochemical properties of Xs8H enzymes will be determined in a yeast expression system through transferring their full-length cDNAs into yeast cells. The functions of the genes will be investigated by regulating their transcript levels in Xanthium sibiricum using RNAi and overexpression techniques. The success of this project will provide the key gene resources for the regulations of xanthatin biosynthesis in Xanthium sibiricum or the biosynthesis of xanthatin using synthetic biology strategy, which will be important for the large-scale production of xanthatin.

倍半萜化合物苍耳素具显著抗肿瘤功效, 其分子中的内酯环为主要活性基团, 而控制该内酯环形成的关键P450基因至今未被克隆, 本项目拟以分离的苍耳腺体细胞为起始研究材料, 克隆此关键P450基因并对其功能进行分析。围绕前期从苍耳腺体细胞中分离出的Xs8H1﹑Xs8H2与Xs8H3基因, 利用qRT-PCR与HPLC分析技术, 研究Xs8H成员基因在苍耳植物不同发育时期的表达模式, 关联分析Xs8H成员基因的表达与苍耳素生物合成之间的相关性; 利用RACE-PCR技术, 分离出Xs8H1﹑Xs8H2与Xs8H3基因的全长cDNAs, 借助酵母表达系统对Xs8H蛋白的生化功能进行鉴定; 利用RNA干扰与基因过表达技术, 在苍耳植物体内对Xs8H成员基因的功能进行分析。本项目的研究成果将为调控苍耳素的生物合成或为苍耳素的合成生物学研究提供必需的基因资源, 进而将促进苍耳素的产业化规模发展。

倍半萜化合物苍耳素具显著抗肿瘤功效，其分子中的内酯环为主要活性基团，该内酯环生物合成机制至今未被解析，本项目旨在解析苍耳素分子内酯环形成分子机理，取得了以下研究结果：..1、结合植物化学以及电镜扫描技术，发现了苍耳素主要在植物叶片表层的腺体细胞中合成、明确了苍耳植物开花前期腺体细胞最为丰富，相关结果已发表在国际SCI杂志Molecules上(Li et al., Molecules, 2014, 19, 12898-12908)；..2、建立了苍耳腺体细胞转录组文库（苍耳腺体细胞转录组原始数据已提交至NCBI公共数据平台，编号为SRP056511）， 发现了可能控制苍耳素内酯环形成的4个关键P450氧化酶基因(Xs8H1-4)，相关成果在国际SCI杂志Plant and cell physiology (Li et al., Plant and cell physiology, 2016, 57(3):630-641 ) 以及Frontiers in Plant Science (Li et al., Frontiers in Plant Science，2016, 7: 1317)上进行了发表；..3、项目组将这些候选P450酶基因在酵母细胞中进行了诱导表达，并以人工制备的Germacrene A acid (GAA) 为底物，在体外进行了生物化学功能分析，发现了Xs8H2(该基因被国际P450命名委员会重新命名为CYP71BL6)能够转化GAA生成了一个疑似内酯环化的产物；利用高效液相制备技术，将上述产物进行了分离纯化，并与复旦大学唐慧儒教授课题组合作，对该产物进行了核磁共振结构解析研究，结果发现该产物确实为一种内酯环，确认为12,8α内酯环，该结构与苍耳素内酯环空间结构一致；另外，项目组在体外模拟体内情况，对内酯环形成机理进行仿生实验，成功解析了内酯环的形成机理-即在底物GAA的C8位置进行羟基化，C8-OH随即攻击C12位置羧基，在不需任何生化酶的情况下即形成内酯，相关成果发表在植物学权威杂志The Plant Journal 上(Gou et al., the Plant Journal, 2018, 93: 92-106)。