Identification and quality evaluation of germplasm resources of commercial Scutellaria baicalensis based on DNA barcode and HPLC / 中国中药杂志

Scutellaria baicalensis is a commonly used Chinese medicinal herb. In this study, we identified the germplasm resources of commercial S. baicalensis samples based on trnH-psbA, petA-psbJ, and ycf4-cemA sequences according to the available chloroplast genome sequencing results, and measured the content of baicalin by HPLC. Through the above means we determined the best DNA barcode that can be used to detect the germplasm resources and evaluate the quality of commercial S. baicalensis samples. A total of 104 samples were collected from 24 provinces, from which DNA was extracted for PCR amplification. The amplification efficiencies of trnH-psbA, petA-psbJ, and ycf4-cemA sequences were 100%, 59.62%, and 25.96%, respectively. The results of sequence analysis showed that 5, 4, and 2 haplotypes were identified based on trnH-psbA, petA-psbJ, and ycf4-cemA sequences, respectively. However, the sequences of haplotypes in commercial samples were different from that of the wild type, and the joint analysis of three fragments of S. baicalensis only identified 6 haplotypes. Furthermore, the phylogenetic analysis and genetic distance analysis indicated that trnH-psbA could be used to identify S. baicalensis from adulterants. The above analysis showed that trnH-psbA was the best fragment for identifying the germplasm resources of commercial S. baicalensis samples. We then analyzed the haplotypes(THap1-THap5) of commercial S. baicalensis samples based on trnH-psbA and found that THap2 was the main circulating haplotype of the commercial samples, accounting for 86.55% of the total samples, which indicated the scarce germplasm resources of commercial S. baicalensis samples. The content of baicalin in all the collected commercial S. baicalensis samples exceeded the standard in Chinese Pharmacopoeia and had significant differences(maximum of 12.21%) among samples, suggesting that the quality of commercial S. baicalensis samples varied considerably. However, there was no significant difference in baicalin content between different provinces or between different haplotypes. This study facilitates the establishment of the standard identification system for S. baicalensis, and can guide the commercial circulation and reasonable medication of S. baicalensis.

Cloning and temporal-spatial expression analysis of dfr gene from Scutellaria baicalensis with different colors / 生物工程学报

Dihydroflavanol-4-reductase (Dfr) is a key enzyme that regulates the synthesis of anthocyanin and proanthocyanidin in the flavonoid biosynthesis pathway. To investigate the difference of dfr gene in Scutellaria baicalensis Georgi with different colors in the same ecological environment, three complete full-length sequences of dfr gene were cloned from the cDNA of S. baicalensis with white, purple-red and purple colors using homologous cloning and RACE techniques. The three genes were named Sbdfr1, Sbdfr2 and Sbdfr3, respectively, and their corresponding structures were analyzed. The results showed that all three Dfr proteins have highly conserved NADPH binding sites and substrate-specific binding sites. Phylogenetic analysis showed that they are closely related to that of the known S. viscidula (ACV49882.1). Analysis of key structural domains and 3D models revealed differences in the catalytically active regions on the surface of all three Dfr proteins, and their unique structural characteristics may provide favorable conditions for studying the substrate specificity of different Dfr proteins. qRT-PCR analysis shows that dfr was expressed at different level in all tissues except the roots of S. baicalensis in full-bloom. During floral development, the expression level of dfr in white and purple-flowered Scutellaria showed an overall upward trend. In purple-red-flowered Scutellaria, the expression first slowly increased, followed by a decrease, and then rapidly increased to the maximum. This research provides a theoretical basis for further exploring the mechanism and function of Dfr substrate selectivity, and are of great scientific value for elucidating the molecular mechanism of floral color variation in S. baicalensis.