Construction And Evaluation Of CRISPR-Cas9 Genome Editing Systems In Salvia Miltiorrhiza And Cucumber

Genome editing technologies represented by CRISPR-Cas9 have developed rapidly in recent years,and are widely used in basic research on gene function in animals and plants.However,research on the basis and application of plant genome editing is mainly focused on important crops and model plants such as rice,corn,tomato,Arabidopsis and tobacco.Limited efforts have been made to develop effective CRISPR-Cas9 genome editing tools based on the genome structure and expression characteristics of other plant materials.This severely restricts the application of gene function research and germplasm innovation based on genome editing strategies in these plant materials.Salvia miltiorrhiza and cucumber are widely used as medicinal plants and vegetable crops respectively,and are also important model plant materials in the study of secondary metabolism and fruit development.However,the existing research related to genome editing is very limited,especially the lack of targeted development of CRISPR-Cas9 genome editing tools for them.Relying entirely on the CRISPR-Cas9 genome editing tools used in Arabidopsis thaliana,tomato,and even rice,the core evaluation indicators of editing tools such as editing efficiency and editing specificity are poor,which seriously restricts the in-depth study of related research.Based on this bottleneck problem,this dissertation focused on the construction of an effective CRISPR-Cas9 genome editing tool for salvia miltiorrhiza and cucumber based on the effective analysis of their genome structure and expression characteristics.In order to provide effective and feasible genome editing tools for further functional gene basic research and molecular breeding application of salvia miltiorrhiza and cucumber.The main contents and results of this study are as follows:1.Based on different assembly strategies of the core elements of the Cas9 editing system,various types of double-unit and single-unit Salvia miltiorrhiza Cas9 genome editing backbone vectors were constructed.Based on the instantaneous transformation system of hairy roots of Salvia miltiorrhiza,it was confirmed that the editing efficiency of the two-unit system（77.5%）was significantly better than that of the single-unit system（21.1%）.Among them,the double-unit backbone vector with the At Ubi10 promoter driving the expression of Cas9 and the At U6 promoter driving the expression of sg RNA has the best editing efficiency.At the same time,based on the strategy of inducing T0 to regenerate seedlings by hairy roots,a reliable editing material of Salvia miltiorrhiza was obtained.2.Based on the confirmation of the double-unit genome editing backbone vector of Salvia miltiorrhiza,its multi-site co-editing ability was tested.Based on the transient transformation results of the hairy roots,the co-editing efficiency of the system in the four endogenous genes of Salvia miltiorrhiza was 6.9%-44.8%.The root system with three genes knocked out at the same time was obtained,which provided a feasible reference for the subsequent creation of multiplex gene mutants of Salvia miltiorrhiza.3.In order to further improve the efficiency of transformant editing events,a two-unit backbone vector with the At Ubi10 promoter driving Cas9 expression and the At U6 promoter driving sg RNA expression was upgraded and constructed: A Hyg selection pressure unit linked by a 2A element is fused downstream of the Cas9 expression unit;GFP-Poly A unit is fused upstream of the sg RNA expression unit.The upgraded two-unit editing backbone vector can realize the co-expression of Cas9-Hyg and GFP-sg RNA,effectively increasing the proportion of Cas9 and sg RNA effective expression events in positive transformants,so as to achieve transient and stable transformation in hairy roots and leaf of Salvia miltiorrhiza.The editing efficiency has been improved.4.Based on the effective construction of the Salvia miltiorrhiza Cas9 genome editing backbone vector,the cucumber high-expression endogenous promoter Cs CRE02,which was isolated and identified earlier,was used to effectively increase the expression levels of Cas9 and sg RNA,and the GRF4-GIF1 plant regeneration-promoting transcription factor expression unit was introduced.In order to explore the feasibility of realizing the effective acquisition of cucumber genome editing events based on the comprehensive improvement of Cas9,sg RNA expression efficiency and cucumber transformation and regeneration efficiency,it will provide a useful reference for the subsequent development and effective application of cucumber genome editing tools.