人参叶和根cDNA文库构建及表达序列标签分析
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摘要
人参作为一种传统的药用植物已有几千年的应用历史,现代药物化学和药理学研究证实了人参中的各种有效成分对中枢神经系统、心血管系统、消化系统、内分泌系统和免疫系统均具有明显的调节作用,但是目前关于人参有效成分的生物合成途径及其调控的基础研究相对缺乏。为了保存人参基因资源,并在基因水平上研究人参有效成分生物合成的调控机理,我们分别构建了人参叶和根组织的全长cDNA文库,并对文库中部分基因序列进行了分析。
本论文分别以六年生人参叶和四年生人参根为研究材料,用Trizol法提取总RNA后,应用Clontech公司的SMART技术构建了两个表达型λ噬菌体全长cDNA文库。经检测人参叶原始cDNA文库含6.55×105个克隆,重组率为81%,插入片段长度在0.3-2.5 kb之间。人参根原始cDNA文库含4.80×105个克隆,重组率为80%,插入片段长度在0.3-2.0kb之间。两者均符合cDNA文库构建的标准要求。
通过对文库中200个重组克隆的序列分析,获得了124条有效ESTs,经DNAStar软件中的Seqman程序聚类处理归纳出了79条非冗余单拷贝序列。通过NCBI提供的在线BLAST程序,在蛋白水平和核酸水平找到61条序列的同源物。之后在dbEST数据库中登录了38条人参EST序列。对测通全长的26条单拷贝序列进行阅读框查找和蛋白质结构域分析后,对12条单拷贝序列进行了功能注释,并提交GenBank登录。
人参叶和根组织cDNA文库的成功构建,不仅保存了人参基因资源,获得的大量表达序列标签,扩充了人参基因数据资料,而且为人参中有明确功能的特异基因的克隆、表达以及新基因的克隆与功能研究奠定了物质基础。
Ginseng (Panax ginseng C.A.Meyer), considered to be one of the most potent medicinal plants, is used in traditional medicine. The reputed medicinal and herbal qualities of Ginseng have led to its popularity, primarily in the Orient, but increasingly worldwide. The various kinds of secondary metabolite, the active components of Ginseng, have an obvious regulatory bioactive effects on central nervous sysytem, cardiovascular system, alimentary system, endocrine system, immune system and so on. Although many reports have been published regarding the pharmacological effects of secondary metabolite, little is known about the genes and biochemical pathways in secondary metabolite biosynthesis. In order to delineate these biosynthesis pathways and their regulation using biochemical and molecular biological technique, and preserve the gene resource information of Ginseng, we constructed full-longth cDNA libraries of ginseng leaf and root tissues, and analyzed partial expressed sequence tags.
The total RNA was prepared with Trizol reagent separately from 6-year-old ginseng leaf and 4-year-old ginseng root tissues. The full-longth cDNA expression library was constructed by SMART technology. The primary titer of the cDNA library of leaf was 6.55×105 pfu and the rate of recombinant was 81%, the insert size ranged from 0.3~2.5kb. Correspondingly, that of the root primary cDNA library was separately 4.80×105 pfu, 80% and 0.3~2.0kb.
Therefore, cDNA libraries of ginseng leaf and root tissues have been successfully constructed. 124 high-quality expressed sequence tags were obtained by single-pass sequencing of 200 randomly chosen recombinant clones, 79 unique sequences were formed after assembly by Seqman program of DNAStar software. 61sequences exhibited homology to previously known sequences of GenBank in the level protein or nucleotide by analysis of blastx and blastn. Subsequently, 38 ESTs were submitted to dbEST. Open reading frame and putative conserved domains were detected in 12 new Ginseng sequences, and then submitted to GenBank.
Successful construction of the full-length cDNA expresssion libraries of Ginseng leaf and root tissues, is essential for cloning of genes known and is also an initial key for screening and cloning of new genes, furthermore, is a key work for building the gene resource information databank of Ginseng.
本论文分别以六年生人参叶和四年生人参根为研究材料,用Trizol法提取总RNA后,应用Clontech公司的SMART技术构建了两个表达型λ噬菌体全长cDNA文库。经检测人参叶原始cDNA文库含6.55×105个克隆,重组率为81%,插入片段长度在0.3-2.5 kb之间。人参根原始cDNA文库含4.80×105个克隆,重组率为80%,插入片段长度在0.3-2.0kb之间。两者均符合cDNA文库构建的标准要求。
通过对文库中200个重组克隆的序列分析,获得了124条有效ESTs,经DNAStar软件中的Seqman程序聚类处理归纳出了79条非冗余单拷贝序列。通过NCBI提供的在线BLAST程序,在蛋白水平和核酸水平找到61条序列的同源物。之后在dbEST数据库中登录了38条人参EST序列。对测通全长的26条单拷贝序列进行阅读框查找和蛋白质结构域分析后,对12条单拷贝序列进行了功能注释,并提交GenBank登录。
人参叶和根组织cDNA文库的成功构建,不仅保存了人参基因资源,获得的大量表达序列标签,扩充了人参基因数据资料,而且为人参中有明确功能的特异基因的克隆、表达以及新基因的克隆与功能研究奠定了物质基础。
Ginseng (Panax ginseng C.A.Meyer), considered to be one of the most potent medicinal plants, is used in traditional medicine. The reputed medicinal and herbal qualities of Ginseng have led to its popularity, primarily in the Orient, but increasingly worldwide. The various kinds of secondary metabolite, the active components of Ginseng, have an obvious regulatory bioactive effects on central nervous sysytem, cardiovascular system, alimentary system, endocrine system, immune system and so on. Although many reports have been published regarding the pharmacological effects of secondary metabolite, little is known about the genes and biochemical pathways in secondary metabolite biosynthesis. In order to delineate these biosynthesis pathways and their regulation using biochemical and molecular biological technique, and preserve the gene resource information of Ginseng, we constructed full-longth cDNA libraries of ginseng leaf and root tissues, and analyzed partial expressed sequence tags.
The total RNA was prepared with Trizol reagent separately from 6-year-old ginseng leaf and 4-year-old ginseng root tissues. The full-longth cDNA expression library was constructed by SMART technology. The primary titer of the cDNA library of leaf was 6.55×105 pfu and the rate of recombinant was 81%, the insert size ranged from 0.3~2.5kb. Correspondingly, that of the root primary cDNA library was separately 4.80×105 pfu, 80% and 0.3~2.0kb.
Therefore, cDNA libraries of ginseng leaf and root tissues have been successfully constructed. 124 high-quality expressed sequence tags were obtained by single-pass sequencing of 200 randomly chosen recombinant clones, 79 unique sequences were formed after assembly by Seqman program of DNAStar software. 61sequences exhibited homology to previously known sequences of GenBank in the level protein or nucleotide by analysis of blastx and blastn. Subsequently, 38 ESTs were submitted to dbEST. Open reading frame and putative conserved domains were detected in 12 new Ginseng sequences, and then submitted to GenBank.
Successful construction of the full-length cDNA expresssion libraries of Ginseng leaf and root tissues, is essential for cloning of genes known and is also an initial key for screening and cloning of new genes, furthermore, is a key work for building the gene resource information databank of Ginseng.
引文
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