Indonesian Journal of Biotechnology
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Morphology and molecular characterization of Vanda tricolor × Vanda limbata orchid hybrid based on VOH1 gene characters
No full textVanda is a monopodial epiphyte orchid that spreads throughout Asia and Southeast Asia reaching 70 species. Indonesia itself has its own endemic Vanda orchid such as Vanda tricolor and Vanda limbata. A hybrid of V. tricolor and V. limbata is predicted to form a new specific character in the flower and leaf. The purpose of this study was to determine the morphological and molecular differences between V. tricolor, V. limbata, and Vanda hybrids resulting from crosses between that two orchids, by analysing the morphological characteristics of the roots, leaves, flowers and the structure of the Vanda Orchid Homeobox1 (VOH1) shoot‐forming gene isolated from V. tricolor, V. limbata, and their hybrids. The morphological analysis was conducted using RHS colour chart, size measurement of plants, and the transversal preparation of the leaf. Molecular analysis was performed by PCR using Dendrobium Orchid Homeobox 1 (DOH1) primers, followed by sequencing and bioinformatic analysis. Morphologically, the flower’s colour of the hybrid is most similar to V. limbata but the flower’s patterns are more similar to V. tricolor meanwhile the leaf colour of the hybrid is brighter than the both parents. The slides illustrate the sclerenchyma tissue is made up of strongly thickened walls containing lignin indicates the presence of homeobox DOH1 gene homolog, namely VOH1. The molecular result displayed by the phylogenetic tree of the VOH1 indicates that the hybrid has more similarities with V. limbata
Exploring the mechanism of Glycyrrhiza glabra and Curcuma domestica against skin photoaging based on network pharmacology
No full textExcessive exposure to UV radiation results in skin photoaging, which may be prevented or treated using natural plant compounds. Herbal cosmetics and medicines have grown in popularity due to the abundance of relatively safe compounds. This research aims to explore the network pharmacology of Glycyrrhiza glabra (GG) and Curcuma domestica (CD) against skin photoaging. Active compounds from GG‐CD were sourced from databases including TCSMP, KnapSack, TCMID, and published literature, while disease targets were collected from GeneCards and OMIM databases. The STRING database was utilized to construct the protein‐protein interaction (PPI) network. Enrichment analyses for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed using Metascape. The herb‐compounds‐target‐pathway‐disease (H‐C‐T‐P‐D) network was visualized using Cytoscape software. A total of 529 compounds, 2,335 active compound targets, and 120 skin aging targets were obtained. GO enrichment revealed 1,635 biological processes, 67 cellular components, and 121 molecular functions. The study suggests that GG and CD have the potential to treat skin photoaging by targeting multiple targets, such as TP53, TNF, AKT1, IL6, and IL‐1B, as well as multiple pathways, such as those in cancer, apoptosis, TNF, IL‐17, and the AGE‐RAGE signaling pathway. Experiment validation is necessary to confirm the preliminary network pharmacology results
A robust in planta Agrobacterium‐mediated transformation in red chili (Capsicum annuum L.)
No full textPlant improvement through in vitro culture and genetic engineering is a significant aspect of breeding programs aimed at producing disease‐resistant cultivars of disease‐prone red chili (Capsicum annuum L.). However, the Capsicum genus is recalcitrant to genetic transformation and in vitro regeneration. Moreover, developing a universal transformation protocol is difficult due to its highly genotype‐dependent nature. Therefore, this study aimed to develop an Agrobacterium‐mediated in planta transformation method applicable to various red chili cultivars. Two open‐pollinated varieties, Tanjung 2 and Ciko, were subjected to transformation. The young seedlings were immersed in transformation medium containing Agrobacterium tumefaciens strain GV3101 harboring the binary vector pCAMBIA1301, which carries the β‐glucuronidase (GUS) gene. GUS histochemical analysis revealed that all the primary transformants of Tanjung 2 and Ciko were identified as chimeric. The average staining in the body of the seedlings was 88.63 + 26.33% in Tanjung 2, and 90.65 + 16.77% in the Ciko variety. More than 50% of the seedlings continued to express GUS in their shoot areas 10 days after Agrobacterium infection, indicating the possibility of transgene inheritance in the following generation. The in planta transformation approach is notably genotype independent, making it a promising standard transformation protocol for different red chili varieties
Revealing disease‐specific endogenous target mimic of microRNA from long non‐coding RNA identification and characterization in Musa spp.
No full textBanana (Musa spp.) is one of the most widely consumed fruits in the world. Unfortunately, the plants are at risk from many disease problems, which mainly derive from microorganism. It is a little known about the relationship between disease‐inducing microorganisms and plants, particularly at the molecular level. This research aimed to characterize long non‐coding RNA (lncRNA) from bananas that may have roles in regulating gene expression related to the disease response mechanism in banana derived from transcriptomic libraries. Furthermore, the detected transcripts were analyzed to identify the endogenous target mimics (eTMs) interaction between lncRNA and microRNA (miRNA) using computational approaches. Data from Cavendish banana (AAA group), Berangan (AAA group), Yunnan Banana (Itinerans), Dajiao (ABB group), and Klutuk (BB group) were used in this research. We found that lncRNA tends to be unsustainable, and most sizes are below 1000 bp (≥ 75%). Based on this result, we investigated the eTMs to determine lncRNA transcripts and miRNA, such as miR397 in Cavendish and miR444 in Klutuk. This transcript would be regulated following exposure to extreme temperatures and disease, indicating the possibility of disease‐specific interaction between bananas and their environment at the molecular level
The efficacy of a chicken antibody for the development of immunoassay‐based rapid detection in sugarcane mosaic virus disease
No full textSugarcane Mosaic Virus (SCMV) infection is one of the most serious problems that can result in severe yield loss of sugarcane. Since the symptoms of SCMV infection are similar to other biotic and abiotic stress symptoms, the development of a rapid diagnostic with high precision is required. The use of laboratory animals such as rabbits is required for antibody production in immunoassay‐based detection. However, due to its many advantages, specific chicken egg yolk immunoglobulin (IgY) has received considerable attention as an alternative antibody production in immunodiagnostics for infectious diseases. In this study, IgY antibody against SCMV recombinant coat protein (CP) was successfully obtained from chicken blood serum and tested to compare its efficacy against antibody from rabbit (IgG) using immunocapture reverse transcription‐polymerase chain reaction (IC‐RT‐PCR). The result showed that IgY and IgG could detect 0.1 g SCMV infected leaves using 1000‐times‐diluted antibodies. The IgY antibody was also confirmed to be reproducible and potentially applicable in plant disease diagnostics using an antibody‐based detection
A novel pilot bioreactor for scaling up biomass and bioactive compounds on Gynura procumbens adventitious root culture
No full textBioreactors for adventitious root culture have been developed to obtain biomass and plant bioactive compounds in large quantities. These technologies provide a great opportunity to produce biomass and bioactive compounds more quickly from Gynura procumbens compared to conventional plant cultivation systems. In previous studies, biomass and bioactive compounds of G. procumbens adventitious roots were successfully increased using a small‐scale bioreactor. In this study, a pilot bioreactor the capacity of 19 L polycarbonate gallon was successfully developed. This bioreactor can be sterilized under the pressure of 0.18 MPa for approximately 60 min. While the bioreactor could not be sterilized when the pressure was less than 0.18 MPa damage may have occurred to the bioreactor vessel at pressures exceeding 0.18 MPa. The results of the chemical grade test as root culture media showed that MS‐Tek provided an optimal root biomass compared to MS‐PA after a 35‐day of the culture period. In addition, the productivity of the total phenolics and flavonoids of adventitious root in MS‐PA was higher than in MS‐Tek. This novel pilot bioreactor is suitable for G. procumbens adventitious root culture, and the technical‐grade chemicals are suitable for improving root biomass production
Inhibition of protease activity and anti‐quorum sensing of the potential fraction of ethanolic extract from Sansevieria trifasciata Prain leaves against Pseudomonas aeruginosa
No full textSansevieria trifasciata is a plant that is commonly utilized in traditional medicine. The leaves of S. trifasciata show antibacterial properties against Pseudomonas aeruginosa. This bacterium is an opportunistic pathogen that can cause serious illness in humans and produce a variety of virulence factors responsible for bacterial pathogenesis with quorum sensing (QS) systems that mediate intracellular communication. Bacteria produce protease through a QS mechanism in which they express signaling molecules to become pathogens. Proteases are extracellular enzymes required for successful infection that mediate biofilm spread through QS and regulate a variety of cellular and physiological functions. This research aimed to evaluate the protease, and anti‐QS activities of the ethanolic extract from S. trifasciata leaves against P. aeruginosa and the expression of QS genes. An azocasein test was used to determine the protease activity in qualitative and quantitative methods. Using real‐time quantitative polymerase chain reaction, a study was conducted to investigate the effect of ethanolic extract from S. trifasciata leaves on selected QS‐regulatory genes at the transcriptional level. The results showed that the potential ethanolic extract from S. trifasciata leaves inhibited the protease enzyme activity by as much as 77.1%. The potential ethanolic extract from S. trifasciata leaves decreased the expressions of lasA, lasB, lasI, lasR, rhlI, and rhlR with 2‐ΔΔCt values of 0.81, 0.93, 0.76, 0.97, 0.90, and 0.55 respectively
Biophysical characterization of folded state type II luciferase‐like monooxygenase
No full textWe noticed that the Priestia megaterium genome contains five Luciferase‐like monooxygenase (LLM) encoding genes, however, their functions are unknown. The objective of this work was to characterize the biophysical properties of the recombinant LLM2 from Priestia megaterium PSA10 through in vitro and in silico approaches. We successfully cloned into the pET vector system and expressed the recombinant LLM2 in Escherichia coli BL21(DE3). The recombinant LLM2 was overproduced and purified in the form of an inclusion body with a molecular weight of ±39.5 kDa when it was analyzed in 15% SDS‐PAGE. The inclusion body of recombinant LLM2 was then refolded and characterized for its biophysical properties by measuring the UV spectrum of 200 to 250 nm wavelength and determining the change of enthalpy (ΔH) and entropy (ΔS) at the melting temperature. The refolded recombinant LLM2 exhibited a strong spectrum at 205 nm, while the unfolded recombinant LLM2 did not. The Tm, ΔHTm, and ΔSTm values of the refolded recombinant LLM2 were determined to be 318.31±4.4 K, 11.76±1.3 kJ.mol‐1, and (3.74±0.48)x10‐2 kJ.mol‐1.K‐1, respectively. The predicted 3D structure of LLM2 showed that the protein contains the TIM‐barrel, resembling the common global fold of bacterial luciferases. Determination of the cofactor preference suggested that the LLM2 preferred FAD for its cofactor
Thrombolytic protease characterization from leaves and fruit flesh of the jernang rattan plant (Daemonorops draco)
No full textThrombolytic agents are used for thrombolytic therapy to dissolve blood clots that form in a blood vessel. All currently used thrombolytic agents have unfavorable shortcomings, such as gastrointestinal bleeding, allergic reactions, and thrombolytic agent resistance, treatment for some of which can be quite expensive. As a result, the search for thrombolytic agents derived from plants is currently taking place. Some plants have been discovered to contain protease enzymes with thrombolytic activity; pharmaceuticals derived from plants are believed to be safer. Jernang rattan (Daemonorops draco) is a plant of the Arecaceae family and is known to produce resin. Jernang rattan resin is also known to have antioxidant, antiseptic, antitumor, antimicrobial, and cytotoxic activity, but very limited information on proteolytic activity of the protease from this plant. This research aims to isolate proteases from the leaves and fruit flesh of the rattan jernang plant (D. draco) and to investigate the proteolytic activity of the isolated proteases. The protease was isolated from the leaves and the fruit flesh, and then partially purified by ammonium sulfate precipitation. The radial caseinolytic assay showed that protease in a 60% ammonium sulfate fraction gave a clear zone, with diameters of 1.4 cm and 1.8 cm for the protease isolated from leaves and fruit flesh, respectively. A Folin‐Ciocalteau assay showed that the enzymes isolated were able to hydrolyze casein and release L‐tyrosine, with activity of 0.158 U/mL and 0.174 U/mL for the protease from the leaves and fruit flesh, respectively. A fibrinogenolytic assay showed that the protease from the fruit flesh hydrolyzed the A‐α, B‐β and the γ chain of human fibrinogen, while the protease from the leaves hydrolyzed the A‐α and γ chain. Both proteases were inhibited by 56% by phenylmethylsulfonyl fluoride (PMSF), indicating that the enzymes are serine proteases. Based on the assay results obtained, it can be concluded that proteases isolated from the leaves and fruit flesh have potential as thrombolytic proteases
Nephroprotective effects of cardamom essential oil (Amomum compactum Soland. Ex Maton) on kidney cells
No full textMany chemotherapeutic agents cause various side effects, including nephrotoxicity. Cardamom essential oil (Amomum compactum Soland. ex Maton) contains compounds that exhibit antioxidant activity, such as 1,8‐cineole, α‐pinene, α‐terpineol, and linalool. This study focused on exploring the potency of cardamom essential oil (CEO) as an anti‐senescent induced by doxorubicin using the Vero kidney cell line. We first obtained the CEO by steam distillation, then evaluated its cytotoxicity using a trypan blue exclusion assay. Moreover, we performed senescence‐associated beta‐galactosidase (SA‐β‐gal) staining and 2’,7’‐dichlorodihydrofluorescein diacetate (DCFDA) staining to measure the effect of CEO on intracellular ROS level and cell senescence, respectively. Analysis of the compounds with gas chromatography‐mass spectrophotometry (GC‐MS) revealed seven compounds with significant abundance, namely 1,8‐cineole (50.82%), ß‐pinene (12.43%), α‐terpineol (8.50%), fenchone (4.10%), α‐pinene (4.00%), sabinene (3.00%), and linalool (1.98%). The cytotoxicity assay of CEO on Vero cells showed an IC50 value of 178 μg/mL. Thus, CEO is considered low cytotoxic for normal kidney cells (>100 μg/mL). Concentrations of 50 and 100 μg/mL CEO reduced the cell senescence induced by doxorubicin. Therefore, CEO has potency as a nephroprotective agent in doxorubicin‐induced senescence