Expression And Antifreeze Feature Analysis Of The Daucus Carota And Tentbrio Molitor Fusion Antifreeze Protein (AFP) Gene In Arabidopsis Thaliana

Intercellular environment would dehydrated and crystallized when plant supper from freeze injury. It is the result that cell would physically damaged and dead. There are two basic elements that plant resists the freeze injury. First, plant has to avoid ice in intracellular; Second, plant has to improve the stabilization of the structure of the cellular membrane and biological macromolecule. However, frozen out of cells and cytolymph supercooling are two useful adaptive mechanism for plant to avoid injury. Antifreeze proteins (AFPs) have the ability to bind to ice crystals and restrict their growth, enabling certain organisms to survive under freezing conditions that would otherwise prove fatal, can improve the cold resistance of the plant efficiently.In this study, AFPs gene cloned by RT-PCR from Daucus carota and Tenebrio molitor (Accession number:AJ131340 and AF159114). Two fusion genes, Dc-TmAFP and Tm-DcAFP, were constructed using overlap extension PCR. Then, Overexpression vectors of four genes were constructed. These vectors transformed into GV3101 used for transformation. Arabidopsis thaliana was transformed via floral-dip method. The kanamycin-resistant seedlings were subjected to further analyses of real-time PCR, and the overexpression confirm that the four genes have been integrated into the genome of transformates.To test the function of the four genes, transgenic plant exposed-10℃for 7 h and then back to 21℃for 5d, the seedlings with 5 or 7 leaves. Compared to the wild type at 0%, overexpression genetype DcAFP, TmAFP, Dc-TmAFP and Tm-DcAFP survived at high as 86.387%,86.079%,85.923% and 78.993%, respectively. Statistical result indicated that overexpression genetype DcAFP, TmAFP, Dc-TmAFP and Tm-DcAFP survival frequency were significantly higher than wild-type plants. Genetype Tm-DcAFP survival frequency were significantly difference compared with those three genetype. We also analysed the blade etiolation rate. Overexperssion genetype DcAFP, TmAFP, Dc-TmAFP and Tm-DcAFP etiolated at 57.97%,41.966%, 86.369% and 54.622%, respectively. Statistical result indicated that overexpression genetype TmAFP etiolation was significantly lower than the others, and genetype Dc-TmAFP was higer than the others. Genetype DcAFP and Tm-DcAFP etiolation were no difference.The results indicated that the AFPs have different functions can improve the cold resistance in many different ways. Hyperactive thermal hysteresis protein (THP) like TmAFP are a great help to depress the freezing point. However, when plant suffer from freeze injury a little longer hyperactive ice re-crystallization inhibition protein (IRIP) like DcAFP keeps stabilization of the fluid in the plant apoplast.