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拟南芥耐盐基因AtNHX5的大豆遗传转化研究

标题: 拟南芥耐盐基因AtNHX5的大豆遗传转化研究
英文标题: The Research on Genetic Transformation of Soybean with Arabidopsis Salt Tolerance Relative Gene AtNHX5
作者: 郑文永
出版时间: 2011-01-01
所在大学: 河北农业大学
关键词: AtNHX5基因,大豆,胚尖,遗传转化,除草剂,整体子叶节
英文关键词: AtNHX5,soybean,embryo tip,genetic transformation,phytocide,entirety cotylcdonary nodes
论文级别: 硕士
学位: 学位论文
导师: 王冬梅
专业: 植物学
提交时间: 2011
摘要: 大豆是世界上重要的粮食和油料作物,但受环境等因素的影响,大豆的产量和品质受到一定制约.在我国由于干旱面积、盐碱地和次生盐碱化耕地的面积逐年增加,可耕种面积逐渐减少,严重制约着我国农作物的生产.因此,抗逆新品种的培育成为近年来的研究热点和重点.利用农杆菌介导法将抗逆基因转入大豆,为培育大豆抗逆新品种开辟了新途径.本研究以河北省优质大豆品种为材料,利用胚尖法以及整体子叶节法进行了拟南芥耐盐基因AtNHX5(CPA1家族的拟南芥Na~+/H~+逆向转运蛋白基因)的遗传转化.获得主要结果如下:1、通过形态学方法初步确定了河北省优质大豆品种冀豆17号、五星2号、nf 58和冀黄13号的潮霉素敏感性,为转化植株的筛选确定适宜浓度.结果表明五星2号和冀豆17号的潮霉素筛选终浓度为10 mg/L,冀黄13号与nf58的筛选终浓度为8 mg/L.2、以五星2号成熟大豆胚尖为材料,利用超声波辅助农杆菌转化法优化了大豆胚尖转化体系.侵染阶段对胚尖进行两次80 W的超声波处理,每次20 s,与对照相比超声波处理明显提高了转化效率.在此基础上,以五星2号、五星3号、冀豆15号、冀豆16号和冀黄13号胚尖为外植体,利用超声波辅助农杆菌介导法进行了拟南芥耐盐基因AtNHX5的遗传转化.对五星2号再生植株在开花期进行PCR验证,初步证明AtNHX5基因已整合到五星2号基因组,转化率为0.16%.3、利用新型整体子叶节法进行了AtNHX5基因转化研究.以带有一片子叶的胚轴为外植体,以带Basta筛选标记以及AtNHX5基因的pCAMBIA3301载体质粒进行转化,再生植株经PCR方法验证,初步证明AtNHX5基因已整合到五星2号基因组,转化率为1.47%.该方法省去了外植体转化后繁琐、耗时的组织培养步骤,再生周期明显缩短,为农杆菌介导的大豆遗传转化提供了有力的技术平台.
英文摘要: Soybean is one of the most important foods and oil crop of the world, but its production and quality is restricted by some factors like environment partly. Since the increase of the area of arid lands, saline lands and secondary salinization of cultivated land year by year, and the gradual decrease of cultivable area, it is a big restriction for the production of field crops in our country. For this reason, the incubation of new antireversional variety is becoming a hot spot and an emphasis as well for the past few years. In this study, established a new pathway for incubating new antireversional variety of Soybean with transporting Stress-resistant gene into Soybean by agroinfection. In this study, used high-quailty Soybean variety of Hebei Province as material, and undertook Arabidopsis thaliana salt-tolerant gene-AtNHX5(Arabidopsis Na~~+/H~~+ antiporter gene in Arabidopsis thaliana CPA1 family) genetic transformation by embryo tip method and entirety cotylcdonary nodes method.1. In this study, first determined the hygromycin- sensitivity of high-quailty Soybean variety of Hebei province such as Jidou 17, Wuxing 2, nf 58, Jihuang 13 by morphology methods to determine the best concentration for plant transformation. The result showed that the final hygromycin screening concentration of Wuxing 2 and Jidou 17 were 10 mg/L, and the final screening concentration of Jihuang 13 and nf 58 were 8 mg/L.2. In this study, used Wuxing 2 maturate Soybean embryo tips as material, and optimized the embryo tip transformation system of soybean with the supersonic assisted Agrobacterium-mediated transformation. On this basis, We used the embryo tip explant of wuxing 2, Wuxing 3, Jidou 15, Jidou 16, Jihuang 13 as receptor systems, undertaked Arabidopsis thaliana salt-tolerant gene AtNHX5 genetic transformation by 80 W supersonic assisted Agrobacterium-mediated transformation.We demonstrated the AtNHX5 had already integrated to the genome of Wuxing 2 soybean initially by regeneration Plants PCR verification, and the transformation efficiency was 0.16%.3. In this study, investigated the AtNHX5 transformation by new entirety cotylcdonary nodes method. Used the Hypocotyl involved one seed leaf as explantation, and transformed with pCAMBIA3301 vector plasmid contained Basta screening mark and AtNHX5. Demonstrated the AtNHX5 had already integrated to the genome of Wuxing 2 soybean initially by regeneration Plants PCR verification, and the transformation efficiency was 1.47%. The method cancelled the tedious and time-consuming tissue culture procedure after transformation of explantation, and the Anagen cycle was decurtated obviously. This study provided an effective technique platform for soybean transformation by Agrobacterium-mediated.