日本蛇根草花青素还原酶基因的克隆及其生物信息学分析
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摘要
花青素还原酶(anthocyanidin reductase, ANR)是原花青素合成途径中的关键酶,同时花青素还原酶对植物花色苷的积累也发挥着重要作用。本研究以日本蛇根草为材料,以其转录组测序结果为基础,设计引物通过RT-PCR方法成功克隆得到日本蛇根草ANR基因完整的cDNA序列,利用生物信息学方法和工具对日本蛇根草ANR基因的功能结构域、生理和化学参数、亲/疏水性、信号肽、二级结构等进行预测和分析。结果表明,该基因cDNA全长为1 020 bp,编码339个氨基酸,预测其蛋白分子量为36.37 kD,等电点为5.92,为亲水蛋白,不含信号肽序列。综上,本研究成功克隆获得日本蛇根草ANR基因并完成其生物信息学分析,研究结果将为解析该基因的功能奠定基础。
Anthocyanidin reductase(ANR) is the key enzyme in the procyanidins biosynthetic pathway, and it also plays an important role in the accumulation of anthocyanin in plants. In this study, we utilized Ophiorrhiza japonica as materials, and designed the primers according to the results of the transcriptome sequencing. The integrated cDNA sequence of ANR gene was cloned successfully by RT-PCR method in Ophiorrhiza japonica. Bioinformatics methods and tools were applied to the functional domains, physical and chemical parameters, hydrophobicity or hydrophilicity, signal peptide, secondary structure and other factors in ANR gene of Ophiorrhiza japonica for prediction and analysis. The results showed that the full length of the gene cDNA was 1 020 bp, which encoded 339 amino acids. The protein molecular weight was predicted to be 36.37 kD and the isoelectric points were 5.92, ANR gene of Ophiorrhiza japonica was a hydrophilic protein with no signal peptide sequence. In summary, we have successfully cloned the ANR gene from Ophiorrhiza japonica and finished its bioinformatics analysis, these results will lay a foundation for the analysis of the function of this gene.
Anthocyanidin reductase(ANR) is the key enzyme in the procyanidins biosynthetic pathway, and it also plays an important role in the accumulation of anthocyanin in plants. In this study, we utilized Ophiorrhiza japonica as materials, and designed the primers according to the results of the transcriptome sequencing. The integrated cDNA sequence of ANR gene was cloned successfully by RT-PCR method in Ophiorrhiza japonica. Bioinformatics methods and tools were applied to the functional domains, physical and chemical parameters, hydrophobicity or hydrophilicity, signal peptide, secondary structure and other factors in ANR gene of Ophiorrhiza japonica for prediction and analysis. The results showed that the full length of the gene cDNA was 1 020 bp, which encoded 339 amino acids. The protein molecular weight was predicted to be 36.37 kD and the isoelectric points were 5.92, ANR gene of Ophiorrhiza japonica was a hydrophilic protein with no signal peptide sequence. In summary, we have successfully cloned the ANR gene from Ophiorrhiza japonica and finished its bioinformatics analysis, these results will lay a foundation for the analysis of the function of this gene.
引文
Alber T.S.,Delseny M.,Devic M.,and Banyul S.,1997,A novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat,Plant Journal,11(2):289-299
Han Y.P.,Sornkanok V.,Ruth E.S.,and Schuyler S.K.,2012,Introduction of apple ANR gene into tobacco inhibits expression of both CHI and DFR gene in flowers,leading to loss of anthocyanin,Journal of Experimental Botany,63(7):2437-2447
Huang X.Y.,2012,Study on the genetic transformation of high quality black rice by grape anthocyanin reductase(ANR)gene,Thesis for M.S,Fujian Agriculture and Forestry University,Supervisor:Zheng J.G.,pp.3-10(黄昕颖,2012,葡萄花青素还原酶(ANR)基因遗传转化优质黑米的研究,硕士学位论文,福建农林大学,导师:郑金贵,pp.3-10)
Huang Y.N.,Ke L.N.,and Cheng Z.X.,2011,Cloning and prokaryotic expression of grape anthocyanin reductase gene ORF,Shandi Nongye Shengwu Xuebao(Journal of Mountain Agriculture and Biology),30(6):533-537(黄艺宁,柯丽娜,程祖锌,2011,葡萄花青素还原酶基因ORF的克隆与原核表达,山地农业生物学报,30(6):533-537)
Li J.,Liang Y.M.,Zhao A.C.,Liu C.Y.,Lv R.H.,Liu X.Q.,and and She M.D.,2016,Cloning and expression analysis of mulberry anthocyanin reductase gene MaANR,Canye Kexue(Science of Sericulture),42(4):570-575(李军,梁燕梅,赵爱春,刘长英,吕蕊花,刘晓清,余茂德,2016,桑树花青素还原酶基因Ma ANR的克隆和表达分析,蚕业科学,42(4):570-575)
Lu W.,2009,Traits and microscopic identification of Ophiorrhiza japonica,Zhongguo Yaoxue Zazhi(Chinese Journal of Pharmaceuticals),5(9):1168-1170(卢伟,2009,日本蛇根草的性状及显微鉴别,中国药学杂志,5(9):1168-1170)
Luo Y.,Wang H.X.,Wang Y.S.,Lu Z.W.,Liu Y.J.,Shan Y.,Chen X.T.,Ye H.,Gao L.P.,and Xia T.,2011,Expression and optimization of anthocyanin reductase gene in Escherichia coli,Chaye Kexue(Journal of Tea Science),31(4):326-332(骆洋,王弘雪,王芸生,卢忠尉,刘亚军,单育,陈啸天,叶辉,高丽萍,夏涛,2011,茶树花青素还原酶基因在大肠杆菌中的表达及优化,茶叶科学,31(4):326-332)
Shen X.F.,and Zeng S.Z.,2014,Charaterization of proanthocyanin related leucoanthocyanidin reductase and anthocyanidin reductase genes in Lycium ruthenicum Murr,Journal of Chinese Pharmaceutical Sciences,23(6):369-377
Xia T.,and Gao L.P.,2009,The progress of biosynthesis pathway of flavonoids and tea catechins and their regulatory research,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),42(8):2899-2908(夏涛,高丽萍,2009,类黄酮及茶儿茶素生物合成途径及其调控研究进展,中国农业科学,42(8):2899-2908)
Yang Q.,Zhao L.,Liu Y.J.,Liu L.,Wang Y.S.,Gao L.P.,and Xia T.,2013,Study on enzymatic characteristics of anthocyanin reductase in tea tree,Chaye Kexue(Journal of Tea Science),33(3):221-228(杨琴,赵磊,刘亚军,刘莉,王云生,高丽萍,夏涛,2013,茶树花青素还原酶的酶学特性研究,茶叶科学,33(3):221-228)
Zhang N.,Hu L.Z.,Chen X.S.,Li Y.,and Chen G.P.,2008,Analysis of plant anthocyanin metabolic pathway and establishment of regulation model,Zhongguo Shengwu Gongcheng Zazhi(Journal of Chinese Biotechnology),28(4):97-105(张宁,胡利宗,陈绪清,侯晓姝,李勇,陈国平,2008,植物花青素代谢途径分析及调控模型建立,中国生物工程杂志,28(4):97-105)
Zhang X.L.,2009,Study of anthocyanin reductase in tea tree,Thesis for M.S,Anhui Agricultural University,Supervisors:Xia T.,and Gao L.P.,pp.6-10(张宪林,2009,茶树中花青素还原酶的研究,硕士学位论文,安徽农业大学,导师:夏涛,高丽萍,pp.6-10)
Han Y.P.,Sornkanok V.,Ruth E.S.,and Schuyler S.K.,2012,Introduction of apple ANR gene into tobacco inhibits expression of both CHI and DFR gene in flowers,leading to loss of anthocyanin,Journal of Experimental Botany,63(7):2437-2447
Huang X.Y.,2012,Study on the genetic transformation of high quality black rice by grape anthocyanin reductase(ANR)gene,Thesis for M.S,Fujian Agriculture and Forestry University,Supervisor:Zheng J.G.,pp.3-10(黄昕颖,2012,葡萄花青素还原酶(ANR)基因遗传转化优质黑米的研究,硕士学位论文,福建农林大学,导师:郑金贵,pp.3-10)
Huang Y.N.,Ke L.N.,and Cheng Z.X.,2011,Cloning and prokaryotic expression of grape anthocyanin reductase gene ORF,Shandi Nongye Shengwu Xuebao(Journal of Mountain Agriculture and Biology),30(6):533-537(黄艺宁,柯丽娜,程祖锌,2011,葡萄花青素还原酶基因ORF的克隆与原核表达,山地农业生物学报,30(6):533-537)
Li J.,Liang Y.M.,Zhao A.C.,Liu C.Y.,Lv R.H.,Liu X.Q.,and and She M.D.,2016,Cloning and expression analysis of mulberry anthocyanin reductase gene MaANR,Canye Kexue(Science of Sericulture),42(4):570-575(李军,梁燕梅,赵爱春,刘长英,吕蕊花,刘晓清,余茂德,2016,桑树花青素还原酶基因Ma ANR的克隆和表达分析,蚕业科学,42(4):570-575)
Lu W.,2009,Traits and microscopic identification of Ophiorrhiza japonica,Zhongguo Yaoxue Zazhi(Chinese Journal of Pharmaceuticals),5(9):1168-1170(卢伟,2009,日本蛇根草的性状及显微鉴别,中国药学杂志,5(9):1168-1170)
Luo Y.,Wang H.X.,Wang Y.S.,Lu Z.W.,Liu Y.J.,Shan Y.,Chen X.T.,Ye H.,Gao L.P.,and Xia T.,2011,Expression and optimization of anthocyanin reductase gene in Escherichia coli,Chaye Kexue(Journal of Tea Science),31(4):326-332(骆洋,王弘雪,王芸生,卢忠尉,刘亚军,单育,陈啸天,叶辉,高丽萍,夏涛,2011,茶树花青素还原酶基因在大肠杆菌中的表达及优化,茶叶科学,31(4):326-332)
Shen X.F.,and Zeng S.Z.,2014,Charaterization of proanthocyanin related leucoanthocyanidin reductase and anthocyanidin reductase genes in Lycium ruthenicum Murr,Journal of Chinese Pharmaceutical Sciences,23(6):369-377
Xia T.,and Gao L.P.,2009,The progress of biosynthesis pathway of flavonoids and tea catechins and their regulatory research,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),42(8):2899-2908(夏涛,高丽萍,2009,类黄酮及茶儿茶素生物合成途径及其调控研究进展,中国农业科学,42(8):2899-2908)
Yang Q.,Zhao L.,Liu Y.J.,Liu L.,Wang Y.S.,Gao L.P.,and Xia T.,2013,Study on enzymatic characteristics of anthocyanin reductase in tea tree,Chaye Kexue(Journal of Tea Science),33(3):221-228(杨琴,赵磊,刘亚军,刘莉,王云生,高丽萍,夏涛,2013,茶树花青素还原酶的酶学特性研究,茶叶科学,33(3):221-228)
Zhang N.,Hu L.Z.,Chen X.S.,Li Y.,and Chen G.P.,2008,Analysis of plant anthocyanin metabolic pathway and establishment of regulation model,Zhongguo Shengwu Gongcheng Zazhi(Journal of Chinese Biotechnology),28(4):97-105(张宁,胡利宗,陈绪清,侯晓姝,李勇,陈国平,2008,植物花青素代谢途径分析及调控模型建立,中国生物工程杂志,28(4):97-105)
Zhang X.L.,2009,Study of anthocyanin reductase in tea tree,Thesis for M.S,Anhui Agricultural University,Supervisors:Xia T.,and Gao L.P.,pp.6-10(张宪林,2009,茶树中花青素还原酶的研究,硕士学位论文,安徽农业大学,导师:夏涛,高丽萍,pp.6-10)