基于转录组测序的石斛生物碱和人参皂苷生物合成相关基因的发掘、克隆及鉴定
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摘要
药用植物是传统中药主要来源,其次生代谢物是新药、新先导化合物、新化学实体的重要来源。随着药用植物在药物开发中的广泛应用,药用植物的需求量不断增加,药用植物长期的无序开发不仅造成药用植物资源紧缺,而且严重破坏生态环境,濒危野生药用植物物种数量也随之快速增加。通过植物生物技术大规模合成药效成分,己成为药物生产和新药开发的重要探索途径。药用植物高通量转录组测序技术的出现,结合关键酶基因的克隆与体外高效表达以及代谢工程大规模生产药用植物药效成分,为揭示药用植物药效成分生物合成途径提供了重要的基因资源。
本研究采用454高通量测序技术,基于石斛生物碱、人参皂苷的研究现状,分别从转录组分析、石斛生物碱及人参皂苷生物合成途径、酶基因的发掘与克隆、蛋白异源表达等方面研究石斛生物碱和人参皂苷的生物合成途径。本文的研究内容主要包括以下几个方面：
1)铁皮石斛(Dendrobium offlcinale Kimura et Migo)为兰科珍稀名贵药用植物,为《中国药典》收载的正品石斛之一。由于缺乏基因组和转录组数据,铁皮石斛生物碱生物合成途径尚不明确。本研究应用454GS FLX Titaninm高通量测序技术对铁皮石斛的转录组进行测序。获得553,084条EST序列,平均长度417bp,拼接组装后共获得36,407条独立基因(unique sequences)。将这些独立基因与公共数据库(SwissProt, KEGG, TAIR, Nr和Nt)进行比对注释,其中25,473条序列获得注释。通过分析KEGG数据库注释结果,发现25个参与生物碱合成的酶基因,将其中5个参与生物碱骨架合成的酶基因(共11个转录本)进行实时荧光定量PCR分析,发现这5个基因在石斛叶中的表达量均高于茎中表达量。在454转录组数据库中还发现了193条CYP450基因,22条氨基转移酶基因和122条甲基转移酶基因,11条多药耐药(MDR)基因和964条转录因子。通过对EST-SSR分析,从36,407条独立基因中共发现含了不同重复基元SSRs的序列有1,063条,二基元重复类型是最丰富类型,有179条含SSR位点的序列可以在KEGG数据库中得到注释。
2)人参(Panax ginseng C. A. Meyer)是五加科人参属重要药用植物。采用454-GSFLX测序平台对人参根、茎、叶、花的转录组各进行1/2个run的测序,共获得2,423,076条原始序列。通过Newbler软件分别对根、茎、叶和花的转录组数据进行拼接,拼接后在四个组织部位分别获得45,849,6,172,4,041和3,273条单一序列(Unigenes)。合并拼接后共获得178,145条独立基因,包括86,609条重叠序列(contig)和91,536条单一序列(singleton),有105,522条独立基因是第一次发现,其中65.6%的新独立基因是在茎、叶、花的文库中发现的。通过注释共发现223条与人参皂苷骨架合成相关的转录本,还发现了326条CYP450序列和129条糖基转移酶序列。通过同源比对共发现14条microRNAs序列。从178,145条独立基因中共发现了13,044个SSR位点。
3)三七(Panax notoginseng (Burk.) F.H.Chen)为五加科人参属多年生草本植物,通过甲羟戊酸途径(Mevalonic Acid Pathway)合成人参皂苷生物合成的前体物质。本研究通过分析课题组己获得的三七转录组数据,利用RT-PCR方法获得三七PnMVK1、PnUGT02086和PnUGT13895基因的全长cDNA序列；并对这些基因所编码的蛋白进行理化性质、蛋白二级结构及三维结构预测分析；利用实时荧光定量PCR方法检测了PnMVK1基因在三七的根、茎、叶、花中的表达情况,PnMVK1在三七的根中表达丰度最高。将PnUGT02086和PnUGT13895基因通过酶切连接到pET28a载体并转化表达菌株BL21,成功表达并纯化到了PnUGT02086和PnUGT13895蛋白,为进一步验证其功能奠定了基础。
Medicinal plants is a major source of traditional Chinese medicine, while the secondary metabolites are an important source of new drugs, drug leads and new chemical entities. With the extensive application of medicinal plants in drug development, the demand for medicinal plants is increasing. Long-term disorderly development of medicinal plants is not only caused by the scarcity of resources, but also seriously damage to the ecological environment. The number of endangered species of wild medicinal plants also increased rapidly. The use of biotechnology method to synthesize large-scale medicinal components has become important way to explore drug development. Based on the high throughput sequencing technology, the studies of transcriptome can provide important genetic resources for the medicinal natural product biosynthesis, combined with key gene cloning and identification, as well as large-scale production of medicinal components.
In this study, we used the454high-throughput sequencing technology to analyze the transcriptome of Dendrobium officinale and Panax ginseng and discover key genes related to the Dendrobium alkaloids and ginsenoside biosynthesis. Moreover, several genes encoding key enzymes had been cloned and heterologously expressed in E.coli. The content of this study includes the following aspects:
1) Dendrobium officinale Kimura et Migo(Orchidaceae) is a traditional Chinese medicinal plant. The stem contains an alkaloid that is the primary bioactive component. However, the details of alkaloid biosynthesis have not been effectively explored because of the limited number of expressed sequence tags (ESTs) available in GenBank. In this study, we analyzed RNA isolated from the stem of D. officinale using a single half-run on the Roche454GS FLX Titanium platform to generate553,084ESTs with an average length of417bases. The ESTs were assembled into38,951unique putative transcripts. A total of69.97%of the unique sequences were annotated, and a detailed view of alkaloid biosynthesis was obtained. Functional assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG) terms revealed69unique sequences representing25genes involved in alkaloid backbone biosynthesis. A series of qRT-PCR experiments confirmed that the expression levels of5key enzyme-encoding genes involved in alkaloid biosynthesis are greater in the leaves of D. officinale than in the stems. Cytochrome P450s, aminotransferases, methyltransferases, multidrug resistance protein (MDR) transporters and transcription factors were screened for possible involvement in alkaloid biosynthesis. Furthermore, a total of1,061simple sequence repeat motifs (SSR) were detected from36,407unigenes. Dinucleotide repeats were the most abundant repeat type. Of these,179genes were associated with a metabolic pathway in KEGG.
2) Panax ginseng C A. Meyer is one of the most widely used medicinal plants. Two454pyrosequencing runs generated a total of2,423,076reads from P. ginseng roots, stems, leaves and flowers. The high-quality reads from each of the tissues were independently assembled into separate and shared contigs. In the separately assembled database,45,849,6,172,4,041and3,273unigenes were only found in the roots, stems, leaves and flowers database, respectively, In the jointly assembled database,178,145unigenes were observed, including86,609contigs and91,536singletons. Among the178,145unigenes,105,522were identified for the first time, of which65.6%were identified in the stem, leaf or flower cDNA libraries of P. ginseng. After annotation, we discovered223unigenes involved in ginsenoside backbone biosynthesis. Additionally, a total of326potential cytochrome P450and129potential UDP-glycosyltransferase sequences were predicted based on the annotation results, some of which may encode enzymes responsible for ginsenoside backbone modification. A BLAST search of the obtained high-quality reads identified14potential microRNAs in P. ginseng. A total of13,044simple sequence repeats were identified from the178,145unigenes.
3) Panax notoginseng (Burk.) FH Chen is a perennial herb in Ginseng genus. The precursor substances of ginsenoside are biosynthesized through the mevalonic acid pathway. In this study, we found many genes related to ginsenoside biosynthesis based on the research of the P. notoginseng transcriptome data. The full-length cDNA sequences of PnMVKl, PnUGT02086, and PnUGT13895genes were obtained by RT-PCR strategies. The physical and chemical properties, secondary structure and three-dimensional structure of these proteins were predicted and analyzed, including of the structure and putative functions. Pn MVK1was more abundant in P. notoginseng root than other organisms. PnUGT02086and PnUGT13895were ligated into pET28a vector and transformed into BL21. The proteins of PnUGT02086and PnUGT13895were purified from E.coli, which laid the foundation for the further verification of the function for these genes.
本研究采用454高通量测序技术,基于石斛生物碱、人参皂苷的研究现状,分别从转录组分析、石斛生物碱及人参皂苷生物合成途径、酶基因的发掘与克隆、蛋白异源表达等方面研究石斛生物碱和人参皂苷的生物合成途径。本文的研究内容主要包括以下几个方面：
1)铁皮石斛(Dendrobium offlcinale Kimura et Migo)为兰科珍稀名贵药用植物,为《中国药典》收载的正品石斛之一。由于缺乏基因组和转录组数据,铁皮石斛生物碱生物合成途径尚不明确。本研究应用454GS FLX Titaninm高通量测序技术对铁皮石斛的转录组进行测序。获得553,084条EST序列,平均长度417bp,拼接组装后共获得36,407条独立基因(unique sequences)。将这些独立基因与公共数据库(SwissProt, KEGG, TAIR, Nr和Nt)进行比对注释,其中25,473条序列获得注释。通过分析KEGG数据库注释结果,发现25个参与生物碱合成的酶基因,将其中5个参与生物碱骨架合成的酶基因(共11个转录本)进行实时荧光定量PCR分析,发现这5个基因在石斛叶中的表达量均高于茎中表达量。在454转录组数据库中还发现了193条CYP450基因,22条氨基转移酶基因和122条甲基转移酶基因,11条多药耐药(MDR)基因和964条转录因子。通过对EST-SSR分析,从36,407条独立基因中共发现含了不同重复基元SSRs的序列有1,063条,二基元重复类型是最丰富类型,有179条含SSR位点的序列可以在KEGG数据库中得到注释。
2)人参(Panax ginseng C. A. Meyer)是五加科人参属重要药用植物。采用454-GSFLX测序平台对人参根、茎、叶、花的转录组各进行1/2个run的测序,共获得2,423,076条原始序列。通过Newbler软件分别对根、茎、叶和花的转录组数据进行拼接,拼接后在四个组织部位分别获得45,849,6,172,4,041和3,273条单一序列(Unigenes)。合并拼接后共获得178,145条独立基因,包括86,609条重叠序列(contig)和91,536条单一序列(singleton),有105,522条独立基因是第一次发现,其中65.6%的新独立基因是在茎、叶、花的文库中发现的。通过注释共发现223条与人参皂苷骨架合成相关的转录本,还发现了326条CYP450序列和129条糖基转移酶序列。通过同源比对共发现14条microRNAs序列。从178,145条独立基因中共发现了13,044个SSR位点。
3)三七(Panax notoginseng (Burk.) F.H.Chen)为五加科人参属多年生草本植物,通过甲羟戊酸途径(Mevalonic Acid Pathway)合成人参皂苷生物合成的前体物质。本研究通过分析课题组己获得的三七转录组数据,利用RT-PCR方法获得三七PnMVK1、PnUGT02086和PnUGT13895基因的全长cDNA序列；并对这些基因所编码的蛋白进行理化性质、蛋白二级结构及三维结构预测分析；利用实时荧光定量PCR方法检测了PnMVK1基因在三七的根、茎、叶、花中的表达情况,PnMVK1在三七的根中表达丰度最高。将PnUGT02086和PnUGT13895基因通过酶切连接到pET28a载体并转化表达菌株BL21,成功表达并纯化到了PnUGT02086和PnUGT13895蛋白,为进一步验证其功能奠定了基础。
Medicinal plants is a major source of traditional Chinese medicine, while the secondary metabolites are an important source of new drugs, drug leads and new chemical entities. With the extensive application of medicinal plants in drug development, the demand for medicinal plants is increasing. Long-term disorderly development of medicinal plants is not only caused by the scarcity of resources, but also seriously damage to the ecological environment. The number of endangered species of wild medicinal plants also increased rapidly. The use of biotechnology method to synthesize large-scale medicinal components has become important way to explore drug development. Based on the high throughput sequencing technology, the studies of transcriptome can provide important genetic resources for the medicinal natural product biosynthesis, combined with key gene cloning and identification, as well as large-scale production of medicinal components.
In this study, we used the454high-throughput sequencing technology to analyze the transcriptome of Dendrobium officinale and Panax ginseng and discover key genes related to the Dendrobium alkaloids and ginsenoside biosynthesis. Moreover, several genes encoding key enzymes had been cloned and heterologously expressed in E.coli. The content of this study includes the following aspects:
1) Dendrobium officinale Kimura et Migo(Orchidaceae) is a traditional Chinese medicinal plant. The stem contains an alkaloid that is the primary bioactive component. However, the details of alkaloid biosynthesis have not been effectively explored because of the limited number of expressed sequence tags (ESTs) available in GenBank. In this study, we analyzed RNA isolated from the stem of D. officinale using a single half-run on the Roche454GS FLX Titanium platform to generate553,084ESTs with an average length of417bases. The ESTs were assembled into38,951unique putative transcripts. A total of69.97%of the unique sequences were annotated, and a detailed view of alkaloid biosynthesis was obtained. Functional assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG) terms revealed69unique sequences representing25genes involved in alkaloid backbone biosynthesis. A series of qRT-PCR experiments confirmed that the expression levels of5key enzyme-encoding genes involved in alkaloid biosynthesis are greater in the leaves of D. officinale than in the stems. Cytochrome P450s, aminotransferases, methyltransferases, multidrug resistance protein (MDR) transporters and transcription factors were screened for possible involvement in alkaloid biosynthesis. Furthermore, a total of1,061simple sequence repeat motifs (SSR) were detected from36,407unigenes. Dinucleotide repeats were the most abundant repeat type. Of these,179genes were associated with a metabolic pathway in KEGG.
2) Panax ginseng C A. Meyer is one of the most widely used medicinal plants. Two454pyrosequencing runs generated a total of2,423,076reads from P. ginseng roots, stems, leaves and flowers. The high-quality reads from each of the tissues were independently assembled into separate and shared contigs. In the separately assembled database,45,849,6,172,4,041and3,273unigenes were only found in the roots, stems, leaves and flowers database, respectively, In the jointly assembled database,178,145unigenes were observed, including86,609contigs and91,536singletons. Among the178,145unigenes,105,522were identified for the first time, of which65.6%were identified in the stem, leaf or flower cDNA libraries of P. ginseng. After annotation, we discovered223unigenes involved in ginsenoside backbone biosynthesis. Additionally, a total of326potential cytochrome P450and129potential UDP-glycosyltransferase sequences were predicted based on the annotation results, some of which may encode enzymes responsible for ginsenoside backbone modification. A BLAST search of the obtained high-quality reads identified14potential microRNAs in P. ginseng. A total of13,044simple sequence repeats were identified from the178,145unigenes.
3) Panax notoginseng (Burk.) FH Chen is a perennial herb in Ginseng genus. The precursor substances of ginsenoside are biosynthesized through the mevalonic acid pathway. In this study, we found many genes related to ginsenoside biosynthesis based on the research of the P. notoginseng transcriptome data. The full-length cDNA sequences of PnMVKl, PnUGT02086, and PnUGT13895genes were obtained by RT-PCR strategies. The physical and chemical properties, secondary structure and three-dimensional structure of these proteins were predicted and analyzed, including of the structure and putative functions. Pn MVK1was more abundant in P. notoginseng root than other organisms. PnUGT02086and PnUGT13895were ligated into pET28a vector and transformed into BL21. The proteins of PnUGT02086and PnUGT13895were purified from E.coli, which laid the foundation for the further verification of the function for these genes.
引文
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