| Strawberry(Fragaria × ananassa. Duch.) is a perenninal herbaceous plant of the Rosaceae Fragaria. The fruits has important health function with containing a high concentration of flavonoids and significant economic value, therefore they are loved by consumers and growers. However, in order to keep the economic traits of the varieties, people usually use the burying stolons or division propagation to produce the seedlings in production, which leads to the accumulation and spread of virus diseases with an increased hazards. Currently, the use of biological techniques to obtain virus-free strawberry seedling or virus resistant seedling is the only effective way to prevent the virus hazards. Nevertheless, virus-free strawberry seedlings still do not have a strong immunity to the virus, and it will have the possibility of a high frequency of infection during the cultivation process, so it is necessary to replant and replace the virus-free seedlings regularly. This matter not only has brought great inconvenience in production, but also resulted in an increased costs. Therefore, using the biotechnology to cultivate antiviral Strawberry directly, has great practical significance and application value.The CRISPR/Cas9 is a simple and efficient genome editing technology emerging in 2013, which has been widely used in plants and animals. It is primarily based on a bacterium transformed from acquired immune system. The CRISPR/Cas9 can be used for site-directed editing of DNA, and can act simultaneously on multiple target sites or genes. In addition, It has obvious advantages compared with the conventional transgenic method and the silence was more thorough, so more and more researchers have been shown an interest to it. In this study, the'Benihoppe'strawberry and the pX330?pCAMBIA1302?pUC19 plasmid were used as materials to constructed the CRISPR/Cas9 plant vectors successfully by recombinant DNA technology. The PDS gene of cultivation strawberry was choosed as target site, and the CRISPR/Cas9 vector that were constructed has been using for a preliminary verification. The transgenic plant systems act on ORF ? or ORF ? and ORF ? sequence of SVBV virus were also constructed at the same time. The main contents and results are as follows:1.The plasmid pX330?pCAMBIA1302?pUC19 were used as materials to construct the CRISPR/Cas9 vector by the recombinant DNA technology. The results showed that:the mgfp sequence in pCAMBIA1302 has been successfully replaced and the recombinant plasmids was constructed successfully, abbreviated as pCC; the sgRNA sequence was imported into the multiple cloning site region of pUC19 plasmid, and the recombinant plasmid was abbreviated as pSG. pCC and pSG composed two basicvectors of the CRISPR/Cas9.2.The PDS gene of 'Benihoppe' strawberry was selected as target site and the CRISPR/Cas9 vector was constructed. Genetic transformation system was achieved through the leaf disc by agrobacterium-mediated transformation. The results show that:the CRISPR/Cas9 vector acting on PDS gene target site was successfully constructed, mutations can be detected in the PDS gene target site and adjacent areas after agrobacterium infection in callus, no deletions or insertions were discovered. Established genetic transformation system of strawberry were as follows:pre-culture the leaf disc for 3 days; using the agrobacterium suspension with a concentration of OD600= 0.1 infected leaf disc tissue for 20 minutes; cultivating on the co-cultivation medium containing 100?M acetosyringone and 50?M lipoic acid for 2 days; transferred to the pre-selection medium containing 100?M validamycin A?250mg/L Timentin?250mg/L Cefotaxime for 6 days; cultivating on the selection medium with 250mg/L Timentin?250mg/L Carbencine and 5mg/L Hygromycin, then gradually increase to the concentration of Hygromycin to10mg/L.3. Select the ORF IV and ORF VI sequence of SVBV virus as target sites, constructing the CRISPR/Cas9 vector of ORF IV or ORF IV and ORF VI separately, transformed to the 'Benihoppe' strawberry by Agrobacterium. The results showed:the related CRISPR/Cas9 vector were successfully constructed, after identification by PCR and sequencing, the number of the transgenic seedlings act on ORF IV was 6, and the number of the transgenic seedlings act on ORF IV and ORF VI simultaneously was... |
PDF Full Text Request