马占相思纤维素合酶基因AmCesA1的克隆及分析
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摘要
【目的】马占相思是我国南方广泛种植的一种造纸用树。纤维素合酶(CesA)在植物纤维素合成途径中发挥主要调节作用,是控制木材纤维品质和产量的重要因素。本研究克隆马占相思的纤维素合酶基因,研究它对激素的应答,为了解马占相思的纤维素合成和获得高纤维素得率的马占相思良种提供帮助。【方法】采用RT-PCR和RACE技术,从马占相思幼苗中获得1个CesA基因,命名为AmCesA1(AY643519)。利用生物信息学软件对该基因进行分析。使用Southern分析确定该基因的拷贝形式。采用实时荧光定量PCR确定该基因在不同组织中的表达量以及基因在赤霉素(GA_3)、6-BA、茉莉酸甲酯(MeJA)处理后的表达变化。【结果】AmCesA1的cDNA大小为3 793 bp,其开放读码框为3 249 bp,推测这个基因编码1 082个氨基酸;蛋白分子式为C5495H8491N1457O1579S50,分子质量为121.83 kDa。正电荷氨基酸残基数(Arg+Lys)的数目为121,负电荷氨基酸残基数(Asp+Glu)的数目为125;等电点为6.51,为酸性蛋白质;它的不稳定系数是40.82,属于不稳定蛋白质。AmCesA1的氨基酸一级结构分析显示它具有纤维素合酶保守的D,D,D,QxxRW功能域,具有植物纤维素合酶所特有的P-CR区及HVR区和N端的锌指结构,在C端存在6个跨膜区域,但在N端的2个跨膜区域并不明显。二级结构分析显示它具有较多的α-螺旋、无规则卷曲,β-转角很少,β-折叠数目因算法的不同存在较大差异。聚类分析表明,AmCesA1与大豆GmCesA1和蔓花生Ad CesA1相似性较高。但并没有出现预期同木本植物亲缘较近的结果。进一步与拟南芥纤维素合酶家族氨基酸序列比较,发现AmCesA1与拟南芥的AtCesA1和AtCesA10比较相似,其相似性为86%和80%,从而推测它的功能与拟南芥的AtCesA1和AtCesA10相近。Southern分析显示,马占相思基因组中,AmCesA1是以多拷贝形式存在。实时荧光定量PCR表明,AmCesA1广泛表达于根、茎、叶部位,且差异不显著。AmCesA1对GA_3、6-BA、MeJA处理均有响应,其中GA_3处理响应相对最强。【结论】本研究克隆到的马占相思AmCesA1为植物CesA基因家族中的一员,推测其参与初级细胞壁的形成。该基因对赤霉素、6-BA、茉莉酸甲酯处理均有响应,其中基因的表达量在不同的激素处理中都有不同程度的上调。表明该基因参与马占相思对激素应答的正调控。
【Objective】Acacia mangium is widely cultivated in southern China for paper production. Cellulose synthase( CesA) plays a vital role in the synthesis of cellulose,which is an important factor in controlling the quality and yield of wood fiber. In this study,we cloned a cellulose synthase gene of A. mangium,and studied its response to hormones to help the understanding of the synthesis of cellulose and the high fiber yield of A. mangium. 【Method 】 A CesA was obtained from A. mangium seedlings by RT-PCR and RACE,named AmCesA1( AY643519). The gene was analyzed by bioinformatics software. The copy form of the gene was determined through using Southern analysis. The expression level of the gene in different tissues and the expression of gibberellin,6-BA and methyl jasmonate were determined by real-time fluorescence quantitative PCR.【Result】 The molecular size of AmCesA1 is 3 793 bp,and its ORF is 3 249 bp,suggesting that this gene encodes 1 082 amino acids. Protein molecular formula is C5495 H8491 N1457 O1579 S50. The number of positive charge amino acid residues( Arg + Lys) is 121,and the number of negative charge amino acid residues( Asp + Glu) is125. The isoelectric point is 6. 51,which means that it is acidic protein. Its instability coefficient is 40. 82,belonging to the unstable protein. The amino acid primary structure analysis of AmCesA1 showed that it had the conserved D,D,D and QxxRW functional domains of cellulose synthase,and had a unique P-CR region and the HVR region and N-terminal zinc finger structure. There were six transmembrane regions at the C-terminus,but the two transmembrane regions at the N-terminus were not significant. Secondary structure analysis showed that it had more α-helix,random coil,but fewerβ-turn,while β-sheet number varies greatly due to the different algorithms. Cluster analysis showed that AmCesA1 had similarity with Glycine max GmCesA1 and Arachis duranensis Ad CesA1. But it did not appear to be close to the results of woody plants. Further comparison with Arabidopsis thaliana cellulose gene family amino acid sequence indicated a fact that AmCesA1 was similar to AtCesA1 and AtCesA10 in A. thaliana,and its similarity was 86% and 80%,suggesting that it had the same function as A. thaliana AtCesA1 and AtCesA10. Southern analysis showed that AmCesA1 was present in multiple copies of the Acacia tree genome. Real-time quantitative PCR showed that AmCesA1 was widely expressed in roots,stems and leaves,and the differences among them were not significant. AmCesA1 had a response to GA_3,6-BA and MeJA treatments,in which the response to GA_3 was relatively strong.【Conclusion】 AmCesA1 cloned in this study is a member of the plant CesA family,presumably involved in the formation of primary cell walls. The gene was responsive to gibberellin,6-BA and methyl jasmonate,and the expression level of the gene was up-regulated in different hormone treatments,which indicates that the gene was involved in the positive regulation of the hormone response.
【Objective】Acacia mangium is widely cultivated in southern China for paper production. Cellulose synthase( CesA) plays a vital role in the synthesis of cellulose,which is an important factor in controlling the quality and yield of wood fiber. In this study,we cloned a cellulose synthase gene of A. mangium,and studied its response to hormones to help the understanding of the synthesis of cellulose and the high fiber yield of A. mangium. 【Method 】 A CesA was obtained from A. mangium seedlings by RT-PCR and RACE,named AmCesA1( AY643519). The gene was analyzed by bioinformatics software. The copy form of the gene was determined through using Southern analysis. The expression level of the gene in different tissues and the expression of gibberellin,6-BA and methyl jasmonate were determined by real-time fluorescence quantitative PCR.【Result】 The molecular size of AmCesA1 is 3 793 bp,and its ORF is 3 249 bp,suggesting that this gene encodes 1 082 amino acids. Protein molecular formula is C5495 H8491 N1457 O1579 S50. The number of positive charge amino acid residues( Arg + Lys) is 121,and the number of negative charge amino acid residues( Asp + Glu) is125. The isoelectric point is 6. 51,which means that it is acidic protein. Its instability coefficient is 40. 82,belonging to the unstable protein. The amino acid primary structure analysis of AmCesA1 showed that it had the conserved D,D,D and QxxRW functional domains of cellulose synthase,and had a unique P-CR region and the HVR region and N-terminal zinc finger structure. There were six transmembrane regions at the C-terminus,but the two transmembrane regions at the N-terminus were not significant. Secondary structure analysis showed that it had more α-helix,random coil,but fewerβ-turn,while β-sheet number varies greatly due to the different algorithms. Cluster analysis showed that AmCesA1 had similarity with Glycine max GmCesA1 and Arachis duranensis Ad CesA1. But it did not appear to be close to the results of woody plants. Further comparison with Arabidopsis thaliana cellulose gene family amino acid sequence indicated a fact that AmCesA1 was similar to AtCesA1 and AtCesA10 in A. thaliana,and its similarity was 86% and 80%,suggesting that it had the same function as A. thaliana AtCesA1 and AtCesA10. Southern analysis showed that AmCesA1 was present in multiple copies of the Acacia tree genome. Real-time quantitative PCR showed that AmCesA1 was widely expressed in roots,stems and leaves,and the differences among them were not significant. AmCesA1 had a response to GA_3,6-BA and MeJA treatments,in which the response to GA_3 was relatively strong.【Conclusion】 AmCesA1 cloned in this study is a member of the plant CesA family,presumably involved in the formation of primary cell walls. The gene was responsive to gibberellin,6-BA and methyl jasmonate,and the expression level of the gene was up-regulated in different hormone treatments,which indicates that the gene was involved in the positive regulation of the hormone response.
引文
陈英,邵志龙,王浩然,等.2015.杨树Pe AFB基因克隆及表达模式初步分析.林业科学,5(8):26-32.(Chen Y,Shao Z L,Wang H R,et al.2015.Cloning and expression of Pe AFB genes in Populus.Scientia Silvae Sinicae,5(8):26-32.[in Chinese])
李晶,王亦学,郑德刚,等.2009.一种简单高效提取棉花不同组织总RNA的方法.山西农业科学,37(5):17-19.(Li J,Wang Y X,Zheng D G,et al.2009.A simply and high efficiency method of total RNA isolation in upland cotton.Journal of Shanxi Agricultural Sciences,37(5):17-19.[in Chinese])
刘昱翔,陈建荣,彭彦,等.2014.苎麻纤维素合成酶基因Bn Ces A4 c DNA序列的克隆与表达分析.作物研究,28(5):472-478.(Liu Y X,Chen J R,Peng Y,et al.2014.The c DNA cloning and expression analysis on cellulose synthase Bn Ces A4 in Boehmeria nivea.Crop Research,28(5):472-478.[in Chinese])
庞景,童再康,黄华宏,等.2015.杉木纤维素合成酶基因Ces A的克隆及表达分析.浙江农林大学学报,32(1):40-46.(Pang J,Tong Z K,Huang H H,et al.2015.Isolation and expression analysis of cellulose synthase genes in Chinese fir(Cunninghamia lanceolata).Journal of Zhejiang A&F University,32(1):40-46.[in Chinese])
阮维程,潘婷,季孔庶.2015.马尾松纤维素合成酶基因Pm Ces A1的克隆及其分析.分子植物育种,13(4):861-870.(Ruan W C,Pan T,Ji K S.2015.Cloning and analysis on Pm Ces A1gene encoding Pinus massoniana cellulose synthase.Molecular Plant Breeding,13(4):861-870.[in Chinese])
魏凯莉,周厚君,江成,等.2017.杨树次生壁纤维素合酶的表达与互作模式分析.林业科学研究,30(2):245-253.(Wei K L,Zhou H J,Jiang C,et al.2017.Interaction and expression of secondary wall CESAs in Populus.Forest Research,30(2):245-253.[in Chinese])
许雷,刘一星,方连玉.2012.大青杨纤维素合成酶Pu Ces A6基因c DNA的克隆及序列分析.西南林业大学学报,32(5):26-32.(Xu L,Liu Y X,Fang L Y.2012.Cloning and sequence character analysis of full-length c DNA of cellulose synthase Pu Ces A6 from Populus ussuriensis.Journal of Southwest Forestry University,32(5):26-32.[in Chinese])
张保才,周奕华.2015.植物细胞壁形成机制的新进展.中国科学:生命科学,45(6):544-556.(Zhang B C,Zhou Y H.2015.New Advances in the mechanism of plant cell wall formation.Scientia Sinica Vitae,45(6):544-556.[in Chinese])
Arioli T,Peng L C,Betzner A S,et al.1998.Molecular analysis of cellulose biosynthesis in Arabidopsis.Science,279(5351):717-720.
Carroll A,Mansoori N,Li S,et al.2012.Complexes with mixed primary and secondary cellulose synthases are functional in Arabidopsis plants.Plant Physiol,160(2):726-737.
Derbyshire P,Mccann M C,Roberts K.2007.Restricted cell elongation in Arabidopsis hupocatyls is associated with a reduced average pectin esterification level.BMC Plant Biol,7(1):31.
Ellis C,Karafyllidis I,Wasternack C,et al.2002.The Arabidopsis mutant cevl links cell wall signaling to jasmonate and ethylene responses.Plant Cell,14(7):1557-1566.
Fagard M,Desnos T,Desprez T,et al.2000.PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis.Plant Cell,12(12):2409-2424.
Gonneau M,Desprez T,Guillot A,et al.2014.Catalytic subunit stoichiometry within the cellulose synthase complex.Plant Physiol,166(4):1709-1712.
Harris D M,Corbin K,Wang T,et al.2012.Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residuces CESA1A903V and CESA3T9421 of cellulose synthase.Proc Natl Acad Sci USA,109(11):4098-4103.
Hill J L Jr,Hammudi M B,Tien M.2014.The Arabidopsis cellulose synthase complex:a proposed hexamer of CESA trimers in an equimolar stoichipmetry.Plant Cell,26(12):4834-4842.
Hossain Z,Mcgarvey B,Amyot L,et al.2012.DIMINUTO1 affects the lignin profile and secondary cell wall formation in Arabidopsis.Planta,235(3):485-498.
Mizrachi E,Mansfield S D,Myburg A A.2012.Cellulose factories:advancing bioenergy production from forest trees.New Phytol,194(1):54-62.
Morgan J L,Strumillo J,Zimmer J.2013.Crystallographic snapshot of cellulose synthesis and menmbrane translocation.Nature,493(7431):181-186.
Omadjela O,Narahari A,Strumillo J,et al.2013.Bcs A and Bcs B form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis.Proc Natl Acad Sci USA,110(44):17856-17861.
Pear J R,Kawagoe Y,Schreckengost W E,et al.1996.Higher plants contain homologs of the bacterial Cel A genes encoding the catalytic subunit of cellulose synthase.Proc Natl Acad Sci USA,93(22):12637-12642.
Saxena I M,Brown R M Jr.2000.Cellulose synthases and related enzymes.Current Opinion in Plant Biology,3(6):523-531.
Scheible W R,Eshed R,Richmond T,et al.2001.Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidopsis Ixr1 mutants.Proc Natl Acad Sci USA,98(18):10079-10084.
Ong S S,Wickneswari R.2012.Characterization of microRNAs expressed during secondary wall biosynthesis in Acacia mangium.PLo S ONE,7(11):e49662.
Sethapphong L,Davis J K,Slabaugh E,et al.2016.Prediction of the structures of the plant specific regions of vascular plant cellulose synthases and correlated functional analysis.Cellulose,23(1):145-161.
Song D,Shen J,Li L.2010.Characterization of cellulose synthase complexes in Populus xylem differentiation.New Phytologist,187(3):777-790.
Taylor N G.2008.Cellulose biosynthesis and deposition in higher plants.New Phytologist,178(2):239-252.
Taylor N G,Laurie S,Turner S R.2000.Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis.Plant Cell,12(12):2529-2539.
Taylor N G,Scheible W R,Cutler S,et al.1999.The irregular xylem3locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis.Plant Cell,11(5):769-780.
Turner S R,Somerville C R.1997.Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall.Plant Cell,9(5):689-701.
Wu L,Joshi C P.2000.A xylem-specific cellulose synthase gene from aspen(Populus tremuloides)is responsive to mechanical stress.The Plant Journal,22(6):495-502.
Xu P,Donaldson L,Gergely Z,et al.2007.Dual-axis electron tomography:a new approach for investigating the spatial organization of wood cellulose microfibrils.Wood Sci Technol,41(2):101-116.
李晶,王亦学,郑德刚,等.2009.一种简单高效提取棉花不同组织总RNA的方法.山西农业科学,37(5):17-19.(Li J,Wang Y X,Zheng D G,et al.2009.A simply and high efficiency method of total RNA isolation in upland cotton.Journal of Shanxi Agricultural Sciences,37(5):17-19.[in Chinese])
刘昱翔,陈建荣,彭彦,等.2014.苎麻纤维素合成酶基因Bn Ces A4 c DNA序列的克隆与表达分析.作物研究,28(5):472-478.(Liu Y X,Chen J R,Peng Y,et al.2014.The c DNA cloning and expression analysis on cellulose synthase Bn Ces A4 in Boehmeria nivea.Crop Research,28(5):472-478.[in Chinese])
庞景,童再康,黄华宏,等.2015.杉木纤维素合成酶基因Ces A的克隆及表达分析.浙江农林大学学报,32(1):40-46.(Pang J,Tong Z K,Huang H H,et al.2015.Isolation and expression analysis of cellulose synthase genes in Chinese fir(Cunninghamia lanceolata).Journal of Zhejiang A&F University,32(1):40-46.[in Chinese])
阮维程,潘婷,季孔庶.2015.马尾松纤维素合成酶基因Pm Ces A1的克隆及其分析.分子植物育种,13(4):861-870.(Ruan W C,Pan T,Ji K S.2015.Cloning and analysis on Pm Ces A1gene encoding Pinus massoniana cellulose synthase.Molecular Plant Breeding,13(4):861-870.[in Chinese])
魏凯莉,周厚君,江成,等.2017.杨树次生壁纤维素合酶的表达与互作模式分析.林业科学研究,30(2):245-253.(Wei K L,Zhou H J,Jiang C,et al.2017.Interaction and expression of secondary wall CESAs in Populus.Forest Research,30(2):245-253.[in Chinese])
许雷,刘一星,方连玉.2012.大青杨纤维素合成酶Pu Ces A6基因c DNA的克隆及序列分析.西南林业大学学报,32(5):26-32.(Xu L,Liu Y X,Fang L Y.2012.Cloning and sequence character analysis of full-length c DNA of cellulose synthase Pu Ces A6 from Populus ussuriensis.Journal of Southwest Forestry University,32(5):26-32.[in Chinese])
张保才,周奕华.2015.植物细胞壁形成机制的新进展.中国科学:生命科学,45(6):544-556.(Zhang B C,Zhou Y H.2015.New Advances in the mechanism of plant cell wall formation.Scientia Sinica Vitae,45(6):544-556.[in Chinese])
Arioli T,Peng L C,Betzner A S,et al.1998.Molecular analysis of cellulose biosynthesis in Arabidopsis.Science,279(5351):717-720.
Carroll A,Mansoori N,Li S,et al.2012.Complexes with mixed primary and secondary cellulose synthases are functional in Arabidopsis plants.Plant Physiol,160(2):726-737.
Derbyshire P,Mccann M C,Roberts K.2007.Restricted cell elongation in Arabidopsis hupocatyls is associated with a reduced average pectin esterification level.BMC Plant Biol,7(1):31.
Ellis C,Karafyllidis I,Wasternack C,et al.2002.The Arabidopsis mutant cevl links cell wall signaling to jasmonate and ethylene responses.Plant Cell,14(7):1557-1566.
Fagard M,Desnos T,Desprez T,et al.2000.PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis.Plant Cell,12(12):2409-2424.
Gonneau M,Desprez T,Guillot A,et al.2014.Catalytic subunit stoichiometry within the cellulose synthase complex.Plant Physiol,166(4):1709-1712.
Harris D M,Corbin K,Wang T,et al.2012.Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residuces CESA1A903V and CESA3T9421 of cellulose synthase.Proc Natl Acad Sci USA,109(11):4098-4103.
Hill J L Jr,Hammudi M B,Tien M.2014.The Arabidopsis cellulose synthase complex:a proposed hexamer of CESA trimers in an equimolar stoichipmetry.Plant Cell,26(12):4834-4842.
Hossain Z,Mcgarvey B,Amyot L,et al.2012.DIMINUTO1 affects the lignin profile and secondary cell wall formation in Arabidopsis.Planta,235(3):485-498.
Mizrachi E,Mansfield S D,Myburg A A.2012.Cellulose factories:advancing bioenergy production from forest trees.New Phytol,194(1):54-62.
Morgan J L,Strumillo J,Zimmer J.2013.Crystallographic snapshot of cellulose synthesis and menmbrane translocation.Nature,493(7431):181-186.
Omadjela O,Narahari A,Strumillo J,et al.2013.Bcs A and Bcs B form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis.Proc Natl Acad Sci USA,110(44):17856-17861.
Pear J R,Kawagoe Y,Schreckengost W E,et al.1996.Higher plants contain homologs of the bacterial Cel A genes encoding the catalytic subunit of cellulose synthase.Proc Natl Acad Sci USA,93(22):12637-12642.
Saxena I M,Brown R M Jr.2000.Cellulose synthases and related enzymes.Current Opinion in Plant Biology,3(6):523-531.
Scheible W R,Eshed R,Richmond T,et al.2001.Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidopsis Ixr1 mutants.Proc Natl Acad Sci USA,98(18):10079-10084.
Ong S S,Wickneswari R.2012.Characterization of microRNAs expressed during secondary wall biosynthesis in Acacia mangium.PLo S ONE,7(11):e49662.
Sethapphong L,Davis J K,Slabaugh E,et al.2016.Prediction of the structures of the plant specific regions of vascular plant cellulose synthases and correlated functional analysis.Cellulose,23(1):145-161.
Song D,Shen J,Li L.2010.Characterization of cellulose synthase complexes in Populus xylem differentiation.New Phytologist,187(3):777-790.
Taylor N G.2008.Cellulose biosynthesis and deposition in higher plants.New Phytologist,178(2):239-252.
Taylor N G,Laurie S,Turner S R.2000.Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis.Plant Cell,12(12):2529-2539.
Taylor N G,Scheible W R,Cutler S,et al.1999.The irregular xylem3locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis.Plant Cell,11(5):769-780.
Turner S R,Somerville C R.1997.Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall.Plant Cell,9(5):689-701.
Wu L,Joshi C P.2000.A xylem-specific cellulose synthase gene from aspen(Populus tremuloides)is responsive to mechanical stress.The Plant Journal,22(6):495-502.
Xu P,Donaldson L,Gergely Z,et al.2007.Dual-axis electron tomography:a new approach for investigating the spatial organization of wood cellulose microfibrils.Wood Sci Technol,41(2):101-116.