东方山羊豆GoGID基因表达载体构建及紫花苜蓿转化

根据已经克隆得到的东方山羊豆赤霉素受体(GoGID)基因,扩增编码区cDNA.以pBI121为基础载体,采用酶切连接法,构建植物超表达载体pBI121-GoGID.酶切鉴定表明:目的基因已经正确插入载体中,超表达载体构建成功.采用CaCl₂冻融法将重组载体转入农杆菌菌株中.以叶片为外植体,采用农杆菌介导的愈伤组织培养法,转化紫花苜蓿(Medicage sativa),得到抗性苗.对载体携带的nptⅡ基因、GUS基因进行PCR检测均成阳性,表明目的基因已成功导入紫花苜蓿基因组中.同时对转基因植株进行Southern-blot及RT-PCR检测,并均得到目的条带.本研究为进一步分析GoGID基因对紫花苜蓿生物量影响奠定了基础.

Construction of Plant Expression Vector of GoGID Gene and Transformation of Medicago sativa L.

Based on the Galega orientalis Gibberellin Receptor gene (GoGID) sequence, a cDNA fragment was cloned. By the aid of the pBI121 vector, the plant expression vector pBI121-GoGID was constructed using digestion and ligation method. The recombinant vector was verified by enzyme digestion. The plasmid was then transferred to Agrobacterium tumefaciens GV3101 by CaCl₂ freeze-thaw method. Transgenic plants were obtained through somatic embryogenesis from leaves as explants by A.tumefaciens-mediated transformation. The nptⅡ and GUS gene existed in plasmid were tested by PCR in the genome of transgenic plants and all had positive results, indicating that the target gene has bean transferred into plant. The transgenic plants were also tested by Southern-blot and RT-PCR. This study will pave the path for further study of the function of GoGID in alfalfa biomass improvement.