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

Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis

This study describes the cloning, expression and functional characterization of α-humulene synthase, responsible for the formation of the key aromatic compound α-humulene in agarwood originating from Aquilaria malaccensis. The partial sesquiterpene synthase gene from the transcriptome data of A. malaccensis was utilized for full-length gene isolation via a 30 RACE PCR. The complete gene, denoted as AmDG2, has an open reading frame (ORF) of 1671 bp and encodes for a polypeptide of 556 amino acids. In silico analysis of the protein highlighted several conserved motifs typically found in terpene synthases such as Asp-rich substrate binding (DDxxD), metal-binding residues (NSE/DTE), and cytoplasmic ER retention (RxR) motifs at their respective sites. The AmDG2 was successfully expressed in the E. coli:pET-28a(+) expression vector whereby an expected band of about 64 kDa in size was detected in the SDS-PAGE gel. In vitro enzyme assay using substrate farnesyl pyrophosphate (FPP) revealed that AmDG2 gave rise to two sesquiterpenes: α-humulene (major) and β-caryophyllene (minor), affirming its identity as α-humulene synthase. On the other hand, protein modeling performed using AlphaFold2 suggested that AmDG2 consists entirely of α-helices with short connecting loops and turns. Meanwhile, molecular docking via AutoDock Vina (Version 1.5.7) predicted that Asp307 and Asp311 act as catalytic residues in the α-humulene synthase. To our knowledge, this is the first comprehensive report on the cloning, expression and functional characterization of α-humulene synthase from agarwood originating from A. malaccensis species. These findings reveal a deeper understanding of the structure and functional properties of the α-humulene synthase and could be utilized for metabolic engineering work in the future

Identification and molecular characterization of alcohol dehydrogenase (ADH) and its promoter region in sago palm (Metroxylon sagu)

Sago palm (Metroxylon sagu) is one of the main economic crops for the state of Sarawak. It is one of the few plant species that thrive on peat swamp area. The waterlogged condition in such areas causes oxygen deprivation to the root part and triggers the anaerobic pathway. Molecular analysis on sago helps in understanding the ability of the palm to tolerate anaerobic conditions. In this study, proteins from various parts of the sago palm such as the roots (dry and submerged) and leaves were extracted successfully. The proteins were subjected for analysis on agarose and polyacralymide gel, and the presence of alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH) as well as superoxide dismutase (SOD) enzymes was detected through specific staining. The results indicated that ADH is present in all analyzed sago tissues and three variants were detected based on migration on gel. However, the banding patterns and the intensity varied among the tissues with the highest expression of ADH and SOD enzymes being reported in the sago shoots. Apart from that, three putative Adh genes from sago palm denoted as M I, M2 and M3 were isolated through Polymerase Chain Reaction (PCR) method using specific primers in this study. Based on the multiple alignment analysis, the three loci are found to be highly diverged and contain a common 23 bp nucleotide which is conserved in other Adh plant species. Phylogeny analysis indicated that the Adh loci of palm (Arecaceae) are segregated from that of other plant Adh families such as from the grass family (poaceae) and Rosaceae, corresponding to the findings of prevIous phylogenetic analysis. All these results concluded that three unique putative Adh genes from sago palm have been successfully isolated. One sago palm Adh gene, referred to here as M2 was used as template for primer designing for DNA walking to further isolate the upstream region which regulates the transcription of the particular gene. The DNA Walking technique has been adapted since genomic library construction yielded a low number of titer, 1.0 x 105 pfulml. Through DNA Walking, a promoter region was successfully identified. Manual and computational analysis of the promoter region revealed the presence of important elements such as TAT A box, CCAA T box and other putative regulatory elements at their respective locations

Cloning, Expression and Functional Characterization of a Novel α-Humulene Synthase, Responsible for the Formation of Sesquiterpene in Agarwood Originating from <i>Aquilaria malaccensis</i>

This study describes the cloning, expression and functional characterization of α-humulene synthase, responsible for the formation of the key aromatic compound α-humulene in agarwood originating from Aquilaria malaccensis. The partial sesquiterpene synthase gene from the transcriptome data of A. malaccensis was utilized for full-length gene isolation via a 3′ RACE PCR. The complete gene, denoted as AmDG2, has an open reading frame (ORF) of 1671 bp and encodes for a polypeptide of 556 amino acids. In silico analysis of the protein highlighted several conserved motifs typically found in terpene synthases such as Asp-rich substrate binding (DDxxD), metal-binding residues (NSE/DTE), and cytoplasmic ER retention (RxR) motifs at their respective sites. The AmDG2 was successfully expressed in the E. coli:pET-28a(+) expression vector whereby an expected band of about 64 kDa in size was detected in the SDS-PAGE gel. In vitro enzyme assay using substrate farnesyl pyrophosphate (FPP) revealed that AmDG2 gave rise to two sesquiterpenes: α-humulene (major) and β-caryophyllene (minor), affirming its identity as α-humulene synthase. On the other hand, protein modeling performed using AlphaFold2 suggested that AmDG2 consists entirely of α-helices with short connecting loops and turns. Meanwhile, molecular docking via AutoDock Vina (Version 1.5.7) predicted that Asp307 and Asp311 act as catalytic residues in the α-humulene synthase. To our knowledge, this is the first comprehensive report on the cloning, expression and functional characterization of α-humulene synthase from agarwood originating from A. malaccensis species. These findings reveal a deeper understanding of the structure and functional properties of the α-humulene synthase and could be utilized for metabolic engineering work in the future