农杆菌介导的大豆子叶节遗传转化研究  

On Agrobacterium-mediated genetic transformation system of Soybean Cotyledon Node

作　　者：皮照兴[1] 廉玉利[1] 李依娜[1] 崔唱 杨楠[1] PI Zhao-xing LIAN Yu-li LI Yi-na CUI Chang YANG Nan(Chaoyang Teachers College, Chaoyang Liaoning 122000)

机构地区：[1]朝阳师范高等专科学校,辽宁朝阳122000

出　　处：《辽宁师专学报（自然科学版）》2016年第3期86-90,共5页Journal of Liaoning Normal College(Natural Science Edition)

基　　金：辽宁省教育厅科学技术研究项目(L2013431)

摘　　要：大豆遗传转化是目前研究的热点,为提高大豆遗传转化效率提供理论依据,试通过实验确立农杆菌介导大豆遗传转化条件.实验利用含有pCAMBIA3301质粒的农杆菌LBA4404对辽豆-45子叶节进行了侵染,对消毒方法、草铵膦浓度、侵染浓度、侵染时间、乙酰丁香酮浓度、共培养时间等方面进行了优化.实验结果表明:氯气灭菌5h的二次消毒法,可在不影响种子活力的同时把污染率降低为0,确定草铵膦的选择压力为6mg/L.同时确定辽豆-45子叶节遗传转化的最佳条件为:农杆菌侵染菌液的浓度为OD600=0.6,侵染时间为30 min,暗培养条件下共培养时间为96h,且共培养液体培养基添加200μmol/L的乙酰丁香酮有利于转化.经草铵膦抗性筛选、GUS组织化学染色检测、PCR检测,证实pCAMBIA3301质粒的T-DNA已经整合进了辽豆-45基因组,共获得23株转基因大豆.Soybean genetic transformation is a research hotspot in the current study.The aim of this paper is to establish the genetic transformation conditions of Agrobacterium-mediated transformation,and to provide theoretical basis for improving the efficiency of soybean genetic transformation.In the experiment of this study,cotyledon nodes obtained from Liaodou-45 were infected with Agrobacterium tumefacines LBA4404 with pCAMBIA3301.Subsequently,we optimized the double-disinfection method,concentration of glufosinate ammonium,concentration of infection liquid,infection time,and concentration of acetosyringone co-cultivation time.The experiment shows that when the double-disinfection method of chlorine sterilization time was 5h,the zero contamination rate and the highest seed vigor was observed.Determination of selection pressure glufosinate ammonium was 6mg/L.At the same time,the results indicated that the optimal transformation rate of Liaodou-45 cotyledon node was achieved when the concentration of infection liquid at OD600 nm was0.6and the infection time was 30 min,the co-cultivation time was 96 hin dark cultivante.We also found that the addition of 200μmol/L acetosyringone to the co-culture medium was beneficial to the transformation.The result of glufosinate ammonium resistance screening,GUS assay and PCR analysis showed that the T-DNA of pCAMBIA3301 was stably integrated into the Liaodou-45’s genome and expressed in the genome soybean.In the end,we obtained 23 plants transgenic soybean.

关 键 词：大豆 子叶节 农杆菌介导 二次消毒 遗传转化 

分 类 号：S565.1[农业科学—作物学]