农杆菌介导的2mG_2-epsps基因转化玉米自交系18-599R幼胚的研究  

Study on Transformation of 2mG_2-epsps Gene into Immature Embryos of Maize Inbred-line 18-599R by Agrobacterium tumefaciens Mediated

作　　者：贾艾敏[1] 张玲[1] 蒋琴[1] 胡冲[1] 刘坚[1] 

机构地区：[1]四川农业大学玉米研究所,四川温江611130

出　　处：《四川农业大学学报》2015年第3期245-250,共6页Journal of Sichuan Agricultural University

基　　金：转基因专项子项目

摘　　要：【目的】对农杆菌介导的抗草甘膦新基因2 mG2-epsps转化玉米18-599R幼胚的体系进行优化,以获得具有明显草甘膦抗性的转基因植株。【方法】以玉米18-599R幼胚为受体,采用农杆菌介导法,对转化时的预处理条件、菌液浓度×浸染时间、草甘膦筛选浓度设置处理,以优化转化体系;并用PCR、实时荧光定量PCR和喷洒草甘膦鉴定外源基因在转基因植株中的表达情况。【结果】热激处理对抗性愈伤率没有明显的提高作用,离心处理会降低抗性愈伤率;菌液浓度为OD600=0.6,浸染时间为10 min时,平均抗性愈伤率最高,为37.33%;草甘膦浓度为2.0mmol/L是比较理想的筛选浓度;实验共得到46株再生植株,5株PCR检测呈阳性,阳性率为10.87%;实时荧光定量PCR表明外源基因在T2代植株的根、茎、叶中均有表达;对T2代转基因植株喷洒1.5g/L的草甘膦,表现出耐受性。【结论】实验成功将2 mG2-epsps导入18-599R基因组,并获得了明显的草甘膦抗性。【Objective】In this study,a new glyphosate-resistant gene 2mG2-epsps was transformed into immature embryos of maize inbred line 18-599 Rby Agrobacterium tumefaciens mediated to obtain transgenic plants with significant resistance to glyphosate.【Method】2mG2-epsps gene was introduced into 18-599 Rimmature corn embryos by Agrobacterium mediated,transformation system was optimized by control the pretreatment conditions,bacterial concentration ×immersion time and concentration of glyphosate filter setting process.The 2mG2-epsps gene had been expressed in transgenic plant by PCR,real time PCR and spraying glyphosate exogenous gene identification.【Results】Heat shock treatment didn＇t improve the confrontational callus rate,and centrifugation reduced the rate of resistance callus.When the concentration of Agrobacteriumin the inoculation medium was adjusted to OD600=0.6,and the infection time was 10 min,the average rate of resistance callus was up to 37.33%.The ideal concentration of Glyphosate was 2.0mmol/L.Methods using PCR of 46 transgenic plants,5positive transgenic individuals were obtained,with a positive frequency of 10.87%.Real-time PCR showed that the foreign gene was expressed in roots,stems and leaves and inherited to next generation stably.And the T2 generation transgenic plants showed tolerance to glyphosate.【Conclusion】2mG2-epsps gene was successful-ly imported into 18-599 Rgenome,and gained significant glyphosate resistance.

关 键 词：2 mG2-epsps 18-599R 农杆菌介导法 幼胚 

分 类 号：S513[农业科学—作物学]