根癌农杆菌介导的CG1-400-RNase基因转化创制锦橙转基因再生植株

【目的】为了快速有效地利用基因工程的方法获得柑橘无核新种质,【方法】以我国特色多核柑橘优良品种锦橙实生苗上胚轴切段为外植体,采用根癌农杆菌介导法进行能导致种子败育基因CG1-400-RNase的转化;为快速有效地筛选出转化子,在实生苗上胚轴切段转化再生过程中,根据不同发育阶段的组织或器官对抗生素的敏感程度不同采用不同的选择压。【结果】结果表明,在抗性芽再生过程中卡那霉素质量浓度设定为50 mg.L-1,获得的362个抗性芽转入卡那霉素质量浓度为100 mg.L-1的伸长培养基中进行伸长培养后,进行早期PCR检测,获得28个阳性芽;经过不定芽诱导生根或试管嫁接,获得22株完整植株。【结论】再生植株经PCR和Southern杂交检测,获得2株目的基因以单拷贝的形式插入锦橙基因组的转基因植株,为最终获得具有无核性状且可稳定遗传的柑橘新种质奠定了基础。

Creation of transgenic plants in Jincheng orange(Citrus sinensis) with CG1-400-RNase gene by Agrobacterium-mediated transformation

【Objective】 The objective of the study was to quickly and effectively obtain the citrus cultivars with seedless trait by using genetic engineering.Chimeric ribonuclease gene(Barnase) driven by seed-specific promoter(CG1-400) for reducing seed number,was introduced into 'Jincheng' orange,an important commercial and seeded citrus variety in China,【Method】 by Agrobacterium-mediated transformation and using epicotyl segments as explants.【Result】 To screen the tansformants effectively,different concentration of kanamycin was used at different developmental stages since the sensitivities of different tissues and organs to kanamycin were different.The results showed that 362 resistant shoots were obtained when 50 mg·L-1 Kan was added into the shoot-induction medium,28 of which were PCR-positive buds after those were cultivated on the shoot-elongation medium with 100 mg·L-1 Kan.Later twenty two plantlets were obtained by inducing to root or grafting in vitro.【Conclusion】 PCR and Southern blot analysis showed that single copy of CG1-400-RNase gene was integrated into two transgenic plants.The study will facilitate the breeding of transgenic citrus lines with seedless trait.