Sesquiterpenes are a three-isoprene unit compounds which belong to terpenoid family of secondary metabolites. These
volatile compounds are one of the major constituents of essential oils in plants and plays major roles in plant signaling
of defense mechanism. The effects of methyl jasmonate (MeJA) concentrations (100 and 200 μM) on the production
of sesquiterpene compounds after incubation period for 1, 3, and 6 days were investigated in Persicaria minor cell
suspension culture. Headspace Solid-Phase Microextraction (HS-SPME) method was used to absorb volatile compounds
from suspension cells and liquid medium. They were then analyzed through Gas Chromatography-Mass Spectrometry
(GC-MS) to identify sesquiterpene compounds. Among the 15 sesquiterpene compounds identified, α- muurolene was found
in significantly higher concentration in all MeJA treated cultures. The results showed that α-muurolene was detected in
the suspension cells at the highest peak area of 14.17% at 100 μM MeJA treated cultures with 3-day incubation. Analysis
of liquid medium of the treatments identified secretion of α-muurolene into the culture medium, with total peak area of
0.72%. These results showed that sesquiterpene production in MeJA induced P. minor suspension culture depended on
the MeJA concentration and also culture incubation period.
The aim of this study was to establish an effective protocol for callus induction from the seed explants of Solanum dubium and to investigate the callus extract ability in milk clotting activity. The effects of growth regulator, basal media strength and sucrose were studied using different concentrations (0.5, 1.0, 1.5, and 2.0 mg/L) of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthalene acetic acid (NAA) and 2,4-dichlorophenoxy-acetic acid (2,4-D) alone or in combination with 0.5 mg/L of 6-benzylamino purine (BAP). For milk clotting activity, about 50 or 100 μL extracts of seed callus was mixed with 2 mL 50% milk held at 55°C for 5 and 10 min until milk clotting occurred. The results showed that NAA alone or in combination with BAP gave a higher callusing percentage (80 to100%) compared to the other plant growth regulators at the same concentrations. When an auxin was supplied in combination with BAP, a significant increase in callusing percentage or degree of callusing was observed. The time required for callus to be developed was shortened and the quality of the induced callus improved. An increase in callus growth in low sucrose (10 g/L) concentration was found be comparable (88%) to high sucrose concentration (30 g/L; 60%). Crude extracts obtained from S. dubium callus were shown to exhibit milk clotting activity.
This study employed several RNA extraction methods for mangosteen pericarps prior to RNA sequencing. The sequencing platform heavily relies on a high quality RNA yield. However, pericarp tissues contain a lot of phenolic compounds that results in low RNA quality. Hence, we studied several RNA extraction methods to obtain the most suitable method for the best RNA quality from the pericarps of mangosteen. Five different methods including Lopez and Gomez, modified hexadecyltrimethyl ammonium bromide (CTAB) method, several commercial kits from TranszolUP, Favorgen and Qiagen RNeasy were compared. By optimising the CTAB method, it was found to be the best method to obtain pure RNA (high A260/A280 ratio) with the highest yields (up to approximately 600-800 ng/μL concentration). The QC control of these samples using bioanalyzer validated their suitability for the downstream RNA sequencing. This report details the method for extracting high quality and high yield RNA samples from fruit that are rich in polyphenolic compounds such as mangosteen.
Microbial production of natural products using metabolic engineering and synthetic biology approaches often involves
the assembly of multiple gene fragments including regulatory elements, especially when using eukaryotes as hosts.
Traditional cloning strategy using restriction enzyme digestion and ligation are laborious and inflexible owing to the
high number of sequential cloning steps, limited cutting sites and generation of undesired ‘scar’ sequences. In this study,
a homology-based isothermal DNA assembly method was carried out for one-step simultaneous assembly of multiple DNA
fragments to engineer plant phenylpropanoid biosynthesis in Saccharomyces cerevisiae. Rapid construction of yeast
plasmid harboring dual gene expression cassettes was achieved via isothermal assembly of four DNA fragments designed
with 20 bp overlapping sequences. The rate-limiting enzyme of phenylpropanoid pathway, cinnamate 4-hydroxylase
encoded by C4H gene from Polygonum minus was cloned in tandem with yeast promoter and terminator elements of S.
cerevisiae for efficient construction of phenylpropanoid biosynthetic pathway in recombinant yeast. The assembled pAGCAT (C4H-ADH1t-TEF1p) shuttle plasmid and transformation of S. cerevisiae with the plant C4H gene were confirmed
via PCR analysis. Based on these findings, the yeast shuttle plasmid harboring P. minus phenylpropanoid biosynthesis
gene was efficiently constructed to be the starting platform for the production of plant natural products in geneticallyengineered S. cerevisiae.