基于二代测序技术的黄芪萜类合成酶基因挖掘与生物信息学分析
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摘要
目的:对黄芪萜类合成酶(TPS)基因进行挖掘与生物信息学分析,为黄芪萜类化合物特别是三萜皂苷的合成、积累作用及其分子机制研究奠定基础。方法:通过二代测序技术构建黄芪转录组文库,根据基因注释信息进行TPS序列初步筛选,BLAST同源比对确认。运用MEGA 4. 0构建邻接(NJ)系统进化树,采用在线工具开放阅读框(ORF) Finder,Compute p I/Mw,Prot Comp 9. 0进行生物信息学分析。结果:共获得76条TPS序列,其中13条拥有完整的ORF。获得的TPS序列中注释为TPS10序列最多,其次为大根香叶烯D合成酶和单萜合成酶;选取17条代表性TPS序列进行同源性分析,发现黄芪TPS序列分为3个类群,每一类群均包括Ⅰ型和Ⅱ型萜类合成酶序列。此外,挖掘得3条环阿屯醇合酶(CAS)序列,其中原始编号为CL6827. Contig1和Unigene19112的CAS序列具有完整的ORF,所编码蛋白质序列长度分别为795,757个氨基酸,亚细胞定位于内质网,与已报道黄芪CAS序列同源性最高。结论:利用黄芪转录组文库成功挖掘得黄芪单萜、倍半萜及三萜合成环化酶基因,定位于内质网的CAS序列与三萜皂苷主要由细胞质中的甲羟戊酸途径合成特征吻合。
Objective: To identify enzyme genes involved in the terpene biosynthesis in Astragali Radix and to perform bioinformatic analysis for understanding the biosynthesis,accumulation and underlying mechanism of the terpenoids,especially astragaloside. Method: Transcriptome library was constructed using Astragali Radix plant seedlings as raw material by RNA sequencing(RNA-seq),and preliminary screening of terpene synthases(TPS) was performed based on the annotation of unigenes and contigs. The phylogenetic tree was constructed by MEGA 4. 0,and bioinformatic analysis was performed by online tools. Result: A total of 76 TPSs were obtained,of which 13 contained intact open reading frames(ORFs). And the genes annotated as TPS10 were the most,followed by the ones annotated as germacrene D synthase and monoterpene synthase. Seventeen representative sequences of TPSs were selected for alignment analysis,demonstrating that these TPSs were clustered into three groups and each group was consisted of class Ⅰ and class Ⅱ terpenoid synthetases. In addition,three cycloartenol synthases(CASs) responsible for the cycloartenol biosynthesis were identified, two of which had the highest identities to the ones published elsewhere in Astragali Radix. Meanwhile,two CASs contain intact ORFs,and corresponding amino acid length of deduced proteins were 795 aa and 757 aa, respectively. Subcellular localization of all three CASs was predicted in endoplasmic reticulum. Conclusion: TPSs responsible for monoterpenes,sesquiterpenes and triterpene biosynthesis are successfully identified from the transcriptome library.The predicted location of CASs in endoplasmic reticulum is in accordance with the triterpene biosynthesis by cytoplasmic mevalonate(MVA) pathway.
Objective: To identify enzyme genes involved in the terpene biosynthesis in Astragali Radix and to perform bioinformatic analysis for understanding the biosynthesis,accumulation and underlying mechanism of the terpenoids,especially astragaloside. Method: Transcriptome library was constructed using Astragali Radix plant seedlings as raw material by RNA sequencing(RNA-seq),and preliminary screening of terpene synthases(TPS) was performed based on the annotation of unigenes and contigs. The phylogenetic tree was constructed by MEGA 4. 0,and bioinformatic analysis was performed by online tools. Result: A total of 76 TPSs were obtained,of which 13 contained intact open reading frames(ORFs). And the genes annotated as TPS10 were the most,followed by the ones annotated as germacrene D synthase and monoterpene synthase. Seventeen representative sequences of TPSs were selected for alignment analysis,demonstrating that these TPSs were clustered into three groups and each group was consisted of class Ⅰ and class Ⅱ terpenoid synthetases. In addition,three cycloartenol synthases(CASs) responsible for the cycloartenol biosynthesis were identified, two of which had the highest identities to the ones published elsewhere in Astragali Radix. Meanwhile,two CASs contain intact ORFs,and corresponding amino acid length of deduced proteins were 795 aa and 757 aa, respectively. Subcellular localization of all three CASs was predicted in endoplasmic reticulum. Conclusion: TPSs responsible for monoterpenes,sesquiterpenes and triterpene biosynthesis are successfully identified from the transcriptome library.The predicted location of CASs in endoplasmic reticulum is in accordance with the triterpene biosynthesis by cytoplasmic mevalonate(MVA) pathway.
引文
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[6]李燕敏,杨克玉,孙洁雯,等. SPME-GC-MS分析不同产地干黄芪中挥发性风味成分[J].中国酿造,2016,35(5):175-181.
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[10] Kim Y B,Thwe A A,LI X H,et al. Accumulation of astragalosides and related gene expression in different organs of Astragalus membranaceus Bge. var mongholicus(Bge.)[J]. Molecules,2014,19(8):10922-10935.
[11] Park Y J,Thwe A A,LI X H,et al. Triterpene and flavonoid biosynthesis and metabolic profiling of hairy roots,adventitious roots,and seedling roots of Astragalus membranaceus[J]. J Agric Food Chem,2015,63(40):8862-8869.
[12] SUN H F,KANG B L,KANG L P,et al. Involvement of C6-volatiles in quality formation of herbal medicine:a case study in Astragalus membranaceus var. mongholicus[J]. J Appl Bot Food Qual,2017,90(27):214-223.
[13] Thimmappa R,Geisler K,Louveau T,et al. Triterpene biosynthesis in plants[J]. Annu Rev Plant Biol,2014,65(1):225-257.
[14] CHEN F,Tholl D,Bohlmann J,et al. The family of terpene synthases in plants:a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom[J]. Plant J,2011,66(1):212-229.
[15] Aubourg S,Lecharny A,Bohlmann J. Genomic analysis of the terpenoid synthase(At TPS)gene family of Arabidopsis thaliana[J]. Mol Genet Genomics,2002,267(6):730-745.
[2]潘飞,冯毓秀,陈四保,等.黄芪类药用植物皂甙类成分分析[J].中国中药杂志,1995,20(7):391-393.
[3]田华,邓雁如,周坤,等.蒙古黄芪的化学成分研究[J].中国实验方剂学杂志,2016,22(7):70-73.
[4] Kitagawa I,WANG H K,Yoshikawa M. Saponin and sapogenol. XXXⅦ. Chemical constituents of Astragali Radix,the root of Astragalus membranaceus Bunge.(4).AstragalosidesⅦandⅧ[J]. Chem Pharm Bull,1983,31(2):716-722.
[5]张蔷,高文远,满淑丽.黄芪中有效成分药理活性的研究进展[J].中国中药杂志,2012,37(21):3203-3207.
[6]李燕敏,杨克玉,孙洁雯,等. SPME-GC-MS分析不同产地干黄芪中挥发性风味成分[J].中国酿造,2016,35(5):175-181.
[7] Kumari S,Priya P,Misra G. Structural and biochemical perspectives in plant isoprenoid biosynthesis[J].Phytochem Rev,2013,12(2):255-291.
[8] Keeling C I,Weisshaar S,LIN R P,et al. Functional plasticity of paralogous diterpene synthases involved in conifer defense[J]. Proc Natl Acad Sci USA,2008,105(3):1085-1090.
[9] CHEN J,WU X T,XU Y Q,et al. Global transcriptome analysis profiles metabolic pathways in traditional herb Astragalus membranaceus Bge. var. mongolicus(Bge.)Hsiao[J]. BMC Genomics,2015,16(Suppl 7):S15.
[10] Kim Y B,Thwe A A,LI X H,et al. Accumulation of astragalosides and related gene expression in different organs of Astragalus membranaceus Bge. var mongholicus(Bge.)[J]. Molecules,2014,19(8):10922-10935.
[11] Park Y J,Thwe A A,LI X H,et al. Triterpene and flavonoid biosynthesis and metabolic profiling of hairy roots,adventitious roots,and seedling roots of Astragalus membranaceus[J]. J Agric Food Chem,2015,63(40):8862-8869.
[12] SUN H F,KANG B L,KANG L P,et al. Involvement of C6-volatiles in quality formation of herbal medicine:a case study in Astragalus membranaceus var. mongholicus[J]. J Appl Bot Food Qual,2017,90(27):214-223.
[13] Thimmappa R,Geisler K,Louveau T,et al. Triterpene biosynthesis in plants[J]. Annu Rev Plant Biol,2014,65(1):225-257.
[14] CHEN F,Tholl D,Bohlmann J,et al. The family of terpene synthases in plants:a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom[J]. Plant J,2011,66(1):212-229.
[15] Aubourg S,Lecharny A,Bohlmann J. Genomic analysis of the terpenoid synthase(At TPS)gene family of Arabidopsis thaliana[J]. Mol Genet Genomics,2002,267(6):730-745.