马占相思AmCesA2基因的克隆与表达分析
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摘要
纤维素合酶在植物纤维素合成途径中发挥主要作用,是控制木材纤维品质和产量的决定因素。本试验以相思属的马占相思幼苗为材料,运用RACE技术,从中获得了一个Ces A基因,命名为AmCesA2。序列分析表明,AmCesA2的c DNA大小为3 643 bp,开放读码框为3 228 bp,推测编码1 075个氨基酸,该氨基酸具有纤维素合酶保守的D,D,D,QxxRW结构域以及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。此外,AmCesA2的N端具有2个典型的跨膜结构,C端存在6个跨膜区域。聚类分析表明,AmCesA2与银合欢Ll Ces A7相似性最高。荧光定量PCR表明,AmCesA2在马占相思幼苗的茎中表达量最高,在根和叶中也有一定量的表达。用GA3,6-BA,BR这3种激素处理相思树幼苗,均可提高AmCesA2的表达量,且AmCesA2对GA3的响应最为强烈。
Cellulose synthase plays a major role in the plant cellulose synthesis pathway and it is an important determinant controlling wood fiber quality and yield. In this study,a Ces A gene was acquired from Acacia mangium seedlings by RACE,named AmCesA2.Sequence analysis showed that the c DNA of which was 3 643 bp,and its ORF was 3 228 bp. It indicated that AmCesA2 encoded 1 075 amino acids with the D,D,D and QxxRW structural domains of cellulose synthase and unique P-CR region,the HVR region and Nterminal zinc finger structure. Besides,there are two transmembrane structures regions at the N-terminus and six transmembrane regions at the C-terminus. Cluster analysis showed that AmCesA2 had the highest similarity with Ll Ces A7. Fluorescence quantitative PCR demonstrated that AmCesA2 existed in roots and leaves but the highest expression level in stems. AmCesA2 had a positive response to GA3,6-BA and BR treatments,in which the response to GA3 was the most strong.
Cellulose synthase plays a major role in the plant cellulose synthesis pathway and it is an important determinant controlling wood fiber quality and yield. In this study,a Ces A gene was acquired from Acacia mangium seedlings by RACE,named AmCesA2.Sequence analysis showed that the c DNA of which was 3 643 bp,and its ORF was 3 228 bp. It indicated that AmCesA2 encoded 1 075 amino acids with the D,D,D and QxxRW structural domains of cellulose synthase and unique P-CR region,the HVR region and Nterminal zinc finger structure. Besides,there are two transmembrane structures regions at the N-terminus and six transmembrane regions at the C-terminus. Cluster analysis showed that AmCesA2 had the highest similarity with Ll Ces A7. Fluorescence quantitative PCR demonstrated that AmCesA2 existed in roots and leaves but the highest expression level in stems. AmCesA2 had a positive response to GA3,6-BA and BR treatments,in which the response to GA3 was the most strong.
引文
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[3]HOONG Y B,PIZZI A,TAHIR P,et al. Characterization of Acacia mangium polyflavonoid tannins by MALDI-TOF massspectrometry and CP-MAS13C NMR[J]. European Polymer Journal,2010,46(6):1 268-1 277.
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[6]KEEGSTRA K. Plant cell walls[J]. Plant Physiology,2010,154(2):483-486.
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[9]袁红梅,郭文栋,赵丽娟,等.亚麻纤维素合酶超基因家族的生物信息学及表达分析[J].中国农业科学,2016,49(23):4 656-4 668.
[10]郭涛,杨青川,晁跃辉,等.植物纤维素合酶结构与基因表达研究进展[J].草地学报,2016,24(3):501-508.
[11]KAUR S,DHUGGA K S,GILL K,et al. Novel structural and functional motifs in cellulose synthase(Ces A)genes of breadwheat(Triticum aestivum L.)[J]. PLoS One,2016,11(1):e0 147 046.
[12]李秀云,陈晓沛,徐英武,等.毛竹生长过程中纤维素合成酶基因的时空表达和功能预测[J].浙江农林大学学报,2017,34(4):565-573.
[13]高艳,陈光辉,陈秀娟,等.植物细胞壁纤维素生物合成的调控[J].生物技术通报,2014(1):1-7.
[14]MCFARLANE H E,DRING A,PERSSON S. The cell biology of cellulose synthesis[J]. Annual Review of Plant Biology,2014,65:69-94.
[15] WONG H C,FEAR A L,CALHOON R D,et al. Genetic organization of the cellulose synthase operon in Acetobacterxylinum[J]. Proceedings of the National Academy of Sciences of the United States of America,1990,87(20):8 130-8 134.
[16]PEAR J R,KAWAGOE Y,SCHRECKENGOST W E,et al. Higher plants contain homologs of the bacterial cel A genesencoding the catalytic subunit of cellulose synthase[J]. Proceedings of the National Academy of Sciences of the United Statesof America,1996,93(22):12 637-12 642.
[17]DJERBI S,LINDSKOG M,ARVESTAD L,et al. The genome sequence of black cottonwood(Populus trichocarpa)reveals18 conserved cellulose synthase(Ces A)genes[J]. Planta,2005,221(5):739-746.
[18]KRAUSKOPF E,HARRIS P J,PUTTERILL J. The cellulose synthase gene Pr CESA10 is involved in cellulose biosynthesisin developing tracheids of the gymnosperm Pinus radiata[J]. Gene,2005,350(2):107-116.
[19]RANIK M,MYBURG A A. Six new cellulose synthase genes from Eucalyptus are associated with primary and secondary cellwall biosynthesis[J]. Tree Physiology,2006,26(5):545-556.
[20]SONG D L,SHEN J H,LI L G. Characterization of cellulose synthase complexes in Populus xylem differentiation[J]. NewPhytologist,2010,187(3):777-790.
[21]张海平,王学德,邵明彦,等.外源纤维素合酶基因对棉纤维品质的改良作用[J].棉花学报,2008,20(2):110-115.
[22]BAI W Q,XIAO Y H,ZHAO J,et al. Gibberellin overproduction promotes sucrose synthase expression and secondary cellwall deposition in cotton fibers[J]. PLoS One,2014,9(5):e96537.
[23]范玉龙,姜达,李明容,等.适合于高通量测序的槟榔总RNA的提取方法[J].热带生物学报,2015,6(1):87-90.
[24]魏凯莉,周厚君,江成,等.杨树次生壁纤维素合酶的表达与互作模式分析[J].林业科学研究,2017,30(2):245-253.
[25]XIE L Q,YANG C J,WANG X L. Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genesin Arabidopsis[J]. Journal of Experimental Botany,2011,62(13):4 495-4 506.
[26]张保才,周奕华.植物细胞壁形成机制的新进展[J].中国科学:生命科学,2015,45(6):544-556.
[27]黄成,李来庚.植物细胞壁研究与生物质改造利用[J].科学通报,2016,61(34):3 623-3 629.
[28]张凯权,马履一,段劼,等.刺槐纤维素合酶基因(Rp Ces A2)克隆、表达及SNP分析[J].分子植物育种,2017,15(2):474-482.
[29]SETHAPHONG L,DAVIS J K,SLABAUGH E,et al. Prediction of the structures of the plant-specific regions of vascular plantcellulose synthases and correlated functional analysis[J]. Cellulose,2016,23(1):145-161.
[30]庞景,童再康,黄华宏,等.杉木纤维素合成酶基因Ces A的克隆及表达分析[J].浙江农林大学学报,2015,32(1):40-46.
[31]SUZUKI S,LI L G,SUN Y H,et al. The cellulose synthase gene superfamily and biochemical functions of xylem-specificcellulose synthase-like genes in Populus trichocarpa[J]. Plant Physiology,2006,142(3):1 233-1 245.
[32]陈亚娟,王宏芝,李瑞芬,等.毛白杨纤维素合酶基因家族部分成员的克隆及表达[J].林业科学,2011,47(10):70-75.
[33]HUANG D B,WANG S G,ZHANG B C,et al. A gibberellin-mediated DELLA-NAC signaling cascade regulates cellulosesynthesis in rice[J]. The Plant Cell,2015,27(6):1 681-1 696.
[34]陈英,邵志龙,王浩然,等.杨树Pe AFB基因克隆及表达模式初步分析[J].林业科学,2015,51(8):26-32.
[35]ROBERT S,MOUILLE G,HFTE H. The mechanism and regulation of cellulose synthesis in primary walls:lessons fromcellulose-deficient Arabidopsis mutants[J]. Cellulose,2004,11(3/4):351-364.
[36]江海霞,郭栋良,李玉环,等.亚麻快速生长期细胞壁形成相关基因的表达分析[J].中国农业科学,2017,50(13):2 442-2 450.
[37]CECCHETTI V,ALTAMURA M M,BRUNETTI P,et al. Auxin controls Arabidopsis anther dehiscence by regulatingendothecium lignification and jasmonic acid biosynthesis[J]. The Plant Journal,2013,74(3):411-422.
[38]YAMAGUCHI M,MITSUDA N,OHTANI M,et al. VASCULAR-RELATED NAC-DOMAIN 7 directly regulates the expressionof a broad range of genes for xylem vessel formation[J]. The Plant Journal,2011,66(4):579-590.
[39]KIM W C,KO J H,KIM J Y,et al. MYB46 directly regulates the gene expression of secondary wall-associated cellulosesynthases in Arabidopsis[J]. The Plant Journal,2013,73(1):26-36.
[2]LE MAIRE G,NOUVELLON Y,CHRISTINA M,et al. Tree and stand light use efficiencies over a full rotation of single-andmixed-species Eucalyptus grandis and Acacia mangium plantations[J]. Forest Ecology and Management,2013,288:31-42.
[3]HOONG Y B,PIZZI A,TAHIR P,et al. Characterization of Acacia mangium polyflavonoid tannins by MALDI-TOF massspectrometry and CP-MAS13C NMR[J]. European Polymer Journal,2010,46(6):1 268-1 277.
[4]BINI D,DOS SANTOS C A,BOUILLET J P,et al. Eucalyptus grandis and Acacia mangium in monoculture and intercroppedplantations:evolution of soil and litter microbial and chemical attributes during early stages of plant development[J]. AppliedSoil Ecology,2013,63:57-66.
[5]DANISH M,HASHIM R,IBRAHIM M N M,et al. Effect of acidic activating agents on surface area and surface functional groupsof activated carbons produced from Acacia mangium wood[J]. Journal of Analytical and Applied Pyrolysis,2013,104:418-425.
[6]KEEGSTRA K. Plant cell walls[J]. Plant Physiology,2010,154(2):483-486.
[7] NODA S,KOSHIBA T,HATTORI T,et al. The expression of a rice secondary wall-specific cellulose synthase gene,Os Ces A7,is directly regulated by a rice transcription factor,Os MYB58/63[J]. Planta,2015,242(3):589-600.
[8]KUMAR M,TURNER S. Plant cellulose synthesis:CESA proteins crossing kingdoms[J]. Phytochemistry,2015,112:91-99.
[9]袁红梅,郭文栋,赵丽娟,等.亚麻纤维素合酶超基因家族的生物信息学及表达分析[J].中国农业科学,2016,49(23):4 656-4 668.
[10]郭涛,杨青川,晁跃辉,等.植物纤维素合酶结构与基因表达研究进展[J].草地学报,2016,24(3):501-508.
[11]KAUR S,DHUGGA K S,GILL K,et al. Novel structural and functional motifs in cellulose synthase(Ces A)genes of breadwheat(Triticum aestivum L.)[J]. PLoS One,2016,11(1):e0 147 046.
[12]李秀云,陈晓沛,徐英武,等.毛竹生长过程中纤维素合成酶基因的时空表达和功能预测[J].浙江农林大学学报,2017,34(4):565-573.
[13]高艳,陈光辉,陈秀娟,等.植物细胞壁纤维素生物合成的调控[J].生物技术通报,2014(1):1-7.
[14]MCFARLANE H E,DRING A,PERSSON S. The cell biology of cellulose synthesis[J]. Annual Review of Plant Biology,2014,65:69-94.
[15] WONG H C,FEAR A L,CALHOON R D,et al. Genetic organization of the cellulose synthase operon in Acetobacterxylinum[J]. Proceedings of the National Academy of Sciences of the United States of America,1990,87(20):8 130-8 134.
[16]PEAR J R,KAWAGOE Y,SCHRECKENGOST W E,et al. Higher plants contain homologs of the bacterial cel A genesencoding the catalytic subunit of cellulose synthase[J]. Proceedings of the National Academy of Sciences of the United Statesof America,1996,93(22):12 637-12 642.
[17]DJERBI S,LINDSKOG M,ARVESTAD L,et al. The genome sequence of black cottonwood(Populus trichocarpa)reveals18 conserved cellulose synthase(Ces A)genes[J]. Planta,2005,221(5):739-746.
[18]KRAUSKOPF E,HARRIS P J,PUTTERILL J. The cellulose synthase gene Pr CESA10 is involved in cellulose biosynthesisin developing tracheids of the gymnosperm Pinus radiata[J]. Gene,2005,350(2):107-116.
[19]RANIK M,MYBURG A A. Six new cellulose synthase genes from Eucalyptus are associated with primary and secondary cellwall biosynthesis[J]. Tree Physiology,2006,26(5):545-556.
[20]SONG D L,SHEN J H,LI L G. Characterization of cellulose synthase complexes in Populus xylem differentiation[J]. NewPhytologist,2010,187(3):777-790.
[21]张海平,王学德,邵明彦,等.外源纤维素合酶基因对棉纤维品质的改良作用[J].棉花学报,2008,20(2):110-115.
[22]BAI W Q,XIAO Y H,ZHAO J,et al. Gibberellin overproduction promotes sucrose synthase expression and secondary cellwall deposition in cotton fibers[J]. PLoS One,2014,9(5):e96537.
[23]范玉龙,姜达,李明容,等.适合于高通量测序的槟榔总RNA的提取方法[J].热带生物学报,2015,6(1):87-90.
[24]魏凯莉,周厚君,江成,等.杨树次生壁纤维素合酶的表达与互作模式分析[J].林业科学研究,2017,30(2):245-253.
[25]XIE L Q,YANG C J,WANG X L. Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genesin Arabidopsis[J]. Journal of Experimental Botany,2011,62(13):4 495-4 506.
[26]张保才,周奕华.植物细胞壁形成机制的新进展[J].中国科学:生命科学,2015,45(6):544-556.
[27]黄成,李来庚.植物细胞壁研究与生物质改造利用[J].科学通报,2016,61(34):3 623-3 629.
[28]张凯权,马履一,段劼,等.刺槐纤维素合酶基因(Rp Ces A2)克隆、表达及SNP分析[J].分子植物育种,2017,15(2):474-482.
[29]SETHAPHONG L,DAVIS J K,SLABAUGH E,et al. Prediction of the structures of the plant-specific regions of vascular plantcellulose synthases and correlated functional analysis[J]. Cellulose,2016,23(1):145-161.
[30]庞景,童再康,黄华宏,等.杉木纤维素合成酶基因Ces A的克隆及表达分析[J].浙江农林大学学报,2015,32(1):40-46.
[31]SUZUKI S,LI L G,SUN Y H,et al. The cellulose synthase gene superfamily and biochemical functions of xylem-specificcellulose synthase-like genes in Populus trichocarpa[J]. Plant Physiology,2006,142(3):1 233-1 245.
[32]陈亚娟,王宏芝,李瑞芬,等.毛白杨纤维素合酶基因家族部分成员的克隆及表达[J].林业科学,2011,47(10):70-75.
[33]HUANG D B,WANG S G,ZHANG B C,et al. A gibberellin-mediated DELLA-NAC signaling cascade regulates cellulosesynthesis in rice[J]. The Plant Cell,2015,27(6):1 681-1 696.
[34]陈英,邵志龙,王浩然,等.杨树Pe AFB基因克隆及表达模式初步分析[J].林业科学,2015,51(8):26-32.
[35]ROBERT S,MOUILLE G,HFTE H. The mechanism and regulation of cellulose synthesis in primary walls:lessons fromcellulose-deficient Arabidopsis mutants[J]. Cellulose,2004,11(3/4):351-364.
[36]江海霞,郭栋良,李玉环,等.亚麻快速生长期细胞壁形成相关基因的表达分析[J].中国农业科学,2017,50(13):2 442-2 450.
[37]CECCHETTI V,ALTAMURA M M,BRUNETTI P,et al. Auxin controls Arabidopsis anther dehiscence by regulatingendothecium lignification and jasmonic acid biosynthesis[J]. The Plant Journal,2013,74(3):411-422.
[38]YAMAGUCHI M,MITSUDA N,OHTANI M,et al. VASCULAR-RELATED NAC-DOMAIN 7 directly regulates the expressionof a broad range of genes for xylem vessel formation[J]. The Plant Journal,2011,66(4):579-590.
[39]KIM W C,KO J H,KIM J Y,et al. MYB46 directly regulates the gene expression of secondary wall-associated cellulosesynthases in Arabidopsis[J]. The Plant Journal,2013,73(1):26-36.