利用RACE(rapid-amplification of cDNA end)方法,以大豆叶片提取总的RNA为模板克隆了大豆Na+/H+逆向转运蛋白(Glycine max Na+/H+antiporter,GmNHX)基因,并将其连接到表达载体PBI121中,构建重组表达载体PBI121-NHX3.分析结果表明:该基...利用RACE(rapid-amplification of cDNA end)方法,以大豆叶片提取总的RNA为模板克隆了大豆Na+/H+逆向转运蛋白(Glycine max Na+/H+antiporter,GmNHX)基因,并将其连接到表达载体PBI121中,构建重组表达载体PBI121-NHX3.分析结果表明:该基因的ORF为1 503 bp,推测编码501个氨基酸.与所选取的10种植物同类蛋白氨基酸序列进行对比,一致性为72%～94%,并具有真核生物单价阳离子(氢离子)反向转运蛋白典型的结构域,将该基因命名为GmNHX3,GenBank接收号为JN872904.通过PCR和酶切鉴定,PBI121-NHX3构建成功.展开更多
AaNhaD, a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica, encodes a Na＋/H＋ antiporter crucial for the bacterium＇s resistance to salt/alkali stresses. However, it remains unknown whether this t...AaNhaD, a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica, encodes a Na＋/H＋ antiporter crucial for the bacterium＇s resistance to salt/alkali stresses. However, it remains unknown whether this type of bacterial gene may be able to increase the tolerance of flowering plants to salt/alkali stresses. To investigate the use of extremophile genetic resources in higher plants, transgenic tobacco BY-2 cells and plants harboring AaNhaDwere generated and their stress tolerance was evaluated. Ectopic expression of AaNhaD enhanced the salt tolerance of the transgenic BY-2 cells in a pH-dependent manner. Compared to wild-type controls, the transgenic cells exhibited increased Na＋ concentrations and pH levels in the vacuoles. Subcellular localization analysis indicated that AaNhaD-GFP fusion proteins were primarily localized in the tonoplasts. Similar to the transgenic BY-2 cells, AaNhaD.overexpressing tobacco plants displayed enhanced stress tolerance when grown in saline-alkali soil. These results indicate that AaNhaD functions as a pH-dependent tonoplast Na＋/H＋ antiporter in plant cells, thus presenting a new avenue for the genetic improvement of salinity/alkalinity tolerance.展开更多
Na+/H+ antiporters have been well documented to enhance plant salt tolerance by regulating cellular ion homeostasis. Here, a putative Na+/H+ antiporter gene homolog GmNHX2 from soybean was cloned and predicted to enco...Na+/H+ antiporters have been well documented to enhance plant salt tolerance by regulating cellular ion homeostasis. Here, a putative Na+/H+ antiporter gene homolog GmNHX2 from soybean was cloned and predicted to encode a protein of 534 amino acids with 10 putative transmembrane domains. GmNHX2 was expressed in all soybean plant tissues but enriched in roots and its expression was induced by NaCl and polyethylene glycol (PEG) treatments. GmNHX2 exhibits greater sequence similarity with LeNHX2 and AtNHX6 than that of AtNHX1 and AtSOS1. Although phylogenetic analysis clustered GmNHX2 with organellar (tonoplast and vesicles) antiporters, the GmNHX2-EGFP (enhanced green fluorescent protein) fusion protein was possibly localized in the plasma membrane or organelle membrane of transgenic plant cells. Furthermore, transgenic Arabidopsis plants expressing GmNHX2 were more tolerant to high NaCl concentrations during germination and seedling stages when compared with wild-type plants. These results suggest that GmNHX2 is a membrane Na+/H+ antiporter and may function to regulate ion homeostasis under salt stress.展开更多
[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the prote...[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.展开更多
目的:嗜盐菌分布广泛且适应能力极强,为加强对嗜盐菌耐盐机制的探索与研究,从盐单胞菌Halomonas alkaliphila DSM 16354^(T)中筛选出潜在的与耐盐有关的基因,对其进行生物信息学分析,并验证相关蛋白的生理功能。方法:利用基因文库筛选...目的:嗜盐菌分布广泛且适应能力极强,为加强对嗜盐菌耐盐机制的探索与研究,从盐单胞菌Halomonas alkaliphila DSM 16354^(T)中筛选出潜在的与耐盐有关的基因,对其进行生物信息学分析,并验证相关蛋白的生理功能。方法:利用基因文库筛选和功能互补相结合的方法,通过与大肠杆菌(Escherichia coli)盐敏感缺陷株KNabc(ΔnhaA、ΔnhaB、ΔchaA)的耐盐功能互补实验,筛选出具有耐盐功能的蛋白编码基因,并通过荧光猝灭恢复实验测定蛋白的逆向转运活性以及底物亲和力。结果:筛选得到两个具有耐盐功能的蛋白编码基因,生物信息学分析结果表明该基因编码来自于DUF1538(domain of unknown function with No.1538 family)家族功能未知的膜蛋白,分别命名为duf1和duf2。系统发育树分析结果表明,来自盐单胞菌DSM 16354^(T)中的DUF1、DUF2属于一个独立的分支,预测这两个蛋白可能是DUF1538家族转运蛋白的新成员。对DUF1和DUF2的生理功能进行分析,发现duf1和duf2单独表达时均不具有耐盐碱能力,而共同表达时则表现出显著的耐盐碱功能,表明DUF1和DUF2两个亚基共同支持了蛋白的耐盐碱功能。蛋白的逆向转运测定活性结果表明双组份蛋白DUF1-2具有Na^(+)(Li^(+)、K^(+))/H^(+)逆向转运活性。结论:筛选得到的基因duf1和duf2共同表达时具有盐碱耐受功能以及逆向转运蛋白活性,这为筛选出新的DUF1538家族转运蛋白基因和进一步探究DUF1538家族转运蛋白功能奠定了基础。展开更多
文摘利用RACE(rapid-amplification of cDNA end)方法,以大豆叶片提取总的RNA为模板克隆了大豆Na+/H+逆向转运蛋白(Glycine max Na+/H+antiporter,GmNHX)基因,并将其连接到表达载体PBI121中,构建重组表达载体PBI121-NHX3.分析结果表明:该基因的ORF为1 503 bp,推测编码501个氨基酸.与所选取的10种植物同类蛋白氨基酸序列进行对比,一致性为72%～94%,并具有真核生物单价阳离子(氢离子)反向转运蛋白典型的结构域,将该基因命名为GmNHX3,GenBank接收号为JN872904.通过PCR和酶切鉴定,PBI121-NHX3构建成功.
基金supported by grants from the National Natural Science Foundation(30771162)the Ministry of Agriculture of China(2009ZX08009-096B)
文摘AaNhaD, a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica, encodes a Na＋/H＋ antiporter crucial for the bacterium＇s resistance to salt/alkali stresses. However, it remains unknown whether this type of bacterial gene may be able to increase the tolerance of flowering plants to salt/alkali stresses. To investigate the use of extremophile genetic resources in higher plants, transgenic tobacco BY-2 cells and plants harboring AaNhaDwere generated and their stress tolerance was evaluated. Ectopic expression of AaNhaD enhanced the salt tolerance of the transgenic BY-2 cells in a pH-dependent manner. Compared to wild-type controls, the transgenic cells exhibited increased Na＋ concentrations and pH levels in the vacuoles. Subcellular localization analysis indicated that AaNhaD-GFP fusion proteins were primarily localized in the tonoplasts. Similar to the transgenic BY-2 cells, AaNhaD.overexpressing tobacco plants displayed enhanced stress tolerance when grown in saline-alkali soil. These results indicate that AaNhaD functions as a pH-dependent tonoplast Na＋/H＋ antiporter in plant cells, thus presenting a new avenue for the genetic improvement of salinity/alkalinity tolerance.
基金Supported by the National Natural Science Foundation of China (Grant No. 30490251)National High Technology Research and Development Program of China (Grant Nos. 2006AA10A110, 2006AA100104)Key Projects in the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (Grant No. 2006BAD13B05)
文摘Na+/H+ antiporters have been well documented to enhance plant salt tolerance by regulating cellular ion homeostasis. Here, a putative Na+/H+ antiporter gene homolog GmNHX2 from soybean was cloned and predicted to encode a protein of 534 amino acids with 10 putative transmembrane domains. GmNHX2 was expressed in all soybean plant tissues but enriched in roots and its expression was induced by NaCl and polyethylene glycol (PEG) treatments. GmNHX2 exhibits greater sequence similarity with LeNHX2 and AtNHX6 than that of AtNHX1 and AtSOS1. Although phylogenetic analysis clustered GmNHX2 with organellar (tonoplast and vesicles) antiporters, the GmNHX2-EGFP (enhanced green fluorescent protein) fusion protein was possibly localized in the plasma membrane or organelle membrane of transgenic plant cells. Furthermore, transgenic Arabidopsis plants expressing GmNHX2 were more tolerant to high NaCl concentrations during germination and seedling stages when compared with wild-type plants. These results suggest that GmNHX2 is a membrane Na+/H+ antiporter and may function to regulate ion homeostasis under salt stress.
基金Supported by Chunhui Plan of Ministry of Education(Z2010101)Open Fund of Food Biotechnology Key Laboratory of Sichuan Province(SZJJ2009-014)Scientific Research Foundation of Xihua University(000022)~~
文摘[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.
文摘目的:嗜盐菌分布广泛且适应能力极强,为加强对嗜盐菌耐盐机制的探索与研究,从盐单胞菌Halomonas alkaliphila DSM 16354^(T)中筛选出潜在的与耐盐有关的基因,对其进行生物信息学分析,并验证相关蛋白的生理功能。方法:利用基因文库筛选和功能互补相结合的方法,通过与大肠杆菌(Escherichia coli)盐敏感缺陷株KNabc(ΔnhaA、ΔnhaB、ΔchaA)的耐盐功能互补实验,筛选出具有耐盐功能的蛋白编码基因,并通过荧光猝灭恢复实验测定蛋白的逆向转运活性以及底物亲和力。结果:筛选得到两个具有耐盐功能的蛋白编码基因,生物信息学分析结果表明该基因编码来自于DUF1538(domain of unknown function with No.1538 family)家族功能未知的膜蛋白,分别命名为duf1和duf2。系统发育树分析结果表明,来自盐单胞菌DSM 16354^(T)中的DUF1、DUF2属于一个独立的分支,预测这两个蛋白可能是DUF1538家族转运蛋白的新成员。对DUF1和DUF2的生理功能进行分析,发现duf1和duf2单独表达时均不具有耐盐碱能力,而共同表达时则表现出显著的耐盐碱功能,表明DUF1和DUF2两个亚基共同支持了蛋白的耐盐碱功能。蛋白的逆向转运测定活性结果表明双组份蛋白DUF1-2具有Na^(+)(Li^(+)、K^(+))/H^(+)逆向转运活性。结论:筛选得到的基因duf1和duf2共同表达时具有盐碱耐受功能以及逆向转运蛋白活性,这为筛选出新的DUF1538家族转运蛋白基因和进一步探究DUF1538家族转运蛋白功能奠定了基础。
