农杆菌介导的马铃薯遗传转化体系的优化及StNTP基因功能的验证

发布时间：2018-06-23 15:24

本文选题：薯块 + 遗传转化　；参考：《华中农业大学》2017年硕士论文

【摘要】：自1983年首次获得转基因株系以来,农杆菌介导的马铃薯遗传转化体系在不断的改进和完善,目前已成功建立不同基因型和外植体类型的转化体系,但仍存在基因型依赖和转化效率低等问题。遗传转化技术的发展与成熟为马铃薯晚疫病抗性机制的研究提供了新的契机。StNTP是调控马铃薯晚疫病抗性机制的NAC转录因子,前期研究发现在本式烟中沉默Nb NTP基因导致晚疫病致病力增强,本研究利用优化的转化体系进一步探究StNTP在马铃薯晚疫病抗性中的作用。选取马铃薯晚疫病感病材料304413.19、304413.89和Désirée及抗病材料301071.3、304413.40,通过观察试管薯形成特点选取幼嫩试管薯为外植体,探索筛选压、分化培养基组成对转化效率的影响,建立相应的转化体系。利用优化的体系获得StNTP转基因阳性株系,对StNTP基因进行晚疫病抗性功能验证。取得研究结果如下:1.对304413.19、304413.40和Désirée试管薯形成特点观测,结果表明304413.19初始结薯时间比Désirée和304413.40提前一周,初步确定自接种起培养55d形成的304413.19试管薯和接种60d的304413.40和Désirée试管薯为外植体优化遗传转化体系。2.基于组织培养条件下的试管薯形成表型,对301071.3和304413.89两种基因型试管薯形成进行两种光周期处理和对301071.3基因型进行蔗糖处理实验,结果表明全黑暗诱导试管薯的提前形成,短日照能增加单株结薯数和单薯块重。60g/L蔗糖浓度有利于301071.3基因型试管薯的形成。3.以304413.19、304413.40和Désirée为受体对卡那(Kan)浓度和分化培养基组成两个因素进行优化。304413.19、304413.40和Désirée最适Kan浓度分别为50mg/L,50mg/L和60mg/L。三个基因型中只有Désirée成功获得转基因株系,最优分化培养基组合为(MS+0.1mg/L IAA+2mg/L ZT+60mg/L Kan+400mg/L Cef),植株再生率和外植体转化率分别为52.27%和11.36%。4.成功构建StNTP超量表达载体,利用优化的Désirée转化体系转入马铃薯受体材料Désirée中,获得StNTP1转基因株系5个,StNTP2转基因株系3个,经晚疫病抗性鉴定,病斑面积分析显示,与对照相比超量株系晚疫病抗性显著增强,说明StNTP增强马铃薯晚疫病抗性。
[Abstract]:Since the first transgenic lines were obtained in 1983, the Agrobacterium tumefaciens mediated transformation system of potato has been continuously improved and improved. At present, the transformation system of different genotypes and explants has been successfully established. However, there are still some problems such as genotype dependence and low efficiency of transformation. The development and maturity of genetic transformation technology provide a new opportunity for the study of resistance mechanism of potato late blight. StNTP is the NAC transcription factor to regulate the resistance mechanism of potato late blight disease. Previous studies showed that silencing NbNTP gene in tobacco could enhance the pathogenicity of late blight disease. This study further explored the role of StNTP in potato late blight resistance by using the optimized transformation system. Potato late blight susceptible materials 304413.89 and D 茅 sir 茅 e and disease-resistant material 301071.3A30444.13.40 were selected. The young tuber was selected as explant by observing the formation characteristics of test-tube tuber, and the effects of selection pressure and the composition of differentiation medium on the transformation efficiency were explored, and the corresponding transformation system was established. StNTP transgenic positive lines were obtained by using the optimized system, and the function of late blight resistance of StNTP gene was verified. The results are as follows: 1. The characteristics of tuber formation in vitro were observed. The results showed that the initial tuber setting time of 304413.19 was one week earlier than that of D 茅 sir 茅 e and D 茅 sir 茅 e. 304413.19 tubers formed from 55 days after inoculation and 304413.40 and D 茅 sir 茅 e from 60 days were identified as the optimal genetic transformation system of explants. Based on the phenotype of tuber formation under tissue culture conditions, two photoperiod treatments and sucrose treatments were carried out for the formation of 301071.3 and 304413.89 genotypes, respectively. The results showed that the formation of tube-tuber was induced in full darkness. Short sunshine could increase the number of tubers per plant and the sucrose concentration of 60 g / L sucrose per plant, which was beneficial to the formation of 301071.3 genotype tube-tuber. The optimum concentration of Kan and the composition of differentiation medium were optimized by using 304413.19 ~ 304413.40 and D 茅 sir 茅 e as receptors. The optimum concentration of Kan was 50 mg / L 50 mg / L and 60 mg / L respectively. Among the three genotypes, only D 茅 sir 茅 e successfully obtained transgenic lines. The optimal differentiation medium was (MS 0.1 mg / L IAA 2 mg / L ZT 60 mg / L Kan 400 mg / L ef). The regeneration rate and explant transformation rate were 52.27% and 11.36% respectively. The overexpression vector of StNTP was successfully constructed and transformed into potato receptor D 茅 sir 茅 e by using the optimized D 茅 sir 茅 e transformation system. Three transgenic lines of StNTP1 were obtained. The results showed that the transgenic lines were resistant to late blight, and the spot area was analyzed. Compared with the control, the resistance to late blight was significantly enhanced by StNTP, which indicated that StNTP enhanced the resistance to late blight in potato.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S532

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