Exploring the optimization of nucleic acid extraction and amplification of thiamine biosynthesis genes from Malaysian seaweeds
Gene Extraction
Laboratory Analysis
Marine Biology
Walk along any Malaysian coastline, and you'll find marine gardens teeming with seaweeds—vibrant red Gracilaria and elegant brown Padina species dancing in the ocean currents.
These aren't just underwater decorations; they're nutritional powerhouses and potential solutions to global health challenges. But behind their humble appearance lies a genetic mystery that scientists are just beginning to unravel: how do these seaweeds produce thiamine, the essential vitamin B1 crucial for all living organisms? 1 6
Malaysia's coastal waters host diverse seaweed species with unique genetic profiles and nutritional value.
Extracting quality genetic material from seaweeds presents unique technical obstacles that require specialized approaches.
Thiamine, or vitamin B1, is far more than just another entry on nutrition labels. This essential molecule serves as a critical cofactor in central metabolic pathways including the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. 6
Thiamine deficiency can lead to severe conditions like beriberi, cardiovascular disease, and neurological disorders. 6
Plants with impaired thiamine biosynthesis show albino leaves and lethal phenotypes. 3
The THIC gene plays a particularly important role as it encodes the first enzyme in the pyrimidine branch of the thiamine biosynthesis pathway. 2 3 Understanding how this gene functions in seaweeds could provide crucial insights into the fundamental biology of these organisms and their remarkable nutritional profiles.
Extracting high-quality nucleic acids from seaweeds represents what many molecular biologists consider a notoriously difficult task. 1 6 The very nature of seaweeds creates perfect storm of technical challenges:
Seaweeds contain viscous polysaccharides like carrageenan and alginate that co-precipitate with nucleic acids during extraction. 6
These secondary metabolites oxidize during extraction, turning samples brown and degrading genetic material. 1
Unique to marine organisms, these compounds interfere with standard extraction protocols and purification methods. 6
The tough cell walls of seaweeds require vigorous disruption, which risks generating heat and further degrading delicate RNA molecules. 6
To tackle these challenges, researchers designed a comprehensive study focusing on two abundant but underutilized Malaysian seaweed species: the red seaweed Gracilaria sp. and the brown seaweed Padina sp., collected from the coastal waters of Port Dickson. 6
Researchers started with a commercial plant DNA extraction kit but introduced crucial modifications. Samples were ground to a fine powder in liquid nitrogen until they resembled white dust—ensuring complete cell disruption. 6
Scientists tested three different extraction methods with modifications: CTAB extraction buffer, guanidine thiocyanate, and sodium dodecyl sulphate (SDS). 1
With extracted nucleic acids in hand, researchers attempted to amplify two key genetic targets: the 18S rRNA gene and the THIC gene fragment specific to thiamine biosynthesis. 6
Coastal waters with rich marine biodiversity
The initial DNA extraction efforts yielded promising results, though with notable differences between the two seaweed species.
| Seaweed Species | DNA Concentration (μg/μl) | Purity (A260/A280 Ratio) | Successful 18S rRNA Amplification | Successful THIC Amplification |
|---|---|---|---|---|
| Gracilaria sp. (Red seaweed) | 240.22 | 2.11 | Yes | Yes |
| Padina sp. (Brown seaweed) | 242.51 | 2.12 | Yes | No |
Table 1: DNA Extraction Results from Malaysian Seaweeds 6
The purity ratios between 2.11-2.12 indicated high-quality DNA largely free from protein contamination, crucial for subsequent molecular applications. 6
With DNA extraction optimized, researchers turned to the more challenging task of RNA isolation.
| Extraction Method | Key Components | Effectiveness for Gracilaria sp. | Effectiveness for Padina sp. | Purity (A260/A280) |
|---|---|---|---|---|
| Method I | CTAB extraction buffer | Moderate | High | 2.0 |
| Method II | Guanidine thiocyanate | Low | Low | N/A |
| Method III | Sodium dodecyl sulphate (SDS) | High | Moderate | 1.8 |
Table 2: Effectiveness of Different RNA Extraction Methods for Seaweeds 1
| Research Aspect | Finding | Significance |
|---|---|---|
| THIC gene amplification from Gracilaria DNA | Successful at 53-57°C annealing temperature | Confirms presence of thiamine biosynthesis genes in red seaweeds |
| THIC gene amplification from Gracilaria cDNA | Successful | Demonstrates functional RNA extraction and reverse transcription |
| THIC gene amplification from Padina | Unsuccessful despite high DNA/RNA quality | Highlights fundamental differences between seaweed types |
| Putative TPP riboswitch in plants | Located at 3' UTR of mRNA | Reveals regulatory mechanism for thiamine biosynthesis 7 |
| Exogenous thiamine application | 5-fold decrease in ThiC expression | Confirms feedback regulation of thiamine pathway 7 |
Table 3: Key Experimental Findings in THIC Gene Research
Seaweed genetic research requires specialized reagents and techniques to overcome the unique challenges posed by these organisms.
Dissolves cell membranes, separates polysaccharides from nucleic acids. Particularly effective for brown seaweeds like Padina. 1
Denatures proteins, disrupts lipid membranes. Optimal for red seaweeds like Gracilaria. 1
Selective precipitation of RNA away from polysaccharides. Helps purify RNA from contaminating carbohydrates. 6
Binds polyphenols. Prevents oxidation and sample browning. 6
The successful optimization of nucleic acid extraction and amplification of thiamine biosynthesis genes from Malaysian seaweeds represents more than just a technical achievement—it opens new doors to understanding the nutritional potential of these marine resources.
While significant challenges remain, particularly for brown seaweeds like Padina, the research demonstrates steady progress toward fully characterizing the molecular machinery behind seaweed nutrition. 1 6
Improved nutritional profiles of cultivated seaweeds for human consumption
Development of natural thiamine supplements derived from marine sources
The journey to unravel seaweed genetics continues, with each wave bringing new discoveries from the shores of Malaysia to laboratories worldwide. As research continues, each extracted gene fragment brings us closer to harnessing the full potential of seaweeds—these remarkable organisms that stand at the intersection of marine biology, human nutrition, and sustainable agriculture.