龙牙楤木三萜皂苷生物合成途径中相关基因的研究
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摘要
龙牙楤木(Aralia elata ( Miq.) Seem)为五加科楤木属的落叶小乔木,传统中草药,根皮、茎皮、叶和嫩芽入药,主要药效成分为齐墩果酸,具有保肝的作用。但龙牙楤木人工栽培周期很长,野生资源完全不能满足临床需求。随着我国提出并实施了“中药基因组计划”,基因工程技术在解决药用植物资源等方面展现出广阔的应用前景。因此,通过生物技术的手段提高龙牙楤木次生代谢产物含量,势在必行。
本研究首先克隆到了龙牙楤木三萜皂苷生物合成途径中的关键酶基因——鲨烯合酶基因,全长1245bp,编码414个氨基酸,将所得序列登录GenBank数据库,登录号为GU354313,生物信息学分析表明SS基因具有完整的开放阅读框,通过比对分析发现龙牙楤木SS基因的氨基酸序列与人参、西洋参、三七、柴胡、甘草和拟南芥同源性分别为95%、91%、96%、83%、89%和72%,同时构建了SS基因的系统进化树。另外生物信息学结果也表明该蛋白为疏水性的膜结合蛋白,本研究又成功构建了原核表达载体pET-28a-AeSS,转化BL21菌株进行原核表达,通过SDS-PAGE和Western blot分析,证明鲨烯合成酶基因在BL21菌株中表达成功。
本研究成功构建了植物表达载体pBI121-AeSS,采用叶盘转化法,利用农杆菌侵染烟草,分子检测初步证明SS基因成功整合进了烟草基因组DNA中;同时成功构建了酵母表达载体pPIC9K-AeSS,采用电转化法转化酵母,通过分子检测证明SS基因成功整合进了酵母基因组DNA中,SDS-PAGE检测分析发现SS基因在毕赤酵母GS115菌株中表达成功。
本研究以根、茎、叶为外植体,设计了60余个培养基诱导愈伤组织的形成,筛选出4个优良培养基,获得了大量愈伤组织,并通过HPLC法测定龙牙楤木侧根、茎、叶、愈伤中的齐墩果酸含量。结果表明:愈伤组织中的齐墩果酸含量最高,茎和叶的含量相近,侧根含量最低。同时还利用Real-time PCR对SS基因在侧根、茎、叶、花中的表达水平进行了分析,分析结果表明:SS基因在茎中的表达量最高,叶次之,侧根最低。HPLC和Real-time PCR的结果表明,SS基因在不同器官表达量的差异与代谢终产物齐墩果酸在该器官的含量有着一定的关联。
Aralia elate ( Miq. ) Seem is deciduous small arbors of Aralia Linn. in Araliaceae. Its root bark, stem bark, leaves and tender bud can be used as traditional herbal medicines. The main medicial composition is oleanane. It is hepatoprotective. But the wild resources cannot completely supply with clinical needs and the period of cultivation is a very long time. With the propose and application in China, "Chinese Genome Project", Genetic engineering shows a great prospect on resources of medicinal plant. Therefore, it is very necessary to improve the content of secondary metabolites in Aralia elate by biotechnology.
The key gene of triterpenoid saponin biosynthesis pathway——Squalene synthase was cloned. Full length is 1245bp and it encodes 414 amino acids. Accession No.is GU354313. Bioinformatics analysis suggested the sequence have full open reading frame. The SS proteins from Aralia elata, Panax ginseng, Panax quinquefolius, Panax notoginseng, Bupleurum chinense, Glycyrrhiza uralensis and Arabidopsis thaliana showed 95%、91%、96%、83%、89% and 72% identities based on amino acid sequences alignment. And phylogenetic trees of gene SS was constructed.It is hydrophobic membrane-bound protein. We constructed the prokaryotic expression vector pET-28a-AeSS. SDS-PAGE and Western blot analysis suggested gene SS is expressed successfully.
We constructed eukaryotes expression vector pBI121-AeSS. The tobacco was infected by leaf disc transformation. Molecular detection proved that gene SS was successfully integrated into the tobacco genome DNA. And we also constructed yeast expression vector pPIC9K-AeSS. The yeast was infected by electroporation methods.. SDS-PAGE analysis gene SS is expressed successfully.
We selected root、stem、leaf and designed more than 60 culture medium to induce the formation of callus. We obtained four good culture mediums. And the content of oleanolic acid of lateral root、stem、leaf and callus were determined by HPLC. The result is that the most content is oleanolic acid in callus. The content of oleanolic acid is similar in stem and leaf. The lateral root is in the least level. We study the expression levels of gene SS in lateral root、stem、leaf and flower by Real-time PCR. The result is that the expression levels of gene SS in stem is the most, secondly leaf, lateral root is the least. The result of HPLC and Real-time RT-PCR suggests: expression differences of different organs have some effect on the content of oleanolic acid of these organs.
本研究首先克隆到了龙牙楤木三萜皂苷生物合成途径中的关键酶基因——鲨烯合酶基因,全长1245bp,编码414个氨基酸,将所得序列登录GenBank数据库,登录号为GU354313,生物信息学分析表明SS基因具有完整的开放阅读框,通过比对分析发现龙牙楤木SS基因的氨基酸序列与人参、西洋参、三七、柴胡、甘草和拟南芥同源性分别为95%、91%、96%、83%、89%和72%,同时构建了SS基因的系统进化树。另外生物信息学结果也表明该蛋白为疏水性的膜结合蛋白,本研究又成功构建了原核表达载体pET-28a-AeSS,转化BL21菌株进行原核表达,通过SDS-PAGE和Western blot分析,证明鲨烯合成酶基因在BL21菌株中表达成功。
本研究成功构建了植物表达载体pBI121-AeSS,采用叶盘转化法,利用农杆菌侵染烟草,分子检测初步证明SS基因成功整合进了烟草基因组DNA中;同时成功构建了酵母表达载体pPIC9K-AeSS,采用电转化法转化酵母,通过分子检测证明SS基因成功整合进了酵母基因组DNA中,SDS-PAGE检测分析发现SS基因在毕赤酵母GS115菌株中表达成功。
本研究以根、茎、叶为外植体,设计了60余个培养基诱导愈伤组织的形成,筛选出4个优良培养基,获得了大量愈伤组织,并通过HPLC法测定龙牙楤木侧根、茎、叶、愈伤中的齐墩果酸含量。结果表明:愈伤组织中的齐墩果酸含量最高,茎和叶的含量相近,侧根含量最低。同时还利用Real-time PCR对SS基因在侧根、茎、叶、花中的表达水平进行了分析,分析结果表明:SS基因在茎中的表达量最高,叶次之,侧根最低。HPLC和Real-time PCR的结果表明,SS基因在不同器官表达量的差异与代谢终产物齐墩果酸在该器官的含量有着一定的关联。
Aralia elate ( Miq. ) Seem is deciduous small arbors of Aralia Linn. in Araliaceae. Its root bark, stem bark, leaves and tender bud can be used as traditional herbal medicines. The main medicial composition is oleanane. It is hepatoprotective. But the wild resources cannot completely supply with clinical needs and the period of cultivation is a very long time. With the propose and application in China, "Chinese Genome Project", Genetic engineering shows a great prospect on resources of medicinal plant. Therefore, it is very necessary to improve the content of secondary metabolites in Aralia elate by biotechnology.
The key gene of triterpenoid saponin biosynthesis pathway——Squalene synthase was cloned. Full length is 1245bp and it encodes 414 amino acids. Accession No.is GU354313. Bioinformatics analysis suggested the sequence have full open reading frame. The SS proteins from Aralia elata, Panax ginseng, Panax quinquefolius, Panax notoginseng, Bupleurum chinense, Glycyrrhiza uralensis and Arabidopsis thaliana showed 95%、91%、96%、83%、89% and 72% identities based on amino acid sequences alignment. And phylogenetic trees of gene SS was constructed.It is hydrophobic membrane-bound protein. We constructed the prokaryotic expression vector pET-28a-AeSS. SDS-PAGE and Western blot analysis suggested gene SS is expressed successfully.
We constructed eukaryotes expression vector pBI121-AeSS. The tobacco was infected by leaf disc transformation. Molecular detection proved that gene SS was successfully integrated into the tobacco genome DNA. And we also constructed yeast expression vector pPIC9K-AeSS. The yeast was infected by electroporation methods.. SDS-PAGE analysis gene SS is expressed successfully.
We selected root、stem、leaf and designed more than 60 culture medium to induce the formation of callus. We obtained four good culture mediums. And the content of oleanolic acid of lateral root、stem、leaf and callus were determined by HPLC. The result is that the most content is oleanolic acid in callus. The content of oleanolic acid is similar in stem and leaf. The lateral root is in the least level. We study the expression levels of gene SS in lateral root、stem、leaf and flower by Real-time PCR. The result is that the expression levels of gene SS in stem is the most, secondly leaf, lateral root is the least. The result of HPLC and Real-time RT-PCR suggests: expression differences of different organs have some effect on the content of oleanolic acid of these organs.
引文
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