红豆杉紫杉烷7-木糖基转移酶基因的功能分析

There are abundent 7-xylosyltaxanes in Taxus, expecially 7-xylosyl-10-deacetyltaxol.Generally, 7-xylosyl-10-deacetyltaxol can reach as high as 0.25% of its dry weight in Taxus.This is more than 10 times higher than that of taxol in Taxus. Generally, 7-xylosyltaxanes can be converted to the anticancer drug taxol via biological or chemical method. However, how 7-xylosyltaxanes are synthesized in Taxus cell and the gene that is reponsible for the formation of 7-xylosyltaxanes has never been studied. On the basis of the preliminary study of our research group, we plan to use the transcriptome data of Taxus chinenesis obtained by Solexa sequencing. The putative xylosy tansferases (XYLT) will be selected as the candidates most likely to be involved in the biosynthesis of 7-xylosyltaxanes. In this study, we will attempt to verify the XYLT enzymes involved in the biosynthesis of 7-xylosyltaxanes by using in vitro and in vivo systems. We will verify the function of XYLT via prokaryotic expression, yeast eukaryotic expression, promoter ananlysis, sub-cellular localization study and amino acid mutation, and find out the specific XYLT gene involved in 7-xylosyltaxanes biosynthesis. Then, we wil study the role of the specific XYLT gene by over-expression, inhibition and cas9 knock-out in Taxus cells by using transgenic approach. This study will not only demonstrate the molecular mechanism of XYLT for 7-xylosyltaxanes formation, but also provide a theoretical basis and target genes for improving greatly the yield of taxol and 7-xylosyltaxanes in Taxus via genetic engineering.

红豆杉富含7-木糖基紫杉烷，特别是7-木糖基-10-去乙酰基紫杉醇可达其干重0.25%，为紫杉醇10倍多。遗憾的是，过去这类化合物被当作废弃物。近年来，人们通过生物和化学法将它们转变成抗癌药紫杉醇等7-羟基紫杉烷，但过程还比较繁琐。如何充分利用它们对于彻底解决紫杉醇药源问题意义重大。7-木糖基紫杉烷是怎样合成的？尤其是参与7-木糖基紫杉烷生物合成（即7-羟基紫杉烷木糖基化）的7-木糖基转移酶基因（XYLT)是什么及其作用机理至今还不清楚。我们拟利用体外（In vitro）表达、启动子分析、亚细胞定位和定点突变等对前期转录组分析所获得的候选XYLT进行功能分析。同时，通过XYLT在红豆杉细胞中（In vivo）的过表达、抑制和敲除试验，阐明7-木糖基紫杉烷生物合成的分子机理，揭示XYLT在7-羟基紫杉烷木糖基化代谢中的重要作用，为大幅度提高紫杉醇和7-木糖基紫杉烷含量提供理论依据和技术支撑。

紫杉醇（taxol）是从红豆杉中分离的一种高效抗癌化合物。紫杉醇等紫杉烷类的C7-木糖基化处于紫杉醇生物合成的分支途径，是影响紫杉醇含量的重要步骤。本项目从红豆杉转录组数据中筛选出12个木糖基转移酶候选基因。生物信息学分析发现它们多为膜蛋白，且富含稀有密码子。qRT-PCR分析结果显示在红豆杉植株中它们的表达具有一定组织特异性。利用原核表达系统进行蛋白表达，采用MBP、SUMO等标签和大肠杆菌Rosetta（DE3）表达菌株得到TcXYLT4、TcXYLT10和TcXYLT12可溶性目的蛋白，体外酶活反应后未检测到目标产物。成功构建了真核表达系统毕赤酵母pPIC9k载体/GS115菌株，但未能获得目的蛋白。利用农杆菌介导法获得了TcXYLT1、TcXYLT2、TcXYLT4、TcXYLT5、TcXYLT6和TcXYLT10的84K杨转基因植株，并且进行了TcXYLT4和TcXYLT5在烟草中的瞬时表达，结果表明它们在异源植物中的蛋白表达量均较低。以拟南芥的糖基转移酶为检索序列，从红豆杉中鉴定出65个尿苷二磷酸糖基转移酶。用目标底物10-去乙酰基紫杉醇（10-Deacetyltaxol, DAT）或目标产物7-木糖基-10-去乙酰基紫杉醇（7-Xylosyl-10-deacetyltaxol, 7-XDT）分别处理红豆杉悬浮细胞，进行转录组测序筛选到在目标底物DAT处理条件下表达量显著上调，而在目标产物7-XDT处理条件下显著下调的5个糖基转移酶基因：TmUGT34、TmUGT9、TmGT1、TmGT2和TmGT3，其中TmUGT34差异最大。在大肠杆菌Rosetta（DE3）菌株和84K杨中进行了TmUGT34的表达。84K杨中TmUGT34在转录水平高表达，仅在大肠杆菌获得可溶性目的蛋白，体外酶活反应未能检测到目标活性。利用农杆菌介导的真空抽滤—瞬时转化红豆杉小苗的检测发现，过表达TmUGT34基因能够显著提高植株体内7-木糖基紫杉醇和7-木糖基-10-去乙酰基紫杉醇的含量。这是首次在红豆杉中鉴定出紫杉烷7-木糖基转移酶。最后，对作用于红豆杉紫杉烷7-木糖基转移酶基因即TmUGT34的转录因子进行了分析，这些研究结果为该基因的定向调控奠定了基础，对于提高红豆杉紫杉醇的含量、降低紫杉醇的生产成本具有重要意义和应用价值。