布渣叶黄烷酮-2-羟化酶基因cDNA克隆及其生物信息学和表达分析
|
摘要
目的从布渣叶中克隆黄酮碳苷合成途径关键酶黄烷酮-2-羟化酶(flavanone 2-hydroxylase,F2H)基因,构建原核表达载体,对其进行生物信息学和表达模式分析。方法根据布渣叶转录组数据中注释为F2H的Unigene设计特异性引物,利用PCR法扩增Mp F2H基因的开放阅读框(open reading frame,ORF),基于在线工具对c DNA序列进行生物信息学分析。同时,依据F2H序列,设计特异引物,采用RT-q PCR方法对其在芽、叶、枝、花、果等组织中的表达进行测定。结果 Mp F2H基因ORF全长为1 557 bp(Genbank登录号KY652921),编码518个氨基酸;相对分子质量54 500,理论等电点5.49,含有信号肽,不含跨膜域,可能定位于叶绿体。同时,Mp F2H基因在不同组织中均有表达,其中在叶中表达量最高,在枝和花中表达量最低。结论 Mp F2H基因在不同组织中表达量差异较大。克隆了Mp F2H基因c DNA,构建了其原核表达载体p ET30a-Mp F2H,为Mp F2H基因的原核表达和功能验证奠定了基础。
Objective To obtain the key enzyme gene involved in flavone C-glycosides biosynthesis pathway, a flavanone 2-hydroxylase(F2 H) gene was cloned from Microcos paniculata, and its bioinformatics analysis and gene expression pattern were also performed. Methods The specific primers were designed according to Unigene in F2 H annotated in the transcriptome data of M. paniculata. The open reading frame(ORF) of Mp F2 H gene was amplified by PCR. Then the PCR product was purified and ligated to p ET30 a, and finally a prokaryotic expression vector p ET30 a-Mp F2 H was constructed. The bioinformation of F2 H gene c DNA sequences was analyzed by some online tools. Using RT-q PCR with suitable primers, the quantitative expression analysis of Mp F2 H gene in different tissues, namely, buds, leaves, twigs, flowers and fruits was carried out. Results The length of Mp F2 H gene ORF was 1 557 bp(Gen Bank accession number KY652921), which encoded a protein with 518 amino acid residues, relative molecular weight of 54 500, theory isoelectric point of 5.49. In which was no transmembrane domain. It was hypothesized that this protein located in chloroplast. Mp F2 H gene was expressed in different tissues, with the highest expression in leaves and the lowest expression in twigs and flowers. Conclusion The expression of Mp F2 H gene varied widely in different tissues. The Mp F2 H gene was cloned from M. paniculata based on p ET30 a-Mp F2 H expression vector. This study will provide the fundamental information for the further preparation and functional research of Mp F2 H protein in flavone C-glycosides biosynthesis pathway.
Objective To obtain the key enzyme gene involved in flavone C-glycosides biosynthesis pathway, a flavanone 2-hydroxylase(F2 H) gene was cloned from Microcos paniculata, and its bioinformatics analysis and gene expression pattern were also performed. Methods The specific primers were designed according to Unigene in F2 H annotated in the transcriptome data of M. paniculata. The open reading frame(ORF) of Mp F2 H gene was amplified by PCR. Then the PCR product was purified and ligated to p ET30 a, and finally a prokaryotic expression vector p ET30 a-Mp F2 H was constructed. The bioinformation of F2 H gene c DNA sequences was analyzed by some online tools. Using RT-q PCR with suitable primers, the quantitative expression analysis of Mp F2 H gene in different tissues, namely, buds, leaves, twigs, flowers and fruits was carried out. Results The length of Mp F2 H gene ORF was 1 557 bp(Gen Bank accession number KY652921), which encoded a protein with 518 amino acid residues, relative molecular weight of 54 500, theory isoelectric point of 5.49. In which was no transmembrane domain. It was hypothesized that this protein located in chloroplast. Mp F2 H gene was expressed in different tissues, with the highest expression in leaves and the lowest expression in twigs and flowers. Conclusion The expression of Mp F2 H gene varied widely in different tissues. The Mp F2 H gene was cloned from M. paniculata based on p ET30 a-Mp F2 H expression vector. This study will provide the fundamental information for the further preparation and functional research of Mp F2 H protein in flavone C-glycosides biosynthesis pathway.
引文
[1]中国药典[S].一部.2015.
[2]李坤平,陈艳芬,岳春华,等.布渣叶保护急性心肌缺血损伤有效部位化学成分研究[J].中草药,2014,45(23):3373-3376.
[3]冯世秀,刘梅芳,魏孝义,等.布渣叶中三萜和黄酮类成分的研究[J].热带亚热带植物学报,2008,16(1):51-56.
[4]邵莹,吴启南,乐巍,等.碳苷黄酮保护心肌缺血损伤作用的研究进展[J].中草药,2015,46(1):128-139.
[5]Xiao J,Capanoglu E,Jassbi A R,et al.Advance on the flavonoid C-glycosides and health benefits[J].Crit Rev Food Sci Nutr,2016,56(Suppl 1):29-45.
[6]Choi J S,Islam M N,Ali M Y,et al.Effects of C-glycosylation on anti-diabetic,anti-Alzheimer’s disease and anti-inflammatory potential of apigenin[J].Food Chem Toxicol,2014,64:27-33.
[7]Paddon C J,Westfall P J,Pitera D J,et al.High-level semi-synthetic production of the potent antimalarial artemisinin[J].Nature,2013,496(7446):528-532.
[8]Yan X,Fan Y,Wei W,et al.Production of bioactive ginsenoside compound K in metabolically engineered yeast[J].Cell Res,2014,24(6):770-773.
[9]赵乐,马利刚,杨方方,等.独行菜磷酸甲羟戊酸激酶La PMK基因克隆、生物信息学分析及原核表达[J].中草药,2016,47(17):3087-3093.
[10]刘昌乐,林爽,何卓儒,等.一测多评法测定布渣叶总黄酮部位中8种黄酮苷成分[J].中草药,2017,48(9):1872-1877.
[11]Sansen S,Yano J K,Reynald R L,et al.Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2[J].J Biol Chem,2007,282:14348-14355.
[12]Brazier-Hicks M,Evans K M,Gershater M C,et al.The C-glycosylation of flavonoids in cereals[J].J Biol Chem,2009,284(27):17926-17934.
[13]Du Y G,Chu H,Chu I K,et al.CYP93G2 is a flavanone2-hydroxylase required for C-glycosylflavone biosynthesis in Rice[J].Plant Physiol,2010,154:324-333.
[14]María F F,Eduardo R,María I C,et al.Identification of a bifunctional maize C-and O-glucosyltransferase[J].J Biol Chem,2013,288(44):31678-31688.
[15]Nagatomo Y,Usui S,Ito T,et al.Purification,molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum(buckwheat)cotyledon[J].Plant J,2014,80(3):437-448.
[16]Hirade Y,Kotoku N,Terasaka K,et al.Identification and functional analysis of 2-hydroxyflavanone C-glucosyltransferase in soybean(Glycine max)FEBS[J].FEBS Lett,2015,589(15):1778-1786.
[17]Melissa B H,Robert E.Metabolic engineering of the flavone-C-glycoside pathway using polyprotein technology[J].Metabolic Eng,2013,16:11-20.
[2]李坤平,陈艳芬,岳春华,等.布渣叶保护急性心肌缺血损伤有效部位化学成分研究[J].中草药,2014,45(23):3373-3376.
[3]冯世秀,刘梅芳,魏孝义,等.布渣叶中三萜和黄酮类成分的研究[J].热带亚热带植物学报,2008,16(1):51-56.
[4]邵莹,吴启南,乐巍,等.碳苷黄酮保护心肌缺血损伤作用的研究进展[J].中草药,2015,46(1):128-139.
[5]Xiao J,Capanoglu E,Jassbi A R,et al.Advance on the flavonoid C-glycosides and health benefits[J].Crit Rev Food Sci Nutr,2016,56(Suppl 1):29-45.
[6]Choi J S,Islam M N,Ali M Y,et al.Effects of C-glycosylation on anti-diabetic,anti-Alzheimer’s disease and anti-inflammatory potential of apigenin[J].Food Chem Toxicol,2014,64:27-33.
[7]Paddon C J,Westfall P J,Pitera D J,et al.High-level semi-synthetic production of the potent antimalarial artemisinin[J].Nature,2013,496(7446):528-532.
[8]Yan X,Fan Y,Wei W,et al.Production of bioactive ginsenoside compound K in metabolically engineered yeast[J].Cell Res,2014,24(6):770-773.
[9]赵乐,马利刚,杨方方,等.独行菜磷酸甲羟戊酸激酶La PMK基因克隆、生物信息学分析及原核表达[J].中草药,2016,47(17):3087-3093.
[10]刘昌乐,林爽,何卓儒,等.一测多评法测定布渣叶总黄酮部位中8种黄酮苷成分[J].中草药,2017,48(9):1872-1877.
[11]Sansen S,Yano J K,Reynald R L,et al.Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2[J].J Biol Chem,2007,282:14348-14355.
[12]Brazier-Hicks M,Evans K M,Gershater M C,et al.The C-glycosylation of flavonoids in cereals[J].J Biol Chem,2009,284(27):17926-17934.
[13]Du Y G,Chu H,Chu I K,et al.CYP93G2 is a flavanone2-hydroxylase required for C-glycosylflavone biosynthesis in Rice[J].Plant Physiol,2010,154:324-333.
[14]María F F,Eduardo R,María I C,et al.Identification of a bifunctional maize C-and O-glucosyltransferase[J].J Biol Chem,2013,288(44):31678-31688.
[15]Nagatomo Y,Usui S,Ito T,et al.Purification,molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum(buckwheat)cotyledon[J].Plant J,2014,80(3):437-448.
[16]Hirade Y,Kotoku N,Terasaka K,et al.Identification and functional analysis of 2-hydroxyflavanone C-glucosyltransferase in soybean(Glycine max)FEBS[J].FEBS Lett,2015,589(15):1778-1786.
[17]Melissa B H,Robert E.Metabolic engineering of the flavone-C-glycoside pathway using polyprotein technology[J].Metabolic Eng,2013,16:11-20.