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大豆GmNHX1基因的克隆、功能初探及遗传转化

标题: 大豆GmNHX1基因的克隆、功能初探及遗传转化
英文标题: Molecular Cloning, Functional Analysis and Genetic Transformation of a Na~+/H~+ Antiporter Gene GmNHX1 from Soybean
作者: 杨郡
出版时间: 2012-01-01
所在大学: 河北农业大学
关键词: GmNHX1基因,拟南芥,大豆,胚尖,遗传转化
英文关键词: GmNHX1,arabidopsis,soybean,embryonic tip,genetic transformation
论文级别: 硕士
学位: 学位论文
导师: 王冬梅
专业: 细胞生物学
提交时间: 2012
摘要: 大豆[Glycine max(L.)Merrill]是重要的油料作物和粮食作物,是世界上食用油和植物蛋白的主要来源.为了适应现代农业发展的要求,培育优质抗逆大豆是大豆育种的发展方向之一.随着分子生物学及基因工程技术的发展,利用转基因技术将抗逆基因转入优质大豆基因组是提高其抗逆性的有效途径.Na+/H+逆向转运蛋白通过将细胞质内的Na+外排或将Na+区隔化到液泡中,来维持细胞质的Na+稳态和Na+/K+比相对稳定,从而减少盐胁迫对植株的伤害.本试验从大豆品种冀豆7号中克隆得到了耐盐基因GmNHX1,将其转入拟南芥验证其与植物耐盐的相关性;在实验室前期工作基础上进一步优化大豆胚尖再生及转化体系;并采用农杆菌介导法将GmNHX1基因转入盐敏感性大豆品种中.结果如下:1、通过RT-PCR从大豆品种冀豆7号中克隆出Na+/H+逆向转运蛋白基因GmNHX1,经生物信息学分析表明该基因与已知功能的液泡膜Na+/H+反向转运蛋白具有较高的同源性.半定量RT-PCR分析表明GmNHX1属于组成型表达,其表达受NaCl胁迫上调.盐胁迫下叶片中GmNHX1的表达明显高于根中,耐盐品种冀豆7号中GmNHX1的本底表达水平比盐敏感性大豆品种冀豆17号低,但在盐胁迫下,冀豆7号GmNHX1的表达量较冀豆17号上调明显.初步表明GmNHX1与大豆响应盐胁迫有关.2、构建了pCAMBIA1300-35S::GmNHX1-eYFP双元表达载体,通过农杆菌介导法转化野生型(columbia型)拟南芥,获得了转GmNHX1的拟南芥,经150 mM NaCl处理证明GmNHX1基因能够明显增加转基因拟南芥的耐盐性.3、在本实验室前期工作基础上对大豆胚尖再生体系进行了优化.结果表明,6-BA浓度在0.2~0.5 mg/L时对供试4个大豆基因型的胚尖再生效果较好,其中冀豆16的再生效果最佳;TDZ浓度在0.2~1.0 mg/L时对4个大豆基因型的胚尖再生效果较好,其中nf37出芽率和植株再生率最佳.4、确定了胚尖再生植株生根时Basta筛选的最适浓度为0.6 mg/L;而土培大豆苗Basta筛选的最适喷洒浓度为200 mg/L.5、利用优化的胚尖再生体系并通过农杆菌介导法将GmNHX1基因转入盐敏品种五星2号中,获得转GmNHX1基因的PCR阳性植株.
英文摘要: Soybean (Glycine max) is an major grain and oil crops, and the main source of edible oil and vegetable protein of the world. In order to meet the development requirements of modern agriculture, soybean resistance enhancement is one of the development direction of soybean breeding. With the development of molecular biology and genetic engineering technology, transferring salt tolerance genes into high-quality soybean genomes is become an effective way to improve soybean salt tolerance. Na+/H+ antiporter protein could export cytosolic Na+ outside the cell or separate Na+ in the vacuole so that to maintain the plant cytoplasm Na+ stability, Na+/K+ relatively ratio, osmotic pressure, and reduce salt stress. In this study, we have cloned the salt tolerance gene GmNHX1 from soybean varieties Jidou 7 and transferred it into Arabidopsis to test gene function on salt tolerance; we further optimized soybean embryonic tips regeneration and transformation system based on laboratory previous work; we transferred GmNHX1 into salt-sensitive soybean varieties with Agrobacterium-mediated transformation method. The results show that:1. We have cloned Na+/H+ antiporter gene GmNHX1 from soybean varieties Jidou 7 by RT-PCR. Bioinformatics analysis indicated GmNHX1 is highly homologous with the function known tonoplast Na+/H+ antiporter. Semi-quantitative RT-PCR analysis indicated that GmNHX1 expression is constitutive and increased under NaCl stress. Under salt stress the GmNHX1 expression level in leaves was significantly higher than that in root. Salt-tolerant varieties Jidou 7 has a lower GmNHX1 background expression level than salt-sensitive soybean varieties Jidou 17, but a higher increase under salt stress. And this could afford a primary evidence that GmNHX1 have a relationship with soybean response to solt strees.2. The binary expression vector pCAMBIA1300-35S::GmNHX1-eYFP was obtained and then transformed into the Arabidopsis theliana. The salt tolerance phenotype in Arabidopsis GmNHX1 overexpression lines under 150mM NaCl indicated GmNHX1 could improve salt tolerance in plants.3. We optimizated the soybean embryonic tip regeneration system. The results show that under 0.2~0.5 mg/L 6-BA the four soybean genotypes had better embryonic tip regeneration rates and regeneration rate of Jidou 16 is the best. Under 0.2~1.0 mg/L TDZ the four soybean genotypes had better embryonic tip regeneration rates; nf37 has a highest shooting rate and plant regeneration rate.4. Determined the optimal Basta screening concentration to the tested soybean varieties embryonic tip regenerated seedlings was 0.6 mg/L; but to the seed germinated seedlings the optimal Basta screening concentration was 200 mg/L.5. We obtained the trans-GmNHX1 gene PCR positive soybean plants by Agrobacterium-mediated embryonic tips genetic transformation using the optimized embryonic tips regeneration system.