马铃薯增强子捕获系群体的创建和分析

发布时间：2018-02-25 01:29

本文关键词： 双单倍体DM 农杆菌转化增强子捕获侧翼序列扩增　出处：《青海大学》2017年硕士论文　论文类型：学位论文

【摘要】：马铃薯作为世界上第四大粮食作物,同时也是中国的第四大粮食作物,在人民生活中占着越来越重要的地位。马铃薯全基因组测序的完成,帮助人们加深对马铃薯遗传规律的了解,加强对其丰富的遗传资源的开发,同时也加快分子育种的运用。双单倍体马铃薯DM(DM13-516-R44)为测序材料之一,本研究以它为材料,通过农杆菌介导将构建好的增强子捕获T-DNA载体导入马铃薯的外植体中,获得转基因植株,得到转基因植株后,利用潮霉素设计引物对生根较好的转化苗进行PCR鉴定,初步构建了马铃薯增强子捕获系群体。此外,利用PCR-walking技术扩增了突变体植株的侧翼序列,并结合已经公布的马铃薯序列对部分侧翼序列进行了分析。主要的研究结果如下:1.初步建立了马铃薯增强子捕获系群体,获得了12000株左右的转化苗,对1500株转化苗进行阳性鉴定,有978个阳性株系。同时,优化了马铃薯DM的转化体系,主要的实验条件为:诱导分化培养基中头孢霉素(cef)的浓度为600mg/L、潮霉素(hyg)为8mg/L,外植体预培养13天、侵染时间8min、农杆菌浓度OD600=0.4、共培养3天。2.利用PCR-walking技术扩增了马铃薯突变体的侧翼序列,对60个阳性突变体进行了扩增,其中28个突变体扩增出单一条带并测序,其测序结果与马铃薯全基因组序列比对后,发现有6个突变体是有效插入,对插入位点的信息做了初步分析。3.将鉴定过的30个阳性突变体植株种植于温室,获得了30个突变体植株的微型种薯,观察到部分突变体的表型变化。将460个已鉴定的阳性突变体植株种植,观察其表型变化。
[Abstract]:Potato, as the world's 4th largest food crop, is also the 4th largest food crop in China, and plays an increasingly important role in people's lives. To help people to understand the genetic law of potato, to strengthen the development of its abundant genetic resources, and to accelerate the application of molecular breeding. DM13-516-R44, a dihaploid potato, is one of the sequencing materials. The constructed enhancer was introduced into potato explants by Agrobacterium tumefaciens to obtain transgenic plants. The transformed plants were identified by PCR with hygromycin primer design. In addition, the flanking sequence of the mutant plant was amplified by PCR-walking technique. Some flanking sequences were analyzed based on the published potato sequences. The main results were as follows: 1. A population of potato enhancer captors was established, and about 12000 transformed seedlings were obtained. There were 978 positive lines in 1500 transformed seedlings. Meanwhile, the transformation system of DM in potato was optimized. The main experimental conditions were as follows: the concentration of cefocefin was 600 mg / L, hygm hyg was 8 mg / L in induced differentiation medium, and the explant was precultured for 13 days. The infection time was 8 min, the concentration of Agrobacterium tumefaciens was OD600,0.4, and co-cultured for 3 days. 2. The flanking sequence of potato mutants was amplified by PCR-walking technique, and 60 positive mutants were amplified, of which 28 mutants were amplified by a single band and sequenced. The results of sequencing were compared with the whole genome sequence of potato. It was found that 6 mutants were effectively inserted. The information of insertion site was analyzed preliminarily. The 30 identified positive mutants were planted in greenhouse. The phenotypic changes of some mutants were observed, and 460 identified positive mutants were planted to observe the phenotypic changes.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S532

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