The Genetic Transformation Of BcUGT3 Gene Of Bupleurum And The Application Of CRISPR/Cas9 Technology In Bupleurum

Bupleuri Radix is one of the most commonly used Chinese traditional medicines in China,and it is also an important raw material for various Chinese patent medicines.Bupleuri Radix contains various chemical components among which the most studied in biosynthesis pathway is saikosaponin.The triterpene saponin backbone is catalyzed by cytochrome P450 monooxygenase,glycosyltransferase and other post-modification enzymes to form saikosaponin in the biosynthetic pathway of saikosaponin.CRISPR/Cas9 gene editing technology has been widely used in gene function research of maize,rice,Arabidopsis and other plants due to its advantages of simplicity,time saving and high efficiency.Based on these foundations,the following two parts of the study are carried out.First,construct the overexpression vector of BcUGT3 gene and transgenic hairy roots were obtained through Agrobacterium-mediated genetic transformation to verify its function,laying the foundation for functional analysis of other glycosyl-transferase genes.Second,make an attempt on applying CRISPR/Cas9 gene editing technology on researches of Bupleurum chinese via genetic transformation system,providing a reference for subsequent applications of the technology in functional studies of BcUGT3 gene and other genes.Specific findings are as follows:1.Using aseptic seedlings of B.chinense as explants,hairy roots were induced by A.rhizogenes SW101-mediated genetic transformation.Hairy roots were regenerated from wounded young rootless seedlings by bacteriostatic culture for about 20 days,at last,20 BcUGT3-pK7WG2D transgenic hairy roots were obtained.Overexpression efficiency of BcUGT3 gene and ?-AS gene were verified by qPCR.The results indicated that BcUGT3 gene may be involved in the biosynthesis of saikosaponin.2.Construct CRISPR/Cas9 vector and transform B.chinense aseptic seedlings by A.rhizogenes SW101-mediated method.20 GFP-1 transgenic hairy roots and 16 GFP-2 transgenic hairy roots were obtained.The hairy roots were amplified and sequenced.Sequencing of 3 root lines had double peaks,indicating that these lines may be successfully edited.After further cloning and sequencing,2 transgenic hairy root lines were found to be edited.One base substitution at 152 bp in GFP-1-10,one base insertion at 327 bp or one base substitution at 220 bp in GFP-2-1.