SsNHX1基因在紫花苜蓿中的表达及其耐盐性研究
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摘要
本研究应用RACE方法成功的从盐生植物-猪毛菜(Salsola soda)中克隆了液泡膜型Na+/H+逆向转运蛋白全基因序列,构建了该基因的植物表达载体并转化紫花苜蓿,并对获得大量转基因苜蓿材料进行了耐盐性研究。
猪毛菜液泡膜型Na+/H+逆向转运蛋白全基因(SsNHXl)序列全长为1934 bp,包含一个长度为1680 bp的开放阅读框(open reading frame,ORF),编码559 aa个氨基酸残基。液泡膜型Na+/H+逆向转运蛋白的高度保守区-抑制剂氨氯吡嗪脒结合位点,Na+/H+交换泵(Na+/H+ exchanger; NHE)和液泡膜型Na+/H+逆向转运蛋白特有的12段跨膜疏水结构域在蛋白序列中被发现。氨基酸序列多重比对分析表明,该蛋白与来自盐爪爪(Kalidium foliatum)、北滨藜(Atriplex gmelini)、紫花苜蓿(Medicago sativa)、棉花(Gossypium hirsutum)、拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)、大麦(Hordeum vulgare)和玉米(Zea mays)的液泡膜型Na+/H+逆向转运蛋白相比,相似性分别达到了90%,87%,77%,76%,7.5%,74%,73%和72%。进化树分析表明,该蛋白与其他液泡膜型Na+/H+逆向转运蛋白亲缘关系较近,而与质膜型Na+/H+逆向转运蛋白亲缘关系较远。在液泡膜型Na+/H+逆向转运蛋白中,与盐生植物-盐爪爪、北滨藜亲缘关系较近,而与甜土植物亲缘关系较远。该基因在酵母中表达能够提高酵母的生长速率,在烟草中表达能够有效的提高T1代转基因种子在含盐培养基中的发芽率,并且生物量和株高都比对照有明显的提高,充分验证了该基因抗盐功能。总之,SsNHXl基因是液泡膜型Na+/H+逆向转运蛋白基因家族的一个全新成员,同时,在植物中过表达此基因能够明显的提高转基因植株的耐盐碱能力。
苜蓿的基因型是影响苜蓿遗传转化的关键因素,只有很少的栽培品种适合建立高效的再生体系。本研究以14份国内外主栽品种作为供试品种,通过比较品种间的诱导能力,筛选到公农1号为最佳的基因型。在此基础上,以公农1号的嫩叶为外植体,建立了一套高效的农杆菌介导苜蓿遗传转化体系,成功将猪毛菜液泡膜型Na+/H+逆向转运蛋白基因导入苜蓿基因组中。Southern杂交证实有6个独立转化系为转基因阳性株系,最终获得了203株阳性植株。
本研究以来自6个独立转化系的苜蓿植株作为耐盐试验材料,进行盐胁迫试验。Northern杂交证实外源基因-猪毛菜液泡膜型Na+/H+逆向转运蛋白基因,在逆境胁迫启动子调控下,随着盐处理浓度的增高而表达量也逐渐增高。耐盐试验表明,与对照相比转基因植株具有明显耐盐能力,最高耐盐能力达到400 mM,而对照在200 mM的条件下逐渐死亡。并且转基因耐盐植株中具有更高Na+,K+的含量。生理分析表明,在盐胁迫条件下,转基因耐盐植株受到的伤害要比对照植株轻的多。转基因植株的超氧化物歧化酶(SOD)活性变化趋势小和脯氨酸含量增长幅度小,另外,转基因植株的相对电导率(REL)变化也较小,这些生理数据表明,猪毛菜液泡膜型Na+/H+逆向转运蛋白基因的表达能帮助转基因植物抵抗盐胁迫,有效的提高了转基因植株的耐盐能力。
In this study, a putative complete vacuolar-type Na+/H+ antiporter gene (SsNHX1) gene was isolated from the halophyte Salsola soda by using the rapid amplification of cDNA ends (RACE) method. The SsNHX1 was constructed into a plant expression vector and transformed to alfalfa(Medicago sativa L.). And we studied on the salt tolerance for transgenic plants.
The sequence of SsNHX1 was 1934 bp in whole length which had one open reading frame (ORF) of 1680 bp, encoding 559 amino acid residues. Highly conserved regions of plant vacuolar Na+/H+ antiporter, including amiloride-binding domain, NHE (Na+/H+ exchanger) domain and 12 transmembrane segments, were found in the deduced amino acid sequence of SsNHX1. Multiple alignment of vacuolar Na+/H+ antiporters showed SsNHX1 shared high identity with vacuolar Na+/H+ antiporters of Kalidium foliatum, Atriplex gmelini, Medicago sativa, Gossypium hirsutum, Arabidopsis thaliana, Oryza sativa, Hordeum vulgare, and Zea mays, the similarity was 90%,87%,77%,76%,75%,74%,73%, and 72%, repectively. Phylogenetic relationship analysis indicated that SsNHX1 had closer affinity with vacuolar Na+/H+ antiporter group, while they were far with plasma membrane Na+/H+ antiporter group. Meanwhile, the SsNHX1 was more closely related to halophyte, Kalidium foliatum and Atriplex gmelini, and it's relationship was far related to glycophyte. The expression of SsNHX1 could improve growth rate of transgenic yeast under salt tolerance. Additionlly, the expression of SsNHX1 could increase the germination rate of T1 seeds of transgenic tabacco, and increase the height and productivity significantly compared with control plants. These results confirmed the salt-tolerant function of SsNHX1. Taken together, these results suggest that SsNHX1 is a new member of the vacuolar Na+/H+ antiporter family, and the overexpression of this gene could obviously enhanced the salt tolerance ability in plant. The genotype of alfalfa was the key factors in genetic transformation, therefore, there were only a few cultivars suitable for the establishment of high efficient regeneration system. In this study,13 main cultivars were selected for expriment among domestic and foreign species. The experiment showed that Gong-Nong 1 was the best genotype for transformation by comparing the inducing capacity. And based on this conclusion, a high efficiency Agrobacterium-mediated genetic transformation of alfalfa system was established and optimized with leaf of Gong-Nong 1 as explants. Moreover, we have successfully transferred the vacuolar Na+/H+ antiporter gene of Salsola soda into alfalfa genome. The Southern blot analysis showed that there were 6 independent transformed lines were positive. Finally, we obtained 203 positive plants in this study.
The six independent transformation lines were taken as material for salt tolerance test. The Northern-blot demonstrated that the expression of SsNHX1 driven by the stress inducible promoter was increased with the treating salt concentration. Salt-stress test showed that the salt-tolerant ability of transgenic alfalfa was enhanced up to 400 mM NaCl compared to control plants died at the stress condition of 200 mM NaCl. And there were higher Na+, K+ content in transgenic plants. The physiological analysis showed that the transgenic alfalfa caused less damage under salt stress compare to control plants. For example, the changes trend of the superoxide dismutase (SOD) activity of transgenic plants was smaller than that of control plants, and proline content was smaller too. In addition, the changes trend of the rate of electrolyte leakage (REL) of transgic plants was also smaller than that of control plants. All these results indicated that the expression of SsNHX1 was much helpful for transgenic plants to resistant to the salt stress, and it could enhance the salt-tolerant ability of transgenic alfalfa effectively.
猪毛菜液泡膜型Na+/H+逆向转运蛋白全基因(SsNHXl)序列全长为1934 bp,包含一个长度为1680 bp的开放阅读框(open reading frame,ORF),编码559 aa个氨基酸残基。液泡膜型Na+/H+逆向转运蛋白的高度保守区-抑制剂氨氯吡嗪脒结合位点,Na+/H+交换泵(Na+/H+ exchanger; NHE)和液泡膜型Na+/H+逆向转运蛋白特有的12段跨膜疏水结构域在蛋白序列中被发现。氨基酸序列多重比对分析表明,该蛋白与来自盐爪爪(Kalidium foliatum)、北滨藜(Atriplex gmelini)、紫花苜蓿(Medicago sativa)、棉花(Gossypium hirsutum)、拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)、大麦(Hordeum vulgare)和玉米(Zea mays)的液泡膜型Na+/H+逆向转运蛋白相比,相似性分别达到了90%,87%,77%,76%,7.5%,74%,73%和72%。进化树分析表明,该蛋白与其他液泡膜型Na+/H+逆向转运蛋白亲缘关系较近,而与质膜型Na+/H+逆向转运蛋白亲缘关系较远。在液泡膜型Na+/H+逆向转运蛋白中,与盐生植物-盐爪爪、北滨藜亲缘关系较近,而与甜土植物亲缘关系较远。该基因在酵母中表达能够提高酵母的生长速率,在烟草中表达能够有效的提高T1代转基因种子在含盐培养基中的发芽率,并且生物量和株高都比对照有明显的提高,充分验证了该基因抗盐功能。总之,SsNHXl基因是液泡膜型Na+/H+逆向转运蛋白基因家族的一个全新成员,同时,在植物中过表达此基因能够明显的提高转基因植株的耐盐碱能力。
苜蓿的基因型是影响苜蓿遗传转化的关键因素,只有很少的栽培品种适合建立高效的再生体系。本研究以14份国内外主栽品种作为供试品种,通过比较品种间的诱导能力,筛选到公农1号为最佳的基因型。在此基础上,以公农1号的嫩叶为外植体,建立了一套高效的农杆菌介导苜蓿遗传转化体系,成功将猪毛菜液泡膜型Na+/H+逆向转运蛋白基因导入苜蓿基因组中。Southern杂交证实有6个独立转化系为转基因阳性株系,最终获得了203株阳性植株。
本研究以来自6个独立转化系的苜蓿植株作为耐盐试验材料,进行盐胁迫试验。Northern杂交证实外源基因-猪毛菜液泡膜型Na+/H+逆向转运蛋白基因,在逆境胁迫启动子调控下,随着盐处理浓度的增高而表达量也逐渐增高。耐盐试验表明,与对照相比转基因植株具有明显耐盐能力,最高耐盐能力达到400 mM,而对照在200 mM的条件下逐渐死亡。并且转基因耐盐植株中具有更高Na+,K+的含量。生理分析表明,在盐胁迫条件下,转基因耐盐植株受到的伤害要比对照植株轻的多。转基因植株的超氧化物歧化酶(SOD)活性变化趋势小和脯氨酸含量增长幅度小,另外,转基因植株的相对电导率(REL)变化也较小,这些生理数据表明,猪毛菜液泡膜型Na+/H+逆向转运蛋白基因的表达能帮助转基因植物抵抗盐胁迫,有效的提高了转基因植株的耐盐能力。
In this study, a putative complete vacuolar-type Na+/H+ antiporter gene (SsNHX1) gene was isolated from the halophyte Salsola soda by using the rapid amplification of cDNA ends (RACE) method. The SsNHX1 was constructed into a plant expression vector and transformed to alfalfa(Medicago sativa L.). And we studied on the salt tolerance for transgenic plants.
The sequence of SsNHX1 was 1934 bp in whole length which had one open reading frame (ORF) of 1680 bp, encoding 559 amino acid residues. Highly conserved regions of plant vacuolar Na+/H+ antiporter, including amiloride-binding domain, NHE (Na+/H+ exchanger) domain and 12 transmembrane segments, were found in the deduced amino acid sequence of SsNHX1. Multiple alignment of vacuolar Na+/H+ antiporters showed SsNHX1 shared high identity with vacuolar Na+/H+ antiporters of Kalidium foliatum, Atriplex gmelini, Medicago sativa, Gossypium hirsutum, Arabidopsis thaliana, Oryza sativa, Hordeum vulgare, and Zea mays, the similarity was 90%,87%,77%,76%,75%,74%,73%, and 72%, repectively. Phylogenetic relationship analysis indicated that SsNHX1 had closer affinity with vacuolar Na+/H+ antiporter group, while they were far with plasma membrane Na+/H+ antiporter group. Meanwhile, the SsNHX1 was more closely related to halophyte, Kalidium foliatum and Atriplex gmelini, and it's relationship was far related to glycophyte. The expression of SsNHX1 could improve growth rate of transgenic yeast under salt tolerance. Additionlly, the expression of SsNHX1 could increase the germination rate of T1 seeds of transgenic tabacco, and increase the height and productivity significantly compared with control plants. These results confirmed the salt-tolerant function of SsNHX1. Taken together, these results suggest that SsNHX1 is a new member of the vacuolar Na+/H+ antiporter family, and the overexpression of this gene could obviously enhanced the salt tolerance ability in plant. The genotype of alfalfa was the key factors in genetic transformation, therefore, there were only a few cultivars suitable for the establishment of high efficient regeneration system. In this study,13 main cultivars were selected for expriment among domestic and foreign species. The experiment showed that Gong-Nong 1 was the best genotype for transformation by comparing the inducing capacity. And based on this conclusion, a high efficiency Agrobacterium-mediated genetic transformation of alfalfa system was established and optimized with leaf of Gong-Nong 1 as explants. Moreover, we have successfully transferred the vacuolar Na+/H+ antiporter gene of Salsola soda into alfalfa genome. The Southern blot analysis showed that there were 6 independent transformed lines were positive. Finally, we obtained 203 positive plants in this study.
The six independent transformation lines were taken as material for salt tolerance test. The Northern-blot demonstrated that the expression of SsNHX1 driven by the stress inducible promoter was increased with the treating salt concentration. Salt-stress test showed that the salt-tolerant ability of transgenic alfalfa was enhanced up to 400 mM NaCl compared to control plants died at the stress condition of 200 mM NaCl. And there were higher Na+, K+ content in transgenic plants. The physiological analysis showed that the transgenic alfalfa caused less damage under salt stress compare to control plants. For example, the changes trend of the superoxide dismutase (SOD) activity of transgenic plants was smaller than that of control plants, and proline content was smaller too. In addition, the changes trend of the rate of electrolyte leakage (REL) of transgic plants was also smaller than that of control plants. All these results indicated that the expression of SsNHX1 was much helpful for transgenic plants to resistant to the salt stress, and it could enhance the salt-tolerant ability of transgenic alfalfa effectively.
引文
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