Indonesian Journal of Biotechnology
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Chitosan suppresses the expression level of WRKY17 on red chili (Capsicum annuum) plant under drought stress
No full textChili pepper plays a significant role in the global market. However, the production is often impeded by drought stress involving WRKY genes as the defense regulator. Chitosan is considered as a promising alternative fertilizer and defense elicitor. Hence, this study aimed to determine the role of chitosan in improving plant growth and survival of red chili pepper against drought stress. At the onset of the generative phase, chili plants were subjected to 1 mg mL‐1 chitosan, 50 percent drought, or chitosan‐drought treatment. Observations were made on several growth parameters, opened stomata, and WRKY gene expression. The results showed that chitosan‐drought treatment decreased plant growth and yielded significantly. The percentage of opened stomata was recorded at 0.56‐fold lower than control. It was followed by the decrease of the relative expression of WRKY17 and WRKY53 genes up to 0.56 and 0.72‐fold lower than control, respectively. Therefore, we suggested that the double treatment of chitosan‐drought might decrease plant growth performance but increase the defense system by suppressing the expression level of the WRKY17 gene. Interestingly, the drought treatment significantly increased WRKY17 expression level up to 7‐fold higher than control. Hence, it was suggested that WRKY17 has a specific role in response to drought stress
Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter
No full textTrichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively
Application of CRISPR/Cas9 genome editing system for molecular breeding of orchids
No full textOrchid is an important ornamental plant in Indonesia due to their natural beauty of flowers. In the tropical forest, orchids are being acquired for trading and commercial market. Thus, the effort is required to proliferate orchid in large quantities for conservation and improve the floral variation for plant breeding. The purpose of this study is to develop a firmed methodology of molecular breeding of orchids using CRISPR/Cas9 KO system. The plant material used was Phalaenopsis amabilis protocorms growth on NP medium+pepton (2 g/L). Protocorm were submerged in the culture of Agrobacterium tumefaciens that Ti‐plasmid had been filled with a T‐DNA construct of a pRGEB32 vector harboring sgRNA with PDS3 sequence. Detection for transformants was confirmed by PCR using HPT primers (545 bp), Cas9 primers (402 bp), PDS primers (280 bp) and trnL‐F (1200 bp) as an internal control. The results showed that 0.96% PDS transformants were obtained from PDS3T2 lines. Several transformant showed pale leaf color compared to non‐transformant plants. This study suggests that the target gene has successfully edited by CRISPR/Cas9 system and could be applied for that functional gene editing in orchids
Cloning and in silico analysis revealed a genetic variation in osmotin-encoding genes in an Indonesian local cacao cultivar
No full textTheobroma cacao L. is an important Indonesian estate crop, which suffers from biotic and abiotic stresses. TcOSM, which encodes osmotin as a response to pathogens and environmental stresses, is, therefore, a focus of interest in this research, aiming to characterize TcOSM in an Indonesian local cacao cultivar. Bioinformatics queries for putative TcOSM were performed against the reference genome of a Criollo-type cacao cultivar. Based on nucleotide sequence determination, our results revealed two genes, TcOSM1 and TcOSM2, which have the highest similarity (≥ 90\%) to the cacao reference genes. Heterozygosity was detected in the TcOSM1-encoding gene, which contained two overlapping peaks in Sanger-sequencing chromatograms. One of the alleles resulted from a single nucleotide change (G to A), leading to a same-sense mutation that did not substitute corresponding alanine residue. Homology modeling using Phyre2 and structural alignment (superimposition) was conducted to examine the influence of genetic variations in TcOSM sequences upon the global protein structures. The result showed no significant changes (RMSD ≤ 0.206 Å, TM-score > 0.5) in tertiary protein structures. Altogether, this research succeeded in characterizing TcOSM while providing a fundamental study for future cacao biotechnology endeavors.
In vitro expression of the recombinant fusion protein of Newcastle disease virus from local Indonesian isolates by using a cell-free protein expression system
No full textThe aim of this work was the in vitro expression of the recombinant fusion (F) protein of Newcastle disease virus (NDV). The pBT7-N-His-Fusion-NDV expression plasmid which carries the recombinant F protein encoding gene from local Indonesian isolates, was prepared and transformed into E. coli BL21 (DE3). To detect bacterial colonies carrying the recombinant plasmid, a restriction endonuclease analysis was performed using the EcoRI restriction endonuclease. These results showed that the pBT-N-His-Fusion-NDV plasmid was successfully isolated with a size of 4.601 bp, and three recombinant plasmids carrying the gene coding for the recombinant F protein of NDV were obtained. Selected recombinant plasmids were then in vitro by using a cell-free protein expression system followed by visualization of the recombinant F protein on a 12% SDS-PAGE gel both by Coomassie Brilliant Blue staining and Western blotting. Recombinant F protein was successfully in vitro expressed by using a cell-free protein expression system as indicated by a specific single protein band with a molecular mass of 25.6 kDa
The potency of Pentagamavunone‐0 (PGV‐0) as chemopreventive agent for the formation and growth of breast cancer as revealed in 3D model
No full textPentagamavunone‐0 (PGV‐0) or 2,5‐bis(4’‐hydroxy‐3‐methoxybenzylidine)‐cyclopentanone is a curcumin analogue that exhibits anticancer activity in breast cancer cells. However, most of previous reports are limited to the use of two‐dimensional (2D) cell culture. The use of three‐dimensional (3D) cell culture model in cancer research can represent the real condition of cancer growth in patients better than the 2D culture. The purpose of this study was to determine the anticancer activity of PGV‐0 on a 3D model of HCC 1954 breast cancer cells. HCC 1954 cells were grown in the 3D culture in the presence of PGV‐0, and the spheroid formation and growth of formed spheroids were observed using microscope at 24 and 96 h, respectively. The cytotoxic effects were measured by MTT assay. PGV‐0 inhibited the formation and growth of spheroids at the concentration as low as 60 µM. The cytotoxic effect of PGV‐0 appeared in a dose‐dependent manner with the IC50 value of 70.9 µM. The results of this study indicate that PGV‐0 has an anticancer activity on a 3D model of HCC 1954 breast cancer cell line. Therefore, the result supported the potency of PGV‐0 as cancer chemopreventive agent
Enhancement of transient erythropoietin protein expression by valproic acid in CHO‐K1 suspension adapted cells
No full textErythropoietin (EPO) is a therapeutic protein that is widely used to increase red blood cell production in chronic kidney failure. EPO protein can be produced quickly with a transient gene expression system (TGE). However, the titer produced using TGE is usually lower than the stable gene expression system (SGE). It has been known that TGE can be improved by histone deacetylase inhibitors (iHDACs) such as valproic acid (VPA). This study was conducted to examine the VPA effect on EPO protein expression in CHO‐K1 suspension adapted cells and to find the optimum concentration of VPA on transient EPO protein production. EPO proteins was quantified using the enzyme‐linked immunosorbent assay (ELISA) method. The optimization of VPA concentrations showed that VPA increased the EPO protein yield by up to 2‐fold in transient EPO production, and the optimum concentration of VPA was 4 mM. VPA optimization was very helpful to obtain the maximum increase in the transiently expressed protein. Furthermore, this study can be used as a model to produce EPO proteins or other recombinant proteins rapidly with TGE of CHO‐K1 suspension adapted cells
Metagenomic analysis of intestinal microbiota in geese from different farming systems in Gunungpati, Semarang
No full textThe diversity of intestinal bacteria in geese correlates with environmental conditions, rearing methods, and consumed feeds. The intestinal bacteria composition is useful for the absorption of nutrition, improving the metabolism, and may be related to the immune system. This study was conducted to examine the intestinal bacteria composition and the diversity of maintained goose in aviaries and barns. This research was an observational exploratory. Five geese were taken purposively from local breeders in Gunungpati District, Semarang City. A total of 5 g of intestinal contents from each sample was used for microbial genome isolation. Then, the genome was amplified to collect 16S rRNA gene region V3-V4. The amplicons were then sequenced using the next generation sequencing (NGS) method (Illumina high-throughput sequencing; paired-end reads) and analyzed using QIIME2 to identify bacterial species. In addition, GC-MS was performed to identify and measure fatty acid contents in the intestinal. The results showed that both rearing and caged goose contained nine phyla of intestinal bacteria. The number of intestinal bacteria of barn geese (SU) reached 32,748 Operational Taxonomy Units (OTU); higher than aviary geese (SK), which was 11,646 OTU. The intestinal bacteria community in barn geese was approved by Phylum TM7 (Saccharibacteria candidate) (53.18%), followed by Firmicutes (32.51%) and Bacteriodetes (5.42%). Whereas on SK Firmicutes was compiled 49.3 4% of total OTU, TM7 (S. candidate) up to 21.17%, and Actinobacteria up to 15.99 %. The abundance of TM7 may contribute to high 9,12-octadecadienoic acid production, while Firmicutes was related to the high production of oleic acid. Based on these data, the reared geese had a more abundant diversity of bacteria than the caged one
Genetic recombination of bovine viral diarrhea virus subgenotype -1a and -1c in persistently infected dairy cattle
No full textThe bovine viral diarrhea virus (BVDV) is a major viral pathogen in cattle worldwide. In Indonesia, diversity in subgenotypes of BVDV-1 has been observed, with the highest proportion of subgenotype -1a, followed by -1c, -1b, and -1d. So far, phylogenetic analysis of BVDV-1 is based on nucleotide sequences of the 5′ UTR and partial NS5B regions. Accuracy in identifying the subgenotype and antigenic type is critical for vaccine development and effective vaccination. The aim of this study was to determine genetic recombination of BVDV through phylogenetic analysis of five different regions (5′ UTR, NPro, E2, NS3, and NS5B) of BVDV in persistently infected dairy cattle. Five isolates were sequenced using next-generation sequencing, and data were analyzed with the CLC Genomic Workbench 9.0 and MEGA-X programs. Phylogenetic analysis based on the 5′ UTR (275 nt), NPro (504 nt), E2 (1,122 nt), NS3 (2,049 nt), and NS5B (2,157 nt) regions indicated that one BVDV isolate from Banyumas, Central Java, could be classified into different subgenotypes based on the E2 region (-1c), but the same subgenotype based on the other four regions (-1a), suggesting the presence of genetic recombination of the BVDV subgenotypes -1a and -1c in persistently infected dairy cattle
Cloning and in silico study of an endoglucanase from a thermophilic bacterium isolated from a hydrothermal vent of West Kawio, Sangihe‐Talaud waters, North Sulawesi, Indonesia
No full textEndoglucanase is used in industries that apply high temperatures, such as bioethanol, detergent, paper, and animal feed industries. Most available endoglucanases have very low stability at high temperatures. Therefore, this study aimed to identfy a new thermostable endoglucanase that is able to maintain its actvity at high temperatures. Five isolates of thermophilic bacteria were previously isolated from the hydrothermal vent of West Kawio, Indonesia. Among them, the DSI2 isolate showed the highest endoglucanase actvity, and was identfed and named as Bacillus safensis DSI2. The EgDSI2 gene was cloned from B. safensis DSI2. EgDSI2 is 1851 bp long encoding a protein of 616 amino acids. The encoded protein, EgDSI2, has high sequence identty to other B. safensis endoglucanases and was predicted with the Compute pI/Mw tool to be 69.41 kDa. EgDSI2 was high in hydrophobic amino acids. The enzyme had higher percentage of Ala andPro, and lower percentage of Gly compared to thermolabile endoglucanases from two Bacillus species. EgDSI2 harbored a catalytc domain belonging to glycosyl hydrolase family 9 (GH9) and a type 3 cellulose‐binding domain (CBM3). Propertes of endoglucanases with GH9‐CBM3 modular organizaton include actvity over a wide pH range, high optmum temperature, and thermostablity. Therefore, EgDSI2 has potental applicatons in the industries