Displaying publications 1 - 20 of 45 in total

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  1. Genome characterization of a breeding line derived from a cross between Oryza sativa and Oryza rufipogon
    Keong BP, Harikrishna JA
    Biochem Genet, 2012 Feb;50(1-2):135-45.
    PMID: 22089543 DOI: 10.1007/s10528-011-9479-8
    A preliminary screening was conducted on BC3F1 and BC4F1 backcross families developed from crossing Oryza sativa (MR219) and O. rufipogon (IRGC105491). Despite earlier results showing that O. rufipogon alleles (wild introgression) contributed to both number of panicles (qPPL-2) and tillers (qTPL-2) at loci RM250, RM208, and RM48 in line A20 of the BC2F2 population, we observed that wild introgression was lost at loci RM250 and RM208 but retained at locus RM48 in BC3F1 and BC4F1. Progeny tests conducted utilizing genotype and phenotype data on both BC4F1 and a reference population, BC2F7 (A20 line), did not show significant differences between groups having the MR219 allele and wild introgression at locus RM48. This suggests that there is no additive and transgressive effect of wild introgression in the BC3F1 and BC4F1 generated. The presence of wild introgression was largely due to gene contamination by cross-pollination during field breeding practices.
  2. Fulltext Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes
    Abu Bakar F, Yeo CC, Harikrishna JA
    Int J Mol Sci, 2016 Apr 20;17(4).
    PMID: 27104531 DOI: 10.3390/ijms17040321
    Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells.
  3. Fulltext Expression of the Streptococcus pneumoniae yoeB chromosomal toxin gene causes cell death in the model plant Arabidopsis thaliana
    Bakar FA, Yeo CC, Harikrishna JA
    BMC Biotechnol, 2015;15:26.
    PMID: 25887501 DOI: 10.1186/s12896-015-0138-8
    Bacterial toxin-antitoxin systems usually comprise of a pair of genes encoding a stable toxin and its cognate labile antitoxin and are located in the chromosome or in plasmids of several bacterial species. Chromosomally-encoded toxin-antitoxin systems are involved in bacterial stress responses and activation of the toxins usually leads to cell death or dormancy. Overexpression of the chromosomally-encoded YoeB toxin from the yefM-yoeB toxin-antitoxin locus of the Gram-positive bacterium Streptococcus pneumoniae has been shown to cause cell death in S. pneumoniae as well as E. coli.
  4. Fulltext Erratum to: expression of the Streptococcus pneumoniae yoeB Chromosomal toxin gene causes Cell Death in the model plant Arabidopsis thaliana
    Bakar FA, Yeo CC, Harikrishna JA
    BMC Biotechnol, 2015;15:101.
    PMID: 26514869 DOI: 10.1186/s12896-015-0220-2
  5. Fulltext The efficacy of molecular markers analysis with integration of sensory methods in detection of aroma in rice
    Yeap HY, Faruq G, Zakaria HP, Harikrishna JA
    ScientificWorldJournal, 2013;2013:569268.
    PMID: 24222741 DOI: 10.1155/2013/569268
    Allele Specific Amplification with four primers (External Antisense Primer, External Sense Primer, Internal Nonfragrant Sense Primer, and Internal Fragrant Antisense Primer) and sensory evaluation with leaves and grains were executed to identify aromatic rice genotypes and their F1 individuals derived from different crosses of 2 Malaysian varieties with 4 popular land races and 3 advance lines. Homozygous aromatic (fgr/fgr) F1 individuals demonstrated better aroma scores compared to both heterozygous nonaromatic (FGR/fgr) and homozygous nonaromatic (FGR/FGR) individuals, while, some F1 individuals expressed aroma in both leaf and grain aromatic tests without possessing the fgr allele. Genotypic analysis of F1 individuals for the fgr gene represented homozygous aromatic, heterozygous nonaromatic and homozygous nonaromatic genotypes in the ratio 20:19:3. Genotypic and phenotypic analysis revealed that aroma in F1 individuals was successfully inherited from the parents, but either molecular analysis or sensory evaluation alone could not determine aromatic condition completely. The integration of molecular analysis with sensory methods was observed as rapid and reliable for the screening of aromatic genotypes because molecular analysis could distinguish aromatic homozygous, nonaromatic homozygous and nonaromatic heterozygous individuals, whilst the sensory method facilitated the evaluation of aroma emitted from leaf and grain during flowering to maturity stages.
  6. Fulltext Bcl-xL silencing induces alterations in hsa-miR-608 expression and subsequent cell death in A549 and SK-LU1 human lung adenocarcinoma cells
    Othman N, In LL, Harikrishna JA, Hasima N
    PLoS One, 2013;8(12):e81735.
    PMID: 24339958 DOI: 10.1371/journal.pone.0081735
    Bcl-xL is an anti-apoptotic protein that is frequently found to be overexpressed in non-small cell lung cancer leading to an inhibition of apoptosis and poor prognosis. Recently, the role of miRNAs in regulating apoptosis and cell survival during tumorigenesis has become evident, with cancer cells showing perturbed expression of various miRNAs. In this study, we utilized miRNA microarrays to determine if miRNA dysregulation in bcl-xL silenced lung adenocarcinoma cells could be involved in regulating cell death. Short interfering RNA-based transfection of A549 and SK-LU1 lung adenocarcinoma cells was successful in inducing a reduction in bcl-xL expression levels, resulting in a decrease in cell viability. A total of 10 miRNAs were found to be significantly differentially expressed when compared between siRNA-transfected and non-transfected cells including hsa-miR-181a, hsa-miR-769-5p, hsa-miR-361-5p, hsa-miR-1304 and hsa-miR-608. When overexpression studies on hsa-miR-608 was performed via transfection of miRNA mimics, cell death was found to be induced in A549 and SK-LU1 cells in comparison to untreated cells. This effect was reversed when knockdown studies involving anti-sense inhibitors were introduced. Combination of siRNA based silencing of bcl-xL (siBcl-xL) followed by anti-sense inhibitor transfection led to a decrease in the apoptotic population of A549 and SK-LU1 cells in comparison to cells only treated with siBcl-xL, illustrating the connection between bcl-xL, hsa-miR-608 and cell death. Gene target prediction analysis implicated the PI3K/AKT, WNT, TGF-β, and ERK signaling pathways as targets of bcl-xL induced miRNA alterations. We have demonstrated that bcl-xL silencing in A549 and SK-LU1 cells leads to the occurrence of cell death through the dysregulation of specific miRNAs. This study also provides a platform for anti-sense gene therapy whereby miRNA expression can be exploited to increase the apoptotic properties in lung adenocarcinoma cells.
  7. High frequency plant regeneration from mature seed of elite, recalcitrant Malaysian indica rice ( Oryza sativa L.) CV. MR 219
    Sivakumar P, Law YS, Ho CL, Harikrishna JA
    Acta. Biol. Hung., 2010 Sep;61(3):313-21.
    PMID: 20724277 DOI: 10.1556/ABiol.61.2010.3.7
    An efficient in vitro plant regeneration system was established for elite, recalcitrant Malaysian indica rice, Oryza sativa L. CV. MR 219 using mature seeds as explant on Murashige and Skoog and Chu N6 media containing 2,4-dichlorophenoxy acetic acid and kinetin either alone or in different combinations. L-proline, casein hydrolysate and L-glutamine were added to callus induction media for enhancement of embryogenic callus induction. The highest frequency of friable callus induction (84%) was observed in N6 medium containing 2.5 mg l(-1) 2,4-dichlorophenoxy acetic acid, 0.2 mg l(-1) kinetin, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate, 20 mg l(-1) L-glutamine and 30 g l(-1) sucrose under culture in continuous lighting conditions. The maximum regeneration frequency (71%) was observed, when 30-day-old N6 friable calli were cultured on MS medium supplemented with 3 mg l(-1) 6-benzyl aminopurine, 1 mg l(-1) naphthalene acetic acid, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate and 3% maltose. Developed shoots were rooted in half strength MS medium supplemented with 2% sucrose and were successfully transplanted to soil with 95% survival. This protocol may be used for other recalcitrant indica rice genotypes and to transfer desirable genes in to Malaysian indica rice cultivar MR219 for crop improvement.
  8. Sequence analysis and characterization of vacuolar-type H+ -ATPase proteolipid transcript from Acanthus ebracteatus Vah1
    Ho CL, Nguyen PD, Harikrishna JA, Rahim RA
    DNA Seq., 2008 Feb;19(1):73-7.
    PMID: 17852357
    The vacuolar-type H+ -ATPase (V-ATPase) is a multimeric enzyme with diverse functions in plants such as nutrient transport, flowering, stress tolerance, guard cell movement and development. A partial sequence of V-ATPase proteolipid was identified among the expressed sequence tags (ESTs) generated from Acanthus ebracteatus, and selected for full-length sequencing. The 876-nucleotide cDNA consists of an open reading frame of 165 amino acids. The deduced amino acid sequence displays high similarity (81%) with its homologs from Arabidopsis thaliana, Avecinnia marina and Gossypium hirsutum with the four transmembrane domains characteristics of the 16 kDa proteolipid subunit c of V-ATPase well conserved in this protein. Southern analysis revealed the existence of several members of proteolipid subunit c of V-ATPase in A. ebracteatus. The mRNA of this gene was detected in leaf, floral, stem and root tissues, however, the expression level was lower in stem and root tissues.
  9. Sheath blight of rice: a review and identification of priorities for future research
    Singh P, Mazumdar P, Harikrishna JA, Babu S
    Planta, 2019 Nov;250(5):1387-1407.
    PMID: 31346804 DOI: 10.1007/s00425-019-03246-8
    MAIN CONCLUSION: Rice sheath blight research should prioritise optimising biological control approaches, identification of resistance gene mechanisms and application in genetic improvement and smart farming for early disease detection. Rice sheath blight, caused by Rhizoctonia solani AG1-1A, is one of the most devasting diseases of the crop. To move forward with effective crop protection against sheath blight, it is important to review the published information related to pathogenicity and disease management and to determine areas of research that require deeper study. While progress has been made in the identification of pathogenesis-related genes both in rice and in the pathogen, the mechanisms remain unclear. Research related to disease management practices has addressed the use of agronomic practices, chemical control, biological control and genetic improvement: Optimising nitrogen fertiliser use in conjunction with plant spacing can reduce spread of infection while smart agriculture technologies such as crop monitoring with Unmanned Aerial Systems assist in early detection and management of sheath blight disease. Replacing older fungicides with natural fungicides and use of biological agents can provide effective sheath blight control, also minimising environmental impact. Genetic approaches that show promise for the control of sheath blight include treatment with exogenous dsRNA to silence pathogen gene expression, genome editing to develop rice lines with lower susceptibility to sheath blight and development of transgenic rice lines overexpressing or silencing pathogenesis related genes. The main challenges that were identified for effective crop protection against sheath blight are the adaptive flexibility of the pathogen, lack of resistant rice varieties, abscence of single resistance genes for use in breeding and low access of farmers to awareness programmes for optimal management practices.
  10. Fulltext Impact of sea-salt on morpho-physiological and biochemical responses in banana (Musa acuminata cv. Berangan)
    Mazumdar P, Lau SE, Singh P, Takhtgahi HM, Harikrishna JA
    Physiol Mol Biol Plants, 2019 May;25(3):713-726.
    PMID: 31168234 DOI: 10.1007/s12298-019-00659-3
    Banana is often grown in coastal-regions, and while known for its sensitivity towards seawater, little is documented on the effect of sea-salt on the growth, physiology and metal homeostasis. Here we report that banana plantlets exposed to sea-salt at extreme (average seawater concentration; 52.7 dS m-1), severe (28.5 dS m-1) or moderate (10.2 dS m-1) salinity levels had reduced root length (2.0-6.0-fold), plant height (1.2-1.6-fold), leaf number (2.0-2.3-fold) and leaf area (3.3-4.0-fold) compared to control plantlets. Degradation of pigments (total chlorophyll: 1.3-12.3-fold, chlorophyll a: 1.3-9.2-fold; chlorophyll b: 1.3-6.9-fold lower and carotenoids: 1.4-3.7-fold lower) reflected vulnerability of photosystems to salt stress. Relative water content showed a maximum decrease of 1.5-fold in salt stress. MDA analysis showed sea-salt exposure triggers 2.3-3.5-fold higher lipid peroxidation. Metal content analysis showed a 73-fold higher Na value from roots exposed to extreme salinity compared to control plantlets. While phenotype was clearly affected, moderate salinity showed no significant alteration of macro (N, P, K and Ca) and micro (Fe, Mn and Cu) metal content. The antioxidant enzymes: SOD (3.2-fold), CAT (1.7-fold) and GR (6-fold) showed higher activity at moderate salinity level compared to control plantlets but lower activity at severe (SOD: 1.3-fold; CAT: 1.5-fold; GR: 2-fold lower) and extreme seawater salinity (SOD: 1.5; CAT: 1.9; GR: 1.3-fold lower). Mild changes in growth and physiology at sea-salt levels equivalent to moderate seawater flooding, indicate that banana will survive such flooding, while extreme seawater inundation will be lethal. This data provides a reference for future salinity-mediated work in banana.
  11. Identification and characterization of Ageratum yellow vein Malaysia virus (AYVMV) and an associated betasatellite among begomoviruses infecting Solanum lycopersicum in Malaysia
    Mahmoudieh M, Noor MRM, Harikrishna JA, Othman RY
    J Appl Genet, 2020 Dec;61(4):619-628.
    PMID: 32808206 DOI: 10.1007/s13353-020-00574-4
    The study describes results of a survey of tomato fields for the presence of begomoviruses from different regions of Peninsular Malaysia. An ORF-based (C2 and C3) study was performed to determine the distribution of begomoviruses associated with a severe leaf curl disease in tomato-growing areas of Peninsular Malaysia. Viral DNA was isolated from symptomatic tomato plants, and begomovirus association was confirmed by PCR using DNA-A degenerate primers. The C2 and C3 sequences of the putative begomoviruses were similar to two corresponded ORFs of different geographically separated strains of begomoviruses: Pepper yellow leaf curl Indonesia virus and Tomato yellow leaf curl Kanchanaburi virus. The present study also identified a unique isolate, Ageratum yellow vein Malaysia virus (AYVMV) among above mentioned survey. It has a single-stranded DNA component and its associated betasatellite. The single-stranded DNA component is consisting of 2750 nt with six open reading frames and an organization resembling that of monopartite geminiviruses. The full length of viral single-stranded DNA component genome obtained using next generation sequencing (NGS) showed the highest sequence identity (99%) with Ageratum yellow vein virus (AYVV-BA). The betasatellite component genome obtained by NGS has 1342 nt and showed the highest sequence identity (91%) with the Pepper yellow leaf curl betasatellite. Following ICTV guidelines, Ageratum yellow vein Malaysia virus was assigned the abbreviation AYVMV with sequence and phylogenetic analysis indicating that it might have evolved by recombination of two or more viral ancestors.
  12. Fulltext dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid
    Lau SE, Schwarzacher T, Othman RY, Harikrishna JA
    BMC Plant Biol, 2015;15:194.
    PMID: 26260631 DOI: 10.1186/s12870-015-0577-3
    The R2R3-MYB genes regulate pigmentation and morphogenesis of flowers, including flower and cell shape, and therefore have importance in the development of new varieties of orchids. However, new variety development is limited by the long breeding time required in orchids. In this study, we identified a cDNA, DhMYB1, that is expressed during flower development in a hybrid orchid, Dendrobium hybrida (Dendrobium bobby messina X Dendrobium chao phraya) then used the direct application of dsRNA to observe the effect of gene silencing on flower phenotype and floral epidermal cell shape.
  13. Fulltext Expression and DNA methylation of SERK, BBM, LEC2 and WUS genes in in vitro cultures of Boesenbergia rotunda (L.) Mansf
    Karim R, Tan YS, Singh P, Khalid N, Harikrishna JA
    Physiol Mol Biol Plants, 2018 Sep;24(5):741-751.
    PMID: 30150851 DOI: 10.1007/s12298-018-0566-8
    The process of somatic embryogenesis and plant regeneration involve changes in gene expression and have been associated with changes in DNA methylation. Here, we report the expression and DNA methylation patterns of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK), BABY BOOM (BBM), LEAFY COTYLEDON 2 (LEC2) and WUSCHEL (WUS) in meristematic block of newly emerged shoots from rhizome, embryogenic and non-embryogenic calli, prolonged cell suspension culture, ex vitro leaf, and in vitro leaf of regenerated plants of Boesenbergia rotunda. Among all seven samples, based on qRT-PCR, the highest level of expression of SERK, BBM and LEC2 was in embryogenic callus, while WUS was most highly expressed in meristematic block tissue followed by embryogenic callus. Relatively lower expression was observed in cell suspension culture and watery callus for SERK, LEC2 and WUS and in in vitro leaf for BBM. For gene specific methylation determined by bisulfite sequencing data, embryogenic callus samples had the lowest levels of DNA methylation at CG, CHG and CHH contexts of SERK, LEC2 and WUS. We observed negative correlation between DNA methylation at the CG and CHG contexts and the expression levels of SERK, BBM, LEC2 and WUS. Based on our results, we suggest that relatively higher expression and lower level of DNA methylation of SERK, BBM, LEC2 and WUS are associated with somatic embryogenesis and plant regeneration in B. rotunda.
  14. Global trends in research and commercialization of exogenous and endogenous RNAi technologies for crops
    Mat Jalaluddin NS, Othman RY, Harikrishna JA
    Crit Rev Biotechnol, 2018 Sep 09.
    PMID: 30198341 DOI: 10.1080/07388551.2018.1496064
    It has only been about 20 years since the first Nobel Prize-winning work on RNA interference (RNAi) in Caenorhabditis elegans was published in the journal Nature. Fast forward to today, and the use of RNA molecules as gene-silencing elements in crops has helped scientists to unveil possible solutions to the global problems of agricultural losses due to pests, viruses, pathogens, and to other abiotic and biotic stresses. The recent proliferation of publications suggests that the technology has gained significant attention and received ample funding support. In this article, an attempt has been made to visualize recent trends in Research & Development (R&D) investment in this field by analyzing top cited scholarly articles, patent trends, and commercialization activity. The publication and citation analysis identified that the development of RNAi-based crops conferring resistance against viruses, fungi, and pests are at the forefront of RNAi research and that Chinese and US institutions are the leaders in this field. The patent landscape analysis for RNAi technology over all aspects related to RNAi-derived crops provides an overview of patenting activity from a geographical, organizational, and legal perspective. Such an exercise is pivotal to industry players and public institutions aiming at creating intellectual property that is commercially appealing. An upswing in commercial interests in this technology in recent years is reflected by a consistent number of patent filings in US, European, and Chinese patent offices, with multinational giant firms as the most prolific patent filers. The expanding RNAi commercialization landscape is supported by a series of strategic partnerships, licensing agreements, and acquisitions created between agribusinesses, public research institutions, and startup companies. From key observations, we would like to highlight that such investments have very positive impacts on the development of RNAi technology. Nonetheless, the success of this technology is dependent on several factors, such as financial requirements, the complexity, and timeframe of the entire development process, as well as stringent regulations imposed by the relevant authorities. In most countries, RNAi-based transgenic crops are still considered as a genetically modified (GM) product, which necessitates the crops to undergo rigorous evaluation before approval is granted. Recent advancements in exogenous RNAi-derived biopesticides have provided a nontransgenic alternative to GM crops. However, challenges still remain in the form of technical hurdles and regulatory ambiguities surrounding this emerging technology. Its full potential remains to be realized.
  15. Beyond the PAR spectra: impact of light quality on the germination, flowering, and metabolite content of Stevia rebaudiana (Bertoni)
    Rengasamy N, Othman RY, Che HS, Harikrishna JA
    J Sci Food Agric, 2022 Jan 15;102(1):299-311.
    PMID: 34091912 DOI: 10.1002/jsfa.11359
    BACKGROUND: Stevia rebaudiana is a high value crop due to the strong commercial demand for its metabolites (steviol glycosides) but has limited geographical cultivation range. In non-native environments with different daylength and light quality, Stevia has low germination rates and early flowering resulting in lower biomass and poor yield of the desired metabolites. In this study, artificial lighting with light-emitting diodes (LEDs) was used to determine if different light quality within and outside of the photosynthetically active radiation (PAR) range can be used to improve germination rates and yields for production of steviol glycosides for the herbal supplement and food industry.

    RESULTS: Plants treated with red and blue light at an intensity of 130 μmol m-2  s-1 supplemented with 5% of UV-A light under a 16-h photoperiod produced the most desirable overall results with a high rate of germination, low percentage of early flowering, and high yields of dry leaf, stevioside and rebaudioside A, 175 days after planting.

    CONCLUSION: While red and blue light combinations are effective for plant growth, the use of supplemental non-PAR irradiation of UV-A wavelength significantly and desirably delayed flowering, enhanced germination, biomass, rebaudioside A and stevioside yields, while supplemental green light improved yield of biomass and rebaudioside A, but not stevioside. Overall, the combination of red, blue and UV-A light resulted in the best overall productivity for Stevia rebaudiana. © 2021 Society of Chemical Industry.

  16. Fulltext DhMYB22 and DhMYB60 regulate pigment intensity and floral organ shape in Dendrobium hybrid
    Khairul-Anuar MA, Mazumdar P, Othman RY, Harikrishna JA
    Ann Bot, 2022 Sep 26;130(4):579-594.
    PMID: 35980362 DOI: 10.1093/aob/mcac103
    BACKGROUND: Flower pigment and shape are determined by the coordinated expression of a set of structural genes during flower development. R2R3-MYB transcription factors are known regulators of structural gene expression. The current study focused on two members of this large family of transcription factors that were predicted to have roles in pigment biosynthesis and organ shape development in orchids.

    METHODS: Phylogenetic analysis was used to identify candidate Dendrobium catenatum R2R3-MYB (DcaMYB) sequences associated with pigment and cell shape development. Gene silencing of candidate DhMYBs in Dendrobium hybrid by direct application of dsRNA to developing flowers was followed by observation of gene expression level and flower phenotypes. Silencing of the structural gene chalcone synthase was used as a comparative control.

    KEY RESULTS: Ten candidate flower-associated DcaMYBs were identified. Flowers treated with dsRNA of DhMYB22 and DhMYB60 sequences were less pigmented and had relatively low expression of anthocyanin biosynthetic genes (F3'H and DFR), lower total anthocyanin concentration and markedly lower levels of cyanidin-3-glucoside and cyanidin-3-rutinoside. Petals of DhMYB22-treated flowers and sepals of DhMYB60-treated flowers showed the greatest colour difference relative to the same organs in untreated flowers. DhMYB22-treated flowers had relatively narrow and constricted lips, while DhMYB60-treated flowers had narrow and constricted sepals. No significant difference in shape was observed for DhCHS-treated or untreated flowers.

    CONCLUSIONS: Our results demonstrate that DhMYB22 and DhMYB60 regulate pigment intensity and floral organ shape in Dendrobium. This is a first report of MYB regulation of floral organ shape in orchids.

  17. Ectopic expression of a Musa acuminata root hair defective 3 (MaRHD3) in Arabidopsis enhances drought tolerance
    Wong GR, Mazumdar P, Lau SE, Harikrishna JA
    J Plant Physiol, 2018 Dec;231:219-233.
    PMID: 30292098 DOI: 10.1016/j.jplph.2018.09.018
    Genetic improvement is an important approach for crop improvement towards yield stability in stress-prone areas. Functional analysis of candidate stress response genes can provide key information to allow the selection and modification of improved crop varieties. In this study, the constitutive expression of a banana cDNA, MaRHD3 in Arabidopsis improved the ability of transgenic lines to adapt to drought conditions. Transgenic Arabidopsis plants expressing MaRHD3 had roots with enhanced branching and more root hairs when challenged with drought stress. The MaRHD3 plants had higher biomass accumulation, higher relative water content, higher chlorophyll content and an increase in activity of reactive oxygen species (ROS) scavenging enzymes; SOD, CAT, GR, POD and APX with reduced water loss rates compared to control plants. The analysis of oxidative damage indicated lower cell membrane damage in transgenic lines compared to control plants. These findings, together with data from higher expression of ABF-3 and higher ABA content of drought-stressed transgenic MaRHD3 expressing plants, support the involvement of the ABA signal pathway and ROS scavenging enzyme systems in MaRHD3 mediated drought tolerance.
  18. Adding value to banana farming: Antibody production in post-harvest leaves
    Singh JKD, Mazumdar P, Othman RY, Harikrishna JA
    J Biotechnol, 2024 May 20;387:69-78.
    PMID: 38582406 DOI: 10.1016/j.jbiotec.2024.04.001
    Banana, a globally popular fruit, is widely cultivated in tropical and sub-tropical regions. After fruit harvest, remaining banana plant materials are low-value byproducts, mostly composted or used as fibre or for food packaging. As an aim to potentially increase farmer income, this study explored underutilised banana biomass as a novel plant tissue for production of a high-value product. Protein scFvTG130 used in this study, is an anti-toxoplasma single chain variable fragment antibody that can be used in diagnostics and neutralising the Toxoplasma gondii pathogen. Using detached banana leaves, we investigated the factors influencing the efficacy of a transient expression system using reporter genes and recombinant protein, scFvTG130. Transient expression was optimal at 2 days after detached banana leaves were vacuum infiltrated at 0.08 MPa vacuum pressure for a duration of 3 min with 0.01% (v/v) Tween20 using Agrobacterium strain GV3101 harbouring disarmed virus-based vector pIR-GFPscFvTG130. The highest concentration of anti-toxoplasma scFvTG130 antibody obtained using detached banana leaves was 22.8 µg/g fresh leaf tissue. This first study using detached banana leaf tissue for the transient expression of a recombinant protein, successfully demonstrated anti-toxoplasma scFvTG130 antibody expression, supporting the potential application for other related proteins using an underutilised detached banana leaf tissue.
  19. Deep parallel sequencing reveals conserved and novel miRNAs in gill and hepatopancreas of giant freshwater prawn
    Tan TT, Chen M, Harikrishna JA, Khairuddin N, Mohd Shamsudin MI, Zhang G, et al.
    Fish Shellfish Immunol, 2013 Oct;35(4):1061-9.
    PMID: 23816854 DOI: 10.1016/j.fsi.2013.06.017
    MicroRNAs (miRNAs) are ~20-22 nucleotides, non protein-coding RNA regulatory genes that post-transcriptionally regulate many protein-coding genes, influencing critical biological and metabolic processes. While the number of known microRNA is increasing, there is currently no published data for miRNA from giant freshwater prawns, Macrobrachium rosenbergii (M. rosenbergii), a commercially cultured and economically important food species. In this study, we identified novel miRNAs in the gill and hepatopancreas of M. rosenbergii. Through a deep parallel sequencing analysis and an in silico data analysis approach, 327 miRNA families were identified from small RNA libraries with reference to both the de novo transcriptome of M. rosenbergii obtained from RNA-Seq and to miRBase (Release 18.0, November 2012). Based on the identified mature miRNA and recovered precursor sequences that form appropriate hairpin structures, three conserved miRNA (miR125, miR750, miR993) and 27 novel miRNA candidates encoding messenger-like non-coding RNA were identified. miR-125, miR-750, G-m0002/H-m0009, G-m0005, G-m0008/H-m0016, G-m0011/H-m0027 and G-m0015 were selected for experimental validation with stem-loop quantitative RT-PCR and were found to be coherent with the expression profile of deep sequencing data as evaluated with Pearson's correlation coefficient (r = 0.835178 for miRNA in gill, r = 0.724131 for miRNA in hepatopancreas). Using a combinatorial approach of pathway enrichment analysis and inverse expression relationship of miRNA and mRNA, four co-expressed novel miRNA candidates (G-m0005, G-m0008/H-m0016, G-m0011/H-m0027, and G-m0015) were found to be associated with energy metabolism. In addition, the expression of the three novel miRNA candidates (G-m0005, G-m0008/H-m0016, and G-m0011/H-m0027) were also found to be significantly reduced at 9 and 24 h post infection in M. rosenbergii challenged with infectious hypodermal and hematopoietic necrosis virus, suggesting a functional role of these miRNAs in crustacean immune defense.
  20. Fulltext Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications
    Yeo CC, Abu Bakar F, Chan WT, Espinosa M, Harikrishna JA
    Toxins (Basel), 2016 Feb 19;8(2):49.
    PMID: 26907343 DOI: 10.3390/toxins8020049
    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.
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