Transcriptome And Metabolite Profiling Unveil The Mechanisms Of Scutellaria Baicalensis Georgi.Petal Coloration

Scutellaria baicalensis Georgi is a perennial medicinal plant of Scutellaria in Labiatae,which is rich in flavonoids such as baicalin and baicalein,and has high medicinal value and economic value.Under natural conditions,the petals of S.baicalensis are purple or blue-purple,but few S.baicalensis plants are found to be white flowers and purple-red flowers in the field.In order to deeply analyze the molecular mechanism of the color difference of S.baicalensis in the same ecological environment,this study used different color petals（purple,purple-red and white）of S.baicalensis as research materials,and screened the key regulatory genes involved in anthocyanin biosynthesis of S.baicalensis by joint analysis of transcriptomics and metabonomics,and cloned and preliminarily verified the key regulatory genes.On the one hand,the research results analyze the mechanism of the change of S.baicalensis color difference from the molecular level;On the other hand,it provides theoretical basis for the rational development and utilization of flavonoids such as anthocyanins in S.baicalensis.The main experimental results are as follows:1.Using metabonomics and transcriptomics techniques,the flavonoid secondary metabolites and genes of three different colors of S.baicalensis were identified and analyzed.The results showed that 168 metabolites were identified and 56.47 Gb clean data were obtained.According to the criteria of |Log₂FC| ≥1 and P < 0.05,the differential metabolites and differentially expressed genes among different colors of S.baicalensis were screened,and the KEGG pathway enrichment analysis showed that they were mainly enriched in flavonoid synthesis pathway and anthocyanin synthesis pathway.2.Through the joint analysis of differential metabolites and differential genes in flavonoid synthesis pathway of S.baicalensis with different colors,it was found that the accumulation of anthocyanin cyanidin3-rutin and delphinidin was related to the change of Scutellaria’s color.Furthermore,through the analysis of key genes in flavonoid biosynthesis pathway,it was found that SbDFR,SbANS and Sb3 GT genes played an important role in anthocyanin accumulation in S.baicalensis.3.Using the petal cDNA of S.baicalensis as a template,the CDS region of SbDFR was cloned from it based on homologous cloning technology,with a total length of 1167 bp,encoding 388 amino acids.CDS region of SbANS is 1050 bp in length and encodes 349 amino acids.The full length of CDS region of Sb3 GT is 1374 bp,encoding 457 amino acids.Bioinformatics analysis showed that they all had binding,active sites and conserved domains in the corresponding protein families,and the secondary structures were composed of four conformations: α-helix,β-turn,extended chain and random coil.They all have the closest genetic relationship with the same Labiatae plants in evolution.4.The anthocyanin content of S.baicalensis in different flower development stages was determined.It was found that the anthocyanin content accumulated continuously with the petal development process,and reached the highest level at the full flowering stage.At the full bloom stage,the anthocyanin content in SbP was the highest,which was 9.05 g/FW,and that in SbPR was 7.80 g/FW,while SbW was the lowest,which was 0.51 g/FW,which was significantly lower than that in SbP and SbPR,and conformed to the change rule of petal apparent color.5.The qRT-PCR was used to analyze the spatio-temporal expression patterns of SbDFR,SbANS and Sb3 GT in S.baicalensis.It was found that SbDFR,SbANS and Sb3 GT genes were expressed in different tissues of S.baicalensis with three colors,but their relative expression levels were highest in petals.At the same time,the expression levels of the three genes in different flower development stages were determined,and it was found that the expression levels of other genes in in F4 stage of different flower designs of Scutellaria baicalensis Georgi were the highest except Sb3 GT in white flowers.6.SbDFR,SbANS and Sb3 GT genes were constructed into the plant expression vector pCAMBIA1302-GFP by homologous recombination technology.Transient expression analysis of tobacco infected by Agrobacterium showed that the anthocyanin content of recombinant plasmids pCAMBIA1302-SbDFR,pCAMBIA1302-SbANS and pCAMBIA1302-Sb3 GT increased after transient expression in tobacco leaves,which preliminarily explained the functional role of SbDFR,SbANS and Sb3 GT genes in anthocyanin synthesis pathway.