农杆菌介导的大白菜遗传转化体系的建立

发布时间：2018-06-17 21:05

本文选题：大白菜 + 再生体系　；参考：《沈阳农业大学》2017年硕士论文

【摘要】：大白菜(Brassicarapa L.ssp.Pekinensis)原产自中国,是中国乃至亚洲重要蔬菜作物。稳定的遗传转化体系将为大白菜分子育种及基因功能验证奠定基础。本研究对大白菜自交系'GT-24'的再生体系进行优化。在此基础上,成功建立农杆菌介导的大白菜'GT-24'的遗传转化体系,并获得了转化植株。结果如下:1.以大白菜'GT-24'为材料,探讨外植体类型、蔗糖、6-BA、AgO3添加浓度对不定芽分化的影响,对'GT-24'离体再生体系进行优化。结果表明:最佳外植体为子叶-子叶柄;最佳芽诱导培养基为 MS+0.5mg/L NAA+5mg/L 6-BA+4mg/L AgN03+7g/L 琼脂+30g/L蔗糖(pH=5.8),不定芽平分化率可达93.5%,较原再生体系提高了 7.28%。2.利用农杆菌介导法,通过对筛选抗生素潮霉素和抑菌抗生素特美汀的适宜浓度、及外植体最佳预培养时间的筛选,建立起遗传化体系。'GT-24'遗传转化过程中,外植体无需预培养,农杆菌菌液OD600值为0.5,侵染时间为15 min,避光共培养2d,抗性芽筛选培养基为 MS+0.5mg/L NAA+5mg/L 6-BA+4mg/L AgN03 +200mg/L TMT+25mg/L Hyg +7g/L琼脂+30g/L蔗糖(pH=5.8)。转基因植株的生根诱导培养基中需添加1 mg/L NAA或者1 mg/L IBA。对抗性植株进行PCR、RT-PCR检测及GUS组织化学染色鉴定,证实获得转基因植株,经重复性试验,平均转化率为2.49%。
[Abstract]:Brassicarapa L.ssp. Pekinensis) originated from China and is an important vegetable crop in China and even in Asia. Stable genetic transformation system will lay a foundation for molecular breeding and gene function verification of Chinese cabbage. The regeneration system of Chinese cabbage inbred line GT-24 'was optimized. On this basis, the genetic transformation system of Chinese cabbage GT-24 'mediated by Agrobacterium tumefaciens was successfully established, and the transformed plants were obtained. The result is as follows: 1. The explant type and the effect of sucrose 6-BAE AgO3 concentration on adventitious bud differentiation were studied by using GT-24'of Chinese cabbage as the material, and the regeneration system of GT-24 'was optimized in vitro. The results showed that the best explants were cotyledons and cotyledon petioles, and the best medium for bud induction was MS 0.5mg / L NAA 5mg / L 6-BA 4mg / L Agar 30g / L AgN03 7g / L Agar 30g / L sucrose (pH5.8g / L). By means of Agrobacterium tumefaciens mediated screening of suitable concentrations of hygromycin and termetin, and the optimal pre-culture time of explants, a genetic system, I. e., GT-24 'genetic transformation, was established, and the explants were not pre-cultured. The OD600 value of Agrobacterium tumefaciens was 0.5, the infection time was 15 min, and the culture medium was MS 0.5mg / L NAA 5mg / L 6-BA 4mg / L AgN03 200mg / L TMT 25mg / L Hyg 7g / L Agar 30g / L sucrose pH 5.8g / L. 1 mg / L NAA or 1 mg / L IBA should be added to the rooting induction medium of transgenic plants. The transgenic plants were confirmed by PCR RT-PCR and Gus histochemical staining. The average conversion rate was 2.49%.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S634.1

【相似文献】