Molecular Phylogeny and Predicted 3D Structure of Plant beta

beta-D-N-Acetylhexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of β-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. We constructed phylogenetic tree of β-hexosaminidases to analyze the evolutionary history and predicted functions of plant hexosaminidases. Phylogenetic analysis reveals the complex history of evolution of plant β-hexosaminidase that can be described by gene duplication events. The 3D structure of tomato β-hexosaminidase (β-Hex-Sl) was predicted by homology modeling using 1now as a template. Structural conformity studies of the best fit model showed that more than 98% of the residues lie inside the favoured and allowed regions where only 0.9% lie in the unfavourable region. Predicted 3D structure contains 531 amino acids residues with glycosyl hydrolase20b domain-I and glycosyl hydrolase20 superfamily domain-II including the (β/α)8 barrel in the central part. The α and β contents of the modeled structure were found to be 33.3% and 12.2%, respectively. Eleven amino acids were found to be involved in ligand-binding site; Asp(330) and Glu(331) could play important roles in enzyme-catalyzed reactions. The predicted model provides a structural framework that can act as a guide to develop a hypothesis for β-Hex-Sl mutagenesis experiments for exploring the functions of this class of enzymes in plant kingdom

Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu

Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zincbinding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group

Emerging genomic technologies for agricultural biotechnology: current trends and future prospects

The current earth’s population of 7.6 billion is expected to reach 8.6 billion by 2030. The increased population will need more food than it can currently produce. However, world agriculture is facing severe challenges such as global climate change, exhausted resources, reduction of arable lands and various pathogens. Advances in genomic technologies may offer potential solutions to these agricultural problems. New genomic technologies such as, Next generation sequencing (NGS), Ribonucleic acid sequencing (RNA-Seq), Clustered Regularly Interspaced Short Palindromic Repeat-Cas9 (CRISPR/Cas9), Transcription activator-like effector nucleases (TALENS) and Oligonucleotide‐directed mutagenesis (ODM) as well as doubled haploids, molecular markers and mapping populations have been developed and utilized for increasing the crop production. Together with the rapidly expanding availability of genome sequence data, these technologies have the potential to transform plant breeding

Multiple Forms of Alcohol Dehydrogenase (Adh) Genes in Sago Palm: A Preliminary Study

Flooding is a worldwide phenomenon in wetland and river areas. Excess water in the soil could produce anoxic soil condition. Sago palms in Sarawak can be found on mainly waterlogged areas. These plants are able and possibly have evolved a system for overcoming the anoxic/hypoxic conditions especially in the root section. Here we report the detection and activity of Adh gene in sago palm. The Adh enzyme was isolated, analysed on polyacrylamide and agarose gel, and detected by specific Adh staining. We found that Adh is present in all sago tissues and three variants are present based on migration on gel. We also utilised the polymerase chain reaction method to generate the PCR products by using primers that are designed from other plant species. Genomic DNA was used for this purpose and four PCR fragments were generated and the nucleotide sequence were determined. Preliminary results of nucleotide sequence analysis indicated that at least three types of Adh genes are present in sago

Expression of Recombinant Alcohol Dehydrogenase in Escherichia coli Strain BL21 (DE3) and In Planta Agrobacterium Transformation of Tomato Seeds

Alcohol dehydrogenase is an enzyme that is involved in various roles in plant such as in plant development, growth and plant responses to abiotic and biotic stresses. A recombinant alcohol dehydrogenase 1 (Adh1) cDNA (r-msAdh1) from Metroxylon sagu has been previously isolated, containing 20 nucleotides derived from Elaeis guineensis at the 5’-end, with a molecular weight of 1.14 kb. The objective of this study is to determine the function of r-msAdh1 via analyses in prokaryotic and eukaryotic hosts. For expression in prokaryotic system, pET-41a(+) with a 8x His tag at the C terminal was used for r-msAdh1 protein purification and expression was achieved using IPTG for four to six hours in Escherichia coli strain BL21 (DE3) incubated at low temperature. The induced BL21 strain produced a small amount of soluble r-msAdh1 protein while large amount was present as insoluble aggregates. Subsequently, the r-msAdh1 cDNA was transformed into tomato seeds (Solanum lycopersicum cv. MT1) via Agrobacterium-mediated in planta transformation. The integration of r-msAdh1 cDNA and the selectable marker were detected in transformed seedlings, T0, using polymerase chain reaction technique. The transformation efficiency was determined to be 33% for r-msAdh1 cDNA and 46% for the selectable marker. For stability analysis of the transgene, eleven T1 generation randomly selected from the transgenic T0 were analyzed for the presence of the cDNA, and all seedlings were found to contain the full length of r-msAdh1 cDNA. However, out of eleven T1 transgenic lines produced, only four seedlings were used for expression analysis using the reverse transcriptase PCR (RT-PCR). Two transgenic lines, T19 and T111, were determined to contain r-msAdh1 cDNA and verified by nucleotide sequencing. Although only a small number of T1 transgenic seedlings was obtained, this study shows that tomato seeds could be used as a target tissue for Agrobacterium-mediated in planta transformation primarily because the protocol is easy, rapid and cheaper compared to tissue culture-based method

Molecular And 3D-Structural Characterization Of Fructose-1,6-Bisphosphate Aldolase Derived From Metroxylon Sagu

Fructose-1,6-bisphosphate aldolase (FBAld) is an enzyme that catalyzes the cleavage of D-fructose-1,6-phosphate (FBP) to D-glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), and plays vital role in glycolysis and gluconeogenesis. However, molecular characterization and functional roles of FBAld remain unknown in sago palm. Here we report a modified CTAB-RNA extraction method was developed for the isolation of good quality RNA (RIN>8) from sago leaves and the isolation of FBAld cDNA from sago palm. The isolated sago FBAld (msFBAld) cDNA has total length of 1288 bp with an open reading frame of 1020 bp and a predicted to encode for a protein of 340 amino acid resides. The predicted protein shared a high degree of homology with Class-I FBAld from other plants. Meanwhile, the msFBAld gene spanned 2322 bp and consisted of five exons. Conserved domain search identified fifteen catalytically important amino acids at the active site and phylogenetic tree revealed localization of msFBAld in the chloroplast. A molecular 3D-structure of msFBAld was generated by homology modeling and a Ramachandran plot with 86.7% of the residues in the core region, 13.4% in the allowed region with no residues in the disallowed region. The modeled structure is a homotetramer containing an /-TIM-barrel at the center. Superimposition of the model with Class-I aldolases identified a catalytic dyad, Lys209-Glu167, which could be involved in the Schiff's base formation and aldol condensation. Apart from that, overproduction of the recombinant msFBAld in Escherichia coli resulted in increased tolerance towards salinity

Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment

Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture usingAgrobacteriumtumefaciens and gene gun systems.Thetransformed embryoids cellswere selected against Basta (concentration 10 to 30mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis.This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots.The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacteriumwhen targetswere bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance

Decolourisation Capabilities of Ligninolytic Enzymes Produced by Marasmius cladophyllus UMAS MS8 on Remazol Brilliant Blue R and Other Azo Dyes

Marasmius cladophyllus was examined for its ability to degradatively decolourise the recalcitrant dye Remazol Brilliant Blue R (RBBR) and screened for the production of ligninolytic enzymes using specific substrates. Monitoring dye decolourisation by the decrease in absorbance ratio of A 592/A 500 shows that the decolourisation of RBBR dye was associated with the dye degradation. Marasmius cladophyllus produces laccase and lignin peroxidase in glucose minimal liquid medium containing RBBR. Both enzyme activities were increased, with laccase activity recorded 70 times higher reaching up to 390 U L-1 on day 12. Further in vitro RBBR dye decolourisation using the culture medium shows that laccase activity was correlated with the dye decolourisation. Fresh RBBR dye continuously supplemented into the decolourised culture medium was further decolourised much faster in the subsequent round of the RBBR dye decolourisation. In vitro dye decolourisation using the crude laccase not only decolourised 76% of RBBR dye in just 19 hours but also decolourised 54% of Orange G and 33% of Congo red at the same period of time without the use of any exogenous mediator. This rapid dye decolourisation ability of the enzymes produced by M. cladophyllus thus suggested its possible application in the bioremediation of dye containing wastewate