【摘要】 为了优化根癌农杆菌(Agrobacterium tumefaciens)介导的棉花(Gossypium hirsutumL.)下胚轴切段的基因转化体系,以苜蓿(Medicago sativaL.)抗菌肽基因(alfla faantifungul peptide,alfAFP)为目标,利用β-葡萄糖甘酸酶基因(gus)和潮霉素抗性基因(Hpt)的检测方法,分析了根癌农杆菌菌种、菌液浓度、浸染下胚轴的时间、共培养中乙酰丁香酮(AS)的浓度、共培养温度和时间等因子对基因转化的影响。优化分析表明,用农杆菌菌株LBA4404,当OD600值为0.5～0.7的菌液浸染5～7日龄的棉花无菌苗下胚轴切段10～15min,在含200μmol/LAS的共培养基中21℃暗培养60h,可更有效地提高转化率。经上述条件处理的下胚轴在含50mg/L潮霉素的愈伤诱导培养基上培养3周可产生转化率最高的愈伤组织。愈伤组织经分化培养获得15株再生棉花,经过PCR和PCR-Southern分析,发现其中12株含有外源基因alfAFP。更多还原

【Abstract】 In order to develop a high effective gene transformation protocol of cotton (Gossypium hirsutum L.) hypocotyl pieces medieted via Agrobacterium tumefaciens, an alflafa (Medicago sativa L.) antifungul peptide gene (alfAFP) was used as a foreign gene, and effect factors of transformation, including Agrobacterium strains, bacterium density, infection time, acetosyringone (AS) concentration in co-culture medium, co-culture time and co-culture temperature, were evaluated by the expression detection of β-glucuronnidase gene (gus) and hygromycin phosphotransferase gene (Hpt) in transgenic callus. Optimum condition for transformation was that cotton hypocotyl pieces from 5 to 7 days-old sterile seedlings were dipped into Agrobacterium LBA4404 suspension (OD600=0.5～0.7) for 10～15 min, then transferred on co-culture medium with 200 μmol/L AS and co-cultured at 21℃ for 60 h in the dark. The most amount of transgenic calli was gained by culturing hypocotyl pieces on callus introduction medium with 50 mg/L hygromycin. 15 regenerative plants were developed from somatic embryos differentiated from transgenic calli on embryogenesis medium. PCR and PCR-Southern detection proved that the antifungal gene was introduced in 12 regenerative plants.更多还原