Development and Analyses of Expressed Sequence Tags from Gracilaria Changii for Functional Genomic Studies

Macroalgae from the genus Gracilaria is the most common agarophytic genus in Malaysia (Phang et al., 1996). This wild population of seaweed has been identified as an important source of raw material for the agar industry. Despite its potential to produce good gel strength agar, Gracilaria sp. was genetically less studied. The aims of this study are to generate and sequence a thousand Expressed Sequence Tag (EST) sequences from G. changii for further cDNA microarray to facilitate functional genomic research. RNA extraction from G. changii is difficult due to poor yield, polysaccharide contamination and gel formation. To circumvent these problems the RNA isolation procedure was modified and repeated more than 150 times (more than 10 kg of fresh samples were used) to obtain high quality RNA for further studies. From the three modified RNA extraction methods, the modified method of Kim et al. (1997) was chosen for rapid RNA isolation from G. changii. This method can be completed within 1 day and many samples can be processed at the same time. The yield was increased from 0.018 μg/g to 1.14 μg/g of tissue with an average purity measured as A(260/280) of 1.90. After the modification, the mRNA was recovered from the total RNA of G. changii at a ratio of 0.5 – 1.0%. Starting from 5 μg of mRNA, a primary cDNA library of 1.14 x 106 clones was constructed and 1.375 x 1010 pfu/mL plaques were established for the amplified library. A total of 1854 cDNA clones were successfully sequenced. The database consists of ESTs with putative functions in protein synthesis (6%), energy (4%), protein destination and storage (3%), metabolism (3%), transportation (2%), transcription (2%), signal transduction (1%), cell structure/maintenance (1%), disease and defence (1%), cell growth and division (1%), intracellular traffic (1%) and other miscellaneous functions (2%). Putative proteins with unknown functions (67%), and novel sequences (6%) that do not show significant matches to the existing sequence databases are also present. Among the ESTs, 1342 sequences (72.38%) were clustered as singleton, and the remaining 512 were clustered into 168 contigs

Transcripts of Gracilaria changii that improve copper tolerance of Escherichia coli

In this study, we used bacterial functional screening to isolate transcripts from a red seaweed, Gracilaria changii Abbott, Zhang et Xia (Xia et Abbott) that improved copper tolerance of Escherichia coli. We have identified several seaweed proteins that may be involved in copper efflux, detoxification and ROS scavenging, such as ATPase, outward-rectifying potassium channel (KCO1), vanadium chloroperoxidase and a high affinity phosphate transporter. All transcripts were shown to be able to enhance the copper tolerance of E. coli up to 4 mM CuCl2. These transcripts may share similar functions in the copper homeostasis of both E. coli and G. changii. In addition, we discovered a few transcripts with uncharacterized function(s) in copper tolerance in both organisms

Transcriptomic analysis of Gracilaria changii (Rhodophyta) in response to hyper- and hypo-osmotic stresses

Osmotic stress is one of the most significant natural abiotic stresses that occur in the intertidal zones. Seaweeds may physiologically acclimate to changing osmolarity by altering their transcriptome. Here, we investigated the transcriptomic changes of Gracilaria changii (B. M. Xia et I. A. Abbott) I. A. Abbott, J. Zhang et B. M. Xia in response to hyper- and hypoosmotic stresses using a cDNA microarray approach. Microarray analysis revealed that 199 and 200 genes from ∼3,300 genes examined were up- and down-regulated by >2-fold in seaweed samples treated at 50 parts per thousand (ppt) artificial seawater (ASW) compared with those at 30 ppt ASW, respectively. The number of genes that were up- and down-regulated by >2-fold in seaweed samples treated at 10 ppt ASW compared with those at 30 ppt ASW were 154 and 187, respectively. A majority of these genes were only differentially expressed under hyper- or hypoosmotic conditions, whereas 67 transcripts were affected by both stresses. The findings of this study have shed light on the expression profiles of many transcripts during the acclimation of G. changii to hyperosmotic and hypoosmotic conditions. This information may assist in the prioritization of genes to be examined in future studies

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

Molecular cloning and characterization of GDP-mannose-3′,5′-epimerase from Gracilaria changii

GDP-mannose-3′,5′-epimerase (GME) is an enzyme involved in the biosynthesis of GDP-l-galactose which is a building unit of agar and cell wall polysaccharides. GME catalyzes the formation of GDP-β-l-galactose and GDP-l-gulose from GDP-mannose. In this study, the gene and transcript encoding GME from the red alga Gracilaria changii (GcGME) were cloned. The structural gene sequence of GcGME is devoid of an intron. The cis-acting regulatory element involved in light response is the most abundant element at the 5′-flanking region of GcGME. The open reading frame of GcGME consists of 1,053 nucleotides with 351 amino acids. This cDNA was cloned into pET32a expression vector for recombinant protein production in Escherichia coli. High yield of soluble recombinant GcGME (55 kDa) was expressed upon isopropyl β-d-1-thiogalactopyranoside induction. The enzyme activity of recombinant GcGME was detected using thin layer chromatography and high-performance liquid chromatography. The transcript abundance of GcGME was the highest in G. changii and the lowest in Gracilaria salicornia corresponding to their agar contents. The characterization of GcGME from G. changii is important to facilitate the understanding of its role in agar production of this seaweed

Isolation of salinity tolerant genes from the mangrove plant, Bruguiera cylindrica by using suppression subtractive hybridization (SSH) and bacterial functional screening

In this study, we have identified and isolated 126 salinity tolerant cDNAs from the root of a mangrove plant, Bruguiera cylindrica (L.) Blume by using suppression subtractive hybridization (SSH) and bacterial functional screening. Sequencing of 51 subtracted cDNA clones that were differentially expressed in the root of B. cylindrica exposed to 20 parts per thousand (ppt) NaCl water revealed 10 tentative unique genes (TUGs) with putative functions in protein synthesis, storage and destination, metabolism, intracellular trafficking and other functions; and 9 unknown proteins. Meanwhile, the 75 cDNA sequences of B. cylindrica that conferred salinity tolerance to Escherichia coli consisted of 29 TUGs with putative functions in transportation, metabolism and other functions; and 33 with unknown functions. Both approaches yielded 42 unique sequencess that have not been reported else where to be stress related and might provide further understanding of adaptations of this plant to salinity stress

Sequence and transcript analyses of antioxidant genes from Acanthus ebracteatus Vahl.

In this study, we report the sequence, Southern analyses and spatial distribution of four cDNA sequences related to reactive oxygen species (ROS) scavenging systems from a mangrove plant, Acanthus ebracteatus. These four complementary DNA (cDNA) sequences encode cytosolic ascorbate peroxidase (AeAPX), monodehydroascorbate reductase (AeMDHR), glutathione-S-transferase (AeGST), and mitochondrial manganese superoxide dismutase (AeMnSOD), respectively. Experimental results indicated that AeAPX and AeGST belong to multigene families, whereas AeMDHAR and AeMnSOD exhibited substantial differences from other members of the same families. Transcript analyses indicated that all these genes were expressed in flowers. However, only AeAPX, AeMDHAR, and AeMnSOD were expressed in all tissues examined. Although both AeMDHAR and AeMnSOD were highly expressed in flowers, the highest expression of AeMnSOD was in the leaf tissue. The expression of AeMDHAR and AeMnSOD in stem was slightly higher than in the root. AeAPX was expressed at low levels in root and stem tissues and its expression in leaf was slightly higher than in the flower. On the other hand, the transcripts of AeGST were not detected in root and leaf. Its expression was almost equal in stem and flower. The information on spatial distribution and predicted subcellular locations of these antioxidant proteins are important for comprehensive analysis and characterization of ROS scavenging and signaling mechanisms in various plant compartments and tissues

Molecular cloning, homology modeling and site-directed mutagenesis of vanadium-dependent bromoperoxidase (GcVBPO1) from Gracilaria changii (Rhodophyta)

Vanadium-dependent haloperoxidases belong to a class of vanadium enzymes that may have potential industrial and pharmaceutical applications due to their high stability. In this study, the 5′-flanking genomic sequence and complete reading frame encoding vanadium-dependent bromoperoxidase (GcVBPO1) was cloned from the red seaweed, Fracilaria changii, and the recombinant protein was biochemically characterized. The deduced amino acid sequence of GcVBPO1 is 1818 nucleotides in length, sharing 49% identity with the vanadium-dependent bromoperoxidases from Corralina officinalis and Cor. pilulifera, respectively. The amino acid residues associated with the binding site of vanadate cofactor were found to be conserved. The Km value of recombinant GcVBPO1 for Br− was 4.69 mM, while its Vmax was 10.61 μkat mg−1 at pH 7. Substitution of Arg379 with His379 in the recombinant protein caused a lower affinity for Br−, while substitution of Arg379 with Phe379 not only increased its affinity for Br− but also enabled the mutant enzyme to oxidize Cl−. The mutant Arg379Phe was also found to have a lower affinity for I−, as compared to the wild-type GcVBPO1 and mutant Arg379His. In addition, the Arg379Phe mutant has a slightly higher affinity for H2O2 compared to the wild-type GcVBPO1. Multiple cis-acting regulatory elements associated with light response, hormone signaling, and meristem expression were detected at the 5′-flanking genomic sequence of GcVBPO1. The transcript abundance of GcVBPO1 was relatively higher in seaweed samples treated with 50 parts per thousand (ppt) artificial seawater (ASW) compared to those treated in 10 and 30 ppt ASW, in support of its role in the abiotic stress response of seaweed

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