拟南芥多效性基因CPR5转化水稻中花11的研究

摘要

利用根癌农杆菌介导法将拟南芥多效性基因CPR5转化水稻中花11,同时优化了其再生体系和遗传转化体系.结果表明:相比27℃、暗培养,32℃、持续光照培养可使诱导率提高到100％,且缩短了诱导周期；预分化培养后,在附加5 mg/L 6-BA和1mg/L NAA的分化培养基上分化率和再生率可分别达到100％和90.50％.同时探索了较高转化频率的条件为A600≈0.05 ～0.1的农杆菌菌液浸染5min,共培养时间为3d,同时添加1张用AAI培养液浸湿的无菌滤纸.对部分转化植株进行PCR、Southern blot和半定量RT-PCR检测,结果表明AtCPR5基因已经整合到水稻基因组中,在所试验的转化植株中均为单拷贝,但表达程度存在差异.%This study investigated the transformation of pleiotropic gene AtCPRS into Zhonghua 11 ( Ory-za saliva L. Subsp. Japonica) mediated by Agrobacterium tumefaciens, and optimized the regeneration system and genetic transformation system. It was showed the induction rate of callus could be increased to 100 % and the period of induction could be shortened under the condition of continuous light and 32 ℃ compared with the condition of dark and 27 ℃. After predifferentiation, the differentiation rate and regeneration rate could be raised to 100 % and 90. 50 % , respectively, by adding 5 mg/16-BA and 1 mg/ 1 NAA in the differential medium. Additionally, the higher transformation efficiency was obtained with the infected concentration of A. Tumefaciens to an Am of 0. 05 ~ 0. 1, 5 minutes of infection and 3 days of cocultivation by placing a filter moistened with AAI medium. Furthermore, the results of PCR, Southern blot and semi-quantitative RT-PCR indicated AtCPRS gene has been integrated into rice genome with single copy, however, the expression level 5 are different in transgenic plantlets.