杨树天冬氨酸转氨酶基因家族鉴定及表达分析
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摘要
【目的】鉴定杨树天冬氨酸转氨酶(ASPAT)基因家族成员,并检测其组织表达特异性,为研究ASPAT在杨树初级氮素同化中的生物学功能提供参考依据。【方法】从杨树基因组数据库中筛选鉴定ASPAT基因家族成员,利用生物信息学软件分析各基因家族成员序列和基因结构及编码蛋白的理化性质、亚细胞定位和保守基序等,并利用实时荧光定量PCR检测正常施氮处理(1 mmol/L NH4NO3)下其在杨树根、茎和叶中的组织表达特异性。【结果】从杨树基因组中共鉴定出9个ASPAT基因家族成员,包括7个真核型ASPAT(AAT)基因(PtASPAT1～PtASPAT7)和2个原核型ASPAT(PAT)基因(PtASPAT8～PtASPAT9),编码区(CDS)序列长度1215～1791 bp,编码的氨基酸数目304～480个,外显子数7～14个,编码蛋白的等电点5.66～8.99,相对分子质量33.11～53.31 kD,脂融指数76.36～93.54,分别定位于叶绿体、线粒体和胞质中,除PtASPAT6和PtASPAT7为不稳定蛋白,其他均为稳定蛋白。拟南芥和杨树的ASPAT蛋白可聚为两大类,Ⅰ类为AAT蛋白,包括PtASPAT1～PtASPAT7和AtASPAT1～AtASPAT5;Ⅱ类为PAT蛋白,包括PtASPAT8、PtASPAT9和拟南芥PAT(AtPAT)。PtASPAT1～PtASPAT6均含有5个保守基序,PtASPAT7缺少2个保守基序,PtASPAT8和PtASPAT9均仅含有1个与AtPAT相同的保守基序。9个杨树ASPAT蛋白均含有磷酸吡哆醛结合位点(SGTHNYSSK)、同源二聚体多肽结合位点(GAVAER)和催化残留活性位点(K)。正常氮素处理下,PtASPAT1基因在杨树根、茎和叶中表达量无明显差异;PtASPAT2～PtASPAT9基因在根部的表达量较在茎和叶中的高,尤其是PtASPAT4和PtASPAT8基因表达量较高。【结论】杨树ASPAT基因家族成员主要在根部表达,尤其是PtASPAT4和PtASPAT8基因在杨树根部的初级氮素同化中发挥重要作用。
【Objective】The purpose of the study was to identify poplar aspartate aminotransferase(ASPAT)gene family members,analyze their bioinformatics and detect their tissue expression specificity,and provide reference for studying the biological function of ASPAT in poplar primary nitrogen assimilation.【Method】Screening and identification of ASPAT gene family members from poplar genome were conducted. Bioinformatics software was used to analyze the sequence and gene structure of each gene family member,and the physicochemical properties,subcellular localization and conserved motifs of the encoded proteins. The tissue expression specificity of poplar roots,stems and leaves was analyzed using realtime fluorescence quantitative PCR by normal nitrogen application(1 mmol/L NH4 NO3).【Result】A total of nine ASPAT genes were identified from the poplar genome,including seven eukaryotic ASPAT(AAT)genes(PtASPAT1-PtASPAT7)and two prokaryotic ASPAT(PAT)genes(PtASPAT8-PtASPAT9). The coding region(CDS)sequence was 1215-1791 bp in length,the number of encoded amino acids 304-480,the number of exons 7-14,the isoelectric point of the encoded protein 5.66-8.99,the relative molecular mass 33.11-53.31 kD,and the fat melting index 76.36-93.54,respectively. They were located in the chloroplast,mitochondria and cytoplasm. Except PtASPAT6 and PtASPAT7 were unstable proteins,others were stable proteins. The ASPAT proteins of Arabidopsis thaliana and poplar could be clustered into two major classes,class I was AAT protein,including PtASPAT1-PtASPAT7 and AtASPAT1-AtASPAT5;class Ⅱ was PAT protein,including PtASPAT8,PtASPAT9 and A. thaliana PAT(AtPAT). PtASPAT1-PtASPAT6 all contained five conserved motifs,PtASPAT7 lacked two conserved motifs,and both PtASPAT8 and PtASPAT9 contained only one conserved motif identical to AtPAT. The nine poplar ASPAT proteins all contained a pyridoxal phosphate binding site(SGTHNYSSK),a homodimeric polypeptide binding site(GAVAER)and a catalytic residual active site(K). Under normal nitrogen treatment,there was no significant difference in the expression of PtASPAT1 in roots,stems and leaves. The expression level of PtASPAT2-PtASPAT9 genes in roots was higher than that in stems and leaves,especially the expression of PtASPAT4 and PtASPAT8 genes.【Conclusion】The members of the ASPAT gene family of poplar are mainly expressed in roots,especially PtASPAT4 and PtASPAT8 play important roles in primary nitrogen assimilation in poplar roots.
【Objective】The purpose of the study was to identify poplar aspartate aminotransferase(ASPAT)gene family members,analyze their bioinformatics and detect their tissue expression specificity,and provide reference for studying the biological function of ASPAT in poplar primary nitrogen assimilation.【Method】Screening and identification of ASPAT gene family members from poplar genome were conducted. Bioinformatics software was used to analyze the sequence and gene structure of each gene family member,and the physicochemical properties,subcellular localization and conserved motifs of the encoded proteins. The tissue expression specificity of poplar roots,stems and leaves was analyzed using realtime fluorescence quantitative PCR by normal nitrogen application(1 mmol/L NH4 NO3).【Result】A total of nine ASPAT genes were identified from the poplar genome,including seven eukaryotic ASPAT(AAT)genes(PtASPAT1-PtASPAT7)and two prokaryotic ASPAT(PAT)genes(PtASPAT8-PtASPAT9). The coding region(CDS)sequence was 1215-1791 bp in length,the number of encoded amino acids 304-480,the number of exons 7-14,the isoelectric point of the encoded protein 5.66-8.99,the relative molecular mass 33.11-53.31 kD,and the fat melting index 76.36-93.54,respectively. They were located in the chloroplast,mitochondria and cytoplasm. Except PtASPAT6 and PtASPAT7 were unstable proteins,others were stable proteins. The ASPAT proteins of Arabidopsis thaliana and poplar could be clustered into two major classes,class I was AAT protein,including PtASPAT1-PtASPAT7 and AtASPAT1-AtASPAT5;class Ⅱ was PAT protein,including PtASPAT8,PtASPAT9 and A. thaliana PAT(AtPAT). PtASPAT1-PtASPAT6 all contained five conserved motifs,PtASPAT7 lacked two conserved motifs,and both PtASPAT8 and PtASPAT9 contained only one conserved motif identical to AtPAT. The nine poplar ASPAT proteins all contained a pyridoxal phosphate binding site(SGTHNYSSK),a homodimeric polypeptide binding site(GAVAER)and a catalytic residual active site(K). Under normal nitrogen treatment,there was no significant difference in the expression of PtASPAT1 in roots,stems and leaves. The expression level of PtASPAT2-PtASPAT9 genes in roots was higher than that in stems and leaves,especially the expression of PtASPAT4 and PtASPAT8 genes.【Conclusion】The members of the ASPAT gene family of poplar are mainly expressed in roots,especially PtASPAT4 and PtASPAT8 play important roles in primary nitrogen assimilation in poplar roots.
引文
杜亚琳,陈海燕,徐丽琳,王琛,范莲雪.2018.黄瓜氮素胁迫相关基因CsAHP1的克隆及功能分析[J].河南农业科学,47(6):92-97.[Du Y L,Chen H Y,Xu L L,Wang C,Fan L X.2018.Cloning and function analysis of cucumber CsAHP1 gene involved in nitrogen tolerance[J].Journal of Henan Agricultural Sciences,47(6):92-97.]
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梁成刚,张青,李敬,熊丹,许光利,汪燕,刘泉,黄鹏,李天.2013.水稻灌浆期高温对天冬氨酸代谢酶活性及其家族氨基酸含量的影响[J].中国水稻科学,27(1):71-76.[Liang C G,Zhang Q,Li J,Xiong D,Xu G L,Wang Y,Liu Q,Huang P,Li T.2013.Effects of high temperature on aspartate metabolic enzyme activity and its family amino acid content during rice filling period[J].Chinese Rice Science,27(1):71-76.]
刘宝林,邹小云,宋来强,官春云.2017.氮肥追施时期对油菜产量、效益及氮素吸收利用的影响[J].江西农业学报,29(11):29-32.[Liu B L,Zou X Yun,Song L Q,Guan C Y.2017.Effects of nitrogen fertilizer topdressing period on yield,benefit and nitrogen absorption and utilization of rapeseed[J].Acta Agriculturae Jiangxi,29(11):29-32.]
刘红江,肖敏,张丽萍,陈留根,张岳芳,郭智,郑建初.2017.前氮后移对水稻氮素吸收和利用效率的影响[J].江苏农业学报,33(3):550-554.[Liu H J,Xiao M,Zhang LP,Chen L G,Zhang Y F,Guo Z,Zheng J C.2017.Nitrogen uptake and use efficiency of rice in response to postponed nitrogen application[J].Jiangsu Journal of Agricultural Sciences,33(3):550-554.]
刘瑞响,曹晓良,陶勇生,张祖新.2012.不同氮素水平下玉米产量与zmAspAT基因表达分析[J].中国农学通报,28(24):22-26.[Liu R X,Cao X L,Tao Y S,Zhang Z X.2012.Analysis of maize yield and zmAspAT gene expression under different nitrogen levels[J].Chinese Agricultural Science Bulletin,28(24):22-26.]
马文奇,张福锁,陈新平.2006.中国养分资源综合管理研究的意义与重点[J].科技导报,24(10):64-67.[Ma W Q,Zhang F S,Chen X P.2006.Significance and focus of research on integrated management of nutrient resources in China[J].Science and Technology Review,24(10):64-67.]
于妍,宋万坤,刘春燕,高运来,李文福,孙殿君,陈庆山,胡国华.2008.植物天冬氨酸代谢途径关键酶基因研究进展[J].生物技术通报,(S1):7-11.[Yu Y,Song W K,Liu CY,Gao Y L,Li W F,Sun D J,Chen Q S,Hu G H.2008.Research progress on key enzyme genes in plant aspartate metabolic pathways[J].Biotechnology Bulletin,(S1):7-11.]
赵会杰,周颖,李华,王斯琪,许海良,蒲文宣,张锦韬,易克,汪耀富.2017.施氮量对烤烟叶片碳同化能力及同化产物分配的影响[J].河南农业大学学报,51(5):603-608.[Zhao H J,Zhou Y,Li H,Wang S Q,Xu H L,Pu W X,Zhang J T,Yi K,Wang Y F.2017.Effect of nitrogen application rate on carbon assimilation capability and assimilation product distribution of flue-cured tobacco leaves[J].Journal of Henan Agricultural University,51(5):603-608.]
周莹.2009.水稻中天冬氨酸转氨酶的分子生物学研究和转基因应用[D].武汉:华中农业大学.[Zhou Y.2009.The reseach of molecular biology and application of aspartate aminotransferase in rice[D].Wuhan:Huazhong Agricultural University.]
Cai H,Zhou Y,Xiao J,Li X,Zhang Q,Lian X.2009.Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice[J].Plant Cell Reports,28(3):527-537.
de la Torre F,Ca?as R A,Pascual M B,Avila C F,Cánovas M.2014.Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants[J].Journal of Experimental Botany,65(19):5527-5534.
de la Torre F,Moya-García A,Suárez M F,Rodríguez-Caso C,Ca?as R A,Sánchez-Jiménez F,Cánovas F M.2009.Molecular modelling and site-directed mutagenesis reveal essential residues for catalysis in a prokaryote-type aspartate aminotransferase[J].Plant Physiology,149(4):1648-1660.
de la Torre F,Santis L D,Crespillo R,Francisco M C.2006.Identification and functional analysis of a prokaryotictype aspartate aminotransferase:Implications for plant amino acid metabolism[J].Plant Journal for Cell&Molecular Biology,46(3):414-431.
Graindorge M,Giustini C,Kraut A,Moyet L,Curien G,Matringe M.2014.Three different classes of aminotransferases evolved prephenate aminotransferase functionality in arogenate-competent microorganisms[J].Journal of Biological Chemistry,289(6):3198-3208.
Igarashi D,Ishizaki T,Totsuka K,Ohsumi C.2009.ASN2 is a key enzyme in asparagine biosynthesis under ammonium sufficient conditions[J].Plant Tissue Culture Letters,26(1):153-159.
Ireland R J,Joy K W.1983.Subcellular localisation of asparaginase and asparagine aminotransferase in Pisum sativum leaves[J].Plant Physiology,72(4):1127-1129.
Krouk G,Crawford N M,Coruzzi G M,Yifang T,Sonnewald U,Frommer W B.2010.Nitrate signaling:Adaptation to fluctuating environments[J].Current Opinion in Plant Biology,13(3):265-272.
Martins M L L,Mourato M P de F B,Mendon?a A P A de V e.2002.Characterization of aspartate aminotransferase isoenzymes from leaves of Lupinus albus L.cv Estoril[J].Journal of Biochemistry and Molecular Biology,35(2):220-227.
Miesak B H,Coruzzi G M.2002.Molecular and physiological analysis of arabidopsis mutants defective in cytosolic or chloroplastic aspartate aminotransferase[J].Plant Physiology,129(2):650-660.
Morino K,Olsen O A,Shimamoto K.2004.Silencing of the aleurone-specific Ltp2-gus gene in transgenic rice is reversed by transgene rearrangements and loss of aberrant transcripts[J].Plant&Cell Physiology,45(10):1500.
Murooka Y,Mori Y,Hayashi M.2002.Variation of the amino acid content of Arabidopsis seeds by expressing soybean aspartate aminotransferase gene[J].Journal of Bioscience&Bioengineering,94(3):225-230.
Robinson D L,Kahn M L,Vance C P.1994.Cellular localisation of nodule-enhanced aspartate aminotransferase in Medicago sativa L.[J].Planta,192(2):202-210.
Schultz C J,Coruzzi G M.1995.The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments[J].The Plant Journal,7(1):61-75.
Sentoku N,Taniguchi M,Sugiyama T,Ishimaru K,Ohsugi R,Takaiwa F,Toki S.2000.Analysis of the transgenic tobacco plants expressing Panicum miliaceum aspartate aminotransferase genes[J].Plant Cell Reports,19(6):598-603.
Silvente S,Camas A,Lara M.2003.Molecular cloning of the cDNA encoding aspartate aminotransferase from bean root nodules and determination of its role in nodule nitrogen metabolism[J].Journal of Experimental Botany,54(387):1545-1551.
Teng W,He X,Tong Y P.2017.Transgenic approaches for improving use efficiency of nitrogen,phosphorus and potassium in crops[J].Journal of Integrative Agriculture,16(12):2657-2673.
Wilkie S E,Lambert R,Warren M J.1996.Chloroplastic aspartate aminotransferase from Arabidopsis thaliana:An examination of the relationship between the structure of the gene and the spatial structure of the protein[J].The Biochemical Journal,319(3),969-976.
Zhou Y,Cai H,Xiao J,Li X,Lian X.2009.Overexpression of aspartate aminotransfer ase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds[J].Theoretical and Applied Genetics,118(7):1381-1390.
李源,张炎,哈丽哈什·依巴提,李青军.2019.新型尿素对膜下滴灌棉花产量及氮肥利用率的影响[J].江苏农业学报,35(1):85-90.[Li Y,Zhang Y,Halihashi·Yibat,Li Q J.2019.Effects of new-type urea on yield and nitrogen use efficiency of drip irrigated cotton under plastic film mulching[J].Jiangsu Journal of Agricultural Sciences,35(1):85-90.]
梁成刚,张青,李敬,熊丹,许光利,汪燕,刘泉,黄鹏,李天.2013.水稻灌浆期高温对天冬氨酸代谢酶活性及其家族氨基酸含量的影响[J].中国水稻科学,27(1):71-76.[Liang C G,Zhang Q,Li J,Xiong D,Xu G L,Wang Y,Liu Q,Huang P,Li T.2013.Effects of high temperature on aspartate metabolic enzyme activity and its family amino acid content during rice filling period[J].Chinese Rice Science,27(1):71-76.]
刘宝林,邹小云,宋来强,官春云.2017.氮肥追施时期对油菜产量、效益及氮素吸收利用的影响[J].江西农业学报,29(11):29-32.[Liu B L,Zou X Yun,Song L Q,Guan C Y.2017.Effects of nitrogen fertilizer topdressing period on yield,benefit and nitrogen absorption and utilization of rapeseed[J].Acta Agriculturae Jiangxi,29(11):29-32.]
刘红江,肖敏,张丽萍,陈留根,张岳芳,郭智,郑建初.2017.前氮后移对水稻氮素吸收和利用效率的影响[J].江苏农业学报,33(3):550-554.[Liu H J,Xiao M,Zhang LP,Chen L G,Zhang Y F,Guo Z,Zheng J C.2017.Nitrogen uptake and use efficiency of rice in response to postponed nitrogen application[J].Jiangsu Journal of Agricultural Sciences,33(3):550-554.]
刘瑞响,曹晓良,陶勇生,张祖新.2012.不同氮素水平下玉米产量与zmAspAT基因表达分析[J].中国农学通报,28(24):22-26.[Liu R X,Cao X L,Tao Y S,Zhang Z X.2012.Analysis of maize yield and zmAspAT gene expression under different nitrogen levels[J].Chinese Agricultural Science Bulletin,28(24):22-26.]
马文奇,张福锁,陈新平.2006.中国养分资源综合管理研究的意义与重点[J].科技导报,24(10):64-67.[Ma W Q,Zhang F S,Chen X P.2006.Significance and focus of research on integrated management of nutrient resources in China[J].Science and Technology Review,24(10):64-67.]
于妍,宋万坤,刘春燕,高运来,李文福,孙殿君,陈庆山,胡国华.2008.植物天冬氨酸代谢途径关键酶基因研究进展[J].生物技术通报,(S1):7-11.[Yu Y,Song W K,Liu CY,Gao Y L,Li W F,Sun D J,Chen Q S,Hu G H.2008.Research progress on key enzyme genes in plant aspartate metabolic pathways[J].Biotechnology Bulletin,(S1):7-11.]
赵会杰,周颖,李华,王斯琪,许海良,蒲文宣,张锦韬,易克,汪耀富.2017.施氮量对烤烟叶片碳同化能力及同化产物分配的影响[J].河南农业大学学报,51(5):603-608.[Zhao H J,Zhou Y,Li H,Wang S Q,Xu H L,Pu W X,Zhang J T,Yi K,Wang Y F.2017.Effect of nitrogen application rate on carbon assimilation capability and assimilation product distribution of flue-cured tobacco leaves[J].Journal of Henan Agricultural University,51(5):603-608.]
周莹.2009.水稻中天冬氨酸转氨酶的分子生物学研究和转基因应用[D].武汉:华中农业大学.[Zhou Y.2009.The reseach of molecular biology and application of aspartate aminotransferase in rice[D].Wuhan:Huazhong Agricultural University.]
Cai H,Zhou Y,Xiao J,Li X,Zhang Q,Lian X.2009.Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice[J].Plant Cell Reports,28(3):527-537.
de la Torre F,Ca?as R A,Pascual M B,Avila C F,Cánovas M.2014.Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants[J].Journal of Experimental Botany,65(19):5527-5534.
de la Torre F,Moya-García A,Suárez M F,Rodríguez-Caso C,Ca?as R A,Sánchez-Jiménez F,Cánovas F M.2009.Molecular modelling and site-directed mutagenesis reveal essential residues for catalysis in a prokaryote-type aspartate aminotransferase[J].Plant Physiology,149(4):1648-1660.
de la Torre F,Santis L D,Crespillo R,Francisco M C.2006.Identification and functional analysis of a prokaryotictype aspartate aminotransferase:Implications for plant amino acid metabolism[J].Plant Journal for Cell&Molecular Biology,46(3):414-431.
Graindorge M,Giustini C,Kraut A,Moyet L,Curien G,Matringe M.2014.Three different classes of aminotransferases evolved prephenate aminotransferase functionality in arogenate-competent microorganisms[J].Journal of Biological Chemistry,289(6):3198-3208.
Igarashi D,Ishizaki T,Totsuka K,Ohsumi C.2009.ASN2 is a key enzyme in asparagine biosynthesis under ammonium sufficient conditions[J].Plant Tissue Culture Letters,26(1):153-159.
Ireland R J,Joy K W.1983.Subcellular localisation of asparaginase and asparagine aminotransferase in Pisum sativum leaves[J].Plant Physiology,72(4):1127-1129.
Krouk G,Crawford N M,Coruzzi G M,Yifang T,Sonnewald U,Frommer W B.2010.Nitrate signaling:Adaptation to fluctuating environments[J].Current Opinion in Plant Biology,13(3):265-272.
Martins M L L,Mourato M P de F B,Mendon?a A P A de V e.2002.Characterization of aspartate aminotransferase isoenzymes from leaves of Lupinus albus L.cv Estoril[J].Journal of Biochemistry and Molecular Biology,35(2):220-227.
Miesak B H,Coruzzi G M.2002.Molecular and physiological analysis of arabidopsis mutants defective in cytosolic or chloroplastic aspartate aminotransferase[J].Plant Physiology,129(2):650-660.
Morino K,Olsen O A,Shimamoto K.2004.Silencing of the aleurone-specific Ltp2-gus gene in transgenic rice is reversed by transgene rearrangements and loss of aberrant transcripts[J].Plant&Cell Physiology,45(10):1500.
Murooka Y,Mori Y,Hayashi M.2002.Variation of the amino acid content of Arabidopsis seeds by expressing soybean aspartate aminotransferase gene[J].Journal of Bioscience&Bioengineering,94(3):225-230.
Robinson D L,Kahn M L,Vance C P.1994.Cellular localisation of nodule-enhanced aspartate aminotransferase in Medicago sativa L.[J].Planta,192(2):202-210.
Schultz C J,Coruzzi G M.1995.The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments[J].The Plant Journal,7(1):61-75.
Sentoku N,Taniguchi M,Sugiyama T,Ishimaru K,Ohsugi R,Takaiwa F,Toki S.2000.Analysis of the transgenic tobacco plants expressing Panicum miliaceum aspartate aminotransferase genes[J].Plant Cell Reports,19(6):598-603.
Silvente S,Camas A,Lara M.2003.Molecular cloning of the cDNA encoding aspartate aminotransferase from bean root nodules and determination of its role in nodule nitrogen metabolism[J].Journal of Experimental Botany,54(387):1545-1551.
Teng W,He X,Tong Y P.2017.Transgenic approaches for improving use efficiency of nitrogen,phosphorus and potassium in crops[J].Journal of Integrative Agriculture,16(12):2657-2673.
Wilkie S E,Lambert R,Warren M J.1996.Chloroplastic aspartate aminotransferase from Arabidopsis thaliana:An examination of the relationship between the structure of the gene and the spatial structure of the protein[J].The Biochemical Journal,319(3),969-976.
Zhou Y,Cai H,Xiao J,Li X,Lian X.2009.Overexpression of aspartate aminotransfer ase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds[J].Theoretical and Applied Genetics,118(7):1381-1390.