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大白菜BcDREB1基因的遗传转化研究

标题: 大白菜BcDREB1基因的遗传转化研究
英文标题: Study on Genetic Transformation of BcDREB1 Gene in Chinese Cabbage
作者: 马茜
出版时间: 2010-01-01
所在大学: 河北农业大学
关键词: 大白菜,拟南芥,农杆菌,BcDREB1基因,遗传转化,再生体系
英文关键词: Chinese cabbage,Arabidopsis thaliana,Agrobacterium-mediated,BcDREB1 gene,Genetic transformation,Regeneration system
论文级别: 硕士
学位: 学位论文
导师: 王彦华
专业: 蔬菜学
提交时间: 2010
摘要: 大白菜是我国原产蔬菜之一,在蔬菜的周年供应中占有重要地位.随着生物技术的发展,利用基因工程技术改良大白菜品种已成为当前研究热点之一.本试验通过农杆菌介导的遗传转化,将已克隆的大白菜BcDREB1基因分别转入大白菜和拟南芥中,得到转化植株,并验证大白菜BcDREB1基因的抗逆性功能,为大白菜抗逆分子育种奠定基础.主要研究结果如下:1.通过分析不同基因型、外植体类型、激素组合及预培养处理条件对大白菜不定芽再生的影响,建立了高效的大白菜离体再生体系.确定最适基因型为大白菜自交系'85-1';最适外植体为带柄子叶;0.5 mg/L 2,4-D预培养1 d;最适分化培养基为MS+0.7mg/LTDZ+ 0.5mg/LNAA+ 5.0mg/LAgNO3的配比组合.2.通过对农杆菌侵染时间、乙酰丁香酮浓度及卡那霉素抗性的筛选,建立并优化了大白菜遗传转化体系.确定了大白菜转化过程中抗性芽筛选最适卡那霉素浓度为10mg/L;农杆菌侵染时间5min,共培养2d;共培养过程中添加乙酰丁香酮150 mg/L利于转化.3.抗生素筛选获得的大白菜T0代转基因植株,经GUS组织化学染色、GUS-PCR检测后得到转基因植株共15株.对大白菜转化植株进行250mmol/LNacl渗透胁迫处理和-5℃低温处理,转化植株的耐盐性和耐寒性明显提高.4.利用Floral-dip转化方法将大白菜BcDREB1基因导入拟南芥中,抗生素筛选获得拟南芥T0代植株25株及T1代植株10株,经GUS组织化学染色、PCR检测后得到的转基因植株共有8株,并收获T2代种子.对拟南芥转化植株进行了耐盐性及耐寒性试验,验证了大白菜BcDREB1基因的耐盐和耐寒功能.
英文摘要: Chinese cabbage is one vegetables originated from our country, occuping an important position in annual supplies of vegetable. With the development of biotechnology, the improvement of Chinese cabbage varieties using genetic engineering technology has become one of interesting research topics currently. In this experiment the BcDREB1 gene cloned from Chinese cabbage was transferred into Chinese cabbage and Arabidopsis thaliana respectively by agrobacterium-mediated genetic transformation technology. And the transformation plants were obtained, which were verified the resistance function with the BcDREB1 gene of Chinese cabbage. This will lay a foundation for resistance molecular breeding in Chinese cabbage. The main research results are as follows:1 Through analyzing the effects of different genotypes, explant types, hormone combinations and pre-cultivation conditions on the shoot regeneration, the regeneration system with high frequency in Chinese cabbage was established, in which the optimal genotype was inbred line'85-1', the optimal explant was cotyledon with petioles, pre-cultivation condition was 0.5 mg/L 2,4-d 1d, the optimal differentiation medium was MS + 0.7 mg/LTDZ + 0.5 mg/LNAA + 5.0 mg/L AgNO3.2 The genetic transformation system of Chinese cabbage was established and optimized through screening the time with agrobacterium infection, the Acetosyringone concentrations and Kanamycin resistance. During the process of genetic transformation in Chinese cabbbage, we selected the optimal conditions of screening resistance buds with kanamycin concentration of 10mg/L, agrobacterium-mediated infection time of 5 min, co-cultivation of 2d. And we found that it is useful for transformation by adding Acetosyringone with 150 mg/L concentration during the process of co-cultivation.3 Fifteen transgenic plants were identified by GUS-histochemical staining, GUS-PCR detection of T0 transgenic plants obtained from the antibiotics screening. The resistance to salt of Chinese cabbage transgenic plants was significantly increased by testing cold resistance with -5℃and osmotic stress resistance with 250mmol/LNacl. 4 Using Floral-dip method, the gene BcDREB1 of the Chinese cabbage was transferred into Arabidopsis thaliana, Twenty five T0 generation plants and 10 T1 generation plants were obtained by screening the antibiotics, in which 8 transgenic plants were identified by GUS-histochemical staining and GUS-PCR detection and T2 generation seeds were harvested. The function in resistance to salt and cold of Chinese cabbage gene BcDREB1 was verified by testing resistance to the salt and cold in Arabidopsis thaliana transformation plants.