云南萝芙木异胡豆苷合成酶基因的全长cDNA克隆与序列分析
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摘要
云南萝芙木是一种重要的药用植物,其中含有多种萜类吲哚生物碱(terpenoid indole alkaloids, TIAs),是治疗高血压、性功能障碍和癌症等疾病的原料药。异胡豆苷合成酶催化色胺和裂环马钱子苷缩合生成TIAs的共同前体物质3α(S)-异胡豆苷。它是云南萝芙木萜类吲哚生物碱次生代谢的合成途径中重要的限速酶。现在尚未见从云南萝芙木中克隆该基因的报道,因此研究云南萝芙木STR基因对于提高TIAs产量有指导意义。本论文主要研究结果如下：
1)云南萝芙木STR基因的全长cDNA克隆。以云南萝芙木新鲜叶片为材料提取RNA,将其反转录的cDNA作为模板,根据NCBI上已有的萝芙木种中STR的核苷酸序列片段,用专业软件Primer Premier5.0设计特异引物,通过PCR扩增出STR基因,克隆产物经过插入pMD18-T载体,以及转化大肠杆菌DH5α菌株等一系列操作之后送至生物公司测序,克隆片断测序结果通过DNAMAN等生物信息学软件的分析,获得了1038bp的STR基因的全长cDNA序列。
2)云南萝芙木STR的生物信息学分析。通过生物信息学软件DNAMAN对测序结果进行分析：STR基因cDNA长度1038p,含有一个1037bp的ORF,编码345个氨基酸。在GenBank序列BLAST比对获知STR与同种属蛇根木的STR蛋白序列相似性分别达到99%,与同科的长春花STR序列相似性达80%。根据STR基因的cDNA序列推导出蛋白质的氨基酸序列,应用一系列生物信息学软件对理化性质、高级结构等进行预测分析,结果显示：STR蛋白等电点5.20,分子量38.4kDa,不稳定系数34.76,属于稳定蛋白质脂肪系数为86.70,平均亲水性-0.132；存在跨膜结构区域,N-端有一长度为27个氨基酸参与分泌的信号肽。二级结构预测指出,延伸链和不规则盘绕是STR蛋白质最大量的结构元件,而α-螺旋和β-转角则散布于整个蛋白质。
Rauvolfia yunnanensis Tsiang is an important medicinal plant, which contains a variety of terpenoid indole alkaloids (terpenoid indole alkaloids, TIAs). It's the bulk drugs for the treatment of diseases such as hypertension, sexual dysfunction and cancer. Strictosidine synthase work as a catalytic. It catalyze tryptamine and secologanin condensation to3α(S)-Strictosidine. STR is the key intermediate of Rauvolfia yunnanensis Tsiang terpenoid indole alkaloids sencondary metabolism'pathway. It haven't been reported from the Rauvolfia yunnanensis Tsiang. So the study on STR of Rauvolfia yunnanensis Tsiang is meaningful for the improve the production of TIAs. The major findings of this paper are as follows:
1) Full-length cDNA cloning of STR gene from Rauvolfia yunnanensis Tsiang. Using the fresh leaf of Rauvolfia yunnanensis Tsiang as the material, to extract total RNA, and transcript it reversed to cDNA as a template. Based on the nucleotide sequence fragments in NCBI, through a professional software, Primer Premier5.0, to designe specific primers. Then amplified by PCR, the fragment was cloned into the vector pMD18-T and then transformed into Escherichia. Coli DH5a and its sequence was obtained by sequencing recombinant plasmid DNA extracted from the transformed strain. After analyzing by DNAMAN,the full-length of a1038bp STR gene cDNA cloning from Rauvolfia yunnanensis Tsiang were obtained.
2) Bioinformatic analysis of STR from Rauvolfia yunnanensis Tsiang. We use the DNAMAN to analysis the sequences of STR genes, the1038bp STR cDNA sequence has a1037bp ORF coding345amino acids. The homology percentage of the STR from Rauvolfia yunnanensis Tsiang with other Rauvolfia species in Genbank reach up to99%、and homology percentage with Catharanthus roseus reach up to80%. The characteristic of physical chemistry and the second structure of the deduced amino acid sequences were predicted and analyzed by Bio-softwares, and the results for STR are:pl value is5.20; molecular weights is38.4kDa; the instability index is34.76; the aliphatic index is86.70; average hydrophilia is 0.132.And27amino acids constructed a signal peptide that attend excrete at its N-end.The prediction of its secondary structure shows that extend strand and random coil is the most quantities in STR protein and α-spiral and β-outer corner distribute in the whole protein.
1)云南萝芙木STR基因的全长cDNA克隆。以云南萝芙木新鲜叶片为材料提取RNA,将其反转录的cDNA作为模板,根据NCBI上已有的萝芙木种中STR的核苷酸序列片段,用专业软件Primer Premier5.0设计特异引物,通过PCR扩增出STR基因,克隆产物经过插入pMD18-T载体,以及转化大肠杆菌DH5α菌株等一系列操作之后送至生物公司测序,克隆片断测序结果通过DNAMAN等生物信息学软件的分析,获得了1038bp的STR基因的全长cDNA序列。
2)云南萝芙木STR的生物信息学分析。通过生物信息学软件DNAMAN对测序结果进行分析：STR基因cDNA长度1038p,含有一个1037bp的ORF,编码345个氨基酸。在GenBank序列BLAST比对获知STR与同种属蛇根木的STR蛋白序列相似性分别达到99%,与同科的长春花STR序列相似性达80%。根据STR基因的cDNA序列推导出蛋白质的氨基酸序列,应用一系列生物信息学软件对理化性质、高级结构等进行预测分析,结果显示：STR蛋白等电点5.20,分子量38.4kDa,不稳定系数34.76,属于稳定蛋白质脂肪系数为86.70,平均亲水性-0.132；存在跨膜结构区域,N-端有一长度为27个氨基酸参与分泌的信号肽。二级结构预测指出,延伸链和不规则盘绕是STR蛋白质最大量的结构元件,而α-螺旋和β-转角则散布于整个蛋白质。
Rauvolfia yunnanensis Tsiang is an important medicinal plant, which contains a variety of terpenoid indole alkaloids (terpenoid indole alkaloids, TIAs). It's the bulk drugs for the treatment of diseases such as hypertension, sexual dysfunction and cancer. Strictosidine synthase work as a catalytic. It catalyze tryptamine and secologanin condensation to3α(S)-Strictosidine. STR is the key intermediate of Rauvolfia yunnanensis Tsiang terpenoid indole alkaloids sencondary metabolism'pathway. It haven't been reported from the Rauvolfia yunnanensis Tsiang. So the study on STR of Rauvolfia yunnanensis Tsiang is meaningful for the improve the production of TIAs. The major findings of this paper are as follows:
1) Full-length cDNA cloning of STR gene from Rauvolfia yunnanensis Tsiang. Using the fresh leaf of Rauvolfia yunnanensis Tsiang as the material, to extract total RNA, and transcript it reversed to cDNA as a template. Based on the nucleotide sequence fragments in NCBI, through a professional software, Primer Premier5.0, to designe specific primers. Then amplified by PCR, the fragment was cloned into the vector pMD18-T and then transformed into Escherichia. Coli DH5a and its sequence was obtained by sequencing recombinant plasmid DNA extracted from the transformed strain. After analyzing by DNAMAN,the full-length of a1038bp STR gene cDNA cloning from Rauvolfia yunnanensis Tsiang were obtained.
2) Bioinformatic analysis of STR from Rauvolfia yunnanensis Tsiang. We use the DNAMAN to analysis the sequences of STR genes, the1038bp STR cDNA sequence has a1037bp ORF coding345amino acids. The homology percentage of the STR from Rauvolfia yunnanensis Tsiang with other Rauvolfia species in Genbank reach up to99%、and homology percentage with Catharanthus roseus reach up to80%. The characteristic of physical chemistry and the second structure of the deduced amino acid sequences were predicted and analyzed by Bio-softwares, and the results for STR are:pl value is5.20; molecular weights is38.4kDa; the instability index is34.76; the aliphatic index is86.70; average hydrophilia is 0.132.And27amino acids constructed a signal peptide that attend excrete at its N-end.The prediction of its secondary structure shows that extend strand and random coil is the most quantities in STR protein and α-spiral and β-outer corner distribute in the whole protein.
引文
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