何首乌中芪合酶、白藜芦醇羟化酶基因的功能研究

2, 3, 5, 4’-tetrahydroxy-stilbene-2-O-β-D-glucoside(THSG) is a characteristic active pharmacological compound in Fallopia multiflora. It has been reported that resveratrol and other trihydroxy stilbenes are synthesized through phenylalanine pathways and the last step of which is catalyzed by stilbene synthase. However, THSG is a kind of tetrahydroxy stilbene and its biosynthetic pathway has not been reported. In our previous study, de novo transcriptome assembly and digital gene expression profiling analyses were performed on Fallopia multiflora using the RNA-seq system, while 2 stilbene synthase genes and 4 resveratrol hydroxylase gene involved in phenylalanine pathways were screened, so we speculate that THSG is synthesized based on resveratrol through hydroxylation and glycosylation. To confirm this speculation, in this study, stilbene synthase genes and resveratrol hydroxylase genes will be transformed into Arabidopsis thaliana and Fallopia multiflora, HPLC and LC-MS/MS will be performed to detect the expression products, and then the genes will be knocked out through CRISPR/Cas9 technology to confirm their function in TSHG synthesis. This study will help to illuminate the biosynthesis pathway of TSHG in Fallopia multiflora.

二苯乙烯苷是我国传统中药材何首乌特有的药效成分。已有研究表明白藜芦醇等三羟基芪类是经苯丙氨酸途径由芪合酶催化合成，而二苯乙烯苷为四羟基芪类，是否经相同的途径合成至今还未见报道。本课题组前期研究通过RNA-Seq技术对何首乌块根进行了转录组测序和数字基因表达谱分析，筛选到苯丙氨酸代谢途径中的2个芪合酶基因和4个白藜芦醇羟化酶基因片段，推测何首乌中二苯乙烯苷的合成可能是由芪合酶催化合成白藜芦醇，后经羟基化和糖苷化修饰而来。为了证实这一推测，本项目首先将芪合酶基因和白藜芦醇羟化酶基因转化拟南芥和何首乌进行超表达，HPLC以及LC-MS/MS检测转基因植株中芪类物质的类型和含量变化，然后通过CRISPR/Cas9技术敲除何首乌中的芪合酶、白藜芦醇羟化酶基因，根据基因敲除后芪类物质的变化，确认何首乌中芪合酶、白藜芦醇羟化酶基因在二苯乙烯苷合成途径中的功能，进而揭示何首乌中二苯乙烯苷的次生代谢途径。

二苯乙烯苷是我国传统中药材何首乌主要的药效成分，但作为次生代谢产物，其在何首乌中的含量较低。二苯乙烯苷属于芪类物质，已有研究表明，植物中芪类物质的生物合成途径是经苯丙氨酸途径由关键酶芪合酶催化合成。为了研究何首乌中二苯乙烯苷的生物合成途径，我们首先克隆了何首乌中的芪合酶基因FmSTS1和4个白藜芦醇羟化酶基因，荧光定量PCR研究证实芪合酶和白藜芦醇羟化酶基因在何首乌块根和老藤中表达量较高，而在幼嫩的茎和叶中含量较低，这种组织表达特性与经HPLC检测的何首乌中二苯乙烯苷的含量分布规律一致。拟南芥本身具有苯丙氨酸途径，但因为不含有芪合酶从而不能产生芪类物质，本研究将芪合酶基因FmSTS1转化拟南芥，经HPLC和质谱分析，转基因拟南芥中有芪类物质白藜芦醇苷的积累，证实了我们克隆的芪合酶能够直接催化合成芪类物质。将芪合酶基因和白藜芦醇羟化酶基因通过2A多肽串联表达后，在拟南芥中的表达产物仍然为白藜芦醇苷，推测原因可能是2A多肽甚至拟南芥中内源羟化酶的存在影响了外源羟化酶基因的表达。进一步将芪合酶基因FmSTS1转化何首乌毛状根进行超表达，结果超表达组的二苯乙烯苷含量提高，通过RNA干扰将芪合酶基因FmSTS1进行抑制后，二苯乙烯苷的含量明显降低，这都说明芪合酶给基因FmSTS1在何首乌二苯乙烯苷的生物合成中具有重要的作用。