农杆菌介导玉米胚性愈伤的遗传转化研究

Agrobacterium tumefaciens-mediated Transformation of Maize Embryonic Calli

利用3种不同类型的农杆菌菌株C58、LBA4404和EHA105携带外源GUS基因分别侵染玉米自交系齐319和18(红)胚性愈伤。结果显示,不同的菌株和自交系间的搭配,其遗传转化效率差异很大,GUS瞬时表达率呈极显著差异(F=24.92**),抗性愈伤率也呈极显著差异(F=19.43**)。其中,EHA105-齐319组合遗传转化效率最高,其GUS瞬时表达率平均为55.5%,最高可达71.1%;其抗性愈伤率平均为14.4%,最高可达20%;对22株转基因T0代抗性植株进行PCR检测,其中PCR呈阳性植株有11株,阳性率为50%。进一步对此22株T0代抗性植株进行叶片组织化学染色分析,结果显示,PCR呈阳性的植株中均有GUS基因表达。从而证明,外源GUS基因在转基因玉米T0代植株中得到稳定表达,而且验证了PCR检测结果和GUS表达分析结果的一致性。

Three types of Agrobocterium strains C58, LBA4404 and EHA105 containing standard binary vector pCAMBIA1301 with GUS as reporter gene were used to inoculate maize embryonic calli of two elite inbred lines Qi 319 and 18 （red）,respectively. The results indicated that the transformation efficiencies including efficiencies of GUS transient expression（F=24.92^＋＋）and regenerated resistant calli（F=19.43^＋＋）were significant different between six combinations of bacteria strains and maize inbred lines. EHA105-Qi 319 was the optimized combination for transformation,and average GUS transient expression efficiency was 55.5%,and some up to 71.1%. The efficiency of average regenerated resistant calli was 14.4%,and some up to 20%. PCR analysis was conducted with 22 regenerated resistant To generation plants and the efficiency of positive plants was 50%. Histochemical GUS assay was conducted with those resistant plants. The results indicated that intense blue staining was observed in the leaves of all PCR positive plants as expected,and no GUS expression was observed in the leaves of PCR negative and wild type maize plants. The results proved that foreign GUS gene was expressed stably in the To generation maize plants and also confirmed the consistency of PCR analysis with histochemical GUS assay.