Peng Di

Methyl jasmonate dramatically enhances the accumulation of phenolic acids in Salvia miltiorrhiza hairy root cultures

The aim of this work was to examine rosmarinic acid and its derivative lithospermic acid B accumulation, as well as related gene transcript and metabolite profiling in Salvia miltiorrhiza Bunge (Lamiacae) hairy root cultures, in response to methyl jasmonate (0.1 mM). Results showed methyl jasmonate dramatically enhanced both rosmarinic acid and lithospermic acid B accumulation, from approximately 3.25 to 6.02%, and 2.94 to 19.3% of dry weight, respectively. Meantime, several rosmarinic acid biosynthetic gene transcripts were coordinately induced, with phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, tyrosine aminotransferase, 4-hydroxyphenylpyruvate reductase and 4-hydroxyphenylpyruvate dioxygenase transcripts displaying the most rapid and substantial increases. Liquid chromatographic-tandem mass spectrometry was used to characterize the profile of metabolites involved in rosmarinic acid biosynthesis pathway, in both control and elicited-treated hairy root cultures. Further canonical correlation analysis constructed a gene-to-metabolite network, locating possible gene candidates which would directly link to phenolic acids (rosmarinic acid and lithospermic acid B) production, and thereby, would help to prompt the possibility of a key gene-based metabolic engineering for the synthesis of active pharmaceutical compounds in S. miltiorrhiza.

The c4h, tat, hppr and hppd Genes Prompted Engineering of Rosmarinic Acid Biosynthetic Pathway in Salvia miltiorrhiza Hairy Root Cultures

Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors.

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

BackgroundIsatis 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.ResultsAnalysis 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.ConclusionsNew 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.

Transgenic tetraploid Isatis indigotica expressing Bt Cry1Ac and Pinellia ternata agglutinin showed enhanced resistance to moths and aphids

Co-expression of multiple genes encoding different kinds of insect resistant proteins has been developed to confer a broader spectrum of pest control. Tetraploid Isatis indigotica Fort was transformed with a plasmid, p3300BP, containing Bacillus thuringiensisCry1Ac gene (Bt) and Pinellia ternata agglutinin gene (Pta) and the selectable marker herbicide resistance gene (Bar) driven by the CaMV35S promoter via Agrobacterium tumefaciens-mediated transformation. The integration and expression of introduced genes in regenerated transgenic plants were confirmed by PCR and Western blot assays. Insect bioassay test demonstrated transgenic lines had significant inhibition to diamondback moths (Plutella xylostella L.) and peach potato aphids (Myzus persicae Sulzer) simultaneously. Our study reported here would be a great motivation for field culture of tetraploid I. indigotica, also providing an efficient molecular breeding strategy to provide insect tolerant plants.