转乙醇酸脱氢酶(GDH)基因棉花的获得及表型鉴定
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摘要
[目的]减少植物光呼吸的消耗能有效提高光合效率,有助于提高植物生物量。将细菌乙醇酸代谢途径导入植物建立光呼吸支路,可以把乙醇酸代谢产生的CO_2直接释放到叶绿体中,促进Rubisco羧化反应,从而提高光合效率。[方法]本研究运用农杆菌介导法,将乙醇酸脱氢酶基因GDH导入常规陆地棉受体材料R15建立光呼吸支路,通过体细胞愈伤诱导法在含抗生素的筛选培养基中获得再生植株。[结果]分子检测结果显示外源基因被成功导入棉花受体,并获得36株转基因材料。通过对转基因植株及对照的光合参数测定及田间表型观察,发现转基因植株比对照净光合速率提高26.99%～37.29%,株高增加10.07%～16.18%,叶片鲜重增加21.59%～44.25%,干重增加11.06%～17.79%,表明GDH降低了棉花光呼吸消耗,增加了植株生物量。[结论]本研究为筛选高光合效率及高产棉花种质资源提供了新的思路。
[Objective]Reducing photorespiration rate can effectively improve photosynthetic efficiency andincrease the plant biomass. Present study was designed to introduce bacterial glycolic acid pathway into plants and to establish a photorespiration bypass which directly release CO_2 into chloroplasts and promote Rubisco carboxylation resulting in photosynthetic efficiency improvement. [Methods]In the study, glycolate dehydrogenase gene(GDH)from E. coliwas transferred into upland cotton variety R15 by Agrobacterium tumefaciens mediated method, and regenerated plants were obtained by somatic callus induction in antibiotic-containing screening medium. [Results]Molecular detection revealed that GDH was successfully introduced into cotton receptor and a total of 36 transgenic plants were obtained. The photosynthetic parameterswere measured and the phenotypes of plants in the field were determined. Results indicated that the net photosynthetic rate of several transgenic plants was enhancedby 26.99%～37.29%, and plant height wasincreased by 10.07%～16.18%. Fresh leaf weight and dry weightwere increased 21.59%～44.25% and 11.06%～17.79%, respectively, compared to that of control plants.These results demonstrated that photorespiration rate of GDH gene modified plants was reduced and plant biomass were significantly improved. [Conclusion]This study provided a new way to screen cotton germplasm resources with high photosynthetic efficiency and yield.
[Objective]Reducing photorespiration rate can effectively improve photosynthetic efficiency andincrease the plant biomass. Present study was designed to introduce bacterial glycolic acid pathway into plants and to establish a photorespiration bypass which directly release CO_2 into chloroplasts and promote Rubisco carboxylation resulting in photosynthetic efficiency improvement. [Methods]In the study, glycolate dehydrogenase gene(GDH)from E. coliwas transferred into upland cotton variety R15 by Agrobacterium tumefaciens mediated method, and regenerated plants were obtained by somatic callus induction in antibiotic-containing screening medium. [Results]Molecular detection revealed that GDH was successfully introduced into cotton receptor and a total of 36 transgenic plants were obtained. The photosynthetic parameterswere measured and the phenotypes of plants in the field were determined. Results indicated that the net photosynthetic rate of several transgenic plants was enhancedby 26.99%～37.29%, and plant height wasincreased by 10.07%～16.18%. Fresh leaf weight and dry weightwere increased 21.59%～44.25% and 11.06%～17.79%, respectively, compared to that of control plants.These results demonstrated that photorespiration rate of GDH gene modified plants was reduced and plant biomass were significantly improved. [Conclusion]This study provided a new way to screen cotton germplasm resources with high photosynthetic efficiency and yield.
引文
[1]李朝霞, 赵世杰,孟庆伟, 光呼吸途径及其功能 [J]. 植物学报, 2003,20 (2): 190-197.
[2]Bowes G, Ogren W L, Hageman R H, et al. Phosphoglycolate production catalyzed by ribulose diphosphate carboxylase[J]. Biochemical and Biophysical Research Communications, 1971. 45(3): 716-722.
[3]Ogren W L, Salvucci M E,Portis A R J. The Regulation of Rubisco Activity[J]. Philosophical Transactions of the Royal Society of London, 1986. 313(1162): 337-346.
[4]Laing W A, William L O, Richard H. Regulation of soybean net photosynthetic CO2 fixation by the interaction of CO2, O2, and ribulose 1,5-diphosphate carboxylase[J]. Plant Physiology, 1974. 54(5): 678-685.
[5]李合生. 《现代植物生理学》(第2版) [M]. 北京: 高等教育出版社, 2006: 21-55.
[6]Sharkey T D. Estimating the rate of photorespiration in leaves[J]. Physiologia Plantarum, 1988. 73(1): 147-152.
[7]Leegood R C, Lea P J, Adcock M D, et al. The regulation and control of photorespiration[J]. Journal of Experimental Botany, 1995. 46: 1397-1414.
[8]Lorimer G. An early Arabidopsis demonstration resolving a few issues concerning photorespiration[J]. Plant Physiology, 2001. 125(1): 20-24.
[9]张树伟, 王霞, 马燕斌,等. 植物光呼吸途径及其支路研究进展[J]. 山西农业大学学报(自然科学版), 2016,36(12): 885-889.
[10]Lord, J. M. "Glycolate oxidoreductase in Escherichia coli." Biochimica et Biophysica Acta-Bioenergetics. 1972,267(2): 227-237.
[11]Eisenhut M, Ruth W, Haimovich M, et al. The photorespiratory glycolate metabolism is essential for cyanobacteria and might have been conveyed endosymbiontically to plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008. 105(44): 17199-17204.
[12]Kebeish R, Niessen M, Thiruveedhi K, et al. Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana[J]. Nature Biotechnology, 2007. 25(5): 593.
[13]N?lke G, Houdelet M, Kreuzaler F, et al. The expression of a recombinant glycolate dehydrogenase polyprotein in potato (Solanum tuberosum) plastids strongly enhances photosynthesis and tuber yield[J]. Plant Biotechnology Journal, 2014. 12(6): 734-742.
[14]Dalal J, Lopez H, Vasani N B, et al. A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa[J]. Biotechnology for Biofuels, 2015. 8(1): 175.
[15]张树伟, 马燕斌, 孙璇,等. 棉花遗传育种现状及展望[J]. 山西农业科学, 2016,44(1): 119-122.
[16]李燕娥, 焦改丽, 吴家和,等. 棉花农杆菌介导高效转化体系[J]. 中国棉花, 2000,27(5):10-11
[17]宋国立, 崔荣霞, 王坤波,等. 改良CTAB法快速提取棉花DNA[J]. 棉花学报, 1998 (5): 273-275.
[18]Lacuesta M, Dever L V, Muňoz-Rueda A, et al. A study of photorespiratory ammonia production in the C4 plant Amaranthus edulis, using mutants with altered photosynthetic capacities[J]. Physiologia Plantarum, 1997,99(3): 447-455.
[19]Murray A J S, Blackwell R D, Lea P J,et al. Metabolism of hydroxypyruvate in a mutant of barley lacking NADH-dependent hydroxypyruvate reductase, an important photorespiratory enzyme activity[J]. Plant Physiology, 1989,91(1): 395-400.
[20]Moreno J I, Martín R, Castresana C, et al. Arabidopsis SHMT1, a serine hydroxymethyltransferase that functions in the photorespiratory pathway influences resistance to biotic and abiotic stress[J]. Plant Journal, 2005,41(3): 451-463.
[21]Raines C A. Transgenic approaches to manipulate the environmental responses of the C3 carbon fixation cycle[J]. Plant Cell and Environment, 2006,29(3): 331-339.
[22]Zelitch I, Schultes N P, Peterson R B, et al. High glycolate oxidase activity is required for survival of maize in normal air[J]. Plant Physiology, 2009,149(1): 195-204.
[2]Bowes G, Ogren W L, Hageman R H, et al. Phosphoglycolate production catalyzed by ribulose diphosphate carboxylase[J]. Biochemical and Biophysical Research Communications, 1971. 45(3): 716-722.
[3]Ogren W L, Salvucci M E,Portis A R J. The Regulation of Rubisco Activity[J]. Philosophical Transactions of the Royal Society of London, 1986. 313(1162): 337-346.
[4]Laing W A, William L O, Richard H. Regulation of soybean net photosynthetic CO2 fixation by the interaction of CO2, O2, and ribulose 1,5-diphosphate carboxylase[J]. Plant Physiology, 1974. 54(5): 678-685.
[5]李合生. 《现代植物生理学》(第2版) [M]. 北京: 高等教育出版社, 2006: 21-55.
[6]Sharkey T D. Estimating the rate of photorespiration in leaves[J]. Physiologia Plantarum, 1988. 73(1): 147-152.
[7]Leegood R C, Lea P J, Adcock M D, et al. The regulation and control of photorespiration[J]. Journal of Experimental Botany, 1995. 46: 1397-1414.
[8]Lorimer G. An early Arabidopsis demonstration resolving a few issues concerning photorespiration[J]. Plant Physiology, 2001. 125(1): 20-24.
[9]张树伟, 王霞, 马燕斌,等. 植物光呼吸途径及其支路研究进展[J]. 山西农业大学学报(自然科学版), 2016,36(12): 885-889.
[10]Lord, J. M. "Glycolate oxidoreductase in Escherichia coli." Biochimica et Biophysica Acta-Bioenergetics. 1972,267(2): 227-237.
[11]Eisenhut M, Ruth W, Haimovich M, et al. The photorespiratory glycolate metabolism is essential for cyanobacteria and might have been conveyed endosymbiontically to plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008. 105(44): 17199-17204.
[12]Kebeish R, Niessen M, Thiruveedhi K, et al. Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana[J]. Nature Biotechnology, 2007. 25(5): 593.
[13]N?lke G, Houdelet M, Kreuzaler F, et al. The expression of a recombinant glycolate dehydrogenase polyprotein in potato (Solanum tuberosum) plastids strongly enhances photosynthesis and tuber yield[J]. Plant Biotechnology Journal, 2014. 12(6): 734-742.
[14]Dalal J, Lopez H, Vasani N B, et al. A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa[J]. Biotechnology for Biofuels, 2015. 8(1): 175.
[15]张树伟, 马燕斌, 孙璇,等. 棉花遗传育种现状及展望[J]. 山西农业科学, 2016,44(1): 119-122.
[16]李燕娥, 焦改丽, 吴家和,等. 棉花农杆菌介导高效转化体系[J]. 中国棉花, 2000,27(5):10-11
[17]宋国立, 崔荣霞, 王坤波,等. 改良CTAB法快速提取棉花DNA[J]. 棉花学报, 1998 (5): 273-275.
[18]Lacuesta M, Dever L V, Muňoz-Rueda A, et al. A study of photorespiratory ammonia production in the C4 plant Amaranthus edulis, using mutants with altered photosynthetic capacities[J]. Physiologia Plantarum, 1997,99(3): 447-455.
[19]Murray A J S, Blackwell R D, Lea P J,et al. Metabolism of hydroxypyruvate in a mutant of barley lacking NADH-dependent hydroxypyruvate reductase, an important photorespiratory enzyme activity[J]. Plant Physiology, 1989,91(1): 395-400.
[20]Moreno J I, Martín R, Castresana C, et al. Arabidopsis SHMT1, a serine hydroxymethyltransferase that functions in the photorespiratory pathway influences resistance to biotic and abiotic stress[J]. Plant Journal, 2005,41(3): 451-463.
[21]Raines C A. Transgenic approaches to manipulate the environmental responses of the C3 carbon fixation cycle[J]. Plant Cell and Environment, 2006,29(3): 331-339.
[22]Zelitch I, Schultes N P, Peterson R B, et al. High glycolate oxidase activity is required for survival of maize in normal air[J]. Plant Physiology, 2009,149(1): 195-204.