融合杀虫基因对甘蔗的遗传转化

目的]利用MAR序列介导转基因表达及融合杀虫基因,以期克服外源基因失活,增强抗虫基因表达能力,延缓昆虫抗性的发生,获得高抗虫转基因甘蔗植株.方法]以甘蔗品种ROC25为材料,进行愈伤组织、芽苗分化和生根的Hyg抗性筛选试验.利用农杆菌介导,将含MAR序列介导融合杀虫基因(AVAc-CpTI)导入甘蔗基因组.结果]愈伤组织增殖、出芽时Hyg的最佳筛选浓度为50 mg/L,生根时Hyg的最佳筛选浓度为30 mg/L.获得13个MAR序列介导转基因表达基因系,48株Southern杂交阳性甘蔗植株;获得8个无MAR序列介导转基因表达基因系,7株Southern杂交阳性甘蔗植株.MAR序列介导转基因表达使转化过程中转基因系的获得数量提高了62.5%.结论]建立了ROC25的潮霉素抗性筛选体系;MAR序列介导融合杀虫基因进行甘蔗遗传转化,可提高甘蔗外源基因转化效率.

Fusion insecticidal gene for genetic transformation of sugarcane

Objective]The aim of this experiment is to produce transgenic sugarcane plants with high insect-resistance by using MAR sequence mediated transgenic expression and fusion insecticidal gene to overcome the exogenous gene inactivation, strengthen gene expression and delay insect resistance. Method]Using the embryogenic calli sugarcane variety ROC25 as the acceptors, its sensitivity of hygromycin to callus proliferation, bud differentiation and rooting were tested. Fusion insecticidal gene (AVAc-CpTI) flanked on both sides of the MARs were introduced into sugarcane by A. tumefaciens infection. Result]The optimum screening concentration of hygromycin for callus proliferation and budding was 50 mg/L; as for rooting, the concentration was 30 mg/L. Thirteen transgenic lines were attained when the vector ligated with MARs in the same direction on both sides of the fused gene, and resulted in 48 transgenic positive sugarcane plants by methods of southern blotting; which was 62.5% higher than that of the control. On the other hand, 8 transgenic lines without MARs, produced only 7 confirmed transgenic sugarcane plants in the control group. Conclusion]The screening system of hygromycin resistance for sugarcane cultivar ROC25 was established. Fusion insecticidal gene (AVAc-CpTI) that flanked on both sides of the MARs were successfully integrated into sugarcane genome by A .tumefaciens infection and transgenic plants were obtained. In the genetic transformation of sugarcane, fusion insecticidal gene that flanked on both sides of the MARs could increase the number of transgenic lines and improve the transformation efficiency of exogenous gene.