Construction Of Over-expression And RNAi-expression Vector Of DkCCDl And Genetic Transformation Study In Micro-Tom Tomato

The essential relationship of careotenoid cleavage oxidases with fruit quality was the CCD1 and CCD4 branches,which were related to the formation of the aroma components of apocarotenoids.Our previous studies have shown that apocarotenoids in aroma components surely exists in persimmon fruit,but it is uncertain that whether CCD1 relates to the formation of apocarotenoids.This study constructed an over-expression vector pCAMBIA1301-CCD1 and a RNAi-expression vector pCAMBIA1301-CCD1-RNAi according to the DkCCD1 gene that has been cloned.The genetic transformation system of Micro-Tom tomato was established.Two vectors were successfully introduced into Micro-Tom tomato by agrobacterium mediated leaf disk transformation.The main results were as follows:1.Total RNA was extracted from 'Xiaofangsh' with CTAB method.According to the target gene by using primers with different restriction,the results of sequence alignment and amino acid sequence alignment showed that the similarity of the gene sequence was more than 99%and the similarity of the amino acid sequence was 100%,which was used in the follow-up experiments of the target gene.The resulting gene was ligated with the binary expression vector pCAMBIA1301.The over-expression vector pCAMBIA1301-CCD1 and the RNAi expr-ession vector pCAMBIA1301-CCD1-RNAi with hairpin structure were constructed successfully.The two vectors were transferred to agrobacterium EHA105 by freeze-thawing,and stored in-80 ? refrigerator.2.On the basis of the regeneration system of Micro-Tom tomato,the marker gene Hyg B carried on the vector and the bacteriostatic agent timetine(Tim)in the screening process were pressure-screened.The results showed that the higher the concentration of Hyg,the greater the effect on explants differentiation,the concentration of 7 mg/L,9 mg/L and 5 mg/L in callus induction,bud differentiation and rooting culture respectively;Tim did not inhibit the growth of explants at a concentration of 450 mg/L,and had little effect on the callus and bud differentiation at the concentration of 500 mg/L,The concentration of bacteriostatic agent was 400 mg/L during the screening and culture.According to the above results,the callus selection medium was MS+ 2 mg/L 6-BA + 0.2 mg/L NAA + 7 mg/L Hyg + 400 mg/L Tim + 0.7%agar+ 3%sucrose.The bud differentiation selection medium was MS + 1 mg/L ZT + 9 mg/L Hyg+ 400 mg/L Tim + 0.7%agar +3%sucrose,the rooting selection medium was MS + 0.1 mg/L NAA + 5mg/L Hyg + 400mg/L + 0.7%agar + 3%sucrose.3.According to the hygromycin gene on the expression vector design primers,the target band size was 729 bp.DNA and RNA of the resistant strains were detected by PCR and RT-PCR,respectively.It was confirmed that the target gene DkCCDl was successfully introduced into the plant and expressed on the basis of the final RT-PCR test positive as the standard of successful transformation,and the transformation rate of the over-expression plants and the RNAi expression plants in the experiment were both 0.23%.