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农杆菌介导的大豆子叶节非组织培养转化体系优化及Bar基因转化研究

标题: 农杆菌介导的大豆子叶节非组织培养转化体系优化及Bar基因转化研究
英文标题: The Research on Optimization of the Agrobacterium-Mediated Genetic Transformation System of Soybean Cotyledonary Node with Non Tissue-Culture and Transformation of Gene Bar
作者: 李丹丹
出版时间: 2012-01-01
所在大学: 河北农业大学
关键词: 大豆,子叶节,非组织培养,遗传转化,草铵膦筛选
英文关键词: Soybean,Cotyledonary node,Non tissue-culture,Genetic transformation,Glufosinate screening
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]作为重要的油料作物和经济作物,是食用油和蛋白的主要作物来源,在食品工业和农业生产中占重要地位.随着基因工程技术的不断发展,利用转基因技术培育大豆新品种、新种质得到了广泛应用.目前,在大豆的遗传转化中农杆菌介导的子叶节转化体系存在着组织培养过程中茎伸长和生根难的问题,因此尝试建立非组织培养的大豆遗传转化体系是解决上述问题的有效途径.本研究以河北省推广的优良大豆品种为材料,以GUS基因在子叶节区的瞬时表达为依据,通过探讨影响农杆菌转化效率的多种因素,优化了大豆子叶节非组织培养遗传转化体系,并利用该体系对冀豆16号进行了Bar基因的遗传转化.通过比较叶片针刺法和涂抹法两种筛选方法的筛选效率,确立了大豆子叶节非组织培养遗传转化体系的有效筛选方法,并且通过针刺法摸索了8个河北省优良大豆品种的草铵膦筛选浓度.主要结果如下:1、通过对蔗糖浓度、表面活性剂、侵染方式、侵染次数、侵染液浓度及大豆基因型的摸索,发现侵染液中附加3%蔗糖、OD600=0.6、以脱脂棉作为菌液附着介质同时不添加表面活性剂Silwet L-77、侵染1次的GUS阳性率最高达到62.13%,冀豆16号为易感大豆基因型.2、针刺法的筛选效率为27.2%,而涂抹法的筛选效率仅为9.1%,从而确定针刺法是适合该转化体系的有效筛选方法.通过针刺法摸索了8个河北省优良大豆品种的草铵膦筛选浓度,根据叶片及植株茎尖枯焦死亡情况初步确定其草铵膦筛选浓度分别为:冀豆15号、五星2号为100 mg/L;冀豆16号、冀豆17号、五星1号、中黄13号为150 mg/L;冀豆7号、nf 58为200 mg/L.3、利用优化的大豆子叶节非组织培养遗传转化体系对河北省优良大豆品种冀豆16号进行了Bar基因的遗传转化,植株再生率达71.2%,草铵膦抗性植株经PCR检测获得T0代阳性植株10个,转化率为2.5%.经PCR和RT-PCR鉴定共获得3株T1代阳性植株,初步证明目的基因已整合到大豆基因组中.
英文摘要: As the important oil and economic crops, soybean [Glycine max (L.) Merrill] is the major source of edible oil and protein, occupying an important position in food industry and agricultural production. With the continuous development of genetic engineering, cultivating the new varieties of soybean by transgenic technology has been widely used. At present, the problems of stem elongation and rooting are still existing in the Agrobacterium-mediated genetic transformation system of soybean cotyledonary node. Establishing a genetic transformation system of soybean with non tissue-culture is an effective way to solve these problems. In this study, we used the high-quality soybean varieties which are popularized in Hebei province as the materials, by investigating the factors influencing the transformation efficiency of Agrobacterium, the genetic transformation system of soybean cotyledonary node with non tissue-culture was optimized according to the transient expression of GUS in the region of cotyledonary node. The gene Bar was transformed into soybean jidou 16 by using the optimized system. An effective screening method of the genetic transformation system of soybean cotyledonary node with non tissue-culture was established by comparing the screening efficiency of glufosinate injected and painted on leaves. The screening concentrations of eight high-quality soybean varieties in Hebei province were studied by glufosinate injected on leaves. The results showed that:1. By investigating the concentrations of sugar, surfactant, ways of infection, times of infection, concentrations of infection medium and soybean genotypes, we found that infection medium contains 3% sugar and none Silwet L-77, OD600=0.6, with the absorbent cotton as attached media, infect one time have the highest percentage of GUS positive, was 62.13%. Jidou 16 is the susceptible soybean genotype.2. The screening efficiency of glufosinate injected on leaves was 27.2%, while the screening efficiency of glufosinate painted on leaves was only 9.1%. Consequently, the screening method with glufosinate injected on leaves was confirmed to be the effective screening method of this genetic transformation system. According to the scorch and death of leaves and stem apexes, the screening concentrations of eight high-quality soybean varieties in Hebei province with glufosinate injected on leaves were preliminary confirmed respectively to be: 100 mg/L (jidou 15, wuxing 2); 150 mg/L (jidou 16, jidou 17, wuxing 1, zhonghuang 13); 200 mg/L (jidou 7, nf 58).3. The gene Bar was transformed into soybean jidou 16 which is a high-quality soybean varieties in Hebei province by using the optimized genetic transformation system of soybean cotyledonary node with non tissue-culture, the regeneration rate was 71.2%. We obtained ten positive plants in T0 generation after detected the transgenic plants which resistant to glufosinate by PCR, the transformation efficiency was 2.5%. Three positive plants of T1 generation were obtained by detection of PCR and RT-PCR, preliminary demonstrated that the target gene was integrated into the soybean genome.