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以哈密大枣无菌苗叶片为试材,以根癌农杆菌LBA4404为媒介将抗寒基因转录因子CBF基因导入哈密大枣,利用PCR、RT-PCR对转化植株进行鉴定,建立了哈密大枣的遗传转化体系。结果表明:在对哈密大枣无菌苗叶片进行遗传转化时,预培养2d,农杆菌菌液OD600为0.5,浸染20min,共培养3d的转化效率为最高。试验共获得7个抗性再生转化系。经PCR鉴定,CBF基因在7个抗性再生转化系中均为阳性;经RT-PCR分析,CBF基因在7个抗性再生转化系中均得到表达。
The leaves of Hami jujuba aseptic seedlings were used as experimental materials, the CBF gene of cold-tolerant gene transcription factor was introduced into Hami jujube via Agrobacterium tumefaciens LBA4404, and the transformed plants were identified by PCR and RT-PCR. Hami jujube Genetic transformation system. The results showed that when the aseptic seedlings of Hami jujube were genetically transformed for 2 days, the Agrobacterium tumefaciens OD600 was 0.5 and the infection time was 20 min. A total of seven resistant regenerated lines were obtained. The CBF gene was positive in all 7 resistant regenerated lines by PCR, and CBF gene was expressed in 7 resistant regenerated lines by RT-PCR analysis.