Cultivation And Protein Analysis Of Low Hydroxyl Transgenic Castor New Varieties

With the deterioration of the global environment and the depletion of petroleum energy,bio-aviation fuels have attracted more and more attention due to their low carbon emissions and renewable characteristics.In this study,a stable in vitro regeneration system of pure castor was constructed.The RcFAH12 gene of castor was knocked out by CRISPR/Cas9 gene editing technology,and functional deletion mutants were developed.It is expected that a new variety of low hydroxyl castor oil acid will be obtained,so that the deoxidation process of castor-based bio-aviation oil will be omitted in the process of industrial preparation,thus greatly reducing the cost of aviation oil preparation,meeting the needs of aviation industry and improving the market application value.The RcFAH12 gene sequence of Castor was cloned successfully.The physical and chemical properties,secondary structure and interaction network of the enzyme were predicted by bioinformatics and database analysis.The published sequence of Genebank and the sequencing results of five pure lines of Castor were compared and analyzed.Data analysis showed that RcFAH12 gene was interrelated with many important proteins in fatty acid synthesis pathways,like FAD2 and DGAT.strongly suggesting that the deletion of RcFAH12 gene sequence would cause changes in fatty acid composition of castor oil.Sequence alignment results showed that RcFAH12 gene was highly conservative,which provided a guarantee for the specific recognition and splicing of CRISPR/Cas9 gene knockout vectors.Ten pure line Castor varieties were screened on the basis of existing research results in the laboratory.The results showed that HP003 and HP005 could be used as the main experimental materials to study the in vitro regeneration system mediated by Agrobacterium tumefaciens.A stable and efficient castor genetic transformation system was constructed by transfecting the pBI121 overexpression vector containing Arabidopsis salt-tolerant gene AtAVP1 into Agrobacterium LBA44.The experimental data showed that HP003 was the best material for genetic transformation system of castor bean embryo tip.The optimum embryogenic culture condition was MS medium containing 0.88?mol/L6-BA+0.20 mg/L IBA.The optimum medium formula for bud induction was MS+2.20 ?mol/L 6-BA+0.25 mg/L IBA+0.1%B5+400 mg/L Cefo.By optimizing the conditions of bud elongation,the optimum conditions for bud elongation were MS medium containing MS+2.20?mol/L 6-BA+0.25 mg/L IBA+0.01 mg/L GA3+0.1%B5+400 mg/L Cefo,MS+0.50 mg/L IBA being the component of the optimum root induction medium.After optimization experiment of regeneration system of transgenic castor,59 plants survived.PCR detection of exogenous gene AtAVP1 proved that 27 castor seedlings were successfully transformed,and the transformation efficiency was 45.76%.Salt tolerance experiments showed that the salt tolerance of transgenic castor plants was significantly higher than that of wild type plants.In this paper,three different CRISPR/Cas9 gene knockout vectors were successfully constructed and transfected into Agrobacterium LBA4404 to prepare transformed engineering strains.Using the optimized genetic transformation system of castor germ tip,a new castor variety with low hydroxyl content was developed.Through screening 67 surviving castor seedlings,30 seedlings were successfully transformed.After sequencing the RcFAH12 gene of transformed plants,only two seedlings edited by pKES401 vector were successful mutants of RcFAH12 gene.The results showed that AtU6-SgRNA-mediated 35S-Cas9 gene knockout vector could be successfully applied to castor genetic transformation,with the transformation efficiency of 44.78%and editing efficiency of 2.99%.