根癌农杆菌介导的高羊茅遗传转化研究

Research on Festuca arundinacea Transformation Mediated by Agrobacterium tumefaciens

采用携带卡那霉素抗性基因nptⅡ和Na+/H+反向运输AtNHX1基因的表达载体pROK2/AtNHX1(带有35S启动子)和pROK2U/AtNHX1(带有ubi1启动子)的根癌农杆菌AGL1和GV3101,对4个品种高羊茅下胚轴来源的胚性愈伤组织进行了遗传转化。胚性愈伤组织经根癌农杆菌感染和共培养后,用50～150mg/L巴龙霉素筛选抗性愈伤组织,获得1126棵再生植株,用10～20mg/L卡那霉素进一步筛选再生植株,总共得到525棵绿色抗性植株。抗性植株的总DNA用AtNHX1基因的特异引物进行PCR检测,其中21棵为PCR阳性,最高转化频率为1.77%。Southern杂交结果证实,外源基因以低拷贝整合到高羊茅的基因组中,实验发现,在不同品种之间转化效率有所差异。

The embryo derived calli from four types of tall fescues ( Festuca arundinacea Schreb) were transformed with two Agrobactrium tumefaciens strains AGL1 and GV3101.AGL1 harbors an intron AtNHX1 expression vector pROK2U containing ubiqutin promoter and npt Ⅱ marker gene. GV 3101 harbors an intron AtNHX1 expression vector pROK2 containing 35S promoter and npt Ⅱgene.After infection and co culture with AGL 1 or GV 3101,the embyogenic calli were selected with 50 150 mg/L paromomycine and 1 126 resistant plants regenerated from the resistant calli.All plants were selected further with 10 20 mg/L Kanamycin and 525 of them remained green.Genome DNA of the resistant plants was checked with specific primers and probe from AtNHX1 gene.The results of PCR assay and Southern blot analysis indicted that exogenous target gene ( AtNHX1 gene) had been transferred into different cultivars of Festuca arundinacea .Different transformation frequencies among the four cultivars were obtained.