【作者】 吴耀生；

【导师】 舒雨雁；

【作者基本信息】 广西医科大学 ， 药理学， 2008， 博士

【摘要】 三萜类物质是植物界一大类次生代谢产物,广泛分布于各种植物中。自然界中的菌类、蕨类、单子叶、双子叶植物和动物及海洋生物中皆有发现,尤以双子叶植物中分布最多,其中菊科、大戟科、楝科、卫矛科、茜草科、橄榄科、唇形科等植物中更为普遍,具有重要的生物学意义。药用植物中以五加科、豆科、桔梗科、远志科、伞形科、毛茛科三萜皂苷种类、含量十分丰富。三萜皂甙呈现结构多变性及生物活性的多样性,在植物中不仅可参与防御病原体和害虫的生理过程,而且具有广泛的药理作用。一些常用的中草药含丰富的三萜皂甙并作为这些中草药的主要有效成分。这些中草药植物中的皂甙有些具有抗肿瘤、抗病毒、降低胆固醇、提高机体免疫能力等多种生物活性,有些则对心血管系统以及神经系统有重要药理作用,均具有重要的药用商业价值。植物三萜皂苷主要通过MVA的合成,经异戊二烯途径生成鲨烯,2,3氧化鲨烯的环化,产生五环或四环三萜的骨架,再经历各种修饰如氧化、取代和糖苷化,受细胞色素P₄₅₀依赖的单加氧酶、糖苷转移酶及其他酶的催化。随着模式植物拟南芥基因组、水稻基因组研究项目的完成,对植物的功能基因的注释日益增多,涉及植物三萜皂苷生物合成通路的各种酶基因克隆研究逐渐增加。三七（Panax notoginseng）为五加科人参属多年生草本植物,曾为广西特产药用植物之一。目前已从三七分离出20多种达玛烷型三萜皂苷,其中主要成分与人参一致。国内外对三七皂苷已作了广泛的药理研究,证实三七皂苷是三七的主要有效成分,具有很大的开发利用前景。本研究主要目的是对广西特产药用植物三七的三萜生物合成通路做较系统研究,对这一通路所涉及关键酶进行克隆分析,认识其基本特征,从而有利于对一些药用植物有效成分三萜生物合成进行调控,促进三萜有效成分的合成与积累;另一方面通过对植物三萜生物合成通路酶分子聚类分析,从分子水平上为研究植物亲缘关系提供理论依据。本研究在已经获得的植物类异戊二烯及三萜生物合成功能酶基因信息的基础上,利用RT-PCR、3’-RACE及5’-RACE技术,从广西靖西县产的三七根中,克隆了FPS、SS、SE、CAS四个三萜生物合成途径中的关键酶基因cDNA,并得到完整的读码框架。上述基因cDNA序列均已登录NCBI的GenBank。本研究还构建了三七FPS、SE的原核表达载体pET32a（+）-FPS及pET32a（+）-SE,分别成功转化至大肠杆菌BL21（DE3）中。pET32a（+）-FPS能够在转化菌中,经IPTG进行有效的诱导表达,表达的重组蛋白分子量与预计的一致。比对分析表明,三七FPS、SS、SE、CAS读码框架推衍的氨基酸序列与人参相应基因推衍的氨基酸序列一致性分别为:FPS98.8%,SS 98.1%,SE 96.7%,CAS 90.1%。从中也可看出,同为五加科人参属植物的三七与人参在三萜生物合成途径中有关功能酶基因有高度同源性。而且随着30碳的鲨烯形成、氧化、环化,所需的酶分子逐渐增大,FPS有343个氨基酸,SS为415个氨基酸,SE为537个氨基酸,CAS为758个氨基酸。这可能是适应底物分子增大,结构变得更复杂的需要。三七三萜生物合成通路中的酶分子氨基酸序列与人参比对分析看出,越接近这一途径末端,差异也逐渐增加。以三七GAPDH为内参基因,通过克隆得到的三七GAPDH部分cDNA及三七SS基因全长cDNA片段序列构建的重组质粒为标准品,建立了三七SS基因转录表达的实时荧光定量PCR（Real-Time RT-PCR）技术,对一年生三七根、芦头、茎不同部位鲨烯合酶的转录表达进行了分析。同时,建立三七不同部位组织三萜总皂苷含量测定的方法,分别测定三七根、芦头、茎、叶的三萜总皂苷含量。总皂甙含量测定结果表明,一年生三七根、芦头、茎、叶4种组织的总皂甙分别为2.73%、11.86%、1.09%、5.96%。对各组结果进行方差分析,P值均为0.0000,表明各组总皂甙含量的差异均有统计学意义,即任意两组的含量均不同:芦头＞叶＞根＞茎。Real-time RT-PCR研究及统计分析表明,一年生三七根、芦头、茎SS基因的表达有差异（P＜0.05）,根的表达量最高,但尚不能确定茎和芦头SS基因的表达量有否差别。研究结果说明SS基因在三七皂甙生物合成途径中具有一定重要作用,SS基因表达量的高低与皂甙含量存在一定的关系。但要充分说明SS基因的表达与皂甙含量之间的关系仍需做进一步的研究。本研究在较为系统地进行了三七三萜合成通路功能酶基因的克隆测序分析,获得其中四个关键酶FPS、SS、SE、CAS的cDNA克隆后,对已知植物三萜或甾醇生物合成途径关键酶的氨基酸序列进行聚类分析,从而探讨三萜生物合成酶基因的进化规律。聚类分析表明,不论是FPS、SS、SE或CAS,五加科的三七、人参与伞形科的积雪草、柴胡均有更密切的聚类关系,揭示五加科人参属与伞形科积雪草属、柴胡属在遗传上亲缘关系较近。这与植物分类学上五加科与伞形科的亲缘关系是一致的。这在分子水平上研究植物物种的进化过程及亲缘关系提供了新的依据。研究结果进一步证明,在同科属的药用植物中,或三萜成分结构类似的植物中,其三萜类皂苷的生物合成途径功能酶基因有高度同源性,与植物进化过程密切相关,同时也可用于指导发掘结构相似的三萜药用植物资源。更多还原

【Abstract】 Triternpenoids are a structurally diverse class of secondary metabolites in abundance widely existing in plant kingdom with very important biological significance In nature,not only triterpenoids occur in monocotyledons,dicotyledons,but also in fungus,ferns,even in animals and marine life,particularly ubiquitous in dicotyledons, such as Asteraceae,Euphorbiaceae,Meliaceae,Celastraceae,Rubiaceae, Burseraceae,Lamiaceae et al.Medicinal plants from Araliaceae,Fabaceae, Campanulaceae,Polygalaceae,Umbelliferae,Ranunculaceae,are abundant in various different structures of triterpenoid saponins with diversal biological activity. Naturally,these triterpenoid saponins play numerous functional roles in defence pathogen and botanic pest in ecological interations.Triterpenoid saponins are major effective compounds in many medicinal plants,which are responsible for biological activities of anticancer,antivirus,lowering cholesterol,elevating immunity,or exert the important pharmacologic actions on cardiovascular system and nervous system, possessing the considerable pharmaceutical and commerce values.The process of biosynthesis of tritepene saponins in various plant species first involves in the formation of MVA,mevalonic acid,to yield squalene（30C）via the isoprene pathway,then 2,3 oxidosqualene cyclization result in the pentacyclic or tetracyclic triterpene backbone,undergoing several kinds of modification,for example oxidation,substitution and glycosylation catalyzed by monooxygenase, glycosyltransferases and other enzymes dependent on cytochrome P₄₅₀.With the completion of mode plant arabidopsis genome and rice genome projects,the anatations to the functional genes in plant species quickly increase,to lead to gradually the acumulation of various enzyme gene cloning sequences involved in triterpenoid biosynthesis.Panax notoginseng,which belongs to Araliaceae,panax perennial herbs,had been one of special local medicinal plants in Guangxi.About 20 kinds of dammarane type of triterpenoid have been isolated from panax notoginseng that the major components are accordance with those in ginseng.Pharmacological actions on the notoginsenosides have been extentively researched no matter internal or external, confirming that notoginsenosides are the main effective components in panax notoginseng,and presenting a good perspective to be developed and utilized.It is the major aim for the project to research systematically the pathway of triterpenoid biosynthesis in Panax notoginseng from Guangxi special local medicinal plants,to analyze the basic characteristics of the key enzymes involved in this pathway in favor of the regulation for the triterpenoid biosynthesis which are effective components in some medicinal plants and to facilitate the synthesis and accumulation of triterpenoid in some medicinal plants.On the other hand,to supply some theory basis for the research of plant phylogenesis on the molecular level through the clustering analysis of enzyme molecules that have been registered in GenBank related to the triterpenoid biosynthesis in some plants.Base on the information about the functional enzyme genes involved in plant isoprenoid and triterpenoid biosynthesis,the cDNAs cloning of FPS,SS,SE,CAS from root of panax notoginseng grown in Jingxi county,Guangxi,were performed with the RT-PCR,3’-RACE and 5’-RACE technology,respectively and integrite open reading frames for these four gene expression have been obtained.All these cDNA squences have been registered in GenBank of NCBI and released publicly Prokaryote expression vectors of notoginseng FPS and SE have been constructed successfully as pET32a（+）-FPS and pET32a（+）-SE,and transferred into BL21（DE3）, respectively,which can be induced expression by IPTG validly in the host cells and the recombinant protein’s molecular weight expressed are coincidence with the predictation.The alignment results revealed that the identities of the induced amino acid sequences of panax notoginseng FPS,SS,SE,CAS compared with those of ginseng are 98.8%,98.1%,96.7%,90.1%,respectively,which has shownthatthefunctional enzyme genes involved in triterpenoid biosynthesis are high homologous between ginseng and notoginseng.With the formation of squalene（30C）,oxidation,cyclization, modification,the molecular weight of enzymes needed for the pathway become gradually larger,for example FPS containing 343 amino acids,SS 415 amina acids, SE 537 amino acids,CAS 758 amino acids.It is presumed that it is needed because the substrate molecules turn to be larger and their structures become more complex with the process of the pathway in advance.Near to the end of the pathway,the differences of enzymes gradually increase accoding to the alignment analysis involved in triterpenoin biosynthesis in panax notoginseng compared with those in ginseng.Panax notoginseng SS transctrition expression analysis were performed with SYBR Green I Real-Time RT-PCR technology using notoginseng GAPDH as internal control and recombinant plasmid pMT18-GAPDH fragmant and pMT18-SS serve as standard.Transcription expression of SS in root,rootstock and stem of one year growth notoginseng were analyzed.Meanwhile the total amount of notoginsenoside in root,rootstack,stem and leaves tissues were messured,respectivley.The messurement for the total saponin in root,rootstock,stem and leaves tissues are 2.73%、11.86%、1.09%、5.96%in order.ANOVA analysis was performed through comparison of any two of several samples,P=0.000 for each comparison and showed that the differences are distinct statistically,the saponin contents are different for any two groups.Transcription analysis and statistic results reveals that the SS expression level in notoginseng root is higher than in stem and rootstock.The amount of total triterperoids in rootstock is higher than in leaf,root,stem in order but it is still difficult to determine whether SS expression are different between in rootstock and in stem.The results show that SS gene play an important role during the process of notoginseng saponin biosynthesis.Perhaps it involves in the expression differences of other key enzymes after SS on the triterpenoid synthesis pathway or/and the directional transportation and accumulation of triterpenoid in different tissues and organs of plants.It is needed to do further research to thoroughly reveal the relationship between SS expression and notoginsenoside contents.After the cDNA clones of notoginseng FPS,SS,SE and CAS involved in triterpenoid biosynthesis were obtained,phylogenetic tree were constructed with the enzymes registered in GenBank related to triterpenoid saponin or sterol biosynthesis in plant species to probe into the evolution rules of triterpenoid biosynthesis enzymes genes.The results reveal that no matter FPS,SS,SE or CAS,there are more close cluster relationship between ginseng,notoginseng in Araliaceae and Centalla asiatica, Bupleurum chinense in Umbelliferae,suggesting nearer genetic relationship evolutionally between Panax and Cenllata,Bupleurum.This is coincidence with the genetic relationship between Araliaceae,Panax and Umbelliferae,Cenllata, Bupleurum in phytotaxonomy,offerring a new evidence for the research on the evolution process of plant species and genetic relationship on molecular level.It is further certificated that the functional enzyme genes of triterpenoid biosynthesis pathway present quite high homologous in the same family and genus plants or in the plants which contain similar triterpenoid components,correlated to the plant evolution process.It could be used to offer some suggestions for digging out some medicinal plant resource containing triterpenoid structure similar.更多还原