茉莉酸甲酯诱导的人参发根培养及SSH文库构建研究
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摘要
人参在中国有着几千年的药用历史,作为名贵药材和补品,在中医学及其他国家民族医学中一直占有重要地位。由于人参生长对环境的要求高,目前普遍采用伐林栽参方式进行人工栽培,对林地破坏严重,环境代价高昂,寻求替代资源成为解决这个难题的一个有效途径。人参发根由发根农杆菌(本实验采用A4菌株)侵染人参根外植体转化而成,属非天然植物。与传统的植物组织培养、细胞悬浮培养相比,植物发根具有生长速度快、活性物质含量高和遗传性状稳定等特点。人参发根培养目的之一就是要获得相对含量较高的人参皂苷,实现人参皂苷工厂化生产。发根中次级代谢产物含量受培养条件、营养因子、前体物和诱导子等因素影响,茉莉酸甲酯作为一种植物信号分子,能够促进次级代谢产物积累,提高发根中人参总皂苷的含量。随着生物工程的日益发展,应用代谢工程手段改变植物细胞生物合成途径,提高次级代谢产物产量已经逐渐成为研究热点。
本文对人参发根三角瓶摇床培养进行考察,通过添加不同浓度的茉莉酸甲酯进行诱导,最终确定浓度在200μmol·L-1时效果最好,在一个培养周期内人参发根生物量增长13.82倍,人参发根总皂苷含量达到2.28%,比对照高35.71%。同时,我们使用实验室自行设计研制的发酵罐进行了人参发根培养实验,结果表明,人参发根在发酵罐中培养30天,生物量平均增长达到23.14倍,人参发根总皂苷含量达到2.04%,人参皂苷单体Rb1、Rd的含量比对照有较大提高,分别提高55.0%和30.1%。人参发根生长迅速,无需添加外源激素,次级代谢产物含量高,茉莉酸甲酯作为诱导子对于提高人参皂苷含量有明显的促进作用,同时表明经改进的发酵罐能够满足人参发根生长需要,有利于进行扩大培养。
人参发根次级代谢产物人参皂苷的种类和含量的差异与生物合成途径中相关基因差异表达调控有关。为研究这些差异表达基因,本实验利用抑制消减杂交技术构建了以茉莉酸甲酯诱导的人参发根为检测子、以对照人参发根为驱动子的抑制消减文库(SSH文库)。采用Trizol法提取两种人参发根材料的总RNA,分离纯化,获得的高质量驱动子和检测子mRNA。采用Clontech公司SSH试剂盒进行差减杂交,构建的人参发根消减文库,经蓝白斑筛选出117个cDNA阳性克隆,文库的重组率在85%以上,满足文库构建要求,其中97个cDNA克隆测序成功。消减文库的差减效率检测表明将18S rRNA基因减弱了212倍,使差异表达cDNA也被富集了同样的倍数。菌落PCR结果表明文库的外源片段的长度分布在200～1,000 bp之间,平均片段长度在500 bp左右。结果表明：所构建的SSH文库在RNA提取质量、插入片断大小、重组率、消减效率等方面均符合文库的质量标准,能够满足下一步实验的要求。
采用质粒提取试剂盒对文库中所有的阳性克隆进行质粒DNA提取。对人参发根构建的抑制消减杂交cDNA文库中的97个EST克隆测序,其数据经过手动去除载体序列,Seqman序列拼接组装,Blastx、Blastn和Interproscan的注释,结合GO功能分类,完成对EST序列统计分析。结果显示：对成功测序的有效克隆进行聚类分析,发现有5个重叠群和79个单拷贝EST,代表了共计84个独立基因。基因注释及功能分类结果表明：已知功能的唯一序列共53个,占63.1%,未知功能唯一序列31个,占36.9%。对84个唯一序列进行功能分类,得到11类。其中有16个克隆与代谢途径相关；31个未知基因；5个克隆与压力反应有关；6个克隆与生物合成有关；7个克隆与tRNA修饰,GTPase,离子结合相关；5个克隆与蛋白质折叠水解有关；1个克隆与转录调控有关；4个克隆与信号传导有关；4个克隆与分泌途径有关；2个克隆与电子传递有关；3个克隆与细胞组织结构有关。
对人参皂苷合成相关的EST序列PRB3、PRB6进行分析,在Genebank上进行同源蛋白比对得到两个同源性达到97%的基因编码产物,分别是鲨烯合酶和鲨烯环氧酶,查找开放阅读框,并进行保守区预测。人参发根SSH文库构建成功,获得了在MeJA诱导下的差异表达基因,对人参皂苷合成途径中关键酶和关键基因的克隆、表达以及新基因的发现和功能研究奠定了物质基础。
Panax Ginseng is always considered as one of the most potent medicinal plant and used widely in traditional medicine. Ginseng hairy root was induced from the explant of ginseng by Agrobacterium rhizogenes(A4 strain in this study)and belonged non-natural plant. Compared with the cell development, hairy root grows quickly, the live material content is high and the hereditary is stability. Ginsenoside is a main effective composition in ginseng hairy root, with many important pharmacological functions, at present, the people wanted to get ginsenosides by culturing ginseng hairy root and achieved to factory production. In order to improve ginsenosides content of ginseng hairy root and satisfy the demand of medication product, we studied factors as nutrition components, culture conditions, precursors and elicitors. We want to change the metabolic pathway to improve the secondary metabolite by adding MeJA in medium of ginseng hairy root. With the development of bioengineering, it gradually becomes the hotspot to improve the product of supersession production by changing plant biosynthetic pathways.
The elicitor MeJA could improve the growth speed of ginseng hairy root when its concentration was at 200μmol·L-1, the results showed the biomass increased 13.82 fold and total ginsenoside content was 2.28 percent,35.71 percent higher than contrast. Meanwhile, hairy root cultured in bioreactor showed that biomass increases 23.14 fold, total ginsenoside content was 2.04 percent, the content ginsenoside Rbi and Rd is obviously higher than contrast. The ginseng hairy root grows quickly without any hormone and the ginsenosides content is high, so the modified bioreactor is suit to cultivate it.
It was assumed that the different content of saponin was the result of differential expression of the genes involved in saponin biosynthes. Therefore, it was very important to clone the genes and investigate their functions. The total RNA was prepared with Trizol reagent separately from ginseng hairy root induced by MeJA and contrast. The SSH library was constructed by suppression subtractive hybridization technology. There were 117 clones from library by blue-white spot screening, and among them 97 clones were sequenced, the recombinant rate of library was above 85%. The efficiency of suppressive subtractive hybridization was 212 fold. The detection of bacterial colony PCR showed the insert fragments size ranged from 0.2-1.0 kb, average size was about 0.5 kb. All these results indicated that SSH library was qualified for further studies. Successful construction of SSH library of ginseng hairy root is essential for cloning of genes known and is also an initial key for screening and cloning of new genes.
97 high-quality expressed sequence tags were obtained by single-pass sequencing from 117 recombinant clones, and 84 unique sequences were formed after cluster analysis by Seqman program of DNAStar software. The sequences exhibited homology to previously known ones of GenBank in the level of protein or nucleotide by analysis of blastx and blastn. Function analysis by Interproscan and the Gene Ontology showed that function-known genes were 53 accounted for 63.1% of tatol genes and classified into 10 categories, most of them related to metabolism and secretory pathway. Function prediction revealed that 16 genes related to metabolism,5 genes related to response to stress,6 genes related to biosynthesis,7 genes related to tRNA modification, GTPase, ion binding et al,5 genes related to protein folding proteolysis,1 gene related to transcription regulation,4 genes related to signal transduction,4 genes related to secretory pathway,2 genes related to electron transport,3 genes related to cell organization. Function-unkown genes were 31 genes and accounted for 36.9%.
EST sequences of PRB3, PRB6 related to ginsenoside synthesis were analyzed by Blastx in Genebank, and homology analysis with squalene synthase and squalene epoxidase enzyme reached 97% respectively. To find the open reading frame and the conserved domain of two predict proteins. Successful construction of SSH library induced by MeJA of Ginseng hairy root, is an initial key for cloning and expressing the genes, furthermore, is essential for finding the new genes and the new functions in ginsenoside analysis pathway.
本文对人参发根三角瓶摇床培养进行考察,通过添加不同浓度的茉莉酸甲酯进行诱导,最终确定浓度在200μmol·L-1时效果最好,在一个培养周期内人参发根生物量增长13.82倍,人参发根总皂苷含量达到2.28%,比对照高35.71%。同时,我们使用实验室自行设计研制的发酵罐进行了人参发根培养实验,结果表明,人参发根在发酵罐中培养30天,生物量平均增长达到23.14倍,人参发根总皂苷含量达到2.04%,人参皂苷单体Rb1、Rd的含量比对照有较大提高,分别提高55.0%和30.1%。人参发根生长迅速,无需添加外源激素,次级代谢产物含量高,茉莉酸甲酯作为诱导子对于提高人参皂苷含量有明显的促进作用,同时表明经改进的发酵罐能够满足人参发根生长需要,有利于进行扩大培养。
人参发根次级代谢产物人参皂苷的种类和含量的差异与生物合成途径中相关基因差异表达调控有关。为研究这些差异表达基因,本实验利用抑制消减杂交技术构建了以茉莉酸甲酯诱导的人参发根为检测子、以对照人参发根为驱动子的抑制消减文库(SSH文库)。采用Trizol法提取两种人参发根材料的总RNA,分离纯化,获得的高质量驱动子和检测子mRNA。采用Clontech公司SSH试剂盒进行差减杂交,构建的人参发根消减文库,经蓝白斑筛选出117个cDNA阳性克隆,文库的重组率在85%以上,满足文库构建要求,其中97个cDNA克隆测序成功。消减文库的差减效率检测表明将18S rRNA基因减弱了212倍,使差异表达cDNA也被富集了同样的倍数。菌落PCR结果表明文库的外源片段的长度分布在200～1,000 bp之间,平均片段长度在500 bp左右。结果表明：所构建的SSH文库在RNA提取质量、插入片断大小、重组率、消减效率等方面均符合文库的质量标准,能够满足下一步实验的要求。
采用质粒提取试剂盒对文库中所有的阳性克隆进行质粒DNA提取。对人参发根构建的抑制消减杂交cDNA文库中的97个EST克隆测序,其数据经过手动去除载体序列,Seqman序列拼接组装,Blastx、Blastn和Interproscan的注释,结合GO功能分类,完成对EST序列统计分析。结果显示：对成功测序的有效克隆进行聚类分析,发现有5个重叠群和79个单拷贝EST,代表了共计84个独立基因。基因注释及功能分类结果表明：已知功能的唯一序列共53个,占63.1%,未知功能唯一序列31个,占36.9%。对84个唯一序列进行功能分类,得到11类。其中有16个克隆与代谢途径相关；31个未知基因；5个克隆与压力反应有关；6个克隆与生物合成有关；7个克隆与tRNA修饰,GTPase,离子结合相关；5个克隆与蛋白质折叠水解有关；1个克隆与转录调控有关；4个克隆与信号传导有关；4个克隆与分泌途径有关；2个克隆与电子传递有关；3个克隆与细胞组织结构有关。
对人参皂苷合成相关的EST序列PRB3、PRB6进行分析,在Genebank上进行同源蛋白比对得到两个同源性达到97%的基因编码产物,分别是鲨烯合酶和鲨烯环氧酶,查找开放阅读框,并进行保守区预测。人参发根SSH文库构建成功,获得了在MeJA诱导下的差异表达基因,对人参皂苷合成途径中关键酶和关键基因的克隆、表达以及新基因的发现和功能研究奠定了物质基础。
Panax Ginseng is always considered as one of the most potent medicinal plant and used widely in traditional medicine. Ginseng hairy root was induced from the explant of ginseng by Agrobacterium rhizogenes(A4 strain in this study)and belonged non-natural plant. Compared with the cell development, hairy root grows quickly, the live material content is high and the hereditary is stability. Ginsenoside is a main effective composition in ginseng hairy root, with many important pharmacological functions, at present, the people wanted to get ginsenosides by culturing ginseng hairy root and achieved to factory production. In order to improve ginsenosides content of ginseng hairy root and satisfy the demand of medication product, we studied factors as nutrition components, culture conditions, precursors and elicitors. We want to change the metabolic pathway to improve the secondary metabolite by adding MeJA in medium of ginseng hairy root. With the development of bioengineering, it gradually becomes the hotspot to improve the product of supersession production by changing plant biosynthetic pathways.
The elicitor MeJA could improve the growth speed of ginseng hairy root when its concentration was at 200μmol·L-1, the results showed the biomass increased 13.82 fold and total ginsenoside content was 2.28 percent,35.71 percent higher than contrast. Meanwhile, hairy root cultured in bioreactor showed that biomass increases 23.14 fold, total ginsenoside content was 2.04 percent, the content ginsenoside Rbi and Rd is obviously higher than contrast. The ginseng hairy root grows quickly without any hormone and the ginsenosides content is high, so the modified bioreactor is suit to cultivate it.
It was assumed that the different content of saponin was the result of differential expression of the genes involved in saponin biosynthes. Therefore, it was very important to clone the genes and investigate their functions. The total RNA was prepared with Trizol reagent separately from ginseng hairy root induced by MeJA and contrast. The SSH library was constructed by suppression subtractive hybridization technology. There were 117 clones from library by blue-white spot screening, and among them 97 clones were sequenced, the recombinant rate of library was above 85%. The efficiency of suppressive subtractive hybridization was 212 fold. The detection of bacterial colony PCR showed the insert fragments size ranged from 0.2-1.0 kb, average size was about 0.5 kb. All these results indicated that SSH library was qualified for further studies. Successful construction of SSH library of ginseng hairy root is essential for cloning of genes known and is also an initial key for screening and cloning of new genes.
97 high-quality expressed sequence tags were obtained by single-pass sequencing from 117 recombinant clones, and 84 unique sequences were formed after cluster analysis by Seqman program of DNAStar software. The sequences exhibited homology to previously known ones of GenBank in the level of protein or nucleotide by analysis of blastx and blastn. Function analysis by Interproscan and the Gene Ontology showed that function-known genes were 53 accounted for 63.1% of tatol genes and classified into 10 categories, most of them related to metabolism and secretory pathway. Function prediction revealed that 16 genes related to metabolism,5 genes related to response to stress,6 genes related to biosynthesis,7 genes related to tRNA modification, GTPase, ion binding et al,5 genes related to protein folding proteolysis,1 gene related to transcription regulation,4 genes related to signal transduction,4 genes related to secretory pathway,2 genes related to electron transport,3 genes related to cell organization. Function-unkown genes were 31 genes and accounted for 36.9%.
EST sequences of PRB3, PRB6 related to ginsenoside synthesis were analyzed by Blastx in Genebank, and homology analysis with squalene synthase and squalene epoxidase enzyme reached 97% respectively. To find the open reading frame and the conserved domain of two predict proteins. Successful construction of SSH library induced by MeJA of Ginseng hairy root, is an initial key for cloning and expressing the genes, furthermore, is essential for finding the new genes and the new functions in ginsenoside analysis pathway.
引文
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