Comparison Of Metabolome And Transcriptome Of Bei-Chaihu And Nan-Chaihu And Construction Of CRISPR/Cas9 Gene Editing System In Isatis Indigotica

Radix Bupleuri(Chaihu in Chinese),which is one of the commonly used Chinese herbal medicines,is derived from the dried roots of plants in the Umbelliferae,including Bupleurum chinense DC and B.scorzonerifolium Willd.It is known for its effects of regulating liver function,relieving depression,and dispersing heat.According to its source,it can be divided into Bei-Chaihu and Nan-Chaihu.The main chemical components of Radix Bupleuri were flavonoids,Saikosaponins(SSs),volatile oil and polysaccharide.There are significant differences in clinical application between Bei-Chaihu and Nan-Chaihu due to their inconsistent chemical composition.The content of Saikosaponin a(SSa)and its analogs is relatively high in the Bei-Chaihu,while the content of flavonoids,volatile oil,Saikosaponin b1(SSbl)and its homologs is relatively high in the Nan-Chaihu.This study analyzed the metabolome and transcriptome of the roots of two Bei-Chaihu(BCYC and BCZC)and one Nan-Chaihu(BSHC),to investigate the differences in chemical compositions between the Bei-Chaihu and Nan-Chaihu and within the Bei-Chaihu,as well as differential expression genes(DEGs).In addition,it utilized both metabolomic and transcriptomic approaches to investigate genes related to the synthesis of differential metabolites.CRISPR/Cas9 is a gene editing technology,which can target and modify specific DNA sequences through the binding of guide RNA(gRNA)to the target DNA.Using CRISPR/Cas9 to edit the phytoene desaturase(PDS)gene by targeted mutation results in a whitening phenotype in the leaves.In this study,a CRISPR/Cas9 gene knockout system was constructed in Ⅰ.indigotica using Agrobacterium-mediated genetic transformation with the PDS gene as a reporter.The main contents and results of this study are described below:1.Differential metabolite analysis between the Bei-Chaihu and Nan-Chaihu(1)A total of 631 metabolites were identified from the three samples of Radix Bupleuri,with flavonoids accounting for 11.73%of the total metabolites,and SSs accounting for 2.69%.Compared with BSHC,135 and 194 diferential metabolites were identifed in BCYC and BCZC,respectively.A total of 163 diferential metabolites were obtained between BCYC and BCZC,including phenolic acids and lipids.(2)The differences in flavonoid metabolites between Bei-Chaihu and Nan-Chaihu were mainly due to isorhamnetin,kaempferol,and quercetin,while the differences in terpenoid metabolites were mainly due to SSs.The abundance of favonoids in Nan-Chaihu was higher than that of Bei-Chaihu,with the latter having higher SSa and Saikosaponin d(SSd)than the former.Pinobanksin was the flavonoid metabolite with the greatest difference detected within the two cultivars of Bei-Chaihu.2.Transcriptome analysis of Bei-Chaihu and Nan-Chaihu(1)Compared with BSHC,6,557 and 5,621 DEGs were found in BCYC and BCZC,respectively.A total of 4,880 DEGs existed between the two cultivars of Bei-Chaihu.(2)After expression annotation analysis of the genes,DEGs between Nan-Chaihu and Bei-Chaihu are mainly annotated to the biosynthesis of unsaturated fatty acids,fatty acid metabolism,and flavonoid biosynthesis pathways.Within Nan-Chaihu,DEGs are mainly annotated to amino sugar and nucleotide sugar metabolism,phenylpropanoid biosynthesis,biosynthesis of unsaturated fatty acids,and flavonoid biosynthesis pathways.3.Construction of CRISPR/Cas9 gene editing system in Isatis indigoticaIn this study,highly homologous PDS gene of Isatis indigotica genome were selected as reporter gene,six gRNAs were designed targeting five exon regions.The recombinant vector PDS-CRISPR/Cas9 was successfully constructed.The recombinant plasmid was introduced into I.indigotica by Agrobacterium tumefaciens mediated genetic transformation method.Twelve edited albino strains were obtained and the gene knockout system of Ⅰ.indigotica by CRISPR/Cas9 technique was constructed.This study greatly enriched the metabolic and transcriptomic information of Radix Bupleuri by sequencing the metabolome and transcriptome of Bei-Chaihu and Nan-Chaihu,providing data support for subsequent metabolic regulation and functional gene verification of Radix Bupleuri.The study successfully constructed a gene knockout system in I.indigotica using CRISPR/Cas9,laying a foundation for the future application of CRISPR/Cas9 in I.indigotica.This study,for the first time,applied CRISPR/Cas9 technology to the medicinal plant I.indigotica.A CRISPR/Cas9 gene knockout system was constructed in I.indigotica using Agrobacterium-mediated genetic transformation with the PDS gene as a reporter.This study laid the foundation for the future application of CRISPR/Cas9 technology in I.indigotica.