Study On Construction Of Plant Expression Vector Carrying DHAR Gene And Its Genetic Transformation In Cyclamen Persicum

Ascorbate acts as an important antioxidants in both enzymatic and non-enzymatic reactions in plant cells. Dehydroascorbate reductase(DHAR) catalyzes the re-reduction of dehydroascorbate(DHA) to ascorbate by glutathione. Thus, DHAR is critical for protection of cellular components against oxidative injury resistance. In this paper, with DHAR gene cloned, the recombinant plasmid pCSB-DHAR carrying DHAR gene was constructed, and introduced into Agrabacterium tumefaciens strain EHA105. Subsequently DHAR gene was transformed to cyclamen by Agrabacterium-mediated method. We initially discussed the transformation and expression of DHAR gene through studying cyclamen genetic transformation, which will pave the way for its breeding via genetic engineering.The results were as follows:1. Construction of plant expression vector carrying DHAR gene and its introduction into Agrobacterium tumefaciens.With the application of DNA recombinant technology, the nucleotides sequence of plasmid pSB166 containing ED35s-Omega-MCS-UTT-TNOS was specifically cloned into plasmid pCAMBIA1303, then plant intermediate expression vector pCSB was successfully constructed by enzyme digestion and electrophoresis.After plasmid pMD18-T-DHAR and plant expression vector pCSB were double-digested by SalⅠand KpnⅠ, the target fragments were collected and purified respectively, after being ligated, recombinant plasmid pCSB-DHAR was constructed.The recombinant plasmid pCSB-DHAR was introduced into Agrobacterium tumefaciens strain EHA105 by means of freezing-thawing. By screening in the medium containing Kan,Str and Rif, PCR and enzyme digestion identification, it was confirmed the result was successful.2. Transient expression of gus gene in vector pCSB-DHAR was detcted and it confirmed that the transfer efficiency was satisfactory.After infected with agrobacterium carrying pCSB-DHAR, transient expression of gus gene in lily leaves was detcted for 2 weeks, it showed that transient expression efficiency was 100% in the 7th day, thus the transient expression efficiency of pCSB-DHAR was satisfactory. 3.Agrobacterium-mediated genetic transformation system was optimized, and the transgenic plantlets of"scarlet 1011"cyclamen cultivar carrying DHAR gene were obtained.Factors afecting the transformation frequency were studied, and some parameters were determinated. The results indicated that better transformation rate was gained when the leaves was precultivated 2 days, the OD600 of Agrobacterium was about 0.6, Agrobacterium infected 15 min, 3 days coculture and 2 weeks of detention screening. The better selective efficiency was gotten when the concentration of hygromycin was gradually enhanced from 2.5 mg·L-1to7.5 mg·L-1.154 resistant plantlets were obtained and 32 of them were positive in PCR assay. It showed preliminarily that the DHAR gene was integreted into the cyclamen genome.