蒺藜苜蓿赤霉素氧化酶基因MtGA20ox的功能解析
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摘要
GA20-氧化酶(GA20-oxidase,GA20ox)是赤霉素(gibberellic acid,GA)合成过程中的关键限速酶。为了对蒺藜苜蓿(Medicago truncatula)中GA20ox基因的功能进行研究,利用生物信息学的方法对蒺藜苜蓿和拟南芥(Arabidopsis thaliana)的GA20ox家族成员进行氨基酸序列比对并构建系统进化树。结果表明,与拟南芥AtGA20ox高度同源的蒺藜苜蓿MtGA20ox有3个,分别为MtGA20ox1、MtGA20ox7和MtGA20ox8,均含有保守的2OG-FeⅡ_Oxy结构域。进一步通过RT-PCR检测MtGA20ox1、MtGA20ox7和MtGA20ox8在不同组织中的表达,结果显示它们的表达都具有组织特异性。同时,亚细胞定位结果表明MtGA20ox1、MtGA20ox7和MtGA20ox8蛋白定位于细胞核和细胞质。为了对MtGA20ox的功能进行深入解析,筛选获得了逆转座子Tnt1插入MtGA20ox7的2个突变体和插入MtGA20ox8的1个突变体,并分别对3个插入突变体进行表型观察和数据统计,发现其与野生型相比株高无明显变化,这可能是由于这3个MtGA20ox基因存在功能冗余。研究结果对深入研究苜蓿中GA20ox的功能以及进一步研究赤霉素对株高的调控具有一定的参考价值。
GA20-oxidase(GA20 ox) is a key rate-limiting enzyme in the synthesis of gibberellic acid(GA). In order to study the function of GA20ox gene in Medicago truncatula, the amino acid sequence alignment of GA20 ox of Arabidopsis thaliana and Medicago truncatula was carried out by bioinformatics methods, and the phylogenetic tree of the families was constructed. The results showed that there were three MtGA20 ox, which were MtGA20 ox1, MtGA20 ox7 and MtGA20 ox8, respectively. They had highly homologous to AtGA20 ox, and each of them contained a conserved 20 G-FeⅡ_Oxy domain. Further, the results of transcript analysis indicated that the expressions of MtGA20 ox1, MtGA20 ox7 and MtGA20 ox8 were tissue specific. Meanwhile, the result of subcellular localization indicated that MtGA20 ox1, MtGA20 ox7 and MtGA20 ox8 proteins were probably localized in nucleus and cytoplasm. In order to comprehend the function of MtGA20ox, two mutants with retrotransposon Tnt1 inserted in MtGA20 ox7 and one mutant with retrotransposon Tnt1 inserted in MtGA20 ox8 were obtained. The phenotypic observations and data statistics of the three mutants manifested that there were no significant changes in the plant height compared with the wild type. This might be due to the functional redundancy of the three MtGA20ox genes. Results of this study had a certain reference value for studying the function of GA20 ox and the regulation of gibberellin on plant height in Medicago truncatula.
GA20-oxidase(GA20 ox) is a key rate-limiting enzyme in the synthesis of gibberellic acid(GA). In order to study the function of GA20ox gene in Medicago truncatula, the amino acid sequence alignment of GA20 ox of Arabidopsis thaliana and Medicago truncatula was carried out by bioinformatics methods, and the phylogenetic tree of the families was constructed. The results showed that there were three MtGA20 ox, which were MtGA20 ox1, MtGA20 ox7 and MtGA20 ox8, respectively. They had highly homologous to AtGA20 ox, and each of them contained a conserved 20 G-FeⅡ_Oxy domain. Further, the results of transcript analysis indicated that the expressions of MtGA20 ox1, MtGA20 ox7 and MtGA20 ox8 were tissue specific. Meanwhile, the result of subcellular localization indicated that MtGA20 ox1, MtGA20 ox7 and MtGA20 ox8 proteins were probably localized in nucleus and cytoplasm. In order to comprehend the function of MtGA20ox, two mutants with retrotransposon Tnt1 inserted in MtGA20 ox7 and one mutant with retrotransposon Tnt1 inserted in MtGA20 ox8 were obtained. The phenotypic observations and data statistics of the three mutants manifested that there were no significant changes in the plant height compared with the wild type. This might be due to the functional redundancy of the three MtGA20ox genes. Results of this study had a certain reference value for studying the function of GA20 ox and the regulation of gibberellin on plant height in Medicago truncatula.
引文
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[11] Spielmeyer W, Ellis M H, Chandler P M. Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellin 20-oxidase gene[J]. Proc. Natl. Acad. Sci. USA, 2002, 99(13): 9043-9048.
[12] Wang Y, Deng D, Ding H, et al.. Gibberellin biosynthetic deficiency is responsible for maize dominant Dwarf11 (D11) mutant phenotype: Physiological and transcriptomic evidence[J]. PLoS ONE, 2013, 8(6): e66466.
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[14] Voorend W, Nelissen H, Vanholme R, et al.. Overexpression of GA20-OXIDASE1 impacts plant height, biomass allocation and saccharification efficiency in maize[J]. Plant Biotechnol. J., 2016, 14(3): 997-1007.
[15] Igielski R, K?pczyńska E. Gene expression and metabolite profiling of gibberellin biosynthesis during induction of somatic embryogenesis in Medicago truncatula Gaertn[J]. PLoS ONE, 2017, 12(7):e0182055.
[16] Luo Y, Dong X, Yu T, et al.. A single nucleotide deletion in Gibberellin20-oxidase1 causes alpine dwarfism in Arabidopsis [J]. Plant Physiol., 2015, 168(3): 930-937.
[17] 郑伶杰,马宏,张媛,等. 苹果矮化砧木GA20氧化酶基因的克隆及其表达分析[J]. 华北农学报,2017,32(1) :53-59.
[18] Tenreira T, Lange M J P, Lange T, et al.. A specific gibberellin 20-oxidase dictates the flowering-runnering decision in diploid strawberry [J]. Plant Cell, 2017, 29(9): 2168-2182.
[19] 赵慧君,仝宗勇,余海忠,等. 苹果赤霉素3-氧化酶1(MdGA3ox1)启动子区的克隆及序列分析[J]. 中国农学通报,2012,28(10):146-150.
[20] 张加强,刘慧春,周江华,等.植物赤霉素氧化酶GA20ox基因的生物信息学分析[J/OL].分子植物育种, http://kns.cnki.net/kcms/detail/46.1068.S.20181012.0907.002.html.
[21] 高世敏,董阳,王武,等. 葡萄赤霉素合成关键基因VvGA20ox2的克隆、亚细胞定位和表达分析[J].江苏农业学报,2018,34(6):1331-1338.
[22] 庞保亚,李强,任仲海,等. 黄瓜CsGA20ox1异源表达促进拟南芥植株发育[J]. 山东农业大学学报(自然科学版),2018, 49(4):578-584.
[23] 中华人民共和国农业农村部. 我国草原面积在世界上的地位[EB/OL]. http://www.moa.gov.cn/ztzl/cyf/200303/t20030306_62346.htm, 2003-03-06.
[24] Rieu I, Ruiz-Rivero O, Fernandez-Garcia N, et al.. The gibberellin biosynthetic genes AtGA20ox1 and AtGA20ox2 act, partially redundantly, to promote growth and development throughout the Arabidopsis life cycle[J]. Plant J., 2008, 53(3): 488-504.
[25] 殷小林,张超,王有成,等.水稻赤霉素3β羟化酶基因(OsGA3ox1)同源基因的生物信息学分析[J/OL].分子植物育种, http://kns.cnki.net/kcms/detail/46.1068.S.20181025.1431.010.html.
[26] Van Damme M, Huibers R P, Elberse J, et al.. Arabidopsis DMR6 encodes a putative 2OG-Fe(Ⅱ) oxygenase that is defense-associated but required for susceptibility to downy mildew[J]. Plant J., 2008, 54(5): 785-793.
[2] Yang S, Zu Y, Li B, et al.. Response and intraspecific differences in nitrogen metabolism of alfalfa (Medicago sativa L.) under cadmium stress[J]. Chemosphere, 2019, 220: 69-76.
[3] 张经荣. 基于SWOT模型的我国苜蓿产业发展对策研究[D]. 兰州:兰州大学,硕士学位论文,2017.
[4] 徐畅. 不同土壤条件下紫花苜蓿牧草产量构成因子的比较分析[D]. 长春:东北师范大学,硕士学位论文,2017.
[5] Asano K, Hirano K, Ueguchi-Tanaka M, et al.. Isolation and characterization of dominant dwarf mutants, Slr1-d, in rice[J]. Mol. Genet. Genomics, 2009, 281(2): 223-231.
[6] Baltazar B M, Scharen A L, Kronstad W E. Association between dwarfing genes‘Rht1’ and ‘Rht2’ and resistance to Septoria tritici Blotch in winter wheat (Triticum aestivum L. em Thell)[J]. Theor. Appl. Genet., 1990, 79(3): 422-426.
[7] 吴建明,陈荣发,黄杏,等. 高等植物赤霉素生物合成关键组分 GA20-oxidase氧化酶基因的研究进展[J]. 生物技术通报,2016,32(7):1-12.
[8] Liu C, Zheng S, Gui J, et al.. Shortened basal internodes encodes a gibberellin 2-oxidase and contributes to lodging resistance in rice[J]. Mol. Plant, 2018, 11(2): 288-299.
[9] Plackett A R, Powers S J, Fernandez-Garcia N, et al.. Analysis of the developmental roles of the Arabidopsis gibberellin 20-oxidases demonstrates that GA20ox1, -2, and -3 are the dominant paralogs[J]. Plant Cell, 2012, 24(3): 941-960.
[10] Qin X, Liu J H, Zhao W S, et al.. Gibberellin 20-oxidase gene OsGA20ox3 regulates plant stature and disease development in rice[J]. Mol. Plant Microbe Interact., 2013, 26(2): 227-239.
[11] Spielmeyer W, Ellis M H, Chandler P M. Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellin 20-oxidase gene[J]. Proc. Natl. Acad. Sci. USA, 2002, 99(13): 9043-9048.
[12] Wang Y, Deng D, Ding H, et al.. Gibberellin biosynthetic deficiency is responsible for maize dominant Dwarf11 (D11) mutant phenotype: Physiological and transcriptomic evidence[J]. PLoS ONE, 2013, 8(6): e66466.
[13] Huang S, Raman A S, Ream J E, et al.. Overexpression of 20-oxidase confers a gibberellin-overproduction phenotype in Arabidopsis[J]. Plant Physiol., 1998, 118(3): 773-781.
[14] Voorend W, Nelissen H, Vanholme R, et al.. Overexpression of GA20-OXIDASE1 impacts plant height, biomass allocation and saccharification efficiency in maize[J]. Plant Biotechnol. J., 2016, 14(3): 997-1007.
[15] Igielski R, K?pczyńska E. Gene expression and metabolite profiling of gibberellin biosynthesis during induction of somatic embryogenesis in Medicago truncatula Gaertn[J]. PLoS ONE, 2017, 12(7):e0182055.
[16] Luo Y, Dong X, Yu T, et al.. A single nucleotide deletion in Gibberellin20-oxidase1 causes alpine dwarfism in Arabidopsis [J]. Plant Physiol., 2015, 168(3): 930-937.
[17] 郑伶杰,马宏,张媛,等. 苹果矮化砧木GA20氧化酶基因的克隆及其表达分析[J]. 华北农学报,2017,32(1) :53-59.
[18] Tenreira T, Lange M J P, Lange T, et al.. A specific gibberellin 20-oxidase dictates the flowering-runnering decision in diploid strawberry [J]. Plant Cell, 2017, 29(9): 2168-2182.
[19] 赵慧君,仝宗勇,余海忠,等. 苹果赤霉素3-氧化酶1(MdGA3ox1)启动子区的克隆及序列分析[J]. 中国农学通报,2012,28(10):146-150.
[20] 张加强,刘慧春,周江华,等.植物赤霉素氧化酶GA20ox基因的生物信息学分析[J/OL].分子植物育种, http://kns.cnki.net/kcms/detail/46.1068.S.20181012.0907.002.html.
[21] 高世敏,董阳,王武,等. 葡萄赤霉素合成关键基因VvGA20ox2的克隆、亚细胞定位和表达分析[J].江苏农业学报,2018,34(6):1331-1338.
[22] 庞保亚,李强,任仲海,等. 黄瓜CsGA20ox1异源表达促进拟南芥植株发育[J]. 山东农业大学学报(自然科学版),2018, 49(4):578-584.
[23] 中华人民共和国农业农村部. 我国草原面积在世界上的地位[EB/OL]. http://www.moa.gov.cn/ztzl/cyf/200303/t20030306_62346.htm, 2003-03-06.
[24] Rieu I, Ruiz-Rivero O, Fernandez-Garcia N, et al.. The gibberellin biosynthetic genes AtGA20ox1 and AtGA20ox2 act, partially redundantly, to promote growth and development throughout the Arabidopsis life cycle[J]. Plant J., 2008, 53(3): 488-504.
[25] 殷小林,张超,王有成,等.水稻赤霉素3β羟化酶基因(OsGA3ox1)同源基因的生物信息学分析[J/OL].分子植物育种, http://kns.cnki.net/kcms/detail/46.1068.S.20181025.1431.010.html.
[26] Van Damme M, Huibers R P, Elberse J, et al.. Arabidopsis DMR6 encodes a putative 2OG-Fe(Ⅱ) oxygenase that is defense-associated but required for susceptibility to downy mildew[J]. Plant J., 2008, 54(5): 785-793.