ACC合成酶基因及其反义基因对西瓜的遗传转化

以2日龄西瓜(Citrullus lanatus(Thunb.)Mansfeld)无菌苗子叶为外植体,通过与根癌农杆菌(Agrobacterium tumefaciens)进行叶盘共培养建立了西瓜的遗传转化系统。所用根癌农杆菌中含有改建后分别携带嵌合NPTⅡ基因和番茄的ACC合成酶基因及其反义基因的质粒。外植体在MSA培养基(MS盐类、B_5维生素、1.0mg/L BA、0.2mg/L IAA)上预培养3～4d后,与根癌农杆菌共培养4d,随后转移外植体至附加100mg/L卡那霉素、300mg/L头孢菌素的MSA培养基上筛选转化芽。将带芽外植体移入含有100mg/L卡那霉素、300mg/L头孢菌素的伸长培养基(MS 0.2mg/L KT)上进行芽伸长,切取2～3cm高的伸长芽移入生根培养基(1/2MS 0.1mg/L NAA)生根。Southern blot结果证明获得转基因植株,乙烯释放指标表明转入的正义和反义ACC合成酶基因得到不同程度的表达。

GENETIC TRANSFORMATION AND PLANT REGENERATION OF WATERMELON USING AGROBACTERIUM TUMEFACIENS

Transgenic watermelon ( Citrullus lanatus (Thunb.) Mansfeld) plants were generated by Agrobacterium-mediated gene transfer. Cotyledon explants excising from 2-day seedlings were first precultured on MSA medium (MS salts B5 vitamins 1.0 mg/L BA 0.2 mg/L IAA) for 3 to 4 d, then cocultured with an Agrobacterium tumefaciens strain (LBA4404) carrying the modified pBI121 plasmid, which contains tomato's sense and anti-sense ACC synthase gene respectively, on original MSA medium. Explants were transferred to MSA medium with 100 mg/L kanamycin and 500 mg/L cefotaxime to select transgenic shoots after 4 d of coculture. Explants with shoot buds elongated on MS medium containing 0.2 mg/L KT, 100 mg/L kanamycin and 300 mg/L cefotaxime and 1 to 3 shoots could be recovered from an explant. All the elongated shoots (about 2 to 3 cm in length) rooted on 1/2 MS medium supplemented with 0.1 mg/L NAA. The transgenic nature of re-generants was demonstrated by plant growth on medium containing kanamycin, expression of the integrated sense and anti-sense ACC synthase gene and Southern blot analysis.