独行菜C4H基因克隆与表达分析
发布时间：2018-07-31 05:12
【摘要】：以独行菜(Lepidium apetalum)为材料,通过分析独行菜转录组数据,设计特异性引物,克隆了独行菜肉桂酸-4-羟化酶(cinnamate-4-hydroxylase,C4H)基因的cDNA序列,命名为LaC4H,Gen Bank登录号为KX064050,并进行生物信息学分析,原核表达、纯化,组织特异性分析和诱导表达分析。结果表明:(1)LaC4H基因开放阅读框(ORF)长1 518 bp,编码505个氨基酸,其蛋白质分子质量为57.73 k D。(2)生物信息学分析显示La C4H蛋白包含细胞色素P450的保守基序和5个特征性底物结合位点,是细胞色素P450超家族成员,系统进化分析显示LaC4H蛋白与拟南芥等十字花科植物C4H蛋白同源性较高。(3)通过构建原核表达载体pET-32a-LaC4H在大肠杆菌BL21(DE3)菌株中成功表达LaC4H重组蛋白,利用Ni~(2+)亲和色谱得到了纯化的LaC4H重组蛋白。(4)荧光定量PCR结果表明LaC4H基因在茎中表达量最高,叶和花中次之,根中表达量最低。经茉莉酸甲酯(methyl jasmonate,MeJA)诱导后,叶中LaC4H表达量明显上升,诱导后48 h达到最高值,叶中LaC4H的表达量与黄酮含量之间呈正相关。这为进一步研究LaC4H基因在独行菜黄酮类化合物生物合成途径中的功能奠定基础。
[Abstract]:The cDNA sequence of cinnamate-4-hydroxylaseC4H gene was cloned by analyzing the transcriptome data and designing a specific primer. The cDNA sequence was named LaC4HG Bank accession number was KX064050, and bioinformatics analysis was carried out. Prokaryotic expression, purification, tissue specific analysis and induced expression analysis. The results showed that: (1) LaC4H gene open reading frame (ORF) was 1 518 BP long, encoding 505 amino acids, and its protein molecular weight was 57.73 k D. (2. Bioinformatics analysis showed that La C4H protein contained the conserved motif of cytochrome P450 and five characteristic substrate binding sites. LaC4H protein is a member of cytochrome P450 superfamily. Phylogenetic analysis shows that LaC4H protein has high homology with C4H protein in Arabidopsis and other cruciferous plants. (3) LaC4H recombinant protein was successfully expressed in Escherichia coli BL21 (DE3) strain by construction of prokaryotic expression vector pET-32a-LaC4H. The purified LaC4H recombinant protein was obtained by Ni ~ (2) affinity chromatography. (4) the results of fluorescence quantitative PCR showed that the expression of LaC4H gene was the highest in stem, second in leaves and flowers, and the lowest in root. After being induced by methyl jasmonate, the expression of LaC4H in leaves increased significantly and reached the highest value 48 h after induction. There was a positive correlation between the expression of LaC4H and the content of flavonoids in leaves. This lays a foundation for the further study of the function of LaC4H gene in the biosynthesis pathway of flavonoids.
【作者单位】： 河南中医药大学药学院;呼吸疾病诊疗与新药研发河南省协同创新中心;
【基金】：国家重点基础研究发展计划“973计划”项目(2013CB531802) 河南省科技攻关计划(162102310468) 河南中医学院博士科研基金(BSJJ2011-07)
【分类号】：R284
，
本文编号：2154519
[Abstract]:The cDNA sequence of cinnamate-4-hydroxylaseC4H gene was cloned by analyzing the transcriptome data and designing a specific primer. The cDNA sequence was named LaC4HG Bank accession number was KX064050, and bioinformatics analysis was carried out. Prokaryotic expression, purification, tissue specific analysis and induced expression analysis. The results showed that: (1) LaC4H gene open reading frame (ORF) was 1 518 BP long, encoding 505 amino acids, and its protein molecular weight was 57.73 k D. (2. Bioinformatics analysis showed that La C4H protein contained the conserved motif of cytochrome P450 and five characteristic substrate binding sites. LaC4H protein is a member of cytochrome P450 superfamily. Phylogenetic analysis shows that LaC4H protein has high homology with C4H protein in Arabidopsis and other cruciferous plants. (3) LaC4H recombinant protein was successfully expressed in Escherichia coli BL21 (DE3) strain by construction of prokaryotic expression vector pET-32a-LaC4H. The purified LaC4H recombinant protein was obtained by Ni ~ (2) affinity chromatography. (4) the results of fluorescence quantitative PCR showed that the expression of LaC4H gene was the highest in stem, second in leaves and flowers, and the lowest in root. After being induced by methyl jasmonate, the expression of LaC4H in leaves increased significantly and reached the highest value 48 h after induction. There was a positive correlation between the expression of LaC4H and the content of flavonoids in leaves. This lays a foundation for the further study of the function of LaC4H gene in the biosynthesis pathway of flavonoids.
【作者单位】： 河南中医药大学药学院;呼吸疾病诊疗与新药研发河南省协同创新中心;
【基金】：国家重点基础研究发展计划“973计划”项目(2013CB531802) 河南省科技攻关计划(162102310468) 河南中医学院博士科研基金(BSJJ2011-07)
【分类号】：R284
，
本文编号：2154519
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