Study On Vector Construction And Transformation Of Rosa Chinensis TCP9 Gene Based On CRISPR/Cas9 Technique

In order to explore the effect of TCP9 gene on the regulation of flower organ development,CRISPR / Cas9 technique was used to construct the plant binary expression vector.The results showed that the plant binary expression vector containing TCP9 gene was successfully constructed.The transformation of Arabidopsis thaliana was carried out by Agrobacterium tumefaciens-mediated inflorescence infection with Arabidopsis thaliana as recipient material.Arabidopsis thaliana transformed plants were obtained.The callus transformation was carried out by Agrobacterium tumefaciens-mediated transformation on the embryogenic callus and the axillary buds of the Rose chinensis.The genetic transformation system was established.main research contents and results were as follows:1.In this study,the mutant TCP9 gene was edited by CRISPR / Cas9 technique,and the target gene fragment was amplified by internal and external bicyclic 4 primer PCR.The target gene fragment was digested with T4 ligase.The specific mutant TCP9 gene fragment was inserted into the p HSE401 plasmid vector.The sequencing assay showed that the plant binary expression vector pHSE401-TCP9 was successfully constructed.2.Using Arabidopsis thaliana as recipient material,The Arabidopsis thaliana inflorescence was transformed into Arabidopsis thaliana by Arabidopsis thaliana-mediated inflorescence infestation,and the plants were transformed into plants,and the floral organ phenotypes of the transformed plants were significantly different.The performance of the petals increased,the number of stamens decreased,pistil stigma bent.It is concluded that the TCP9 gene has a certain ability of regulating the development of plant floral organ.3.By the effect of Kan and Cef on the embryogenic callus of the Rosa chinensis,In order to ensure the normal development of embryogenic callus and prevent excessive growth of Agrobacterium,The medium containing Kan 50 mg / L and Cef 200 mg / L was selected as the ideal screening pressure for the antibiotic in the embryogenic callus transformation system.