香榧中不存在紫杉醇的化学及分子生物学证据  

Chemical and Molecular Biological Evidence of No Taxol in Torreya grandis Fort. ex Lindl. cv. Merrillii

作　　者：王帅[1] 赵中槐 董清华[3] 邵芬娟[1] 芦强[1] 邱德有[1] 

机构地区：[1]中国林业科学研究院林业研究所,北京100091 [2]浙新香榧专业合作社,新昌312500 [3]北京农学院植物科学系,北京102206

出　　处：《基因组学与应用生物学》2016年第11期3172-3177,共6页Genomics and Applied Biology

基　　金：中国林业科学研究院林业研究所中央级公益性科研院所基本科研业务费专项(RIF2014-01)资助

摘　　要：香榧(Torreya grandis Fort.ex Lindl.cv.Merrillii)是红豆杉科榧树属的一个种。由于香榧与红豆杉属植物具有较近的亲缘关系,研究者认为香榧也可以合成紫杉醇,并利用HPLC法验证其中含有紫杉醇。但他们仅仅依靠HPLC中出峰时间来判断,并没有质谱的分析结果。本研究利用LC-MS对香榧茎叶的化学提取物进行分析发现香榧并不能合成紫杉醇。为了进一步证明香榧不能合成紫杉醇,我们利用欧洲红豆杉(Taxus baccata L.)紫杉醇生物合成关键基因-紫杉二烯合成酶基因(Taxadiene synthase gene,Tb TS)TBLASTN比对香榧本地转录组,从中找出并克隆得到与Tb TS同源性最高的Tgr TSL1基因。利用原核表达对Tgr TSL1基因进行功能分析,发现该基因不具有紫杉二烯合成酶编码基因的功能。这样,我们从化学和生物学两个方面对香榧无法合成紫杉醇这个结论进行了证明。这为以后通过植物学和化学分类学等研究穗花杉的分类学地位提供了新的有用信息。Torreya grandis Fort. ex Lindl. cv. Merrillii is one member of Torreya in Taxaceae. Due to the close genetic relationship between Torreya grandis and Taxus spp., some researchers thought that taxol could be synthesized in T. grandis and testified this hypothesis using HPLC method. This conclusion was just reached basing on the retention time of the peak in the extract of Torreya grandis corresponding to that of the taxol standard, but not from any mass spectra data. In this study, we checked the extraction of the stems and leaves of Torreya grandis with LC-MS, and found that there was no taxol in the extraction. In order to testify our conclusion, TgrTSL1 gene, which had the highest homology with TbTS, was isolated and cloned by using the taxadiene synthase of Taxus baccata to TBLASTN the transcriptome of T. grandis. Functional analysis of TgrTSL1 with prokaryotic expression showed that the TgrTSL1 protein cannot catalyze GGPP （geranylgeranyl pyrophosphate） to taxadiene, key intermedia in taxol biosynthesis pathway. Taking together, our chemical and molecular biological evidence proved that that T. grandis cannot product taxol. Our results will be helpful to the research work on the taxonomic study of T. grandis by botany and chemotaxonomy.

关 键 词：香榧 紫杉醇 紫杉二烯合成酶 LC—MS 原核表达 

分 类 号：S664.5[农业科学—果树学]