农杆菌介导的抗菌肽基因转化阳春砂愈伤组织的研究

目的确定遗传转化条件,将抗菌肽CN基因转入阳春砂愈伤组织。方法以阳春砂愈伤组织为受体材料,以农杆菌EHA105/pCAMBIA1305.2-CN工程细胞介导抗菌肽CN基因转化阳春砂,设计正交实验,通过分析gus报告基因的瞬时表达比较转化影响因素,并对目的基因CN进行PCR检测。结果农杆菌浓度、侵染时间、共培养温度、共培养时间及乙酰丁香酮浓度对转化率的影响有显著差异性。阳春砂愈伤组织遗传转化的最佳条件组合是:农杆菌浓度为OD600=0.5,侵染时间10 min,共培养温度24℃,共培养时间4 d,共培养培养基添加乙酰丁香酮200μmol.L-1。gus基因瞬时表达呈阳性的愈伤组织DNA经目的基因CN的PCR检测可见清晰的目的条带。结论建立了农杆菌介导的阳春砂愈伤组织的转化条件,目的基因CN已转入阳春砂愈伤组织。

Agrobacterium - mediated Transformation of Antimicrobial Peptide Gene into the Callus of Amonurn villosum Lour

ObjectiveTo establish an agrobacterium-mediated transformation system for the antimicrobial peptide gene into the callus of Amomum villosum Lour. MethodsWith the callus of A.villosum Lour.used as the gene transformation acceptors,the antimicrobial peptide fusion gene of CN was transferred by Agrobacterium tumefaciens EHA105 harboring expression vector pCAMBIA1305.2-CN.The influencing factors of transformation were investigated by orthogonal design and histochemical assay of transient expression of gus gene.The foreign CN gene was analyzed by PCR. ResultsThe highest transformation efficiency was obtained when A.tumefaciens cell density was OD600=0.5 with 10 minutes of infection,co-culture for 4 days at 24℃,AS at 200 μmol·L-1.CN gene was detected by PCR in the callus with blue dots. ConclusionThe transformation system of A.villosum Lour.by A.tumefaciens was established,and the antimicrobial peptide gene CN was induced into the callus of A.villosum Lour.