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目前在植物遗传转化过程中常用的抗生素类标记基因可能存在潜在的安全隐患,因此探讨使用安全筛选剂替代抗生素类筛选剂便成为植物遗传转化筛选的新途径。本研究构建了同时含有花生(Arachis hypogaea L.)β-1,3葡聚糖酶基因启动子(Ah-Glu-P)以及大肠杆菌木糖异构酶基因(xylose isomerase gene,xyl A)的安全表达载体p CAMBIA1301-xyl A-Ah-Glu-P。利用农杆菌(Agrobacterium tumefaciens)介导法将p CAMBIA1301-xyl A-Ah-Glu-P转化花生子叶外植体,利用木糖作为筛选剂进行筛选,当培养基中蔗糖浓度为10 g/L、木糖浓度为20 g/L时,外植体再生率为16.1%,再生苗阳性率达到78.4%,转基因阳性植株经嫁接驯化后移栽田间,存活率达74.5%。该转化方法以花生子叶作为外植体、利用木糖作为筛选剂进行筛选,与利用其他外植体进行转化及抗生素筛选相比较,具有操作简单快速、安全无污染以及转化效率高的特点,是一种安全、高效的遗传转化方法。
At present, there are potential safety hazards of antibiotic marker genes commonly used in plant genetic transformation. Therefore, exploring the use of safe screening agents instead of antibiotic screening agents will become a new approach for plant genetic transformation screening. In this study, we constructed a recombinant plasmid containing both the Ah-Glu-P β-1,3-glucanase gene (Arachis hypogaea L.) and the xylose isomerase gene (xyl A) Safe expression vector p CAMBIA1301-xyl A-Ah-Glu-P. P CAMBIA1301-xyl A-Ah-Glu-P was transformed into explants of peanut cotyledons using Agrobacterium tumefaciens-mediated method and screened using xylose as a screening agent. When the sucrose concentration in the medium was 10 g / L, When the concentration of xylose was 20 g / L, the regeneration rate of explants was 16.1%, and the positive rate of regenerated seedlings reached 78.4%. The transgenic plants were grafted and transplanted into the field with the survival rate of 74.5%. The transformation method takes peanut cotyledon as explant and uses xylose as a screening agent for screening. Compared with using other explants for transformation and antibiotic screening, the transformation method has the characteristics of simple and quick operation, safety and pollution-free and high transformation efficiency A safe and efficient genetic transformation method.