Construction Of The Plant Expression Vector With Restructuring Brazzein Gene And Tansformation Of Pepino（Solanum Muricatum）

Pepino(Solanum muricatum Ait.) is a perennial herb of Solanaceae Solanum, which has close genetic relationship with tomato and potato. Pepino fruit was famous for the advantages of high protein, low fat, low sugar and so on, which was beneficial to the treatment of hypertension and diabetes and thus it is one of the ideal nutritional and healthy fruits. However, the sensory evaluation of pepino cannot meet needs of consumers, especially the fruit flavor is too delicate and sweet taste is lower, resulting in the neglect of its superior in nutrition and health by people. In addition, the asexual reproduction, which has caused some serious poblems,such as virus disease, together with its weaker foundation of germplasm improvement led to the deterioration of flavor, deterioration of quality and the decline in production. Therefore, it is an urgent requirement to cultivate a new pepino variety with more desirable flavor, stronger resistance and meeted sweet-tasting to the public.According to the bioinformatics methods, the genetic relationship and CIS-regulatory elements conservative analysis of E8 gene non-coding region were analysised for Solanaceae plants to confirmed the genetic evolutionary relationships between the tomato ethylene response fruit specific E8 promoter with the pepino. In view of the deficiency in the flavor characteristics, the sweet Protein Brazzein and E8 promoter were subcloned by PCR. The plant expression vectors were constructed by the E8 promoter and 35 S promoter drivers both recombinant sweet protein and the herbicide resistant gene.The transformation of the pepino was successfully established to lay the foundation for the pepino germplasm improvement and innovation. The results obtained are as follows:1. The genetic relationship and CIS-regulatory elements conservative analysis of E8 gene non-coding region were analysised for Solanaceae plants in this study. The results showed that the non-coding region of E8 gene contain several extremely conserved motif, such as ethylene-responsive(ERE), the Me JA-responsiveness and drought-inducibility elements. Accordingly, the fruit-specific promoter E8 genes were more specific to ginseng fruit flavor of Solanum muricatum Ait as we expected.2. Considering that codon preference, the sweet protein Brazzein was optimal designed. The sweet Protein Brazzein and E8 promoter were subcloned by PCR, and the homology comparison results revealed that the cloned Brazzein and E8 promoter had higher homology with the genes reported in the Gen Bank. Also the spatial structure of Brazzein were analysised by model 2KGQ.3. The plant bivalent expression vectors were generated by intermediate vector p HANNIBAL and plant expression vector p CEPSPS. In addition, these plant expression vectors contain sweet protein Brazzein gene and herbicide resistance gene EPSPS, which driven by fruit-specific E8 promoter and 35 S promoter.4. Using pepino leaf as explants, the genetic transformation system of pepino was successfully established based on MS medium and optimized hormone combinations. As a result, Fifty-four transgenic plants were obtained through agrobacterium-mediated protocol and 14 positive plants were screened in pepino plantlets, in which traget genes were integrated into the genome in pepino.