菊花CmPAL基因的克隆及表达分析
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摘要
[目的]本文旨在克隆菊花中苯丙氨酸解氨酶基因(PAL),并探讨其在不同组织的表达特性以及在胁迫和激素处理下的响应模式。[方法]以菊花‘神马’为试验材料,依据其转录组数据库信息,克隆CmPAL全长,并利用生物信息学方法对其进行分析。利用荧光定量PCR检测该基因在不同组织中的相对表达量,在蚜虫和干旱胁迫以及不同激素处理下的表达变化。[结果]CmPAL基因开放阅读框(ORF)全长2 154 bp,编码氨基酸717个,理论等电点(p I)为5.75,预测蛋白相对分子质量为77.73×10~3。系统进化分析表明该基因编码的蛋白与黄花蒿和泡黄金菊的PAL亲缘关系最近,同源性分别为97.94%和91.88%,具有较高的保守性。CmPAL在菊花根中表达量最高,管状花中次之。蚜虫胁迫和茉莉酸甲酯(MeJA)可诱导CmPAL基因的表达,水杨酸(SA)、独角金内酯类似物GR24则抑制CmPAL的表达。干旱胁迫3 h处理组CmPAL基因表达量高于对照组。[结论]菊花‘神马’CmPAL基因在菊花的根中表达量最高,并受蚜虫和MeJA诱导,受SA和独角金内酯类似物GR24抑制,表明该基因可能参与多种胁迫防御反应和激素应答。
[Objectives]The purpose of this research is to clone the phenylalanine ammonia-lyase gene( PAL) in chrysanthemum and dissect it roles in response to different stresses and phytohormones. [Methods]The full-length of CmPAL was obtained from chrysanthemum'Jinba'based on its RNA-seq library. Bioinformatic technologies were used to analyze the gene. The relative expression levels of CmPAL in different tissues of plant were detected by RT-qPCR. The expression profiles of CmPAL under aphids,drought,and different plant hormones were analyzed. [Results]CmPAL gene had an open reading frame( ORF) of 2 154 bp encoding 717 amino acids. The theoretical isoelectric point( p I) was 5.75,and the predicted protein molecular weight was 77.73×10~3. Phylogenetic analysis showed that it had the closest relationship with Artemisia annua and Chrysanthemum boreale,and the amino acid sequence identity was 97.94% and 91.88%,respectively. CmPAL had the highest expression level in root,followed by that in tubular floret.CmPAL was induced by aphid and methyl jasmonate( MeJA),while it was inhibited by salicylic acid( SA) or the strigolactone analogue GR24. The expression level of CmPAL at 3 h after drought treatment was higher than that of the control group.[Conclusions]CmPAL in chrysanthemum'Jinba'was of the highest expression level in root. The expression of CmPAL positively responded to aphid and MeJA,while negatively to SA and GR24,which suggested that CmPAL might be involved in multiple stress defense and phytohormone responses.
[Objectives]The purpose of this research is to clone the phenylalanine ammonia-lyase gene( PAL) in chrysanthemum and dissect it roles in response to different stresses and phytohormones. [Methods]The full-length of CmPAL was obtained from chrysanthemum'Jinba'based on its RNA-seq library. Bioinformatic technologies were used to analyze the gene. The relative expression levels of CmPAL in different tissues of plant were detected by RT-qPCR. The expression profiles of CmPAL under aphids,drought,and different plant hormones were analyzed. [Results]CmPAL gene had an open reading frame( ORF) of 2 154 bp encoding 717 amino acids. The theoretical isoelectric point( p I) was 5.75,and the predicted protein molecular weight was 77.73×10~3. Phylogenetic analysis showed that it had the closest relationship with Artemisia annua and Chrysanthemum boreale,and the amino acid sequence identity was 97.94% and 91.88%,respectively. CmPAL had the highest expression level in root,followed by that in tubular floret.CmPAL was induced by aphid and methyl jasmonate( MeJA),while it was inhibited by salicylic acid( SA) or the strigolactone analogue GR24. The expression level of CmPAL at 3 h after drought treatment was higher than that of the control group.[Conclusions]CmPAL in chrysanthemum'Jinba'was of the highest expression level in root. The expression of CmPAL positively responded to aphid and MeJA,while negatively to SA and GR24,which suggested that CmPAL might be involved in multiple stress defense and phytohormone responses.
引文
[1] Vogt T. Phenylpropanoid biosynthesis[J]. Molecular Plant,2010,3(1):2-20.
[2] Irisarri P,Zhebentyayeva T,Errea P,et al. Differential expression of phenylalanine ammonia lyase(PAL)genes implies distinct roles in development of graft incompatibility symptoms in Prunus[J]. Scientia Horticulturae,2016,204:16-24.
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[4] Shang Q M,Li L,Dong C J. Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L.[J]. Planta,2012,236(4):1093-1105.
[5] Kim D S,Hwang B K. An important role of the pepper phenylalanine ammonia-lyase gene(PAL1)in salicylic acid-dependent signalling of the defence response to microbial pathogens[J]. Journal of Experimental Botany,2014,65(9):2295-2306.
[6] Cass C L,Peraldi A,Dowd P F,et al. Effects of PHENYLALANINE AMMONIA LYASE(PAL)knockdown on cell wall composition,biomass digestibility,and biotic and abiotic stress responses in Brachypodium[J]. Journal of Experimental Botany,2015,66(14):4317-4335.
[7] Zhang Y,Fu X,Hao X,et al. Molecular cloning and promoter analysis of the specific salicylic acid biosynthetic pathway gene phenylalanine ammonia-lyase(AaPAL1)from Artemisia annua[J]. Biotechnology and Applied Biochemistry,2016,63(4):514-524.
[8] Xia X,Shao Y,Jiang J,et al. Gene expression profiles responses to aphid feeding in chrysanthemum(Chrysanthemum morifolium)[J]. BMC Genomics,2014,15(1):1050-1066.
[9]王银杰.独脚金内酯调控菊花抗蚜性机制研究[D].南京:南京农业大学,2017.Wang Y J. The roles of strigolactone in resistance of chrysanthemum to aphid[D]. Nanjing:Nanjing Agricultural University,2017(in Chinese with English abstract).
[10] Li P,Song A,Gao C,et al. The over-expression of a chrysanthemum WRKY transcription factor enhances aphid resistance[J]. Plant Physiology and Biochemistry,2015,95:26-34.
[11] Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods,2001,25(4):402-408.
[12] Yang H Y,Dong T,Li J F,et al. Molecular cloning,expression,and subcellular localization of a PAL gene from Citrus reticulata under iron deficiency[J]. Biologia Plantarum,2016,60(3):482-488.
[13] Ohl S,Hedrick S A,Chory J,et al. Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis[J]. The Plant Cell,1990,2(9):837-848.
[14] de Jong F,Hanley S J,Beale M H,et al. Characterisation of the willow phenylalanine ammonia-lyase(PAL)gene family reveals expression differences compared with poplar[J]. Phytochemistry,2015,117:90-97.
[15] Elkind Y,Edwards R,Mavandad M,et al. Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene[J]. Proc Natl Acad Sci USA,1990,87(22):9057-9061.
[16] Li W,Tian Z,Yu D. WRKY13 acts in stem development in Arabidopsis thaliana[J]. Plant Science,2015,236:205-213.
[17] Dar S A,Wani A R,Rather B A,et al. Resistance against insect pests by plant phenolics and their derivative compounds[J]. Chem Sci Rev Lett,2017,6(22):1073-1081.
[18] Villada E S,Gonzalez E G,López-SeséA I,et al. Hypersensitive response to Aphis gossypii Glover in melon genotypes carrying the Vat gene[J].Journal of Experimental Botany,2009,60(11):3269-3277.
[19] Napal G N D,Palacios S M. Bioinsecticidal effect of the flavonoids pinocembrin and quercetin against Spodoptera frugiperda[J]. Journal of Pest Science,2015,88(3):629-635.
[20] Nakabayashi R,Yonekura-Sakakibara K,Urano K,et al. Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids[J]. The Plant Journal,2014,77(3):367-379.
[21] Zhu L J,Deng X G,Zou L J,et al. Enhancement of stress tolerance in cucumber seedlings by proanthocyanidins[J]. Biologia Plantarum,2017,61(2):323-332.
[22] Ahmad P,Rasool S,Gul A,et al. Jasmonates:multifunctional roles in stress tolerance[J]. Frontiers in Plant Science,2016,7:813.
[23] Glowacz M,Roets N,Sivakumar D. Control of anthracnose disease via increased activity of defence related enzymes in‘Hass’avocado fruit treated with methyl jasmonate and methyl salicylate[J]. Food Chemistry,2017,234:163-167.
[24] Schouteden N,Lemmens E,Stuer N,et al. Direct nematicidal effects of methyl jasmonate and acibenzolar-S-methyl against Meloidogyne incognita[J].Natural Product Research,2017,31(10):1219-1222.
[25] Caarls L,Pieterse C M J,van Wees S. How salicylic acid takes transcriptional control over jasmonic acid signaling[J]. Frontiers in Plant Science,2015,6:170.
[26] Kusniercayk A,Winge P E R,Jrstad T S,et al. Towards global understanding of plant defence against aphids-timing and dynamics of early Arabidopsis defence responses to cabbage aphid(Brevicoryne brassicae)attack[J]. Plant,Cell and Environment,2008,31(8):1097-1115.
[27] Thaler J S,Humphrey P T,Whiteman N K. Evolution of jasmonate and salicylate signal crosstalk[J]. Trends in Plant Science,2012,17(5):260-270.
[28] Bu Q,LüT,Shen H,et al. Regulation of drought tolerance by the F-box protein MAX2 in Arabidopsis[J]. Plant Physiology,2014,164(1):424-439.
[29] PiisilaM,Keceli M A,Brader G,et al. The F-box protein MAX2 contributes to resistance to bacterial phytopathogens in Arabidopsis thaliana[J].BMC Plant Biology,2015,15(1):53-70.
[30] Cooper J W,Hu Y,Beyyoudh L,et al. Strigolactones positively regulate chilling tolerance in pea and in Arabidopsis[J]. Plant,Cell and Environment,2018,41(6):1298-1310.
[2] Irisarri P,Zhebentyayeva T,Errea P,et al. Differential expression of phenylalanine ammonia lyase(PAL)genes implies distinct roles in development of graft incompatibility symptoms in Prunus[J]. Scientia Horticulturae,2016,204:16-24.
[3] Dixon R A,Paiva N L. Stress-induced phenylpropanoid metabolism[J]. The Plant Cell,1995,7(7):1085-1097.
[4] Shang Q M,Li L,Dong C J. Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L.[J]. Planta,2012,236(4):1093-1105.
[5] Kim D S,Hwang B K. An important role of the pepper phenylalanine ammonia-lyase gene(PAL1)in salicylic acid-dependent signalling of the defence response to microbial pathogens[J]. Journal of Experimental Botany,2014,65(9):2295-2306.
[6] Cass C L,Peraldi A,Dowd P F,et al. Effects of PHENYLALANINE AMMONIA LYASE(PAL)knockdown on cell wall composition,biomass digestibility,and biotic and abiotic stress responses in Brachypodium[J]. Journal of Experimental Botany,2015,66(14):4317-4335.
[7] Zhang Y,Fu X,Hao X,et al. Molecular cloning and promoter analysis of the specific salicylic acid biosynthetic pathway gene phenylalanine ammonia-lyase(AaPAL1)from Artemisia annua[J]. Biotechnology and Applied Biochemistry,2016,63(4):514-524.
[8] Xia X,Shao Y,Jiang J,et al. Gene expression profiles responses to aphid feeding in chrysanthemum(Chrysanthemum morifolium)[J]. BMC Genomics,2014,15(1):1050-1066.
[9]王银杰.独脚金内酯调控菊花抗蚜性机制研究[D].南京:南京农业大学,2017.Wang Y J. The roles of strigolactone in resistance of chrysanthemum to aphid[D]. Nanjing:Nanjing Agricultural University,2017(in Chinese with English abstract).
[10] Li P,Song A,Gao C,et al. The over-expression of a chrysanthemum WRKY transcription factor enhances aphid resistance[J]. Plant Physiology and Biochemistry,2015,95:26-34.
[11] Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods,2001,25(4):402-408.
[12] Yang H Y,Dong T,Li J F,et al. Molecular cloning,expression,and subcellular localization of a PAL gene from Citrus reticulata under iron deficiency[J]. Biologia Plantarum,2016,60(3):482-488.
[13] Ohl S,Hedrick S A,Chory J,et al. Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis[J]. The Plant Cell,1990,2(9):837-848.
[14] de Jong F,Hanley S J,Beale M H,et al. Characterisation of the willow phenylalanine ammonia-lyase(PAL)gene family reveals expression differences compared with poplar[J]. Phytochemistry,2015,117:90-97.
[15] Elkind Y,Edwards R,Mavandad M,et al. Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene[J]. Proc Natl Acad Sci USA,1990,87(22):9057-9061.
[16] Li W,Tian Z,Yu D. WRKY13 acts in stem development in Arabidopsis thaliana[J]. Plant Science,2015,236:205-213.
[17] Dar S A,Wani A R,Rather B A,et al. Resistance against insect pests by plant phenolics and their derivative compounds[J]. Chem Sci Rev Lett,2017,6(22):1073-1081.
[18] Villada E S,Gonzalez E G,López-SeséA I,et al. Hypersensitive response to Aphis gossypii Glover in melon genotypes carrying the Vat gene[J].Journal of Experimental Botany,2009,60(11):3269-3277.
[19] Napal G N D,Palacios S M. Bioinsecticidal effect of the flavonoids pinocembrin and quercetin against Spodoptera frugiperda[J]. Journal of Pest Science,2015,88(3):629-635.
[20] Nakabayashi R,Yonekura-Sakakibara K,Urano K,et al. Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids[J]. The Plant Journal,2014,77(3):367-379.
[21] Zhu L J,Deng X G,Zou L J,et al. Enhancement of stress tolerance in cucumber seedlings by proanthocyanidins[J]. Biologia Plantarum,2017,61(2):323-332.
[22] Ahmad P,Rasool S,Gul A,et al. Jasmonates:multifunctional roles in stress tolerance[J]. Frontiers in Plant Science,2016,7:813.
[23] Glowacz M,Roets N,Sivakumar D. Control of anthracnose disease via increased activity of defence related enzymes in‘Hass’avocado fruit treated with methyl jasmonate and methyl salicylate[J]. Food Chemistry,2017,234:163-167.
[24] Schouteden N,Lemmens E,Stuer N,et al. Direct nematicidal effects of methyl jasmonate and acibenzolar-S-methyl against Meloidogyne incognita[J].Natural Product Research,2017,31(10):1219-1222.
[25] Caarls L,Pieterse C M J,van Wees S. How salicylic acid takes transcriptional control over jasmonic acid signaling[J]. Frontiers in Plant Science,2015,6:170.
[26] Kusniercayk A,Winge P E R,Jrstad T S,et al. Towards global understanding of plant defence against aphids-timing and dynamics of early Arabidopsis defence responses to cabbage aphid(Brevicoryne brassicae)attack[J]. Plant,Cell and Environment,2008,31(8):1097-1115.
[27] Thaler J S,Humphrey P T,Whiteman N K. Evolution of jasmonate and salicylate signal crosstalk[J]. Trends in Plant Science,2012,17(5):260-270.
[28] Bu Q,LüT,Shen H,et al. Regulation of drought tolerance by the F-box protein MAX2 in Arabidopsis[J]. Plant Physiology,2014,164(1):424-439.
[29] PiisilaM,Keceli M A,Brader G,et al. The F-box protein MAX2 contributes to resistance to bacterial phytopathogens in Arabidopsis thaliana[J].BMC Plant Biology,2015,15(1):53-70.
[30] Cooper J W,Hu Y,Beyyoudh L,et al. Strigolactones positively regulate chilling tolerance in pea and in Arabidopsis[J]. Plant,Cell and Environment,2018,41(6):1298-1310.