【作者】 马晓红；

【导师】 武天龙；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 大豆是世界上重要的油料作物以及植物蛋白来源之一,因此大豆基因工程育种具有重要的意义。实验采用一种新的技术方法即大豆整个子叶节为外植体,利用直接芽再生方式建立了一种新的快速有效的再生体系,并在此平台上用双价抗虫基因对大豆进行了遗传转化。成熟大豆种子消毒后在添加BA 0.4 mg L-1的MSB5培养基中培养了5~7 d得到无菌苗,切取整个子叶节外植体,将外植体转入不同芽诱导培养基中培养14 d后,转入MSB5培养基上进行芽伸长培养,至芽伸长到3~4 cm,转至含有IBA 0.5 mg L-1的MSB5培养基上进行生根培养,炼苗并移栽到温室得到可育植株。实验中,对芽的形态发生过程以及细胞内淀粉和蛋白质含量的相对变化进行了观察分析,发现分生细胞内蛋白质含量随着芽发育的不同阶段呈周期性变化;研究了BA或CPPU对大豆整个子叶节芽诱导的作用,确定了BA在整个子叶节芽诱导过程中效果好于CPPU;利用正交设计方法对三种植物激素以及大豆基因型对芽诱导的影响进行研究,结果表明,BA、KT、IBA对芽诱导效果都有极显著影响,其中BA的影响最大,大豆品种合丰25在MSB5 + BA 3.0 mg L-1 + IBA 0.2 mg L-1 +KT 0.5 mg L-1的培养基中诱导出芽效果最好,每个外植体可以得到30~35个芽,并且大部分芽都可以伸长并生根。为了验证新建立的再生体系的效果,将此再生体系与传统的大豆子叶节再生体系、近几年应用较多的大豆胚尖再生体系在再生频率、出芽数目、芽伸长情况以及再生周期等方面进行研究比较,结果表明,大豆整个子叶节再生体系在外植体再生频率以及出芽数量上优于其它两种体系。本实验尝试采用大豆整个子叶节为外植体建立了一种快速有效的再生体系,与子叶节、胚尖再生体系相比较,提高了再生效率和不定芽的数量,减小了品种特异性对大豆再生体系的影响,并且大大缩短了再生周期。研究结果表明,使用这个再生平台,再生效率达到95%以上,可以在60天内获得大量的再生小苗。利用根癌农杆菌介导的转化方法对三个大豆品种的整个子叶节外植体进行双价抗虫基因的遗传转化,并对转化体系中几个因素进行优化,建立了基于整个子叶节再生平台的大豆遗传转化体系。结果表明,不同农杆菌菌株对大豆的转化效率不同,EHA105的转化效率高于LBA4404和GV3101;不同大豆品种对农杆菌浸染的敏感性存在差异,其中合丰48的效果较好;转化过程中采用Cef 300 mg L-1 + Cb 200 mg L-1和PPT 5.0 mg L-1进行除菌和筛选,效果较好。应用这个转化体系,转化频率在5.50%~12.07%之间。经过分子验证以及抗除草剂、抗蚜虫实验证明,外源基因已经整合到大豆基因组中,T1代遗传分析结果表明外源基因在后代中可以稳定遗传和表达,并对大豆蚜虫具有较强的抗性。实验证明,大豆整个子叶节再生体系可以有效地用于农杆菌介导的遗传转化并得到稳定表达的转基因植株。更多还原

【Abstract】 Soybean [Glycine max (L.) Merrill] is one of the most important protein and oil crops worldwide. There is great interest to improve soybean crops by genetic transformation techniques. A rapid and efficient shoot organogenesis and regeneration system was established for soybeans using whole cotyledonary node explants. Based on this protocol, genetically transformed soybeans were produced by co-cultivating whole cotyledonary node explants with Agrobacterium tumefaciens carrying a binary vector (EHA105/pCAMBIA3300) coding for CryIA and pta genes.Whole cotyledonary node explants were obtained from aseptic seedlings cultured on MSB5 medium supplemented with N6-benzyladenine (BA) at the concentration of 0.4 mg L-1 for 5~7 d. Then the explants were transferred to various shoot induction media for 14 d. Shoots were elongated on the MSB5 medium to 3~4 cm. The fertile plants were collected after rooting on MSB5 medium containing indole-3-butyric acid (IBA) 0.5 mg L-1. Plantlets with well-developed roots were hardened and planted in greenhouse to maturity. The process of shoot development was also observed; BA or N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) were used to shoot regeneration and BA was more effective; The effects of the plant growth regulators BA, kinetin (KT), IBA as well as the explants’genotype on shoot induction were evaluated by using a standard orthogonal design, BA exhibiting the greatest benefit and the best combination for shoot regeneration was MSB5 medium supplemented with 3.0 mg L-1 BA, 0.2 mg L-1 IBA and 0.5 mg L-1 KT on Hefeng 25 genotype. Under these most favorable conditions, one explant could regenerate 30~35 shoots. Overall, most of the regenerated shoots elongated and rooted well. In comparison with traditional cotyledonary node and embryonic tip regeneration system, whole cotyledonary node regeneration system appeared superior in shoot regeneration frequency and number of shoot.In the present study, we first report a more efficient and successful regeneration system for soybean using whole cotyledonary nodes as explants. Using this system led to a significant increase in regeneration efficiency and could overcome genotype-associated problems by selecting the proper medium. The results showed that shoot regeneration frequency could reach upwards of 95% and the procedure enabled the production of a large number of regenerated plantlets in a relatively short time (60 days).An efficient transformation system was established for soybean using Agrobacterium-mediated transformation with three cultivars and some important factors that affected transformation efficiency were identified. The results showed that the effect of different Agrobacterium strains on transformation were different and EHA105 was proved to be a better transformer than GV3101 and LBA4404; Hefeng 48 showed a high frequency of transformation; Cef 300 mg L-1 + Cb 200 mg L-1 was used for inhibiting Agrobacterium growth and PPT 5.0 mg L-1 was used to selection of transgenic cells. Using this system, the efficiency of transgenic plant production ranged from 5.50% to 12.07%. PCR and Southern analyses confirmed the stable integration of the insect resistant genes in the primary transgenic plants. The results of T1 plants analysis showed the inheritance and stable integration of transgenes. Insect bioassays showed that the transgenic soybean plants exhibited aphid resistance activity.The results showed that the whole cotyledonary node regeneration system can be used for effective Agrobacterium-mediated transformation for soybean.更多还原