[Cloning and bioinformatic analysis and expression analysis of beta-glucuronidase in Scutellaria baicalensis].

The ß-Glucuronidase gene (sbGUS) cDNA firstly from Scutellari abaicalensis leaf was cloned by RT-PCR, with GenBank accession number KR364726. The full length cDNA of sbGUS was 1 584 bp with an open reading frame (ORF), encoding an unstable protein with 527 amino acids. The bioinformatic analysis showed that the sbGUS encoding protein had isoelectric point (pI) of 5.55 and a calculated molecular weight about 58.724 8 kDa, with a transmembrane regions and signal peptide, had conserved domains of glycoside hydrolase super family and unintegrated trans-glycosidase catalytic structure. In the secondary structure, the percentage of alpha helix, extended strand, ß-extended and random coil were 25.62%, 28.84%, 13.28% and 32.26%, respectively. The homologous analysis indicated the nucleotide sequence 98.93% similarity and the amino acid sequence 98.29% similarity with S. baicalensis (BAA97804.1), in the nine positions were different. The expression level of sGUS was the highest in root based on a real-time PCR analysis, followed by flower and stem, and the lowest was in stem. The results provide a foundation for exploring the molecular function of sbGUS involved in baicalcin biosynthesis based on synthetic biology approach in S. baicalensis plants.

Overexpression of cinnamate 4-hydroxylase and 4-coumaroyl CoA ligase prompted flavone accumulation in Scutellaria baicalensis hairy roots.

Scutellaria baicalensis Georgi, a species of the Lamiaceae family, is considered as one of the 50 fundamental herbs used in traditional Chinese medicine. In order to enhance flavone (baicalein, baicalin, and wogonin) content in S. baicalensis roots, we overexpressed a single gene of cinnamate 4-hydroxylase (C4H) and 4-coumaroyl coenzyme A ligase (4CL) using an Agrobacterium rhizogenes-mediated system. SbC4H- and Sb4CL-overexpressed hairy root lines enhanced the transcript levels of SbC4H and Sb4CL compared with those in the control and also increased flavones contents by approximately 3- and 2.5-fold, respectively. We successfully engineered the flavone biosynthesis pathway for the production of beneficial flavones in S baicalensis hairy roots. The importance of upstream gene C4H and 4CL in flavone biosynthesis and the efficiency of metabolic engineering in promoting flavone biosynthesis in S. baicalensis hairy roots have been indicated in this study.

Molecular cloning and characterization of tyrosine aminotransferase and hydroxyphenylpyruvate reductase, and rosmarinic acid accumulation in Scutellaria baicalensis.

Rosmarinic acid (a-O-caffeoyl-3,4-dihydroxyphenylacetic acid, RA) is a caffeoyl ester widely distributed in plants. cDNA clones encoding tyrosine aminotransferase (TAT1 and 2) and hydroxyphenylpyruvate reductase (HPPR) have been isolated from Scutellaria baicalensis. The open reading frames (ORFs) of SbTAT1 and 2 were 1230 and 1272 bp long and encoded 409 and 423 amino acid residues, respectively. HPPR corresponded to a 942-bp ORF and 313 amino acid residues of translated protein. To study the molecular mechanisms of TAT and HPPR and investigate RA accumulation in S. baicalensis, we examined the transcript levels of TAT isoforms and HPPR with quantitative real-time PCR and analyzed the RA content in different organs by using high-performance liquid chromatography. The transcript levels of SbTATI SbTAT2, and SbHPPR in the flowers were higher than those in other organs. RA was also highly accumulated in the flowers and with a trace amount in the roots. No RA was detected in the leaves and stems of S. baicalensis. The amount of accumulated RA in the flowers was 28.7 times higher than that in the roots. Our results will be helpful in elucidating the mechanisms of RA biosynthesis in S. baicalensis.

Cloning and characterization of a cDNA encoding calcium/calmodulin-dependent glutamate decarboxylase from Scutellaria baicalensis.

Gamma-aminobutyric acid (GABA), synthesized by glutamate decarboxylase (GAD), plays an important role in plants. To study the molecular mechanism of GAD regulation and to examine the levels of GABA in Scutellaria baicalensis, we isolated cDNA clones (SbGAD1 and 2) encoding GAD from S. baicalensis. The open reading frames of SbGADI and 2 were 1,503 and 1,494 bp long and had 450 and 497 amino acid residues, respectively. Quantitative real-time RT-PCR analysis was performed to show the variation of transcript levels among different organs of S. baicalensis. Transcript levels of SbGAD1 and 2 were highest in the root and flower, respectively. The GABA content of different parts (ranked in descending order) was as follows: leaf > flower > stem > root. We concluded that the expression pattern of SbGAD1 and 2 did not match the accumulation pattern of GABA in different organs. We presume that GABA biosynthesis might be more controlled by SbGAD2 than SbGADI. These data will aid in future studies that seek to understand the mechanisms underlying GABA biosynthesis, an important amino acid that is synthesized by the GAD enzyme. To explain adequately the GABA biosynthesis mechanisms in S. baicalensis, the enzyme activities of SbGAD1 and 2 should be determined in the near future.

Different extraction pretreatments significantly change the flavonoid contents of Scutellaria baicalensis.

CONTEXT: Scutellaria baicalensis Georgi (Labiatae) is one of the most commonly used medicinal herbs, especially in traditional Chinese medicine. However, compared to many pharmacological studies of this botanical, much less attention has been paid to the quality control of the herb's pretreatment prior to extract preparation, an issue that may affect therapeutic outcomes. OBJECTIVE: The current study was designed to evaluate whether different pretreatment conditions change the contents of the four major flavonoids in the herb, i.e., two glycosides (baicalin and wogonoside) and two aglycones (baicalein and wogonin). MATERIALS AND METHODS: A high-performance liquid chromatography assay was used to quantify the contents of these four flavonoids. The composition changes of four flavonoids by different pretreatment conditions, including solvent, treatment time, temperature, pH value and herb/solvent ratio were evaluated. RESULTS: After selection of the first order time-curve kinetics, our data showed that at 50 °C, 1:5 herb/water (in w/v) ratio and pH 6.67 yielded an optimal conversion rate from flavonoid glycosides to their aglycones. In this optimized condition, the contents of baicalin and wogonoside were decreased to 1/70 and 1/13, while baicalein and wogonin were increased 3.5- and 3.1-fold, respectively, compared to untreated herb. DISCUSSION AND CONCLUSION: The markedly variable conversion rates by different pretreatment conditions complicated the quality control of this herb, mainly due to the high amount of endogenous enzymes of S. baicalensis. Optimal pretreatment conditions observed in this study could be used obtain the highest level of desired constituents to achieve better pharmacological effects.

[Physiological ecology responses of Scutellaria baicalensis to drought and rewatering].

To study the physiological ecology responses of Scutellaria baicalensis to drought and rewatering of short period, we tested and analyzed photosynthesis and chlorophyll fluorescence parameters of S. baicalensis leaves processed by different ways of water treatment in drought and rewatering period, characteristic indexes of physiology and biochemistry of root SOD, POD, PAL, C4H, etc. and accumulation dynamic change of root baicalin and baicalein. The result showed that along with the worsening drought, P(n), T(r), G(s) and F(v)/F(m) of S. baicalensis declined in different water supply, and F(o) increased. The response of SOD and POD's activity in S. baicalensis root to drought in I and II was earlier than it in III. The response time and increase range of baicalin accumulation existed differences in different water supply, and the indexes regained after rewatering. Therefore, photosynthesis of S. baicalensis changed and it destroyed the antioxidant metabolism balance when soil water content decreased resulting from drought. The synergistic effect of defence mechanism launched by S. baicalensis, SOD, POD, PAL, C4H, baicalin and baicalein reduced active oxygen's damage to the cell.

[Effect of water deficit on gene expression of enzymes related with hydrogen peroxide detoxification system in Scutellaria baicalensis].

OBJECTIVE: To analysis the effects of water deficit on the transcript level of SOD, APX, DHAR and MDHAR genes in Scutellaria baicalensis. METHOD: Three-month-old S. baicalensis was in glasshouse under water deficit stress, and the transcript level of SOD, APX, DHAR and MDHAR genes were analysis utilized semi-quantitative RT-PCR. RESULT: Compared with the control group, a significant decline of the transcriptional level of APX gene was observed at 70 days after water deficit. The transcript level of DHAR gene was reduced at 30 and 50 days after water deficit. And MDHARI gene was significant declined at 50 days. CONCLUSION: AsA which is an important antioxidant plays a major role in hydrogen peroxide clear system under water deficit, and maybe have an antagonistic effect to the accumulation of baicalein.

Flavonoids and antioxidative enzymes in temperature-challenged roots of Scutellaria baicalensis Georgi.

The active compounds in the roots of Scutellaria baicalensis Georgi, a traditional Chinese medicinal plant, are mainly flavonoids which have anti-inflammatory, antitumour, and anti-HIV activity, respectively. The increasing annual average temperature has rendered the S. baicalensis plants grown in some ancient producing regions no longer suitable for their medicinal usage. Hydrogen peroxide plays an important role in root responses to abnormal temperature in S. baicalensis. Baicalin and baicalein and antioxidative enzymes were anticipated to detoxify H2O2 in S. baicalensis. Here, we show that abnormal temperatures (10 and 40 degrees C) decreased the content of flavonoids as compared with the normal temperature (30 degrees C), and the transcripts of UDP-glucuronate:baicalein 7-O-glucuronosyltransferase and beta-glucuronidase involved in the interconversion between baicalin and baicalein were affected by the 40-degrees C treatment. High temperature also increased the activities of catalase and peroxidase. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the transcript levels of peroxidase 2, peroxidase 3, monodehydroascorbate reductase 2, and dehydroascorbate reductase were significantly increased under high-temperature conditions. The respective genes would be candidates for improvement of the adaptation of S. baicalensis plants to abnormal temperatures and for regulation of the contents of the active compounds.

[Correlation between H2O2 scavenging system and flavonoids accumulation of Scutellaria baicalensis].

OBJECTIVE: Study on correlation between H2O2 scavenging system and flavonoids accumulation of Scutellaria baicalensis. METHOD: The content of baicalin and baicalein in suspension cell of S. baicalensis was determined by HPLC. The content of total flavonoids and H2O2, the activity of POD and PAL was detected by UV spectrophotometry. RESULT: The content of total flavonoid and the activity of PAL increased significantly in 12 days after 40 degrees C, dark and PEG stress. Around 12 days after NPA, NPA +40 degrees C, 40 degrees C, NPA + dark, dark and PEG stress, the content of baicalin declined and the content of baicalein rise, the activity of POD showed an increasing trend, and level of H2O2 remain stable. CONCLUSION: Moderate environmental stress could promote the accumulation of total flavonoids in S. baicalensis, baicalin convert to baicalein by POD, and maintaining the stability of H2O2 content to avoid oxidative damage.

[Preliminary functional analysis of intron in chalcone synthase gene from Scutellaria baicalensis].

OBJECTIVE: To study the function of the chalcone synthese gene introns in Scutellaria baicalensis, and clarify preliminarily their role in abiotic stress. METHOD: The CHS introns with specific primers were cloned and bioinformatic method was applied to predict the cis-elements in the intron of CHS. The introns were subcloned into binary vector, pCAMBIA-1301 before being transferred to tobacco. Then the activity of GUS of the transgenic tobacco seeds was analyzed. RESULT: Seven cis-elements were found in the introns. Under the dark and high temperature GUS expression rose at the first (3 h), but then declined (9 h). ABA and MeJA regulated insignificantly the GUS activity in normal temperature; treatment of 10% PEG induced GUS expression. CONCLUSION: CHS introns could be play a role in the regulation of S. baicalensis phenylpropanoid biosynthetic pathway.