Collection And Evaluation Of Scutellaria Baicalensis Germplasm Resources And Preliminary Research On R2R3-MYB Family Members

The dry root of Scutellaria baicalensis Georgi,belonging to family Labiatae,is a well-known traditional Chinese medicine in China.Modern pharmacological research shows that the main medicinal ingredients of S.baicalensis are flavonoids such as baicalin.The latest study found that baicalin can inhibit the replication of new coronavirus(COVID-19)effectively.In recent years,there are more and more reports about S.baicalensis,but no research about the metabolic regulation of baicalin and other flavonoids.R2R3-MYB-is one of the biggest transcription factors(TFs)family of higher plantspecificly and play important roles in many processes of growth and development,including response to regulation of biotic and abiotic stress,primary and secondary metabolism,plant cell fate and identity etc.The genes encoding R2R3-MYB have been identified in many plants.However,little is known about the R2R3-MYB genes in S.baicalensis.In this study,we collected S.baicalensis from different producing areas of the China,and initially studied the differences in germplasm resources of S.baicalensis with different flower colors and the stable internal reference genes of S.baicalensis were selected.Based on the whole genomic data of S.baicalensis,95 R2R3-MYB genes in S.baicalensis were identified and the bioinformatics analysis was performed 8 R2R3-MYB were cloned according to previous reports and the results of gene family analysis,and constructed them to overexpression vectors,respectively.The function of SbMYB12 was studied.The main results are as follows:1.We collected 58 batches samples of S.baicalensis from 50 origins of nine provinces across the country and measured the content of five flavonoids including baicalin,wogonin,baicalein,wogonin and pallidin by HPLC.With five flavonoids as the evaluation criteria,the quality of S.baicalensis in different producing areas was analyzed.Meanwhile,we analyzed and compared the quality of S.baicalensis from different provinces.The studies showed that the content of flavonoids such as anthocyanins and baicalin in different flower colors(purple,pink,and white)of S.baicalensis have extremely significant differences.2.To screen appropriate reference genes in S.baicalensis,we applied four different methods--GeNorm,NormFinder,BestKeeper,and RefFinder--to evaluate the stability of 10 candidates.Expression was examined by qRT-PCR for various tissue types(roots,stems,leaves and flowers),hormone treatment(MeJA,SA and ABA)and abiotic stress(heavy metals,salt,drought,cold and woundings).For all tested samples,?-TUB?UBC and PP2A proved to be the most stable,and ACT11 is the most unstable gene,followed by GAPDH.Comprehensive analysis indicated that the most stable internal reference genes was ?-TUB in different tissues and at different abiotic stress.Under different hormone treatment,ACT7 was the best internal reference gene.All of these results provide a foundation for accurate quantification of expression levels of interested genes in S.baicalensis.3.We identified 95 putative R2R3-MYB genes from the whole genome of S.baicalensis.Their gene structure,conserved motifs,gene duplication,evolutionary relationship and expression analysis were systematically analyzed.Among all 95 R2R3-MYB genes,93 R2R3-MYB genes were located on nine chromosomes,and two R2R3-MYB genes were located on unassembled chromosome scaffolds.The analysis of the R2R3-MYB conserved domains of S.baicalensis showed that the R2 and R3 MYB domains of S.baicalensis had a high degree of sequence conservation and also had certain differences.The analysis of gene structure and sequence composition showed that in the same subfamily,conserved motifs and exon-intron structures are often the same or similar.The analysis of the cis-acting elements of the promoter shows that the R2R3-MYB promoter region of S.baicalensis contains MeJA,ABA,GA,SA,MYB binding sites(participating in drought induction),low temperature,defense and response to stress and many other response elements.The analysis of gene duplication events found that there were limited tandem repeats(1)and fragment duplications(26)in S.baicalensis,indicating that they played an important role in the amplification of S.baicalensis R2R3-MYB gene.Collinearity analysis showed that the homology between S.baicalensis and dicotyledonous plants was higher than that of monocotyledonous plants.GO analysis revealed a variety of biological functions of S.baicalensis R2R3-MYB protein.Based on the transcriptome data of four different tissues,we revealed the differential expression of some S.baicalensis R2R3-MYB genes in plant tissues.In addition,gene expression analysis showed that many of S.baicalensis R2R3-MYB genes are involved in plant abiotic stress tolerance(cold,drought)and hormone induction(ABA,MeJA).These results will lay a solid foundation for further research on the functional characteristics of S.baicalensis R2R3-MYB gene.4.Eight MYBs(SbMYB12,SbMYB18,SbMYB32,SbMYB46,SbMYB47,SbMYB60,SbMYB70 and SbMYB74)were cloned by PCR and constructed into the expression vector pK7WG2R and the yeast self-activation verification vector pGBKT7-GW(BD)through the Gateway technology.Experiments show that six proteins including SbMYB12,SbMYB18,SbMYB32,SbMYB47,SbMYB60 and SbMYB70 all have transcriptional autoactivation activity.At the same time,we also constructed four genes including SbMYB12,SbMYB32,SbMYB60 and SbMYB74 onto the subcellular localization vector pHBT-GFP-NOS to transform Arabidopsis protoplasts,and the GFP signals showed that:they are all located in the nucleus.Through Agrobacterium rhizogenes-mediated transgenic technology,we successfully obtained the SbMYB12 overexpression transgenic hairy roots and.the content of flavonoid components including baicalin in transgenic hairy roots were determined by HPLC.It was found that,compared with the CK control group,the content of baicalin,wogonin and total flavonoids in SbMYB12 overexpression strains increased significantly.The qRT-PCR analysis of the enzyme genes in the flavonoid synthesis pathway in S.baicalensis showed that compared with the CK control group,the expression of the enzyme gene SbPAL2?SbPAL3?SbCCL7-4?SbCCL7-5?Sb4CL-2?Sb4CL-3?SbCHI-2?SbFNS-2?SbF6H-1?SbF6H-2 and SbUGT-1 in the baicalin synthesis pathway in the S.baicalensis SbMYB12 overexpression line was significantly up-regulated.Therefore,SbMYB12 positively regulates the synthesis of baicalin by up-regulating the expression of genes in the baicalin synthesis pathway.The results of this study enriched our understanding of the R2R3-MYB family in S.baicalensis,and laid the foundation for the future study of S.baicalensis at the molecular level.