Construction And Optimizationthe System Of CRISPR/Cas9 Editing And Genetic Transformation Of BcIAA13 In Bupleurum

Radix Bupleuri is a traditional Chinese medicinal material,and it has been used for2000 years.The Chinese Pharmacopoeia recorded that Radix Bupleuri had the functions of evacuating and antipyretic,soothing liver and relieving depression,and lifting yang qi.It was mainly used to treat cold and fever,cold and heat exchanges,chest pain,irregular menstruation and other diseases.With the increase of public demand for Radix Bupleuri in recent years,wild Bupleurum resources have been plundered.Breeding excellent new varieties can effectively alleviate market demand and protect wild Bupleurum resources.Saikosaponin,the main medicinal component of Bupleurum,is stored in the phloem of root,and the content of lateral root is significantly higher than that of main root.Therefore,it is necessary to cultivate Bupleurum with slender main roots and multiple lateral roots in order to obtain high-yielding new varieties with stable efficacy.As a new gene editing tool,CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/Cas9）has the advantages of simple operation,low cost and short cycle.It shows great potential in directional transformation of plants and cultivation of new varieties.In this study,CRISPR/Cas9 technology was used to knock out the auxin response protein BcIAA13 related to lateral root development in order to obtain the gene-edited Bupleurum,which laid a solid foundation for the establishment of a simple and efficient CRISPR/Cas9 gene editing system and the application of new germplasm creation.At the same time,the identification and expression analysis of heme oxygenase（HO）genes related to lateral root development of Bupleurum were carried out to provide theoretical basis for further breeding new varieties of Bupleurum by molecular breeding.The main research results of this paper are as follows:1.Construction of Crispr/Cas9 expression vectorAccording to the BcIAA13 c DNA sequence of Bupleurum chinense,the primers were designed by online design software,and the target sites were assembled into the sg RNA skeleton by overlapping PCR.The p HEC401-BcIAA13 CRISPR expression vector was constructed.2.Optimization of protoplasm preparation system and instantaneous transformation of B.chinense.The hypocotyl callus of B.chinense was used as the material to explore the related factors affecting the protoplast preparation,and the optimal system was obtained as follows:enzymolysis at 28°C for 6 h under dark conditions,centrifugation at 200 g/min for 5 min to purify and collect protoplasts.The yield was about 8.09×10~5/g,and the activity was about 90.2%.The protoplasts isolated and purified were used as the transformation objects.Under the mediation of PEG4000 with a concentration of 20%,the vector with green fluorescent protein was transferred into the protoplasts isolated from B.chinense callus,and the transformation efficiency was 45%.3.The targeting ability of sg RNA was detected by transient transformation system.The constructed p HEC401-DT1T2 vector was transformed into B.chinense protoplasts to detect the targeting ability of the vector to the protoplast genome.The results showed that the target gene was successfully edited with base substitution in the sequences near target 1 and target 2,and the editing efficiency was 19.4%.4.Optimization forsystem of genetic transformation system of B.chinense.The constructed p HEC401-DT1T2 was transformed into Agrobacterium rhizogenes and then infected with B.chinense tissue.The optimal conditions were obtained by optimizing the system as follows:the hypocotyls were used as materials,pre-cultured for 10days,immersed in the concentration of OD₆₀₀=0.3,and screened and differentiated for 30 days with hygromycin concentration of 8 mg.The adventitious roots could be differentiated and proliferated after 60 days.Cas protein detection and target sequence sequencing were performed on the acquired resistant adventitious roots.The results showed that the adventitious roots contained Cas9 protein and the base mutation and deletion occurred at the target site.It indicated that BcIAA13 was knocked out and CRISPR/Cas9 knockout system was established.5.Identification and expression analysis of heme oxygenase family in B.chinenseAccording to the transcriptome data of root development at seedling stage of CBC1 and CHC1,five Bc HO genes were identified from Bupleurum and their expression patterns were analyzed.The identification results showed that all Bc HOs contained a conserved Heme Oxgenase domain.The HO from Bupleurum contains a unique Motif 5,and the function of this structure remains to be explored.Phylogenetic analysis of the seven full-length HO sequences of Arabidopsis thaliana,Oryza sativa and Daucus carota showed that the five HOs of Bupleurum belonged to the HO1subfamily.The expression of HO gene decreased with the appearance of lateral roots at5 d whole root,15 d root tip and mature zone,indicating that HO was involved in the development of lateral roots of CBC1 and CHC1.