AtSOS基因在紫花苜蓿中的表达及其耐盐性研究

doi:10.11686/cyxb2017281

摘要/Abstract

摘要： 本研究采用基因工程技术改良紫花苜蓿耐盐性,通过种植转基因耐盐紫花苜蓿达到改良土壤的目的。以紫花苜蓿子叶节为外植体,通过农杆菌介导法将来源于拟南芥的AtSOS1-AtSOS2-AtSOS3多基因表达载体导入阿尔冈金紫花苜蓿中,经PCR检测、抗除草剂筛选和RT-PCR鉴定,获得了能稳定表达的转基因株系。以转基因的紫花苜蓿和野生型紫花苜蓿为材料进行盐处理,每个处理重复3次,测定其生理生化指标、株高、Na⁺和K⁺含量、细胞膜透性、叶绿素含量。结果显示,在不同盐浓度处理下,所有植株的株高均有所增长,但在100和200 mmol·L^-1的NaCl处理下,转基因植株的长势显著高于野生型植株;随着处理时间的增加,所有植株的叶绿素含量均呈先上升后下降的趋势,且野生型植株叶绿素含量均低于转基因植株;在100和200 mmol·L^-1的NaCl处理下,转基因植株的细胞膜透性、超氧化物歧化酶活性和脯氨酸含量的增加量均小于野生型植株,而过氧化物酶、过氧化氢酶活性和可溶性糖含量的增加量均大于野生型;各植株中丙二醛含量均下降,且野生型植株下降的更为明显;盐处理后,转基因植株根系中Na⁺的积累比野生型植株少,而K⁺的吸收多于野生型植株。转AtSOS基因的紫花苜蓿通过发挥AtSOS途径的作用,促进了植物体将细胞内的Na⁺外排,从而减轻盐胁迫对植物体的伤害,提高了转基因植株的耐盐性。

关键词: 紫花苜蓿, AtSOS基因, 遗传转化, 耐盐性

Abstract: In this study, alfalfa was genetically engineered to improve its salt tolerance. The SOS1-SOS2-SOS3 genes from Arabidopsis thaliana were incorporated into Algonquin using tumefaciens-mediate transformation technology. Transgenic plants were identified by PCR, herbicide resistance screening and RT-PCR. The salt resistance of the transgenic plants was evaluated by exposing them to different concentration (100, 200 and 300 mmol·L^-1) of NaCl for 6 days and comparing them to unmodified plants. Physiological and biochemical indexes, plant height, Na⁺ and K⁺ content, cell membrane permeability and chlorophyll content were measured. The results showed that under different salt treatments, the height of all plants increased but transgenic plant height was significantly higher than that of unmodified plants at 100 and 200 mmol·L^-1 NaCl. After salt exposure the chlorophyll content of all plants initially increased before declining but the chlorophyll content of unmodified plants was lower than that of transgenic plants. Under 100 and 200 mmol·L^-1 NaCl treatments, membrane permeability, superoxide dismutase activity and proline content of transgenic plants were lower than that of unmodified plants. However, peroxidase activity, catalase activity and soluble sugar were higher than that of unmodified plants. Malondialdehyde content declined in all plants but the decline was greater in unmodified plants. The accumulation of Na⁺ in roots of transgenic plants was less than in unmodified plants, while K⁺ content was more than that of unmodified plants. Transgenic plants displayed increased Na⁺ efflux, reducing cellular ion toxicity, alleviating the damage from salt stress.

Key words: Medicago sativa, AtSOS genes, genetic transformation, salt-resistance

麻冬梅, 秦楚. AtSOS基因在紫花苜蓿中的表达及其耐盐性研究[J]. 草业学报, 2018, 27(6): 81-91.

MA Dong-mei, QIN Chu. The expression of the salt tolerance gene AtSOS in Medicago[J]. Acta Prataculturae Sinica, 2018, 27(6): 81-91.

参考文献

[1] Allakhverdiev S I, Sakamoto A, Nishiyama Y, et al. Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in synechococcus sp. Plant Physiology, 2000, 123(3): 1047-1056
[2] Zhu J K.Salt and drought stress signal transduction in plants. Annual Review of Plant Biology, 2003, 53(53): 247-273.
[3] Wang Z Q.Chinese salty soil. Beijing: Science Press, 1993.
王遵亲. 中国盐渍土. 北京: 科学出版社, 1993.
[4] Zhao X, Han J C, Wang H Y, et al. Research progress of saline soil improvement technology. Chinese agronomy bulletin, 2016, (8): 113-116.
赵宣, 韩霁昌, 王欢元, 等. 盐渍土改良技术研究进展. 中国农学通报, 2016, (8): 113-116.
[5] Yan H, Zhao W, Yin S J, et al. The physiological responses of Leymus chinensis to different saline alkali stresses. Acta Prataculturae Sinica, 2006, (6): 49-55.
颜宏, 赵伟, 尹尚军, 等. 羊草对不同盐碱胁迫的生理响应. 草业学报, 2006, (6): 49-55.
[6] Xu Z Q, Jia J F, Hu Z D.Agrobacterium strain A-4 transformed alfalfa suspension cultures. Journal of Biomedical Engineering, 1997, (1): 55-59.
徐子勤, 贾敬芬, 胡之德. 发根农杆菌A-4菌株转化苜蓿悬浮培养物. 生物工程学报, 1997, (1): 55-59.
[7] Cords H P.Weeds and alfalfa hay quality. Weed Science, 1973, 21(5): 400-401.
[8] Chang S, Nan L, Wang X, et al. Alfalfa carbon and nitrogen sequestration patterns and effects of temperature and precipitation in three agro-pastoral ecotones of northern China. Plos One, 2012, 7(11): e50544.
[9] Zhi W, Ke Q, Kim M D, et al. Transgenic alfalfa plants expressing the sweetpotato orange gene exhibit enhanced abiotic stress tolerance. Plos One, 2015, 10(5): e0126050.
[10] Bagavathiannan M V, Begg G S, Gulden R H, et al. Modelling the dynamics of feral alfalfa populations and its management implications. Plos One, 2012, 7(6): e39440.
[11] Postnikova O A, Shao J, Nemchinov L G.Analysis of the alfalfa root transcriptome in response to salinity stress. Plant & Cell Physiology, 2013, 54(7): 1041.
[12] Xu B.Study cloning and functional analysis of P5CS gene in Puccinellia chinampoensis and alfalfa transformation. Beijing: Chinese Academy of Agricultural Sciences, 2012.
徐博. 朝鲜碱茅P5CS基因的克隆及其转化紫花苜蓿的研究. 北京: 中国农业科学院, 2012
[13] Liu Y, Cai H, Liu J, et al. Transformation of GsCRCK gene into Nong Jing No.1 alfalfa and its salt tolerance analysis. Acta Prataculturae Sinica, 2013, (2): 150-157.
刘莹, 才华, 刘晶, 等. GsCRCK基因转化农菁1号苜蓿及其耐盐性分析. 草业学报, 2013, (2): 150-157.
[14] Jin T C, Meng D W, Wang Y, et al. Using AH1 gene transformation to improve the salt tolerance of alfalfa. Molecular Plant Breeding, 2016, (7): 1724-1729.
金太成, 孟大伟, 王悦, 等. 利用AH1基因的遗传转化改良紫花苜蓿耐盐性. 分子植物育种, 2016, (7): 1724-1729.
[15] Zhang L Q.Using transgenic technology to create new germplasm of alfalfa salt tolerance. Hohhot: Inner Mongolia University, 2011.
张立全. 利用转基因技术创建紫花苜蓿耐盐新种质的研究. 呼和浩特: 内蒙古大学, 2011.
[16] Zhang Z L, Qu W J.Experimental instruction of plant physiology. Beijing: Higher Education Press, 2003.
张志良, 瞿伟菁. 植物生理学实验指导. 北京: 高等教育出版社, 2003.
[17] Murray M G, Thompson W F.Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 1980, 8(19): 4321-4325.
[18] Xin Z, Browse J.Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(95): 7799-7804.
[19] Emani C, Sunilkumar G, Rathore K S.Transgene silencing and reactivation in sorghum. Plant Science, 2002, 162(2): 181-192.
[20] Wu G, Xia Y W.Plant transgene silencing and its countermeasure. Biotechnology, 2000, 10(2): 27-32.
吴刚, 夏英武. 植物转基因沉默及对策. 生物技术, 2000, 10(2): 27-32.
[21] Mao X H.Genetic engineering improvement of salt tolerance in Medicago sativa L. Ji’nan: Shandong University, 2009.
毛秀红. 苜蓿耐盐的基因工程改良研究. 济南: 山东大学, 2009.
[22] Wei Z W, Wang H S.Cold acclimation and cold hardiness property of various types of turfgrasses. Pratacultural Science, 1997, 15(2): 62-66.
魏臻武, 王槐三. 两种草坪草抗寒特性及其超氧化物酶的作用. 草业科学, 1997, 15(2): 62-66.
[23] Jiang M, Zhang J.Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings. Plant & Cell Physiology, 2001, 42(11): 1265-1273.
[24] Liu Z L, Zhu Y L, Hu C M, et al. The effect of NaCl stress on the growth, antioxidant enzyme activity and active oxygen metabolism of grafted eggplant. Acta Ecology, 2007, (3): 537-541.
刘正鲁, 朱月林, 胡春梅, 等. 氯化钠胁迫对嫁接茄子生长、抗氧化酶活性和活性氧代谢的影响. 应用生态学报, 2007, (3): 537-541.
[25] Wang X Q, Zhang J W, Wei J H, et al. Preliminary study on physiological mechanism of salt tolerance in barley under salt stress. Acta Agronomica Sinica, 2007, 22(1): 17-21.
王雪青, 张俊文, 魏建华, 等. 盐胁迫下野大麦耐盐生理机制初探. 华北农学报, 2007, 22(1): 17-21.
[26] Zhu J K, Liu J, Xiong L.Genetic analysis of salt tolerance in Arabidopsis: Evidence for a critical role of potassium nutrition. Plant Cell, 1998, 10(7): 1181.
[27] Qi Z, Spalding E P.Protection of plasma membrane K+ transport by the salt overly sensitive1 Na+-H+ antiporter during salinity stress. Plant Physiology, 2004, 136(1): 2548-2555.