农杆菌介导佛手遗传转化主要影响因素的研究

Factors Effecting the Transformation of Citrus medica L.var. sarcodactlis Mediated by Agrobacterium

摘要:

采用根癌农杆菌介导的佛手叶盘转化法,在建立转海藻糖合酶基因（TPS）佛手体系过程中,对影响农杆菌转化频率的各种因素进行了研究.结果表明,佛手叶盘需在黑暗条件下MT培养基上预培养2-3 d,与农杆菌共培养3 d较合适;农杆菌菌液浓度OD₆₀₀约为0.6-0.8,感染时间20 min;抑制农杆菌生长的抗生素浓度以头孢霉素（Cef）250 mg L^-1和羧苄青霉素（Cb）250 mg L^-1且延迟筛选时间4 d最好;共培养基中添加100 μ mol/L乙酰丁香酮（AS）和400 mg L^-1半胱氨酸（L-Cys）对佛手遗传转化有明显的促进作用.经GUS报告基因和PCR技术检测,初步证实TPS基因已整合到佛手基因组中,且GUS报告基因瞬时表达率为5.9%。

Abstract:

Agrobacterium-mediated transformation of Citrus medicaL, var. sarcodactlis was performed by leaf disc. The binary vector harbors the gene of interest TPS and NUFII gene driven by CaMV 35S promoter. The presence of TPS gene integration in the genome was detected and confirmed by GUS assay and PCR, respectively. Transformation parameters optimized were bacterial concentration, pre-culture period, immersion time, concentrations of antibiotics （cefotaxime, Cef and carbenicilline, Cb） and reagents （acetosyringone, AS and L-cysteine, L-Cys）. Results were obtained based on the percentage of GUS positive transient expression. A. tumefaeiens strain LBA4404 at OD_{600 nm} 0.6 showed the highest virulence on the leaf explants of C. medica with 20 rain of immersion. Two to three days ofpre-culture on MT medium in dark and 3 days of co-cultivation were optimum for C. medica. The mixture containing 250 ml L^-1 Cefand 250 ml L^-1 Cb gave the highest rate of callus induction for C. medica. Addition of 100 μ mol/L AS and 400 mol/L L-Cys enhanced the transformation efficiency of C. medica. Under the optimal condition, the percentage of GUS transient expression was 5.9%.transformation; Trehalose synthase gene.