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研究以携带有CYP71E1-PMDC141[含β-葡萄糖苷酸酶基因(GUS)]质粒的EHA105菌株侵染玉米优良自交系A188幼胚,经7 d共培养,统计GUS基因的转化效率,对农杆菌介导玉米幼胚的外植体大小、农杆菌浓度、侵染时间和热预处理时间等影响因子进行优化。结果表明,幼胚为1.0~1.2 mm时,GUS基因的转化效率最高为35.3%;农杆菌菌液的侵染浓度为OD_(660)=0.8时,GUS基因的转化效率最高为46.8%;侵染时间为5 min时,GUS基因的转化效率最高为46.4%;43℃热预处理3 min时,GUS基因的转化效率最高为45.4%。因此选择幼胚1.0~1.2 mm、菌液浓度OD_(660)=0.8、侵染时间5 min及43℃热预处理3 min作为GUS基因最佳转化条件。
In this study, the immature embryos of maize elite inbred line A188 were inoculated with EHA105 strain carrying CYP71E1-PMDC141 [β-glucuronidase gene (GUS)] plasmid and co-cultured for 7 days to calculate the transformation efficiency of GUS gene. Bacillus-mediated maize immature embryo explant size, Agrobacterium concentration, infection time and heat pretreatment time and other factors to optimize. The results showed that the highest transformation efficiency of GUS gene was 35.3% when the young embryo was 1.0-1.2 mm, and the highest transformation efficiency of GUS gene was 46.8% when the concentration of Agrobacterium tumefaciens was OD 660. The highest transformation efficiency of GUS gene was 46.4% when the dyeing time was 5 min. The highest transformation efficiency of GUS gene was 45.4% after heat pretreatment at 43 ℃ for 3 min. Therefore, the optimal transformation conditions of GUS gene were selected from 1.0 to 1.2 mm embryo, OD 660 of bacterial liquid concentration 0.8, infection time 5 min and heat pretreatment at 43 ℃ for 3 min.