甘露糖正向筛选体系的建立及在拟南芥遗传转化中的应用

Establishment of Positive Selection System of Mannose and Application of Genetic Transformation in Arabidopsis thaliana

为了研究甘露糖正向筛选体系在拟南芥转化中的有效性,本研究参照GenBank上公布的PMI基因序列,通过PCR从大肠杆菌克隆了6-磷酸甘露糖异构酶(6-phosphomannose isomerase,PMI)基因,替换植物表达载体pCAMBIA1301中的潮霉素磷酸转移酶(hygromycin phosphotransferase,HPT)基因,并将以CaMV35S和AmCBL1P两种启动子调控的沙冬青AmCBL1基因成功地构建到重组载体pPMI上,并导入根癌农杆菌E-HA105中,然后通过农杆菌花序浸染法转入拟南芥。将获得的阳性植株通过GUS组织化学法鉴定、氯酚红(chlorophenol red,CPR)法及PCR检测,证明PMI基因已经转入拟南芥中。在不使用抗生素和除草剂的情况下这种筛选体系为转基因拟南芥筛选提供了更加安全有效的方法,并且为以后AmCBL1基因的功能验证及植物抗逆性的改良奠定了基础。

In order to study on validity of positive selection system of mannose in the transformation of Arobidopsis thaliana,we cloned the PMI gene from Escherichia coli by using PCR according to the sequence of PMI gene in GenBank.The PMI gene replacement the HPT gene of plant expression vector pCAMBIA1301,and named vector pPMI.As well as,the AmCBL1 gene of Ammopiptanthus mongolicus regulated by 35S promoter and promoter AmCBL1P,was cloned into recombinant vector pPMI and transformed into Agrobacterium EHA105.Transgenic plants were obtained through the flower dipping method mediated by Agrobacterium tumefacien,GUS histochemical assay,CPR assay and PCR analysis indicated that PMI gene had really been introduced into the genomes of Aro-bidopsis thaliana.This selection system would provides an efficient way for selecting transgenic plants in Arobidopsis thaliana under the conditions of without antibiotics and herbicides,and it would lay a basis for verifying of AmCBL1 gene function and improving plant tolerance to biotic and abiotic stresses.