Overexpression of Arabidopsis thaliana gibberellic acid 20 oxidase (AtGA20ox) gene enhance the vegetative growth and fiber quality in kenaf (Hibiscus cannabinus L.) plants.

Kenaf (Hibiscus cannabinus L.; Family: Malvaceae), is multipurpose crop, one of the potential alternatives of natural fiber for biocomposite materials. Longer fiber and higher cellulose contents are required for good quality biocomposite materials. However, average length of kenaf fiber (2.6 mm in bast and 1.28 mm in whole plant) is below the critical length (4 mm) for biocomposite production. Present study describes whether fiber length and cellulose content of kenaf plants could be enhanced by increasing GA biosynthesis in plants by overexpressing Arabidopsis thaliana Gibberellic Acid 20 oxidase (AtGA20ox) gene. AtGA20ox gene with intron was overexpressed in kenaf plants under the control of double CaMV 35S promoter, followed by in planta transformation into V36 and G4 varieties of kenaf. The lines with higher levels of bioactive GA (0.3-1.52 ng g(-1) fresh weight) were further characterized for their morphological and biochemical traits including vegetative and reproductive growth, fiber dimension and chemical composition. Positive impact of increased gibberellins on biochemical composition, fiber dimension and their derivative values were demonstrated in some lines of transgenic kenaf including increased cellulose content (91%), fiber length and quality but it still requires further study to confirm the critical level of this particular bioactive GA in transgenic plants.

Investigating the role of hypothetical protein (AAB33144.1) in HIV-1 virus pathogenicity: A comparative study with FDA-Approved inhibitor compounds through In silico analysis and molecular docking.

Aim and objective: Due to the a lot of unexplored proteins in HIV-1, this research aimed to explore the functional roles of a hypothetical protein (AAB33144.1) that might play a key role in HIV-1 pathogenicity. Methods: The homologous protein was identified along with building and validating the 3D structure by searching several bioinformatics tools. Results: Retroviral aspartyl protease and retropepsin like functional domains and motifs, folding pattern (cupredoxins), and subcellular localization in cytoplasmic membrane were determined as biological activity. Besides, the functional annotation revealed that the chosen hypothetical protein possessed protease-like activity. To validate our generated protein 3D structure, molecular docking was performed with five compounds where nelfinavir showed (-8.2 kcal/mol) best binding affinity against HXB2 viral protease (PDB ID: 7SJX) and main protease (PDB ID: 4EYR) protein. Conclusions: This study suggests that the annotated hypothetical protein related to protease action, which may be useful in viral genetics and drug discovery.

Overexpression of the oil palm (Elaeis guineensis Jacq.) TAPETUM DEVELOPMENT1-like Eg707 in rice affects cell division and differentiation and reduces fertility.

The functional analysis of the TAPETUM DEVELOPMENT1-like analog Eg707 of oil palm was carried out in rice by over-expressing Eg707 under the control of a double cauliflower mosaic virus 35S promoter. Ectopic expression of Eg707 in rice induced dark green and matured compact brownish calli compared to pale wild type and negative control calli. Regenerated transgenic rice plants exhibited a reduction in organ size and plant height, rolled, erect leaves, less tillers, increased chlorophyll content, and reduced fertility with smaller green seeds. At the molecular level Eg707 overexpression caused an increase in the transcription of SAPK9, a SnRK2 protein kinase family member that is activated by ABA and hyperosmotic stress. Together, the results show that ectopic Eg707 expression influences cell division and differentiation, presumably via altered hormone homeostasis.

Sequence analysis and potentials of the native RbcS promoter in the development of an alternative eukaryotic expression system using green Microalga Ankistrodesmus convolutus.

The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS) has been used to drive the expression of ß-glucuronidase (gusA) gene in this microalga. Besides the determination of the transcription start site by 5'-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2), which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif), which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus.

Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalgae Ankistrodesmus convolutus.

Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus.

Cloning and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) cDNA from green microalga Ankistrodesmus convolutus.

An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.

Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda.

Green microalga Ankistrodesmus convolutus Corda is a fast growing alga which produces appreciable amount of carotenoids and polyunsaturated fatty acids. To our knowledge, this is the first report on the construction of cDNA library and preliminary analysis of ESTs for this species. The titers of the primary and amplified cDNA libraries were 1.1×10(6) and 6.0×10(9) pfu/ml respectively. The percentage of recombinants was 97% in the primary library and a total of 337 out of 415 original cDNA clones selected randomly contained inserts ranging from 600 to 1,500 bps. A total of 201 individual ESTs with sizes ranging from 390 to 1,038 bps were then analyzed and the BLASTX score revealed that 35.8% of the sequences were classified as strong match, 38.3% as nominal and 25.9% as weak match. Among the ESTs with known putative function, 21.4% of them were found to be related to gene expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress response, and the remaining 45.8% were classified as novel genes. Analysis of ESTs described in this paper can be an effective approach to isolate and characterize new genes from A. convolutus and thus the sequences obtained represented a significant contribution to the extensive database of sequences from green microalgae.

Studies on the genetic variation of the green unicellular alga Haematococcus pluvialis (Chlorophyceae) obtained from different geographical locations using ISSR and RAPD molecular marker.

Haematococcus pluvialis (Flotow) is a unicellular green alga, which is considered to be the best astaxanthin-producing organism. Molecular markers are suitable tools for the purpose of finding out genetic variations in organisms; however there have been no studies conducted on ISSR or RAPD molecular markers for this organism. The DNA of 10 different strains of H. pluvialis (four strains from Iran, two strains from Finland, one strain from Switzerland and three strains from the USA) was extracted. A genetic similarity study was carried out using 14 ISSR and 12 RAPD primers. Moreover, the molecular weights of the bands produced ranged from 0.14 to 3.4 Kb. The PCA and dendrogram clustered the H. pluvialis strains into various groups according to their geographical origin. The lowest genetic similarity was between the Iran2 and USA2 strains (0.08) and the highest genetic similarity was between Finland1 and Finland2 (0.64). The maximum numbers of bands produced by the ISSR and RAPD primers were 35 and 6 bands, respectively. The results showed that ISSR and RAPD markers are useful for genetic diversity studies of Haematococcus as they showed geographical discrimination.

Comparison of Optimization-Modelling Methods for Metabolites Production in Escherichia coli.

The metabolic network is the reconstruction of the metabolic pathway of an organism that is used to represent the interaction between enzymes and metabolites in genome level. Meanwhile, metabolic engineering is a process that modifies the metabolic network of a cell to increase the production of metabolites. However, the metabolic networks are too complex that cause problem in identifying near-optimal knockout genes/reactions for maximizing the metabolite's production. Therefore, through constraint-based modelling, various metaheuristic algorithms have been improvised to optimize the desired phenotypes. In this paper, PSOMOMA was compared with CSMOMA and ABCMOMA for maximizing the production of succinic acid in E. coli. Furthermore, the results obtained from PSOMOMA were validated with results from the wet lab experiment.

Rapid and effective method of RNA isolation from green microalga Ankistrodesmus convolutus.

The rapid and effective method for the isolation of RNA from green microalga Ankistrodesmus convolutus based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride is developed. This procedure avoids the use of toxic chaotropic agents and phenol while high concentration of dithiothreitol is used to inhibit RNase activity and prevent oxidative cross-linking of nucleic acids by phenolics. The extraction procedure was able to produce high quality and intact RNA from A. convolutus. The yield of total RNA was 0.69-0.73 mg/g of fresh weight, with A(260)/A(280) ratio of 1.79-1.86. The obtained RNA was of sufficient quality and suitable for downstream application such as RT-PCR and cDNA library construction. The procedure may also have wider applicability for total RNA isolation from other green microalgae species.

A non-dominated sorting Differential Search Algorithm Flux Balance Analysis (ndsDSAFBA) for in silico multiobjective optimization in identifying reactions knockout.

Metabolic engineering is defined as improving the cellular activities of an organism by manipulating the metabolic, signal or regulatory network. In silico reaction knockout simulation is one of the techniques applied to analyse the effects of genetic perturbations on metabolite production. Many methods consider growth coupling as the objective function, whereby it searches for mutants that maximise the growth and production rate. However, the final goal is to increase the production rate. Furthermore, they produce one single solution, though in reality, cells do not focus on one objective and they need to consider various different competing objectives. In this work, a method, termed ndsDSAFBA (non-dominated sorting Differential Search Algorithm and Flux Balance Analysis), has been developed to find the reaction knockouts involved in maximising the production rate and growth rate of the mutant, by incorporating Pareto dominance concepts. The proposed ndsDSAFBA method was validated using three genome-scale metabolic models. We obtained a set of non-dominated solutions, with each solution representing a different mutant strain. The results obtained were compared with the single objective optimisation (SOO) and multi-objective optimisation (MOO) methods. The results demonstrate that ndsDSAFBA is better than the other methods in terms of production rate and growth rate.

Detection of tdh and trh genes in Vibrio parahaemolyticus isolated from Corbicula moltkiana prime in West Sumatera, Indonesia.

The occurrence of Vibrio parahaemolyticus in raw Corbicula moltkiana Prime from Lake Singkarak and Pasar Raya Padang market and in cooked samples in West Sumatera, Indonesia, was studied. Thirteen raw and seven cooked bivalve samples were positive using CHROMAgar Vibrio medium. All 47 V parahaemolyticus isolates were positive for toxR gene but negative for trh. However, 36% (17/47) of V parahaemolyticus strains were positive for tdh gene. Antibiotic profiling showed that 76% and 38% of isolates from raw and cooked bivalves respectively were resistant to ampicillin. Using RAPD-PCR analysis, most of the strains were clustered according to their source of isolation but some of the strains from raw and cooked samples were clustered together. These results indicate that pathogenic V parahaemolyticus isolates are present in Corbicula moltkiana Prime in West Sumatera, Indonesia, suggesting that V parahaemolyticus may also be present in seafood in other regions of Indonesia.

Detection of Vibrio parahaemolyticus in cockle (Anadara granosa) by PCR.

This study aimed to determine the occurrence of Vibrio parahaemolyticus in cockles (Anadara granosa) at a harvesting area and to detect the presence of virulent strains carrying the thermostable direct hemolysin (tdh) and TDH-related hemolysin genes (trh) using PCR. Of 100 samples, 62 were positive for the presence of V. parahaemolyticus with an MPN (most probable number) value greater than 3.0 (>1100 MPN per g). The PCR analysis revealed 2 samples to be positive for the tdh gene and 11 to be positive for the trh gene. Hence, these results demonstrate the presence of pathogenic V. parahaemolyticus in cockles harvested in the study area and reveal the potential risk of illness associated with their consumption.

Characterization of vibrio parahaemolyticus isolated from coastal seawater in peninsular Malaysia.

Twenty-one Vibrio parahaemolyticus isolates representing 21 samples of coastal seawater from three beaches in peninsular Malaysia were found to be sensitive to streptomycin, norfloxacin and chloramphenicol. Resistance was observed to penicillin (100%), ampicillin (95.2%), carbenicilin (95.2%), erythromycin (95.2%), bacitracin (71.4%), cephalothin (28.6%), moxalactam (28.6%), kanamycin (19.1%), tetracycline (14.3%), nalidixic acid (9.5%) and gentamicin (9.5%). Plasmids of 2.6 to 35.8 mDa were detected among plasmid-containing isolates. All isolates carried the Vp-toxR gene specific to V. parahaemolyticus and were negative for the tdh gene, but only one isolate was positive for the trh gene. DNA fingerprinting of the isolates using ERIC-PCR and PFGE showed that the isolates belong to two major clonal groups, with several isolates from different locations in the same group, indicating the presence of similar strains in the different locations.

Optimising the production of succinate and lactate in Escherichia coli using a hybrid of artificial bee colony algorithm and minimisation of metabolic adjustment.

Metabolic engineering is a research field that focuses on the design of models for metabolism, and uses computational procedures to suggest genetic manipulation. It aims to improve the yield of particular chemical or biochemical products. Several traditional metabolic engineering methods are commonly used to increase the production of a desired target, but the products are always far below their theoretical maximums. Using numeral optimisation algorithms to identify gene knockouts may stall at a local minimum in a multivariable function. This paper proposes a hybrid of the artificial bee colony (ABC) algorithm and the minimisation of metabolic adjustment (MOMA) to predict an optimal set of solutions in order to optimise the production rate of succinate and lactate. The dataset used in this work was from the iJO1366 Escherichia coli metabolic network. The experimental results include the production rate, growth rate and a list of knockout genes. From the comparative analysis, ABCMOMA produced better results compared to previous works, showing potential for solving genetic engineering problems.

Overexpression of monodehydroascorbate reductase from a mangrove plant (AeMDHAR) confers salt tolerance on rice.

Monodehydroascorbate reductase (MDHAR), an important enzyme of the ascorbate-glutathione cycle, is involved in salt tolerance of plants through scavenging of reactive oxygen species (ROS). In this study, a cDNA encoding MDHAR from the mangrove plant Acanthus ebracteatus was introduced into rice to examine its role in salt tolerance. Three stable transgenic lines (MT22, MT24 and MT25) overexpressing AeMDHAR were selected in vitro using hygromycin and confirmed by PCR, quantitative reverse-transcription (qRT) PCR and enzyme assay. The transgenic line MT24 was predicted to possess a single copy of the transgene while the other two transgenic lines were predicted to have multiple transgene integrations. The AeMDHAR transcripts were detected only in transgenic rice lines but not in untransformed rice. The abundance of AeMDHAR transcripts in transgenic lines MT22 and MT25 was approximately 2.75 times the amount found in MT24. The transgenic rice lines overexpressing AeMDHAR showed a significant increase in MDHAR enzyme activity compared to untransformed plants under both NaCl and control conditions. All transgenic lines showed better yield attributes such as a higher tiller number and increased 1000-grain weight compared to non-transgenics. They also showed tolerance to salt at germination and seedling stages. The transgenic line MT24, which harbors a single copy of AeMDHAR, displayed a lower rate of sterility, a higher number of tillers and longer panicle compared to untransformed plants when subjected to salt stress.

A novel transcript of oil palm (Elaeis guineensis Jacq.), Eg707, is specifically upregulated in tissues related to totipotency.

In this study, we report the molecular characterization of clone Eg707 isolated from cell suspension culture of the oil palm. The deduced polypeptide of clone Eg707 is highly similar to an unknown protein from Arabidopsis thaliana. The presence of an Ald-Xan-dh-C2 superfamily domain in the deduced protein sequence suggested that Eg707 protein might be involved in abscisic acid biosynthesis. Eg707 might be present as a single copy gene in the oil palm genome. This gene is highly expressed in tissue cultured materials compared to vegetative and reproductive tissues, suggesting a role of this gene during oil palm somatic embryogenesis or at the early stages of embryo development. Expression analysis of Eg707 by RNA in situ hybridization showed that Eg707 transcripts were present throughout somatic embryo development starting from proembryo formation at the embryogenic callus stages till the maturing embryo stages. Since proembryo formation within the embryogenic callus is one of the first key factors in oil palm somatic embryo development, it is suggested that Eg707 could be used as a reliable molecular marker for detecting early stage of oil palm somatic embryogenesis.

Isolation and characterization of differentially expressed transcripts from the suspension cells of oil palm (Elaeis guineensis Jacq.) in response to different concentration of auxins.

Oil palm suspension cultures were initiated by transferring the gel-like friable embryogenic tissue onto liquid medium supplemented with auxins. In this study, transcripts that were differentially expressed in oil palm suspension cells cultured at different auxin concentrations were examined using suppression subtractive hybridization. Total RNA was first isolated from oil palm suspension cells proliferated in liquid medium with different hormone concentrations for 6 months. Four different hormone combinations: T1 (0.1 mg/l 2,4-D and 1.0 mg/l NAA), T2 (0.4 mg/l 2,4-D and 1.0 mg/l NAA), T3 (1.0 mg/l NAA), and T4 (0.4 mg/l 2,4-D) were used for the treatments. The first and second subtractions were performed using samples T1 and T2 in forward and reverse order. The other two subtractions were forward and reverse subtractions of T3 and T4, respectively. Reverse northern analyses showed that 14.13% of these clones were preferentially expressed in T1, 13.70% in T2, 14.75% in T3, and 15.70% in T4. Among the 294 cDNA clones that were sequenced, 61 contigs (assembled from 165 sequences) and 129 singletons were obtained. Among the 61 contigs, 10 contigs consist of sequences from treatment T1, 8 contigs were from treatment T2, 10 contigs were contains sequences of treatment T3 and 13 contigs contains sequences of treatment T4. Northern analyses of five transcripts that were shown to be differentially expressed in the oil palm suspension cells by reverse northern analysis revealed that transcripts 16A1 (a putative lignostilbene-alpha,beta-dioxygenase, EgLSD) and 16H12 (a putative ethylene responsive 6, EgER6) were differentially expressed in oil palm suspension cells treated with different levels of auxin.

Morphological and Inter Simple Sequence Repeat (ISSR) markers analyses of Corynespora cassiicola isolates from rubber plantations in Malaysia.

Morphological features and Inter Simple Sequence Repeat (ISSR) polymorphism were employed to analyse 21 Corynespora cassiicola isolates obtained from a number of Hevea clones grown in rubber plantations in Malaysia. The C. cassiicola isolates used in this study were collected from several states in Malaysia from 1998 to 2005. The morphology of the isolates was characteristic of that previously described for C. cassiicola. Variations in colony and conidial morphology were observed not only among isolates but also within a single isolate with no inclination to either clonal or geographical origin of the isolates. ISSR analysis delineated the isolates into two distinct clusters. The dendrogram created from UPGMA analysis based on Nei and Li's coefficient (calculated from the binary matrix data of 106 amplified DNA bands generated from 8 ISSR primers) showed that cluster 1 encompasses 12 isolates from the states of Johor and Selangor (this cluster was further split into 2 sub clusters (1A, 1B), sub cluster 1B consists of a unique isolate, CKT05D); while cluster 2 comprises of 9 isolates that were obtained from the other states. Detached leaf assay performed on selected Hevea clones showed that the pathogenicity of representative isolates from cluster 1 (with the exception of CKT05D) resembled that of race 1; and isolates in cluster 2 showed pathogenicity similar to race 2 of the fungus that was previously identified in Malaysia. The isolate CKT05D from sub cluster 1B showed pathogenicity dissimilar to either race 1 or race 2.