Using SmPAL1 Gene To Regulate The Synthesis Of Salvianolic Acid In Salvia Miltiorrhiza

Salvia miltiorrhiza is a dicotyledonous plant belonging to the genus Salvia of the Lamiaceae.Its dried roots and rhizomes are traditional Chinese herbal medicines in China.CRISPR/Cas9 is a site-specific gene editing technology.This study was based on the CRISPR/Cas9 technologyto determine the selection concentration of hygromycin used in this technology and the type and concentration of antibacterial antibiotics suitable for Salvia miltiorrhiza.The material was Salvia miltiorrhiza with higher content of salvianolic acids.Under CRISPR/Cas9 technology system,we explored the four factors of ultrasonic pretreatment time,acetosyringone concentration,infestation concentration and infection time by single-factor experiments and orthogonal experiments to optimize a Agrobacterium-mediated genetic transformation system of Salvia miltiorrhiza.Then the SmPAL1 gene was used as a target gene to be edited by CRISPR/Cas9.The target sites of SmPAL1 gene were tested in vitro for its target efficiencies and CRISPR/Cas9 vector was constructed.The CRISPR/Cas9 vector was transferred to Salvia miltiorrhiza by Agrobacterium-mediated method and obtained new transgenic plants.The main results are as follows:1.A stable and highly efficient genetic transformation system of Salvia miltiorrhiza was obtained through optimization of callus induction,selection of hygromycin concentration,selection of antibacterial antibiotics,orthogonal experiments and single-factor experiments on the four factors of ultrasonic pretreatment time,infection concentration,acetosyringone concentration,and infection time.2.Three CRISPR target sequences(SmPAL1-g1,SmPAL1-g2,SmPAL1-g3)were designed and the efficiencies were 53.3%,76.6% and 10.0%.SmPAL1-g1 and SmPAL1-g2 were constructed into vector VK005-03 and named as VK005-03-g1 and VK005-03-g2.3.We obtained 16 hygromycin positive transgenic plants and the phenotype of some plantswere abnormal and total phenolic content was reduced.