农杆菌介导的DREB1A基因转化多花黑麦草及其转化体系的优化

Agrobactrium tumefaciens mediated DREB1A gene transfer to Lolium multiflorum and optimization of its genetic transformation

采用携带卡那霉素抗性基因nptII和GUS基因的ubiquitin启动子驱动的表达载体pBI121/DREB1A的根癌农杆菌AGL1,对多花黑麦草幼胚来源的胚性愈伤组织进行了遗传转化,并优化了各种影响因素。胚性愈伤组织经根癌农杆菌感染和共培养后,用50 mg/L巴龙霉素筛选抗性愈伤组织,待抗性愈伤组织在IB分化培养基上分化成苗后用25 mg/L卡那霉素进一步筛选再生植株,获得了部分抗性植株。抗性植株的总DNA用DREB1A基因的特异引物进行PCR检测,转化频率为2.14%,PCR-Southern blot进一步验证了转化植株基因组中含有该外源基因。各种影响转化效率因素的优化实验表明,当转化时菌液浓度的OD600为2.0、侵染时间为1 h、共培养时间为2 d、共培养温度为21℃及在共培养期间使用乙酰丁香酮等,均可明显提高转化频率。

The embryo-derived calli from Lolium multiflorum Lam were transformed with Agrobactrium tumefaciens AGL1 harboring intron-DREB1A expression vector pBI121 containing ubiqutin promoter, npt Ⅱ marker gene and GUS gene. After infection and co-culture with AGL1, the embyogenic calli were selected with 50 mg/L paromomycine and plants regenerated from the resistant calli. All plants were selected further with 25 mg/L kanamycin, some of which remained green. The genome DNA of the resistant plants was checked with specific primers and probes from the DREB1A gene. The results of PCR and PCR-Sonthem blot assay indicated that the DREB1A gene was transferred into Lolium rnultiflorurn Lain with a transformation frequency of 2.14 %. It was found that the trangormation efficiency can be efficiently improved using the following transformation conditions： Agrobacterium cell density of OD600 =2.0, incubation for 1 hour, coculture at 21 ℃ for 2 days and application of As in coculture.