Research On The Accumulation Of Baicalin And The Key Enzyme Genes For Synthesis Of Scutellaria Baicalensis And Scutellaria Glutinosa

Scutellaria baicalensis and Scutellaria viscidula are both perennial herbs of the genus Scutellaria.S.viscidula,which has close phylogenetic relationship with S.baicalensis,is used as Traditional Chinese Medicine for its dry roots.The main active constituents are flavonoids(such as baicalin,baicalein).It has broad pharmacological effects such as anti-inflammation,bacteriostatic action and anti-tumor.S.baicalensis is the authentic product included in Chinese Pharmacopoeia(2015 Edition),but with the shortage of S.baicalensis wild resources and the rapid development of Chinese medicine modernization.As an adulterants or raw materials for extracting active constituents of medicinal plants,S.viscidula has been paid growing attention.There were many related researches on germplasm collection,resource evaluation and active component pharmacology of the genus Scutellaria medicinal plants in the earlier period.However,the molecular mechanism of baical in synthesis and interspecies difference between S.baicalensis and S.viscidula was reported rarely.In this study,we chose S.baicalensis and S.viscidula as experimental materials.The accumulation and change of baicalin in different parts(root,stem and leaves)during different growing stages of two species were comparatively analyzed.The conserved sequences of key enzyme genes CLL-7,FNS?-1 and FNS?-2 that related to the biosynthesis pathway of flavonoids in S.viscidula were cloned based on homological analysis.The full-length cDNA of the key enzyme gene cloned from S.viscidula was analyzed using bioinformatic analysis.The relative expression of CLL-7,FNS ?-1 and FNS II-2 of the two species was determined by Real-time PCR.Based on the analysis of relative expression of key enzyme genes and the content of baicalin,the synthesis mechanism of baicalin and the possible molecular mechanism of interspecific difference in Scutellaria were preliminarily revealed.The main research results of this study were as below:1.The content of baicalin in roots,stems and leaves during different growing stages of the two species was determined by high performance liquid chromatography(HPLC).The results showed that baicalin contents of S.baicalensis and S.viscidula in different parts were similar,and the content of roots was significantly higher than that of stems or leaves(P<0.05).The content of baicalin in the roots of S.baicalensis was significantly higher than that in the roots of S.viscidula.The content of baicalin in the roots of the two species showed a trend of first decline and then rise during different growth stages.In roots,the baicalin content of S.baicalensis in May and of S.viscidula in October were highest,and the content of the vegetative growth period was lowest.These results indicate that the synthesis and accumulation of baicalin will change with different growth stages.2.According to the transcriptome data of S.baicalensis and the biosynthesis pathway of baicalin,three key enzyme genes CLL-7,FNS ?-1 and FNS ?-2 were identified as the target genes of this study.According to the published sequences from S.baicalensis in NCBI,the conserved sequences of SvCLL-7,SvFNS ?-1 and SvFNS ?-2 in S.viscidula were cloned based on homological analysis.The results showed that the homology of the three key enzyme genes from S.baicalensis and S.viscidula was 99%,indicating that there were two flavonoid metabolic pathways in S.viscidula.3.The relative expression of CLL-7,FNS ?-1 and FNS?-2 from S.baicalensis and S.viscidula was determined by Real-time PCR.The results showed that the expression of the three key enzyme genes in roots was significantly higher than that in stems and leaves of the two species(P<0.05).Also,differences exist among the expression of the three key enzyme genes in different months of different parts.Specifically,the relative expression of three genes in S.baicalensis was greater than that of S.viscidula,indicating inter-species specificity.4.The correlation statistical analysis between the relative expression of genes and the content of baicalin at the same time during the growth of two species was conducted.The results showed that there were significant positive correlations between the three key enzyme genes and the content of baicalin.There was also a significant positive correlation between each gene.The correlation coefficients of CLL-7 and FNSII-2 in the two species were both very high,while the other correlation coefficients were low.The above results indicated that CLL-7 and FNSII-2 play an important role in the synthesis of baicalin.5.The cDNA sequence of the length with the encoding frame length 1518 bp of S.baicalensis was obtained by the combination of RACE and sequence cloning,compared with SbFNS?-2.Blast analysis found that the identity of the nucleotide and amino acid sequence was up to 99%.It contained three highly conserved regions of the heme combination region,proline rich region,and oxygen binding region.It belonged to the homologous sequence of the CYP93 family FNS?,and was named SvFNS?-2 and submitted to the NCBI gene bank(GenBank ID:MG737856).Bioinformatics analysis predicts that SvFNS?-2 gene can encode stable hydrophilic protein,which is an intracellular membrane-bound protein synthesized by free ribosomes.The secondary and tertiary structures of SvFNS?-2 gene are mainly composed of alpha helix.6.The phylogenetic analysis of SvFNS?-2 was performed based on the amino acid sequence by the software MEGE 5.1.Phylogenetic analysis suggested that SvFNS?-2 and SbFNS?-2 polymerize into the same branch.SbFNS?-1,PfFNS?and PbFNS also polymerize into the same branch.In addition,the two branches converge into a larger branch.In the phylogenetic tree,the same family is clustered into one branch,which is consistent with the traditional taxonomy.It provides a theoretical basis for biological evolution.The elongated branch length of SvFNS?-2 and SbFNS?-2 suggests accelerated evolution.Phylogenetic analysis suggests that SbFNS?-2 may have diverged from SbFNS?-1 following an early gene duplication event,and then differentiate into different metabolic pathways and evolve into different gene functions.In this study,the accumulations and changes of baicalin in different parts of S.baicalensis and S.viscidula during different growth stages were analyzed.The sequences SvFNS?-2 key enzyme genes related to the biosynthesis pathway of flavonoids in S.viscidula were cloned.The relationship between the gene expression and the baicalin content in the two species of baicalin synthesis pathway was preliminarily analyzed.These results will lay foundation for further study of the molecular mechanism of baicalin synthesis in S.baicalensis and the development and utilization of medicinal resources in the genus Scutellaria.