[Cloning and expression analysis of a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferasegene(IiHCT) from Isatis indigotica].

Based on the transcriptome data, we cloned the open reading frame of IiHCT gene from Isatis indigotica, and then performed bioinformatic analysis of the sequence. Further, we detected expression pattern in specific organs and hairy roots treated methyl jasmonate( MeJA) by RT-PCR. The IiHCT gene contains a 1 290 bp open reading frame( ORF) encoding a polypeptide of 430 amino acids. The predicted isoelectric point( pI) was 5.7, a calculated molecular weight was about 47.68 kDa. IiHCT was mainly expressed in stem and undetectable in young root, leaf and flower bud. After the treatment of MeJA, the relative expression level of IiHCT increased rapidly. The expression level of IiHCT was the highest at 4 h and maintained two fold to control during 24 h. In this study, cloning of IiHCT laid the foundation for illustrating the biosynthesis mechanism of phenylpropanoids in I. indigotica.

The dirigent multigene family in Isatis indigotica: gene discovery and differential transcript abundance.

BACKGROUND: Isatis indigotica Fort. is one of the most commonly used traditional Chinese medicines. Its antiviral compound is a kind of lignan, which is formed with the action of dirigent proteins (DIR). DIR proteins are members of a large family of proteins which impart stereoselectivity on the phenoxy radical-coupling reaction, yielding optically active lignans from two molecules of E-coniferyl alcohol. They exist in almost every vascular plant. However, the DIR and DIR-like protein gene family in I. indigotica has not been analyzed in detail yet. This study focuses on discovery and analysis of this protein gene family in I. indigotica for the first time. RESULTS: Analysis of transcription profiling database from I. indigotica revealed a family of 19 full-length unique DIR and DIR-like proteins. Sequence analysis found that I. indigotica DIR and DIR-like proteins (IiDIR) were all-beta strand proteins, with a signal peptide at the N-terminus. Phylogenetic analysis of the 19 proteins indicated that the IiDIR genes cluster into three distinct subfamilies, DIR-a, DIR-b/d, and DIR-e, of a larger plant DIR and DIR-like gene family. Gene-specific primers were designed for 19 unique IiDIRs and were used to evaluate patterns of constitutive expression in different organs. It showed that most IiDIR genes were expressed comparatively higher in roots and flowers than stems and leaves. CONCLUSIONS: New DIR and DIR-like proteins were discovered from the transcription profiling database of I. indigotica through bioinformatics methods for the first time. Sequence characteristics and transcript abundance of these new genes were analyzed. This study will provide basic data necessary for further studies.

[Accumulation law of epigoitrin in roots of Isatis indigotica of different breed types].

OBJECTIVE: To study the distribution law of epigoitrin in roots of Isatis indigotica of different breed types and provide a scientific basis for screening of high-quality Isatis indigotica breed. METHODS: Determined the contents of epigoitrin in tap root and lateral root of Chinese-cabbage-leaf Isatis, cabbage-leaf Isatis, mustard-leaf Isatis and tetraploid Isatis by HPLC. Also, compared the contents of epigoitin in xylem and phloem of Isatis indigotica. RESULTS: Contents of epigoitrin in the tap root and lateral root of Isatis indigotica of the different breed types were significant different. In four breed types of Isatis indigotica, contents of epigoitrin in the tap root and phloem were higher than those in the lateral root and xylem, respectively. CONCLUSION: Contents of epigoitrin in the lateral root of Isatis indigotica are higher than those of tap root and epigoitrin distributes mainly in phloem.

[Discussion on the botanical origin of Isatidis radix and Isatidis folium based on DNA barcoding].

This paper aimed to investigate the botanical origins of Isatidis Radix and Isatidis Folium, and clarify the confusion of its classification. The second internal transcribed spacer (ITS2) of ribosomal DNA, the chloroplast matK gene of 22 samples from some major production areas were amplified and sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. Phylogenetic study was performed using MEGA 4.0 software in accordance with the Kimura 2-Parameter (K2P) model, and the phylogenetic tree was constructed using the neighbor-joining methods. The results showed that the length of ITS2 sequence of the botanical origins of Isatidis Radix and Isatidis Folium was 191 bp. The sequence showed that some samples had several SNP sites, and some samples had heterozygosis sites. In the NJ tree, based on ITS2 sequence, the studied samples were separated into two groups, and one of them was gathered with Isatis tinctoria L. The studied samples also were divided into two groups obviously based on the chloroplast matK gene. In conclusion, our results support that the botanical origins of Isatidis Radix and Isatidis Folium are Isatis indigotica Fortune, and Isatis indigotica and Isatis tinctoria are two distinct species. This study doesn't support the opinion about the combination of these two species in Flora of China.

[Study on the n-BuOH extract's fingerprint chromatography and bacteriostatic activity of Radix Isatis and correlation analysis].

The fingerprint chromatography and bacteriostatic activity of the n-BuOH extract of Radix Isatis were investigated. The chemical differemce in Radix Isatis from various sources was probed with system cluster analysis and it was co-researched with the bacteriostatic activity using correlation analysis in order to find the baterio static substances in Radix Isatis.

[Study on the optimal extraction process of polysaccharide from Baphicacanthus cuaia (Nees) Bremek with the uniform design].

OBJECTIVE: To study the optimal extraction process of polysaccharide from Baphicacanthus cuaia (Nees) Bremek. METHODS: The optimal extraction process was selected with uniform design and statistical analysis system (SAS). The content of polysaccharide in the extract was determined to investigate the influence of temperature of extraction, the time of extraction and the solid-liquid ratio on it. RESULTS: The optimal extraction process was extraction temperature 90 degrees C with 2 hours and with 1:50 (g/ml) of Baphicacanthus cuaia (Nees) Bremek weight to distilled water. The optimum extraction rates of Polysaccharide from Baphicacanthus cuaia (Nees) Bremek and Isatis indigotica Fort were 3.26% and 12.76%.

[Quality evaluation of indigowoad root and leaf by chemical pattern recognition].

27 samples of indigowoad root and 5 samples of indigowoad leaf from different species and in different habital were collected. The chemical constituents were analyzed by HPLC, and ten original chemical features as a whole obtained. Meanwhile, extrinsic bacteriostasis experiments were carried out for each of the 32 samples. Canonical correlation analysis was applied to the intercorrelation between the chemical and the pharmacologic data, and five chemical features were found to be the effective constituents. In the end, the 32 samples were divided into five classes by the fuzzy clustering technique ISODATA(interachive self-organizing data analysis technique A).

[Quantitative analysis of 4(3)-quinazolinedione in 4 "daqingye", leave of Isatis indigotica, Baphicacanthus cusia, Polygonum tinctorium and Clerodendron cyrtophyllum].

Using 4(3)-quinazolinedione, the active constituent of "Daqingye", as the quality control criteria, 4 samples of the herbal drug collected from Isatis indigotica, Baphicacanthus cusia, Polygonum tinctorium and Clerodendron cyrtophyllum were comparetively analyzed by RP-HPLC. The results showed that the average 4(3)-quinazolinedione contents (microgram/g) were 30.7, 7.2, 4.4 and 27.5 respectively. This method was proved to be quick, simple and reproducible, and provided a reliable basis for the quality control and evaluation of Chinese Traditional Drug "Daqingye".