【作者】 李杨阳；

【导师】 陈全家； 曲延英；

【作者基本信息】 新疆农业大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 本研究通过诱导不同基因型海岛棉下胚轴的体细胞胚胎发生,进一步优化海岛棉再生体系并且获得再生植株,并以新海30号胚性愈伤组织为受体材料,通过农杆菌介导法转化NAC转录子家族,获得转基因再生植株。为今后新疆海岛棉抗旱分子育种奠定了基础。本研究取得的主要结果如下：1.通过愈伤诱导时期激素的配比,IBA含量的不同和糖源的不同对海岛棉胚性愈伤组织分化能力的影响,活性炭对再生苗生根的影响,进一步优化海岛棉的植株再生体系。结果表明愈伤诱导的最佳激素配比为0.1 mg/L2,4-D+0.1 mg/L KT;胚性愈伤诱导率最佳的培养基是加入含量为0.05 mg/L的IBA,糖源为30 g/L葡萄糖；对再生苗生根的效果最好的是培养基中加入0.1%的活性炭。2.为了进一步筛选优化的根癌农杆菌介导海岛棉外源基因转化体系,提高海岛棉外源基因转化率。以生理状态基本一致的海岛棉新海30号的胚性愈伤组织为受体材料,以绿色荧光蛋白基因(GFP)作为报告基因,将转化的时间和共培养条件作为试验要素,筛选最适宜的浸染时间和共培养条件范围。结果表明,以GFP为报告基因进行根癌农杆菌介导的海岛棉外源基因转化体系的最佳浸染时间为15 mmin,共培养时间为24 h。3.农杆菌介导海岛棉新海30号的胚性愈伤组织,获得转NAC转录子家族基因的转基因植株。转化条件为：新海30号胚性愈伤组织预处理10 d后,于OD值为0.5的菌液中浸染15 mmin后共培养24 h。处理完毕后,将胚性愈伤组织转入添加50 mg/L卡那霉素及400 mg/L头孢霉素的培养基中诱导转基因体细胞胚胎分化,可获得15%左右的转基因体细胞胚胎,其中5%可获得正常再生植株。通过PCR检测,初步证明NAC转录子家族基因中的NAC2、NAC3、NAC6已转化到再生的棉花植株中。更多还原

【Abstract】 This study is further to optimize plant regeneration system of the Sea Island cotton and obtain regenerated plants by inducing somatic embryogenesis of Sea Island cotton hypocotyl of different genotypes; And with the embryogenic callus as receptor, transform NAC transcripts family with agrobacterium mediation and obtain transgenic plant, in order to lay the foundation for molecular breeding of drought-resistance. The major results are as follows:1. By exploring effects of the ratio of hormones, different IBA contents and different carbohydrate sources to differentiatial ability of Sea Island cotton embryogenic callus, activated carbon contents on the rooting of regenerated plants, further to optimize plant regeneration system of the Sea Island cotton. The results showed that the best hormones ratio was 0.1 mg/L 2,4-D+0.1 mg/L KT, the best media was 0.05 mg/L IBA,30 g/L glucose and 0.1% activated carbon.2. To further screen optimized agrobacterium-mediated gene transfer system for raising transformation efficiency of exogenous gene in Sea Island cotton, appropriate time of infection and co-cultivation had been confirmed by treating embryogenic callus of Xinhai 30 with the same physiological state and GFP gene respectively as receptor materials and reporter gene, and the time of transformation and co-cultivate were treated as experimental factors. The results indicated that the most suitable time of infection and co-culivate for agrobacterium-mediated gene transfer system of Sea Island cotton was 15 min and 24 h, respectively.3. Transgenic plants of NAC transcripts family were obtained by embryogenic callus of Sea Island cotton Xinhai 30 using the agrobacterium-mediated method. Embryogenic callus of Xinhai 30 were pretreated 10 d, co-culture 24 h after soaking 15 min in the bacilli of OD value was 0.5. After that, transferring the embryogenic callus to media contained 50 mg/L kanamycin and 400 mg/L cephamycin for inducing differentiation of somatic embryos, which was able to obtain 15% transgenic somatic embryos, and 5% of which enabled to obtain normal regenerated plants. PCR tests showed that NAC2、NAC3、NAC6 of NAC Transcripts Family had been transferred into regenerated cotton plants.更多还原