以木糖作为筛选剂的花生子叶外植体遗传转化  

The Genetic Transformation of Peanut(Arachis hypogaea L.) Cotyledon Explants Using Xylose as Screening Reagent

作　　者：宋汝涛 陈新利[1] 于明洋[1] 隋炯明[1] 王晶珊[1] 乔利仙[1] 

机构地区：[1]青岛农业大学生命科学学院/山东省高校植物生物技术重点实验室,青岛266109

出　　处：《农业生物技术学报》2015年第9期1141-1148,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31471542;No.31301356);山东省科技发展计划(No.2014GNC110002);山东省农业良种工程

摘　　要：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。Those antibiotics and herbicides marker genes generally used at present in plant genetic transformation might be potential treats and dangers for human being and environment, so it is urgent to search and develop new secure screening reagents instead of antibiotics and herbicides reagents. New recombinant plasmid pCAMBIA1301-xylA-Ah-Glu-P with promoter of peanut （Arachis hypogaea L.） 13-1,3- glucanase gene （Ah-Glu-P） and xylose isomerase gene （xylA） from Escherichia coli was constructed successfully, pCAMBIA1301-xylA-Ah-Glu-P was then transformed to cotyledon explants of peanut variety Huayu23 by Agrobacterium mediated method using xylose as screening reagent. The frequency of regeneration seedling was 16.1% and the frequency of transformed positive plants reached to 78.4%, when the concentration of sucrose and xylose in medium was 10 and 20 g/L, respectively. The transgenic positive plants were then transplanted to field by grafting and domesticating and the survival rate of transplanted plants was up to 74.5%. The transformation method with cotyledons as explants, with xylose as screening reagent, has characteristics of simple and rapid, safe and pollution-free and higher transgenic positive frequency. So it was a safe and efficient genetic transformation method compared with other transformation methods using other explants and antibiotics screening reagent.

关 键 词：花生 木糖异构酶基因 子叶外植体 安全标记 遗传转化 

分 类 号：S565.403.5[农业科学—作物学]