ABSTRACT
WRKY, a class of conserved transcription factors in plants, plays important roles in plant growth, development and secondary metabolism. In the present study, 65 WRKY members were identified from de novo transcriptome sequencing data of three different tissues (root, stems and leaves) of Baphicacanthus cusia. BcWRKY proteins contained from 221 to 706 amino acids and the isoelectric point is from 4.68 to 9.68. Molecular weights range from 25 711.8 to 75 475 Da. The main secondary structures of BcWRKYs protein are random coil. A subcellular localization prediction indicated that the putative BcWRKY proteins were enriched in the nuclear region. Phylogenetic analysis showed that BcWRKYs could be categorized into three groups and five subgroups (Group IIa, Group IIb, Group IIc, Group IId and Group IIe) in Group II. Structural analysis found that all BcWRKY proteins contained a highly conserved motif WRKYGQK. Finally, the transcriptional profiles of ten BcWRKY genes highly expressed in root, stem and leaf tissues under abscisic acid (ABA), methyl jasmonate (MeJA), or salicylic acid (SA) treatment were systematically investigated using qRT-PCR analysis. Results showed that a total of ten BcWRKY genes were differentially expressed in response to ABA, MeJA, and SA treatment. This work would be provided a basis for further elucidating the molecular mechanism of WRKY transcription factors in the biosynthesis of indole alkaloids in B. cusia.
ABSTRACT
Objective: To clone the tryptophan synthase gene named as BcTSB (GenBank accession number AYM45644.1) involved in the synthesis pathway of indole alkaloids from Baphicacanthus cusia, meanwhile, the bioinformatics analysis and expression analysis were also performed. Method: The open reading frame (ORF) of BcTSB gene was obtained by the database of prophase Baphicacanthus cusia transcriptome. The function of the BcTSB gene was preliminarily predicted by a series of bioinformatics tools. The entire protein-coding cDNA of BcTSB was cloned into the prokaryotic expression vector pET32a, then the recombinant plasmid was transformed into E. coli BL21 (DE3) cells, with IPTG induction. SDS-PAGE was used to investigate the situation of expression. The expression of the gene in root, stem and leaf was determined by using real-time PCR (qRT-PCR). Results: The open reading frame (ORF) of cloned BcTSB gene was 1 452 bp, and encoding 483 amino acids, it was predicted by bioinformatics analysis as hydrophilic protein, being located in the chloroplasts. Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 52 kDa, because prokaryotic expression vector pET32a contained 18 kDa label, SDS-PAGE results showed that a protein band at 70 000 was in consistent with molecular weight of the predicted protein. The QRT-PCR revealed that BcTSB gene was expressed in different tissues of B. cusia, the expression level of BcTSB in stems was much higher than that in roots and leaves. Conclusion: In this study, BcTSB gene of B. cusia was cloned and its expression was analyzed successfully, which laid an experimental foundation for further study on the function and regulation of the gene.
ABSTRACT
In this paper, through the collection and collation of ancient herbs, medical books and prescriptions, combined with modern literature, the historical changes of the name, origin, position, medicinal parts, collection, processing and processing of bluegrass were systematically combed and verified.It can be seen from the research that bluegrass was first used as medicine by the fruit, namely blueberry, which was originally Polygonum tinctorium. Since the Ming and Qing Dynasties, blueberry was rarely used, and it has been no longer used medicinally. In the Wei and Jin Dynasties, the medicinal parts extended to the stems and leaves, and most of them used juice as medicine.Since the Tang Dynasty, origin has been extended to Isatis indigotica, Baphicacanthus cusia, Indigofera tinctoria, Compositae plant Wulan, etc. In the Song Dynasty, the medicinal parts extended to the roots, and the "Banlangen" began to appear, and gradually became the main medicinal parts of blue medicinal materials, the main base of which was B. cusia. Since the Qing Dynasty, I. indigotica, a Cruciferae, has gradually become a genuine indigo root, while B. cusia has become a southern indigo root. It was the first mineral dye imported from abroad for thrush, and then used as medicine, also known as clam powder. Because it was found that it had the same effect with the extract of bluegrass, it was also named indigo naturalis in China, which has lasted till now. The main stream of Isatidis Folium in the past dynasties is the dry stem and leaf of Clerodendrum cyrtophylum. Since the Qing Dynasty, the stem and leaf of Isatis indigotica, P. tinctorium and other blue grasses have been gradually mixed as substitutes and gradually become the mainstream.
Subject(s)
China , Clerodendrum , Isatis , Medicine, Chinese Traditional , Plants, MedicinalABSTRACT
Chorismate synthase(CS, EC:4.2.3.5) catalyses 5-enolpyruvy-shikimate-3-phosphate to form chorismate, which is the essential enzyme for chorismate biosynthesis in organisms. The amino acid sequences of CS from 79 species of higher plants were reported in GenBank at present. 125 amino acid sequences of CS from Baphicacanthus cusia and other 78 species of plants were predicted and analyzed by using various bioinformatics software, including the composition of amino acid sequences, signal peptide, leader peptide, hydrophobic/hydrophilic, transmembrane structure, coiled-coil domain, protein secondary structure, tertiary structure and functional domains. The phylogenetic tree of CS protein family was constructed and divided into eight groups by phylogenetic analysis. The homology comparison indicated that B. cusia shared a high homology with several plants such as Sesamum indicum, Nicotiana tabacum, Solanum tuberosum and so on. The open reading frame(ORF) of all samples is about 1 300 bp, the molecular weight is about 50 kDa, the isoelectric point(pI) is 5.0-8.0 which illustrated that CS protein is slightly basic. The ORF of CS we cloned in B. cusia is 1 326 bp, the amino acid residues are 442, the molecular weight is 47 kDa and pI is 8.11. The CS in B.cusia showed obvious hydrophobicity area and hydrophilicity area, no signal peptide, and may exists transmembrane structure areas. The main secondary structures of CS protein are random coil and Alpha helix, also contain three main structural domains which are an active structural domain, a PLN02754 conserved domain and a FMN binding site. The acquired information in this study would provide certain scientific basis for further study on structure-activity relationship and structure modification of CS in plants in the future.
Subject(s)
Acanthaceae , Amino Acid Sequence , Computational Biology , Phosphorus-Oxygen Lyases , Chemistry , Phylogeny , Plant Proteins , Chemistry , Protein Structure, SecondaryABSTRACT
Baphicacanthus cusia ,widely distributed in the Southwest ,Southern and Eastern regions of China ,is an im-portant medicinal plant of Acanthaceae family .Indigo made from stem and leaf of Baphicacanthus cusia in Fujian has the best quality in China ,and is known as “Jian Indigo naturalis” ,which is the genuine medicinal of Fujian Province .The rhizoma of Baphicacanthus cusia could be used as medicine ,called“Nanbanlangen” ,which together with indigo were included in the“Chi-nese Pharmacopoeia” .Indigo and its original plant both contain indirubin ,which has anti-cancer activity .Indirubin is an active ingredient of Huangdai Pian and Danggui Longhui Wan ,two kinds of traditional Chinese medicine ,which have been successful-ly used in the treatment of malignancies such as chronic myelogenous leukemia .The international advances in the biological characteristics ,genetic diversity ,cultivation technology and molecular biology of Baphicacanthus cusia germplasm resources were summarized .The main problems in Baphicacanthus cusia germplasm resources research are indicated ,which could pro-vide references for the further study and application of Baphicacanthus cusia germplasm resources .
ABSTRACT
Objective To establish a RP-HPLC method for determining indigo and indirubin in Baphicacanthus cusia from different producing areas and medicinal parts. Methods The separation was achieved by an Agilent TC-C18 Column (4.6 mm×250 mm, 5 μm) at 25 ℃ using methanol-water (75??25) as mobile phase at a flow rate of 1 mL??min-1.The detection wavelength was 290 nm. Results Indigo had a good linear relationship with peak area at range of 0. 051 3-0.820 8 μg (r=0.999 3).The recovery rate was 99.00% and RSD was 1.30% (n=6).Indirubin had a good linear relationship with peak area at range of 0.049 5-0.792 0 μg (r=0.999 9).The recovery rate was 98.88% and RSD was 1.51% (n=6). Conclusion The contents of the two components are obviously different in Baphicacanthus cusia because of different places or medicinal parts. The proposed method is simple, rapid and reliable. This method for determination of indigo and indirubin in Baphicacanthus cusia by RP-HPLC provides a basis for quality control of Baphicacanthus cusia.