Research On CRISPR Transformation Technology Based On Rose RcTCP Gene

TCP gene family is a unique transcription factor in plants and plays an important regulatory role in plant growth and development.To explore the effect of TCP9 gene on floral organ development in Rosa chinensis,In this study,CRISPR/Cas9 gene editing technique was used to construct plant expression vectors,The results showed that plant expression vector which contained the TCP9 gene of the R.chinensis had been constructed successfully.R.chinensis has been used as as acceptor material,the leaves of R.chinensis has transformed instantaneously by Agrobacterium-mediated method and leaf injection method which expressing eGFP.The genetic transformation system of Rosa chinensis was preliminarily established by using tissue culture seedlings as recipient materials.main research contents and results were as follows:1.In this study,CRISPR/Cas9 gene editing technique was used,a pair of complementary primers was designed according to conserved sequence of R.chinensis TCP9 gene and ligated in the plasmid vector pKSE402 with T4 ligase.Sequencing and other detection showes that plant expression vector containing the TCP9 of the R.chinensis gene had been successfully constructed.2.R.chinensis was used as receptor material,the leaves of Rosa chinensis were instantaneously transformed by Agrobacterium-mediated leaf injection,and the leaves expressing eGFP were obtained.These results indicated that the constructed plant expression vector containing the TCP9 gene of the R.chinensis could be expressed in R.chinensis.3.In order to ensure the normal growth of axillary buds of Rosa after being infected by Agrobacterium tumefaciens,to prevent the proliferation of Agrobacterium tumefaciens and to screen resistant buds,Kan and Cef were used to test the interaction of axillary buds of R.chinensis,The axillary buds were placed in bud induction medium with different concentrations of Kan and Cef,Observe the germination and growth of axillary buds.Finally,the medium containing 150 mg/L Kan and 200 mg/L Cef was selected as the screening and bacteriostasis medium for Rosa axillary buds.