【作者】 包爱科；

【导师】 王锁民；

【作者基本信息】 兰州大学 ， 草业科学， 2006， 硕士

【摘要】 土壤盐碱化和荒漠化是人类面临的世界性问题,种植耐盐抗旱植物是改良此类土地的一条有效的途径。紫花苜蓿（Medicago sativa）是一种优良的豆科牧草,在全世界广泛种植。但是,紫花苜蓿耐盐抗旱能力较弱,较难适应高盐干旱的生长环境。因此,通过转基因技术培育耐盐抗旱紫花苜蓿品种,对改良和利用我国西北地区大面积盐荒地,从而促进该地区畜牧业的发展以及生态环境的改善都具有重要意义。 拟南芥（Arabidopsis thaliana）V-H⁺-PPase基因AVP1的超表达,可以增强转基因植株的耐盐性和抗旱性。这是因为在盐胁迫下,植物液泡膜质子泵（V-H⁺-ATPase和V-H⁺-PPase）形成的H⁺跨膜电化学梯度驱动Na⁺/H⁺逆向转运蛋白将细胞中过多的Na⁺区隔到液泡中,从而减轻Na⁺对细胞的毒害作用,并维持细胞的渗透势。本研究的目的是将AVP1基因转入紫花苜蓿,并优化转化体系,初步筛选出阳性植株,为最终获得具有较强耐盐性和抗旱性的紫花苜蓿品种打下坚实的基础。 以我国农艺性状优良的紫花苜蓿品种和田苜蓿为材料,用根癌农杆菌（Agrobacterium tumefaciens）介导AVP1基因对和田苜蓿进行遗传转化,探讨了影响苜蓿转化的一些主要因素,初步建立和优化了苜蓿遗传转化体系。主要结果如下:（1） 200mg·L^-1Carb就能完全抑制农杆菌GV3101菌株的生长;而且,Carb对和田苜蓿胚性愈伤组织的诱导无明显影响。（2） Kan对下胚轴诱导愈伤组织的最佳选择压为75mg·L^-1,对诱导体胚的最佳选择压为50 mg·L^-1;选择方式为延迟选择。（3） 优化后的转化体系是:下胚轴预培养3～4d,在OD₆₀₀=0.5～0.7菌液中负压断续处理下侵染3～4×2min/30s,共培养3～4d。（4） 向共培养基中加入AS对转化无促进作用,高浓度AS甚至对转化有抑制作用。（5） 现已得到42株Kan抗性植株。更多还原

【Abstract】 Today, soil salinity and desertification has became the cosmopolitan problem in the earth. Planting salt- and drought-tolerant plants is an crucial approach to ameliorate salinity and desertification land. Alfalfa （Medicago sativa）, a perennial high quality legume, is widely planted worldwide, and it is difficult to grow under salinity and drought conditions for its lower salt- and drought-resistant. Therefore, breeding salt- and drought-tolerant alfalfa cultivar via biotechnology has the vital significance for improving soil quality, developing graziery, and reconstructing ecological environment in Northwest China.Overexpression of the AVP1 gene in the model plant Arabidopsis thaliana results in that transgenic plants are much more resistant to high concentrations of NaCl and to water deprivation than the isogenic wild-type strains because the plant tonoplast proton pumps, the V-H⁺-ATPase and the V-H⁺-PPase, establish an electrochemical H⁺-gradient across the tonoplast that drives the transport of Na⁺ against the concentration gradient from the cytoplasm into the vacuole by Na⁺/H⁺ antiporter. Undr salt stress, vacuolar compartmentalization of Na⁺ reduces the toxic effects of this cation in the cytosol and at the same time increases the osmotic pressure of the plant. The aims of this work was to transfer the AVP1 gene into alfalfa mediated by Agrobacterium tumefaciens, to optimize the transformation system, and to screen out the positive plants, which is first critical step to obtain salt- and drought-tolerant transgenic alfalfa cultivars.Chinese local alfalfa Hetian cultivar was selected as material, which has the superordinary agronomic characters. The A VP1 gene was transformed into Hetian cultivar through Agrobacterium-mediated. We have studied many factors which affect transformation efficiency and established and optimized the transformation system. The main results were as following: （1） 200mg/L Carb could inhibit the reproduction of the agrobacterium strain GV3101. No obvious effect of Carb on embryogenic callus induction was found. （2） The optimum selection pressure of kanamycin for hypocotyls callus and somatic embryo induction were 75 mg · L^-1 and 50 mg · L^-1, respectively. Lingeringly screening was prior to prophase screening. （3） The pre-culture period was 3⁴d, thebacterial concentration was OD6oo=0.5⁰.7, the negative pressure treatment was 3⁴x2min/30s, and the co-culture period was 3⁴d, all of which were essential components of the optimum transformation system. （4） It was fond that AS can’t induce transformation and even high concentration AS inhibit transformation in co-culture medium. （5） Up to now, 42 kanamycin-resistant regeneration plants have been obtained.更多还原