根癌农杆菌介导的玉米幼胚遗传转化体系

摘要

为建立以潮霉素为选择标记的农杆菌(Agrobacterium tumefaciens)介导的玉米高效遗传转化体系,本研究利用携带pMDC141-CYP79A1(含GUS基因)质粒的农杆菌浸染玉米A188幼胚,共培养7d后通过GUS基因瞬时表达率,研究农杆菌的菌液浓度、热激时间和浸染时间等因素对玉米幼胚遗传转化效率的影响.研究结果表明:随着农杆菌的菌液浓度的增加、热激时间和浸染时间的延长,GUS表达效率均呈现先增后减的趋势;当农杆菌菌液的OD600为0.8、热激预处理时间3min和浸染时间5min时,其GUS瞬时表达率均最高,分别为46.8％、46.4％和51.7％.并在最佳条件下将GUS基因转入到玉米幼胚,以潮霉素为选择标记,进行3次选择培养,将重新分化的抗性愈伤组织进一步分化成苗,获得56株潮霉素抗性植株,经PCR分子鉴定,获得22株阳性植株,初步建立根癌农杆菌介导的高效玉米幼胚遗传转化体系,为以抗潮霉素基因作为筛选标记的玉米遗传转化和基因功能验证提供技术基础,获得的抗潮霉素转化植株为玉米育种提供新材料.%In order to establish efficient Agrobacterium tumefaciens mediated transformation system, the marker of hygromycin for maize A188 was used as the receptor material,Agrobacterium that carried pMDC141-CYP79A1 plasmids was used to infect the immature embryos. The genetic transformation efficiency of maize immature embryo on Agrobacterium-tumefaciens concentration, heat shocking time and infection time with Agrobacterium were studied. The GUS activity analysis of immature embryos indicated that Agrobacterium-tumefaciens concentration, pretreatment time of heat shocking time, infection time of GUS transient expression rate increased firstly and then decreased. With Agrobacterium-tumefaciens suspension OD600 nm of 0.8, pretreat time of heating time of 3 min on 43 ℃ and infection time of 5 min, the highest GUS transient expression rates were 46.8％, 46.4％ and 51.7％, respectively. Under the optimal condition, the GUS gene was transformed into immature embryos, hygromycin was used to act selective marker, maize immature embryos were selected for 3 times and the redifferentiation of resistant callus was further differentiated into seedlings, and 56 resistant plant were obtained, 22 positive through PCR detection. An Agrotacteriun-mediated genetic transformation system of immature embryos was established. A technical basis was provided for the genetic transformation of maize and gene function verification, and a new material for maize breeding was obtained from the transgenic plant.