Psc-AFP from Psoralea corylifolia L. overexpressed in Pichia pastoris increases antimicrobial activity and enhances disease resistance of transgenic tobacco.

Psc-AFP, isolated from the seeds of Psoralea corylifolia L., is an antimicrobial protein with trypsin inhibitor activity. Its encoding gene was cloned by 3'- rapid amplification of cDNA ends (RACE) combined with Y-shaped adaptor-dependent extension (YADE) method. The gene Psc-AFP encodes a protein of 203 amino acids with a deduced signal peptide of 24 residues. The growth inhibition effect exerted by the heterologously expressed Psc-AFP in Pichia pastoris revealed that the recombinant Psc-AFP inhibited mycelium growth of Aspergillus niger, Rhizoctonia solani, and Alternaria brassicae and conidial germination of Alternaria alternata. The recombinant Psc-AFP also showed protease inhibitor activity manifested by the inhibition of trypsin. The transgenic tobacco bioassays confirmed that overexpressing Psc-AFP significantly enhanced the disease resistance of tobacco and that some of the transgenic lines were almost fully tolerant to Ralstonia solanacearum and A. alternata, whereas no apparent alteration in plant growth and development was observed. Collectively, these results indicate that the recombinant Psc-AFP is an active antimicrobial protein, with protease inhibitor activity that can be successfully produced in the yeast and tobacco and, therefore, maybe a potential antimicrobial candidate for practical use.

Selection and validation of reference genes for quantitative real-time PCR normalization in Psoralea corylifolia (Babchi) under various abiotic stress.

Psoralea corylifolia L. is a popular herb and has long been used in traditional Ayurvedic and Chinese medicine owing to its extensive pharmacological activities, especially in the treatment of various shin diseases. To date, the systematic evaluation and selection of the optimum reference genes for gene expression analysis of P. corylifolia were not reported. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a method for gene expression quantification. Selecting appropriate reference genes is the prerequisite for accurate normalization of RT-qPCR results. This study assessed the expression stability of 10 candidate reference genes under different abiotic stresses. First, amplification primers for RT-qPCR were designed and received testing and optimization. Then, expression data from each candidate gene were evaluated based on three statistical algorithms, and their results were further integrated into a comprehensive ranking based on the geometric mean. Additionally, one target gene, i.e., 1-aminocyclopropane-1-carboxylate oxidase (ACO), was used to validate the effectiveness of the selected reference. Our analysis suggested that thioredoxin-like protein YLS8 (YLS8), TIP41-like family protein (TIP41), and cyclophilin 2 (CYP2) genes provided superior expression normalization under different abiotic stresses. Overall, this work constitutes the first effort to select optimal endogenous controls for RT-qPCR studies of gene expression in P. corylifolia. It also provides a reasonable normalization standard and basis for further analysis of the gene expression of bioactive components in P. corylifolia.

Next generation DNA sequencing technology delivers valuable genetic markers for the genomic orphan legume species, Bituminaria bituminosa.

BACKGROUND: Bituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. B. bituminosa can be considered a genomic 'orphan' species with very few genomic resources available. New DNA sequencing technologies provide an opportunity to develop high quality molecular markers for such orphan species. RESULTS: 432,306 mRNA molecules were sampled from a leaf transcriptome of a single B. bituminosa plant using Roche 454 pyrosequencing, resulting in an average read length of 345 bp (149.1 Mbp in total). Sequences were assembled into 3,838 isotigs/contigs representing putatively unique gene transcripts. Gene ontology descriptors were identified for 3,419 sequences. Raw sequence reads containing simple sequence repeat (SSR) motifs were identified, and 240 primer pairs flanking these motifs were designed. Of 87 primer pairs developed this way, 75 (86.2%) successfully amplified primarily single fragments by PCR. Fragment analysis using 20 primer pairs in 79 accessions of B. bituminosa detected 130 alleles at 21 SSR loci. Genetic diversity analyses confirmed that variation at these SSR loci accurately reflected known taxonomic relationships in original collections of B. bituminosa and provided additional evidence that a division of the botanical variety bituminosa into two according to geographical origin (Mediterranean region and Canary Islands) may be appropriate. Evidence of cross-pollination was also found between botanical varieties within a B. bituminosa breeding programme. CONCLUSIONS: B. bituminosa can no longer be considered a genomic orphan species, having now a large (albeit incomplete) repertoire of expressed gene sequences that can serve as a resource for future genetic studies. This experimental approach was effective in developing codominant and polymorphic SSR markers for application in diverse genetic studies. These markers have already given new insight into genetic variation in B. bituminosa, providing evidence that a division of the botanical variety bituminosa may be appropriate. This approach is commended to those seeking to develop useful markers for genomic orphan species.

Characterization of isoflavone synthase gene from Psoralea corylifolia: a medicinal plant.

Isoflavones are known to possess medicinal properties and implicated in plant-pathogen interaction. We have for the first time isolated and functionally characterized an isoflavones synthase (IFS) gene from a traditionally acclaimed medicinal plant Psoralea corylifolia abundantly growing in tropical and subtropical regions. The IFS catalyzes the exclusive reaction of phenylpropanoid pathway in leguminous plants to produce isoflavones. The full-length cDNA (PcIFS) of the gene comprised 1,563 bp and putatively encodes a polypeptide of 520 amino acid residues. The gene is expressed ubiquitously although at varying levels in different parts of the plant. The expression analysis suggests that the gene is responsive to methyl jasmonate, salicylic acid and wounding. Overexpression of PcIFS in non-leguminous tobacco plant led to the accumulation of isoflavones in petal tissue, suggesting it a functional gene from P. corylifolia involved in isoflavones biosynthesis.

Amplified fragment length polymorphism and metabolomic profiles of hairy roots of Psoralea corylifolia L.

A reproducible protocol for establishment of hairy root cultures of Psoralea corylifolia L. was developed using Agrobacterium rhizogenes strain ATCC 15834. The hairy root clones exhibited typical sigmoid growth curves. Genomic and metabolomic profiles of hairy root clones along with that of untransformed control were analysed. Hairy root clones, Ps I and Ps II, showed significant differences in their amplified fragment length polymorphism (AFLP) profiles as compared to that of control, besides exhibiting Ri T-DNA-specific bands. These results amply indicate the stable integration of Ri T-DNA into the genomes of these clones. Further, the variations observed between clones in the AFLP profiles suggest the variable lengths and independent nature of Ri T-DNA integrations into their genomes. An isoflavonoid, formononetin, and its glycoside were present only in the hairy root clones while they were absent in the untransformed control. Variations observed in the metabolite profiles of these clones may be attributed to the random T-DNA integrations and associated changes caused by them in the recipient genomes. GC/MS analyses revealed the production of three and six clone-specific compounds in Ps I and Ps II, respectively, suggesting that the clones are dissimilar in their secondary metabolism. HPLC/UV-MS analyses disclosed substantial increases in the total isoflavonoids produced in Ps I (184%) and Ps II (94%) compared to untransformed control.

In vitro isolation, elicitation of psoralen in callus cultures of Psoralea corylifolia and cloning of psoralen synthase gene.

Psoralen, an important furanocoumarin occurring abundantly in seeds of Psoralea corylifolia is used as an anticancerous compound against leukemia and other cancer cell lines. Evaluation and isolation of psoralen from the calluses derived from different plant parts, viz. cotyledons, nodes, leaves and roots have been done in the present case for the first time. Amongst all, a maximum of 1934.75 µg/g f.w. of psoralen was recorded in callus derived from cotyledons, followed by 1875.50 and 1465.75 µg/g f.w. of psoralen in node and leaf derived calluses, respectively. Amount of psoralen enhanced further when cotyledonary calluses were exposed to different concentrations of organic elicitors (yeast extract, proline, inositol, casein hydrolyzate (CH), glycine, glutamine and sucrose) and precursors of psoralen (umbelliferone, cinnamic acid and NADPH). Isolation of psoralen was done using methanol as solvent through column chromatography and TLC. FT-IR and NMR further characterized and confirmed the structure of psoralen. In addition, the putative gene, psoralen synthase involved in psoralen synthesis pathway has been isolated, cloned and sequenced which comprised 1237 bp length. BLAST analysis of the gene sequence of psoralen synthase revealed that its nucleotide sequence showed 93% homology with psoralen synthase isolated from Ammi majus. This is the first report of isolation, cloning and characterization of psoralen synthase from Psoralea corylifolia.