NANNOCHLOROPSIS: sp. is a green alga that is widely used in the aquaculture industry as a feed in Malaysia, but genetic engineering studies of this alga are still underexplored even though there is a growing interest in microalgae genetic engineering for various industrial purposes. This study aims to investigate the efficiency of three transformation methods normally done on microalgae, namely polyethylene glycol (PEG), electroporation, and glass beads on Malaysian indigenous Nannochloropsis sp. using two commercially available plasmids, pUC19 and pGEM-T easy vector as well as an amplicon of ampicillin resistance (AMPR) gene. In this study, out of three transformation methods tested, positive transformants of Nannochloropsis sp. were successfully obtained via electroporation method. Further verification via polymerase chain reaction (PCR) and sequencing confirmed that the electroporation method was found to be the sole successful method in producing transgenic lines of our locally isolated Nannochloropsis sp. Results from this study proved the efficiency of electroporation for delivery of transgene to this green alga which has been reported to be tedious. The described method also provides the gateway for developing Nannochloropsis sp. as a delivery system to aquatic organism due to its importance in the industry.
Fatty acid profile from crude extracts of local sea cucumber Stichopus chloronotus was determined using gas chromatography (GC) technique. The extracts were prepared separately in methanol, ethanol, phosphate buffer saline (PBS), and distilled water as part of our study to look at the affinity of these solvents in extracting the lipid from sea cucumber. The PBS and distilled water extractions indicate water-soluble components, while the organic fractions are extracted in methanol and ethanol as organic solvents. Furthermore, water extraction is the conventional method practiced in Malaysia. In our analysis the C14:0 (myristic), C16:0 (palmitic), C18:0 (stearic), C18:2 (linoleic), C20:0 (arachidic), and C20:5 (eicosapentaenoic, EPA) were significantly different (p < 0.01) in the four solvent extractions. However, the PBS extraction contained a much higher percentage of EPA (25.69%) compared to 18.89% in ethanol, 7.84% in distilled water, and only 5.83% in methanol, and variances were significantly different (p < 0.01 ). On the other hand, C22:6 (docosahexaenoic acid or DHA) is much higher in water extraction (57.55%), in comparison to the others where only 3.63% in PBS and 1.20% in methanol, and this difference is significant at p < 0.01. No DHA was detected in ethanol extractions. Subsequently, C18:1 (oleic acid) was only detected in PBS (21.98%) and water extraction (7.50%). It is interesting that palmitic acid, C16:() was higher in methanol (20.82%) and ethanol (2.18%), while 12.55% was detected in PBS and only 2.20% in water extraction: and again this was significantly different at p < 0.01. Although our results have shown that all four solvents were different in terms of their ability to extract fatty acids, the major component for tissue repair was well preserved. Probably this is one of the important precocious steps when working with a delicate sea cucumber, in both experimental and/or at the preparative stages. Freshness of the sea cucumber samples is important when undertaking this type of experiment. Finally, we believe that the local sea cucumber S. chloronotus contains all the fatty acids required to play a potential active role in tissue repair.
Obtaining high-quality nucleic acid extracted from seaweeds is notoriously difficult due to contamination with polysaccharides and polyphenolic compounds after cell disruption. Specific methods need to be employed for RNA isolation in different seaweed species, and therefore studies of the thiamine biosynthesis pathway have been limited. Two selected Malaysian species which are highly abundant and underutilized, namely Gracilaria sp. and Padina sp., representing the red and brown seaweeds, respectively, were collected to develop optimized total RNA extraction methods. Prior to that, DNA was extracted, and amplification of the 18S rRNA gene and the THIC gene (encoding the first enzyme in the pyrimidine branch of the thiamine biosynthesis pathway) from the DNA template was successful in Gracilaria sp. only. RNA was then extracted from both seaweeds using three different existing methods, with some modifications, using cetyltrimethylammonium bromide, guanidine thiocyanate and sodium dodecyl sulphate. Methods I and III proved to be efficient for Padina sp. and Gracilaria sp., respectively, for the extraction of highly purified RNA, with A260/A280 values of 2.0 and 1.8. However, amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase and the THIC gene was successful in only Gracilaria sp. cDNA derived from extracted RNA. Further modifications are required to improve the exploitation of nucleic acids from brown seaweeds, which has been proven to be difficult. This work should pave the way for molecular studies of seaweeds generally and for the elucidation, specifically, of the thiamine biosynthesis pathway.
The oil palm (Elaeis guineensis) is an important crop in Malaysia but its productivity is hampered by various biotic and abiotic stresses. Recent studies suggest the importance of signalling molecules in plants in coping against stresses, which includes thiamine (vitamin B1). Thiamine is an essential microelement that is synthesized de novo by plants and microorganisms. The active form of thiamine, thiamine pyrophosphate (TPP), plays a prominent role in metabolic activities particularly as an enzymatic cofactor. Recently, thiamine biosynthesis pathways in oil palm have been characterised but the search of novel regulatory element known as riboswitch is yet to be done. Previous studies showed that thiamine biosynthesis pathway is regulated by an RNA element known as riboswitch. Riboswitch binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA. TPP binds specifically to TPP riboswitch to regulate thiamine biosynthesis through a variety of mechanisms found in archaea, bacteria and eukaryotes. This study was carried out to hunt for TPP riboswitch in oil palm thiamine biosynthesis gene. Riboswitch detection software like RiboSW, RibEx, Riboswitch Scanner and Denison Riboswitch Detector were utilised in order to locate putative TPP riboswitch in oil palm ThiC gene sequence that encodes for the first enzyme in the pyrimidine branch of the pathway. The analysis revealed a 192 bp putative TPP riboswitch located at the 3' untranslated region (UTR) of the mRNA. Further comparative gene analysis showed that the 92-nucleotide aptamer region, where the metabolite binds was conserved inter-species. The secondary structure analysis was also carried out using Mfold Web server and it showed a stem-loop structure manifested with stems (P1-P5) with minimum free energy of -12.26 kcal/mol. Besides that, the interaction of riboswitch and its ligand was determined using isothermal titration calorimetry (ITC) and it yielded an exothermic reaction with 1:1 stoichiometry interaction with binding affinities of 0.178 nM, at 30°C. To further evaluate the ability of riboswitch to control the pathway, exogenous thiamine was applied to four months old of oil palm seedlings and sampling of spear leaves tissue was carried out at days 0, 1, 2 and 3 post-treatment for expression analysis of ThiC gene fragment via quantitative polymerase chain reaction (qPCR). Results showed an approximately 5-fold decrease in ThiC gene expression upon application of exogenous thiamine. Quantification of thiamine and its derivatives was carried out via HPLC and the results showed that it was correlated to the down regulation of ThiC gene expression. The application of exogenous thiamine to oil palm affected ThiC gene expression, which supported the prediction of the presence of TPP riboswitch in the gene. Overall, this study provides the first evidence on the presence, binding and the functionality of TPP riboswitch in oil palm. This study is hoped to pave a way for better understanding on the regulation of thiamine biosynthesis pathway in oil palm, which can later be exploited for various purposes especially in manipulation of thiamine biosynthesis pathways in combating stresses in oil palm.