异叶天南星苯丙氨酸解氨酶基因的克隆与蛋白结构分析
|
摘要
以异叶天南星(Arisaema heterophyllum Blume)为材料,采用逆转录PCR(RT-PCR)法扩增其苯丙氨酸解氨酶(Phenylalanine ammonia-lyase,PAL)基因Ah PAL,获得该基因的开放读码框(ORF),并通过系统的生物信息学软件分析Ah PAL的结构和理化性质。结果显示,Ah PAL的ORF全长为2184 bp,编码727个氨基酸;Ah PAL与郁金香(Tulipa fosteriana W. Irving) PAL的亲缘关系最近,序列相似性达88%。空间结构模型分析结果显示,Ah PAL为同型四聚体,每个单体由3个结构域组成,其中MIO结构域含有PAL酶家族的保守序列和Ala-Ser-Gly三肽活性中心,是Ah PAL酶活性的决定性结构域。利用荧光定量PCR方法检测3个Ah PAL Unigene在根、块茎和叶中的表达情况,发现它们在根中表达量均最高,而在叶和块茎中表达较低。
Phenylalanine ammonia-lyase( PAL) is the first key enzyme in the synthesis of flavonoids and the rate-limiting enzyme of the phenylpropanoid pathway,which is associated with secondary metabolites,growth and development,as well as defense of plants. In this study,the mRNA of Arisaema heterophyllum Blume was used as a template and the full-length open reading frame( ORF) of the phenylalanine ammonia-lyase gene( Ah PAL) was amplified by real-time polymerase chain reaction( RT-PCR). The structure and physicochemical properties of Ah PAL were then analyzed by bioinformatics. Results showed that the ORF of Ah PAL was 2,184 bp in length,encoding a protein with 727 amino acids. Furthermore,Ah PAL had the closest relationship with PAL of Tulipa fosteriana W. Irving,with a sequence identity of 88%. Structure analysis indicated that Ah PAL was a homotypic tetramer,and each monomer consisted of three domains. Among these domains,the MIO domain was highly important for the activity of Ah PAL,containing a conserved sequence of the PAL family and Ala-Ser-Gly triad residues. The expression levels of three Ah PAL unigenes in the roots,tubers,and leaves were detected by quantitative RT-PCR( qRT-PCR),with higher expression found for all three in the roots compared with that in the leaves and tubers.
Phenylalanine ammonia-lyase( PAL) is the first key enzyme in the synthesis of flavonoids and the rate-limiting enzyme of the phenylpropanoid pathway,which is associated with secondary metabolites,growth and development,as well as defense of plants. In this study,the mRNA of Arisaema heterophyllum Blume was used as a template and the full-length open reading frame( ORF) of the phenylalanine ammonia-lyase gene( Ah PAL) was amplified by real-time polymerase chain reaction( RT-PCR). The structure and physicochemical properties of Ah PAL were then analyzed by bioinformatics. Results showed that the ORF of Ah PAL was 2,184 bp in length,encoding a protein with 727 amino acids. Furthermore,Ah PAL had the closest relationship with PAL of Tulipa fosteriana W. Irving,with a sequence identity of 88%. Structure analysis indicated that Ah PAL was a homotypic tetramer,and each monomer consisted of three domains. Among these domains,the MIO domain was highly important for the activity of Ah PAL,containing a conserved sequence of the PAL family and Ala-Ser-Gly triad residues. The expression levels of three Ah PAL unigenes in the roots,tubers,and leaves were detected by quantitative RT-PCR( qRT-PCR),with higher expression found for all three in the roots compared with that in the leaves and tubers.
引文
[1]徐大林,朴花子,陈正爱.异叶天南星的药理作用研究进展[J].延边大学医学学报,2013(4):322-324.Xu DL,Piao HZ,Chen ZA.Research development in Arisaema heterophyllum Blume.on pharmacological activities[J].Journal of Medical Science Yanbian University,2013(4):322-324.
[2]何达海,丁克毅,王晓玲.天南星属植物化学成分研究进展[J].西南民族大学学报(自然科学版),2014,40(6):861-865.He DH,Ding KY,Wang XL.Chemical constituent research progress on plants belonging genus Arisaema[J].Journal of Southwest University for Nationalities(Natural Science Edition),2014,40(6):861-865.
[3]张帅.药用植物天南星中有效成分的筛选和药理学研究[D].郑州:郑州大学,2013.
[4]闵巍巍,张作法.黄酮类化合物的药理作用[J].蚕桑通报,2007,38(4):1-3.Min WW,Zhang ZF.Research development in flavonoids on pharmacological activities[J].Bulletin of Sericulture,2007,38(4):1-3.
[5]MrvováN,kandík M,KuniakováM.Modulation of BV-2microglia functions by novel quercetin pivaloyl ester[J].Neurochem Int,2015,90:246-254.
[6]Li Y,Ma C,Qian M,Wen ZM,Jing HY,et al.Butein induces cell apoptosis and inhibition of cyclooxygenase-2expression in A549 lung cancer cells[J].Mol Med Rep,2014,9:763-767.
[7]Min N,Leong PT,Lee RCH,Khuan JSE,Chu JJH.A flavonoid compound library screen revealed potent antiviral activity of plant-derived flavonoids on human enterovirus A71 replication[J].Antiviral Res,2018,150:60-68.
[8]黄小贞,赵德刚.植物苯丙氨酸解氨酶表达调控机理的研究进展[J].贵州农业科学,2017,45(4):16-20.Huang XZ,Zhao DG.Research progress in regulation and control mechanism of phenylalanine ammonia lyasein plants[J].Guizhou Agricultural Sciences,2017,45(4):16-20.
[9]徐晓梅,杨署光.苯丙氨酸解氨酶研究进展[J].安徽农业科学,2009,37(31):15115-15119.Xu XM,Yang SG.Advances in the studies of phenylalanine ammonia-lyase[J].Journal of Anhui Agricultural Science,2009,37(31):15115-15119.
[10]乔枫,耿贵工,张丽,金兰,陈志.枸杞苯丙氨酸解氨酶基因的克隆与表达分析[J].中国农业大学学报,2017,22(12):64-73.Qiao F,Geng GG,Zhang L,Jin L,Chen Z.Molecular cloning and expression pattern of LcPAL from Lycium chinense[J].Journal of China Agricultural University,2017,22(12):64-73.
[11]Zhang C,Wang X,Zhang F,Dong LD,Wu JJ,et al.Phenylalanine ammonia-lyase2.1 contributes to the soybean response towards Phytophthora sojae infection[J].Sci Rep,2017,7:7242.
[12]虞光辉,王桂平,王亮,尹华燕,牟晶晶,等.小麦PAL基因的克隆及赤霉菌诱导下的表达分析[J].植物遗传资源学报,2015,16(5):1055-1061.Yu SH,Wang GP,Wang L,Yin HY,Mou JJ,et al.Cloning of PAL genes and their response to FHB in wheat[J].Journal of Plant Genetic Resources,2015,16(5):1055-1061.
[13]Zhu Q,Xie X,Lin H,Sui SZ,Shen RX,et al.Isolation and functional characterization of a phenylalanine ammonia-Lyase gene(SsPAL1)from Coleus(Solenostemon scutellarioides(L.)Codd)[J].Molecules,2015,20:16833-16851.
[14]赵志常,高爱平,陈业渊,黄建峰,党志国,等.芒果PAL基因的克隆与序列分析[J].安徽农业大学学报,2015,42(5):825-830.Zhao ZJ,Gao AP,Chen YY,Huang JF,Dang ZG,et al.Cloning and sequence analysis of the PAL gene from mango(Mangifera indica)[J].Journal of Anhui Agricultural University,2015,42(5):825-830.
[15]Ritter H.Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase[J].Plant Cell,2004,16:3426-3436.
[16]Jun SY,Sattler SA,Cortez GS,Vermerris W,Sattler SE,et al.Biochemical and structural analysis of substrate specificity of a phenylalanine ammonia-Lyase[J].Plant Physiol,2018,176:1452-1468.
[17]Khan W,Prithiviraj B,Smith DL.Chitosan and chitin oligomers increase phenylalanine ammonia-lyase and tyrosine ammonialyase activities in soybean leaves[J].Plant Physiol,2003,160:859-863.
[18]Nag S.Kumaria S.In silico characterization and transcriptional modulation of phenylalanine ammonia lyase(PAL)by abiotic stresses in the medicinal orchid Vanda coerulea Griff.ex Lindl[J].Phytochemistry,2018,156:176-183.
[19]Ghasemi S,Kumleh HH.Changes in the expression of some genes involved in the biosynthesis of secondary metabolites in Cuminum cyminum L.under UV stress[J].Protoplasma,2019,256(1):279-290.
[20]Li CL,Bai YC,Chen H,Zhao HX,Shao JR,et al.Characterization and functional analysis of a phenylalanine ammonia-lyase gene(Ft PAL)from Fagopyrum tataricum Gaertn[J].Plant Mol Biol Rep,2012,30(5):1172-1182.
[21]Lin W,Liu A,Weng C,Li H,Sun SW,et al.Cloning and characterization of a novel phenylalanine ammonia-lyase gene from Inonotus baumii[J].Enzyme Microb Technol,2018,112:52-58.
[22]Calabrese JC,Jordan DB,Boodhoo A,Sariaslani S,Vannelli T.Crystal structure of phenylalanine ammonia lyase:multiple helix dipoles implicated in catalysis[J].Biochemistry,2004,43:11403-11416.
[23]MacDonald MJ,D’Cunha GB.A modern view of phenylalanine ammonia lyase[J].Biochem Cell Biol,2007,85:273-282.
[24]Pilbák S,Tomin A,Rétey J.The essential tyrosine-containing loop conformation and the role of the C-terminal multihelix region in eukaryotic phenylalanine ammonia-lyases[J].FEBS J,2006,273:1004-1019.
[25]Ma WL,Wu M,Wu Y,Ren ZM,Zhong,Y.Cloning and characterisation of a phenylalanine ammonia-lyase gene from Rhus chinensis[J].Plant Cell Rep,2013,32(8),1179-1190.
[26]Xu F,Deng G,Cheng S,Zhang W,Huang X.Molecular cloning,characterization and expression of the phenylalanine ammonia-lyase gene from Juglans regia[J].Molecules,2012,17(12),7810-7823.
[27]Liu RR,Xu SH,Li JL,Hu YL,Lin ZP.Expression profile of a PAL gene from Astragalus membranaceus var.mongholicus and its crucial role in flux into flavonoid biosynthesis[J].Plant Cell Rep,2006,25:705-710.
[28]Zhao S,Tuan PA,Li X,Kim YB,Kim H,et al.Identification of phenylpropanoid biosynthetic genes and phenylpropanoid accumulation by transcriptome analysis of Lycium chinense[J].BMC Genomics,2013,14:802.
[2]何达海,丁克毅,王晓玲.天南星属植物化学成分研究进展[J].西南民族大学学报(自然科学版),2014,40(6):861-865.He DH,Ding KY,Wang XL.Chemical constituent research progress on plants belonging genus Arisaema[J].Journal of Southwest University for Nationalities(Natural Science Edition),2014,40(6):861-865.
[3]张帅.药用植物天南星中有效成分的筛选和药理学研究[D].郑州:郑州大学,2013.
[4]闵巍巍,张作法.黄酮类化合物的药理作用[J].蚕桑通报,2007,38(4):1-3.Min WW,Zhang ZF.Research development in flavonoids on pharmacological activities[J].Bulletin of Sericulture,2007,38(4):1-3.
[5]MrvováN,kandík M,KuniakováM.Modulation of BV-2microglia functions by novel quercetin pivaloyl ester[J].Neurochem Int,2015,90:246-254.
[6]Li Y,Ma C,Qian M,Wen ZM,Jing HY,et al.Butein induces cell apoptosis and inhibition of cyclooxygenase-2expression in A549 lung cancer cells[J].Mol Med Rep,2014,9:763-767.
[7]Min N,Leong PT,Lee RCH,Khuan JSE,Chu JJH.A flavonoid compound library screen revealed potent antiviral activity of plant-derived flavonoids on human enterovirus A71 replication[J].Antiviral Res,2018,150:60-68.
[8]黄小贞,赵德刚.植物苯丙氨酸解氨酶表达调控机理的研究进展[J].贵州农业科学,2017,45(4):16-20.Huang XZ,Zhao DG.Research progress in regulation and control mechanism of phenylalanine ammonia lyasein plants[J].Guizhou Agricultural Sciences,2017,45(4):16-20.
[9]徐晓梅,杨署光.苯丙氨酸解氨酶研究进展[J].安徽农业科学,2009,37(31):15115-15119.Xu XM,Yang SG.Advances in the studies of phenylalanine ammonia-lyase[J].Journal of Anhui Agricultural Science,2009,37(31):15115-15119.
[10]乔枫,耿贵工,张丽,金兰,陈志.枸杞苯丙氨酸解氨酶基因的克隆与表达分析[J].中国农业大学学报,2017,22(12):64-73.Qiao F,Geng GG,Zhang L,Jin L,Chen Z.Molecular cloning and expression pattern of LcPAL from Lycium chinense[J].Journal of China Agricultural University,2017,22(12):64-73.
[11]Zhang C,Wang X,Zhang F,Dong LD,Wu JJ,et al.Phenylalanine ammonia-lyase2.1 contributes to the soybean response towards Phytophthora sojae infection[J].Sci Rep,2017,7:7242.
[12]虞光辉,王桂平,王亮,尹华燕,牟晶晶,等.小麦PAL基因的克隆及赤霉菌诱导下的表达分析[J].植物遗传资源学报,2015,16(5):1055-1061.Yu SH,Wang GP,Wang L,Yin HY,Mou JJ,et al.Cloning of PAL genes and their response to FHB in wheat[J].Journal of Plant Genetic Resources,2015,16(5):1055-1061.
[13]Zhu Q,Xie X,Lin H,Sui SZ,Shen RX,et al.Isolation and functional characterization of a phenylalanine ammonia-Lyase gene(SsPAL1)from Coleus(Solenostemon scutellarioides(L.)Codd)[J].Molecules,2015,20:16833-16851.
[14]赵志常,高爱平,陈业渊,黄建峰,党志国,等.芒果PAL基因的克隆与序列分析[J].安徽农业大学学报,2015,42(5):825-830.Zhao ZJ,Gao AP,Chen YY,Huang JF,Dang ZG,et al.Cloning and sequence analysis of the PAL gene from mango(Mangifera indica)[J].Journal of Anhui Agricultural University,2015,42(5):825-830.
[15]Ritter H.Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase[J].Plant Cell,2004,16:3426-3436.
[16]Jun SY,Sattler SA,Cortez GS,Vermerris W,Sattler SE,et al.Biochemical and structural analysis of substrate specificity of a phenylalanine ammonia-Lyase[J].Plant Physiol,2018,176:1452-1468.
[17]Khan W,Prithiviraj B,Smith DL.Chitosan and chitin oligomers increase phenylalanine ammonia-lyase and tyrosine ammonialyase activities in soybean leaves[J].Plant Physiol,2003,160:859-863.
[18]Nag S.Kumaria S.In silico characterization and transcriptional modulation of phenylalanine ammonia lyase(PAL)by abiotic stresses in the medicinal orchid Vanda coerulea Griff.ex Lindl[J].Phytochemistry,2018,156:176-183.
[19]Ghasemi S,Kumleh HH.Changes in the expression of some genes involved in the biosynthesis of secondary metabolites in Cuminum cyminum L.under UV stress[J].Protoplasma,2019,256(1):279-290.
[20]Li CL,Bai YC,Chen H,Zhao HX,Shao JR,et al.Characterization and functional analysis of a phenylalanine ammonia-lyase gene(Ft PAL)from Fagopyrum tataricum Gaertn[J].Plant Mol Biol Rep,2012,30(5):1172-1182.
[21]Lin W,Liu A,Weng C,Li H,Sun SW,et al.Cloning and characterization of a novel phenylalanine ammonia-lyase gene from Inonotus baumii[J].Enzyme Microb Technol,2018,112:52-58.
[22]Calabrese JC,Jordan DB,Boodhoo A,Sariaslani S,Vannelli T.Crystal structure of phenylalanine ammonia lyase:multiple helix dipoles implicated in catalysis[J].Biochemistry,2004,43:11403-11416.
[23]MacDonald MJ,D’Cunha GB.A modern view of phenylalanine ammonia lyase[J].Biochem Cell Biol,2007,85:273-282.
[24]Pilbák S,Tomin A,Rétey J.The essential tyrosine-containing loop conformation and the role of the C-terminal multihelix region in eukaryotic phenylalanine ammonia-lyases[J].FEBS J,2006,273:1004-1019.
[25]Ma WL,Wu M,Wu Y,Ren ZM,Zhong,Y.Cloning and characterisation of a phenylalanine ammonia-lyase gene from Rhus chinensis[J].Plant Cell Rep,2013,32(8),1179-1190.
[26]Xu F,Deng G,Cheng S,Zhang W,Huang X.Molecular cloning,characterization and expression of the phenylalanine ammonia-lyase gene from Juglans regia[J].Molecules,2012,17(12),7810-7823.
[27]Liu RR,Xu SH,Li JL,Hu YL,Lin ZP.Expression profile of a PAL gene from Astragalus membranaceus var.mongholicus and its crucial role in flux into flavonoid biosynthesis[J].Plant Cell Rep,2006,25:705-710.
[28]Zhao S,Tuan PA,Li X,Kim YB,Kim H,et al.Identification of phenylpropanoid biosynthetic genes and phenylpropanoid accumulation by transcriptome analysis of Lycium chinense[J].BMC Genomics,2013,14:802.