Total RNA Extraction for the Red Seaweed Gracilaria changii (Gracilariales, Rhodophyta)

Five different RNA extraction methods have been tried out on the red seaweed, Gracilaria changii collected from the mangrove area at Morib, Selangor, Malaysia. Two methods, one utilising guanidinium thiocyanate, and another using cetyltrimethyl ammonium bromide (CTAB), were found to be potential alternatives to obtain pure RNA. By incorporating sand while grinding the tissue, the method using CTAB was found most suitable to obtain pure RNA (high A260:280nm ratio) with high yield (0.16ug RNA per gram of fresh tissue)

Optimisation of RNA Extraction from Gracilaria changii (Gracilariales, Rhodophyta)

RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65-1.14 microg.g^-1 fresh weight) with high quality (A260:280 ratio 1.80-2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies

Profiling the differentially expressed genes in two rice varieties during rapid grain-filling stages

Grain filling is an important agronomic trait, which directly affects the final yield of rice. Partially filled and empty rice grains are among the factors that limit the yield of MR219, one of the highest yielding rice varieties in Malaysia. In this study, the NSF 20 K rice oligonucleotide array, which contains 20,000 70-mer oligonucleotide probes, was used for direct comparison of the transcriptomes of MR219 and MR84 (a rice variety that has higher percentage of filled grains compared to MR219), during rapid grain-filling period at 5 and 10 days after fertilization (DAF). A total of 155 and 233 genes were differentially expressed in MR219 compared to MR84 at 5 and 10 DAF, respectively; and 9 of these expression ratios were tested using quantitative real-time RT PCR. Among the differentially expressed genes identified were those encoding hexokinase, various sugar transporters, GSDL-like lipase/acylhydrolase, brassinosteroid-insensitive 1-associated receptor kinase 1 precursor and homeobox protein GLABRA2, which were analyzed by real-time RT PCR in this study. The differences demonstrated by these genes in their transcript levels and profiles, between the two rice varieties understudied at different stages of grain filling may contribute to the formulation of hypotheses toward the understanding of poor percentage of filled grains in MR219

Selection of reference genes for transcript profiling of Sargassum polycystum by quantitative real-time polymerase chain reaction

Sargassum species are one of the major alginate-producing seaweed species in Asian countries. Alginate is widely used in food, feed, pharmaceutical and medical industries as thickening and stabilizing agents. To establish a set of consistently expressed genes as reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) studies of Sargassum polycystum (Fucales, Ochrophyta) in samples collected at two distinct time points from the field, four candidate reference genes, namely ribosomal protein L3 (RPL3), ribosomal protein S15 (RPS15), alphatubulin (α-TUB) and eukaryotic translation elongation factor 1 alpha (TEF1α), were analyzed using geNorm and NormFinder. The results showed that RPL3, α-TUB and TEF1α were the most stable genes using both programs, whereas RPS15 gene was shown to be the least stable. Identification of stably expressed reference genes is crucial for qRT-PCR studies to allow accurate quantification of target gene expression levels. In addition, the expression of key enzyme in the final step of alginate biosynthesis pathway mannuronan C5 epimerase-SP01411 (MC5E-SP01411) and mannuronan C5 epimerase-SP02271 (MC5E-SP02271) were differentially expressed in the seaweeds collected at two distinct time points from the field. To our knowledge, this is the first report on validation of reference genes for any Sargassum species. Our data provide a basis for the selection of reference genes for future biological research in related studies

Brassinosteroid insensitive 1-associated kinase 1 (OsI-BAK1) is associated with grain filling and leaf development in rice

Brassinosteroid Insensitive 1 (BRI1)-Associated Kinase I (BAK1) has been reported to interact with BRI1 for brassinosteroid (BR) perception and signal transduction that regulate plant growth and development. The aim of this study is to investigate the functions of a rice OsBAK1 homologue, designated as OsI-BAK1, which is highly expressed after heading. Silencing of OsI-BAK1 in rice plants produced a high number of undeveloped green and unfilled grains compared to the untransformed plants. Histological analyses demonstrated that embryos were either absent or retarded in their development in these unfilled rice grains of OsI-BAK1 RNAi plants. Down regulation of OsI-BAK1 caused a reduction in cell number and enlargement in leaf bulliform cells. Furthermore, transgenic rice plants overexpressing OsI-BAK1 were demonstrated to have corrugated and twisted leaves probably due to increased cell number that caused abnormal bulliform cell structure which were enlarged and plugged deep into leaf epidermis. The current findings suggest that OsI-BAK1 may play an important role in the developmental processes of rice grain filling and leaf cell including the bulliform cells

Alternative strategy in crop protection: protease inhibitors from turmeric

In an effort to meet the increasing demand for food arising from the growing human population, it is important to ensure food security by maintaining the continual supply of crop products and increase their productions. However, crops plantations are often challenged by the presences of pest insects and pathogens that could inflict diseases or feed on the crop plants and lead to massive losses in the crop productions. While chemical pesticides are commonly employed to control pest insects and pathogens problems, it is often associated with numerous negative side effects and excessive usage would cause lasting detrimental effects to the environments and consumers. Alternatively, crop plants with improved traits were produced through the application of biotechnology techniques to provide phytoprotection against pest insects and pathogens. Genes that encode for natural plant defence products, such as protease inhibitors, are genetically engineered into the crop plants and it is reported to be effectively showing insecticidal and anti-pathogenic properties. For this purpose, it is crucial to constantly discover uncharacterized protease inhibitors from novel sources as candidate for phytoprotection as this helps to overcome the adaptation and resistance buildup by the pest insects and pathogens. Turmeric plant is a well-known herbal plant commonly used as traditional medicine and it acts as a suitable novel source for discovery of protease inhibitors. As turmeric’s secondary metabolites are reported to exhibit a wide range of medicinal properties, it could be contributed by protease inhibitors which possessed high anti-pathogenic and inhibitory properties

RNA-sequencing of methyl-jasmonate treated turmeric (Curcuma longa) reveals novel protease inhibitor transcripts

Turmeric (Curcuma longa) has long been known in Southeast Asia as a medicinal plant and been used as folk remedies to treat minor illnesses like diarrhea or skin diseases. Recent studies on turmeric have shown numerous pharmacological properties such as anti-oxidant, anti-inflammatory, anti-pathogenic including anti-viral protease activities. All these beneficial properties make turmeric a suitable candidate for the discovery of novel protease inhibitors (PIs). PIs are commonly found in all organisms to regulate biological processes. In plant, PIs are reported to play important roles in plant defense mechanism. A number of these PIs have been genetically engineered into crop plants to enhance protection against microorganism and pest insects. However over time, the pathogens and pest insects are slowly adapting to the current strategies and overcome the additional defense barrier. Hence, this study was conducted to identify novel PIs genes from methyl jasmonate (MeJA)-treated turmeric plants through the whole transcriptome sequencing approach. From the raw data reads obtained from the RNA-sequencing of MeJA-treated leaf tissues, a single reference transcriptome was assembled de novo using Trinity software. A total of 105,529 contiguous sequences were obtained. Sequence annotation and homology search were performed onto several protein databases such as Uniprot, Pfam, GO and KEGG which resulted in around 50% of the transcripts showed similarity hits to known proteins. The individual expression profile of the transcripts from the control and MeJA-treated turmeric samples were generated and compared in order to identify differential expressed genes. A total of 4274 transcripts had been identified to be differentially expressed where there were 1599 upregulated transcripts and 2715 downregulated transcripts. A total of 21 transcripts showed sequence similarity hits to PIs families and three of the transcripts were identified to be upregulated from the MeJA treatment. These identified PIs transcript can serve as candidate genes for further functional studies and applications

Isolation and characterization of floral transcripts from mangosteen (Garcinia mangostana L.)

The understanding of flower initiation, development, and maturation in mangosteen is of paramount importance to shorten its long juvenile phase and to synchronize its flowering or fruiting time. In this study, we have identified 97 tentative unique genes with higher expression levels in young flower buds compared to young shoots by using suppressive subtraction hybridization and reverse northern analysis. Sequence analysis showed that 63.9% of these transcripts had non-significant matches to sequences in the non-redundant protein database in Gen- Bank, 19.6% had significant matches to unknown proteins while the remaining 16.5% had putative functions in transcription, stress, signal transduction, cell wall biogen-esis, photosynthesis and miscellaneous. The full-length cDNA of GmAGMBP encoding AG-motif binding protein(a zinc finger transcriptional factor), and 3 0 termini cDNA sequences of GmHSA32 and GmBZIP, encoding heat-stress-associated 32 (HSA32) and bZIP transcription factor,respectively; were cloned and further analysed. Real-time PCR analysis revealed that these three genes have different transcript profiles in flowers of different developmental stages and young shoots. The highest abundance of transcripts was achieved in flowers with diameters ranging from 0.5 to 0.9 cm for GmAGMBP and GmBZIP and in flowers with diameters less than 0.5 cm for GmHSA32. Southern analysis suggested that GmAGMBP might be single copy gene while GmHSA3A could possibly belong to a small gene family in the mangosteen genome

Comparison of Mortality Outcomes in Acute Myocardial Infarction Patients With or Without Standard Modifiable Cardiovascular Risk Factors

Background: Acute myocardial infarction (AMI) cases have decreased in part due to the advent of targeted therapies for standard modifiable cardiovascular disease risk factors (SMuRF). Recent studies have reported that ST-elevation myocardial infarction (STEMI) patients without SMuRF (termed "SMuRF-less") may be increasing in prevalence and have worse outcomes than "SMuRF-positive" patients. As these studies have been limited to STEMI and comprised mainly Caucasian cohorts, we investigated the changes in the prevalence and mortality of both SMuRF-less STEMI and non-STEMI (NSTEMI) patients in a multiethnic Asian population. Methods: We evaluated 23,922 STEMI and 62,631 NSTEMI patients from a national multiethnic registry. Short-term cardiovascular and all-cause mortalities in SMuRF-less patients were compared to SMuRF-positive patients. Results: The proportions of SMuRF-less STEMI but not of NSTEMI have increased over the years. In hospitals, all-cause and cardiovascular mortality and 1-year cardiovascular mortality were significantly higher in SMuRF-less STEMI after adjustment for age, creatinine, and hemoglobin. However, this difference did not remain after adjusting for anterior infarction, cardiopulmonary resuscitation (CPR), and Killip class. There were no differences in mortality in SMuRF-less NSTEMI. In contrast to Chinese and Malay patients, SMuRF-less patients of South Asian descent had a two-fold higher risk of in-hospital all-cause mortality even after adjusting for features of increased disease severity. Conclusion: SMuRF-less patients had an increased risk of mortality with STEMI, suggesting that there may be unidentified nonstandard risk factors predisposing SMuRF-less patients to a worse prognosis. This group of patients may benefit from more intensive secondary prevention strategies to improve clinical outcomes

Profiling the transcriptome of Gracilaria changii (Rhodophyta) in response to light deprivation

Light regulates photosynthesis, growth and reproduction, yield and properties of phycocolloids, and starch contents in seaweeds. Despite its importance as an environmental cue that regulates many developmental, physiological, and biochemical processes, the network of genes involved during light deprivation are obscure. In this study, we profiled the transcriptome of Gracilaria changii at two different irradiance levels using a cDNA microarray containing more than 3,000 cDNA probes. Microarray analysis revealed that 93 and 105 genes were up- and down-regulated more than 3-fold under light deprivation, respectively. However, only 50% of the transcripts have significant matches to the nonredundant peptide sequences in the database. The transcripts that accumulated under light deprivation include vanadium chloroperoxidase, thioredoxin, ferredoxin component, and reduced nicotinamide adenine dinucleotide dehydrogenase. Among the genes that were down-regulated under light deprivation were genes encoding light harvesting protein, light harvesting complex I, phycobilisome 7.8 kDa linker polypeptide, low molecular weight early light-inducible protein, and vanadium bromoperoxidase. Our findings also provided important clues to the functions of many unknown sequences that could not be annotated using sequence comparison