Indonesian Journal of Biotechnology
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Phylogenetic analysis of 23 accessions of Indonesian banana cultivars based on Internal Transcribed Spacer 2 (ITS2) region
No full textPisang Kepok (Musa spp. [ABB ’Saba’ subgroup]) has several unique characteristics, such as tolerance to drought and Fusarium Foc (TR4) disease. Currently, the genetic diversity of Pisang Kepok in Indonesia is not well identified, although it is widely cultivated. Information on genetic diversity is essential for developing breeding strategies to achieve efficient cultivar improvement in the future. Aims of this research were to analyze the genetic variation of Pisang Kepok from some islands in Indonesia and to determine the genetic relationship between Pisang Kepok and other accessions banana cultivars based on ITS2 region, as a basis for future research in improving banana quality through molecular breeding. We have conducted the multiple sequence alignment and built the phylogenetic tree analysis using the Bayesian Inference Phylogeny method of one million generations (ngen = 1,000,000). The ITS2 region showed two clade ingroups: first clade consists of banana with B genome (balbisiana), while the second clade consists of banana with only A genome (acuminata). In general, all accessions of Pisang Kepok cultivars were clustered in the B genome of bananas cultivars. In addition, the ITS2 sequences and secondary structures among Pisang Kepok from various regions are identical, suggesting that there was no genetic variation in the ITS2 region of Pisang Kepok from multiple areas in Indonesia
Expression profiling of the CHS8, CHI1A, IFS2, and CHR genes in black soybean seed [Glycine max (L). Merr.] of F4 generation
No full textBlack soybean [Glycine max (L.) Merr.] produces isoflavones as secondary metabolites, which have many benefits for human health and plant defense system. Expression profiling can guide potential work in functional genomics of the isoflavone biosynthesis pathway. Previous studies showed the vital role of the CHS8, CHI1A, and IFS2 genes in isoflavone biosynthesis. However, expression profiling of these genes in the local black soybean varieties is still limited. This study investigated the gene expression levels of the CHS8, CHI1A, IFS2, and CHR genes in local varieties, namely, UP106 (high isoflavone) and UP122 (low isoflavone) and its progenies, i.e., UP106xUP122 and UP122xUP106. Relative gene expression profiling was conducted on the basis of Reverse Transcriptase Polymerase Chain Reaction (RT‐PCR) with ACT2/7 as a housekeeping gene. As a result, the expression level of CHS8 in UP122 is lower than that in UP106. No significant difference in the expression level of CHI1A was observed in all samples. The expression levels of CHS8 and CHI1A in both progenies were higher than that in the parental line, whereas the expression levels of IFS2 in both progenies were lower than that in the parental line. CHS8 and IFS2 expression from UP106xUP122 was higher than that from UP122xUP106, whereas CHI1A expression from UP122xUP106 was higher than that from UP106xUP122. CHR showed a high expression in the reciprocal cross; however, this expression did not exceed from UP106. In conclusion, the crossing between parental lines did not affect the gene expression level in the isoflavone biosynthesis pathway
Cytoprotective activity of carrot and tomato callus extracts and the ex‐ pression of cytokines in UV‐B irradiated fibroblast cells
No full textStudies have suggested that both carrot (Daucus carota L.) and tomato (Solanum lycopersicum L.) callus extracts contain antoxidant compounds that might have the potental to protect cells from free radicals such as H2O2 that contribute to cell damage. The other sources of free radical exposure in human cells, such as UV‐B, should also be examined. UV‐B exposure can trigger increased expression of inflammatory cytokines such as cyclooxygenase‐2 (COX‐2) and tumor necrosis factor‐α (TNF‐α) and the antinflammatory cytokine interleukin‐10 (IL‐10), which causes photoaging. This study was conducted to investigate the cytoprotectve actvity of carrot and tomato callus aqueous extracts by observing cell viability using the MTT assay. Immunocytochemistry methods were used to examine the effects of carrot and tomato callus aqueous extracts on the expression of COX‐2, TNF‐α, and IL‐10 in human dermal fibroblast adult (HDFa) cells exposed to UV‐B light. Carrot and tomato callus aqueous extracts were obtained by the maceration method using aqua bidistilled solvent. Results showed that both carrot and tomato callus aqueous extracts at 0.5 mg/mL exhibited the highest cytoprotective effect in HDFa cells compared to that at other concentratons. Both carrot and tomato callus aqueous extracts could also decrease the expression of COX‐2 and TNF‐α, whereas carrot callus aqueous extract increased the expression of the anti‐inflammatory cytokine IL‐10 in HDFa cells
Physiological, biochemical and HSP70 and HSP90 gene expression profiles of tropical abalone Haliotis squamata in response to Vibrio alginolyticus infection
No full textVibrio spp. have been known responsible for fish diseases in marine and brackish‐water systems in the tropics regions. Heat shock proteins are a highly conserved protein group that is known for its rapid response to environmental stresses, including infection. This study aimed to investigate physiological and biochemical responses of tropical abalone Haliotis squamata to Vibrio alginolyticus infection. Abalones were infected with V. alginolyticus by intramuscular injection at a dose of 105,106,107 cfu/abalone. The expression of HSP70 and HSP90 genes, the activity of superoxide dismutase, phenol oxidase and catalase enzymes, histology, falling and mortality were observed at 12, 24, 48, 72, and 96 hours post‐infection (hpi). The different expression of HSPs was found in this study. While the expression of HSP70 was downregulated after infection, the expression of HSP90 was upregulated at 12 hpi and followed by downregulated after 24 hpi for 106 cfu infection, but expressed at a normal level for 105 infection treatment. The expression ofsuperoxide dismutase and catalase increased within 12 hpi, and the expression of phenol oxidase increased after 24 hpi. V. alginolyticus is virulent with LD50 of less than 105 cfu on H. squamata with an average weight of 5.13 g, and caused enlargement of hemolymph sinus and development intraepithelial and intramuscular abscesses
Computational modeling of AGO-mediated molecular inhibition of ARF6 by miR-145
No full textInhibition of ADP-ribosylation factor 6 messenger RNA (ARF6 mRNA) by microRNA-145 (miR-145), mediated by Argonaute (AGO) protein, has been found to play essential roles in several types of cancer and cellular processes. This study aimed to model the molecular interaction between miR-145 and ARF6 mRNA with AGO protein. The sequences of miR-145 and the 3’ untranslated region (UTR) of ARF6 mRNA were retrieved from miRTarBase, followed by miRNA target-site and structure predictions were done using RNAhybrid, RNAfold, and simRNAweb, respectively. The interaction between the miRNA-mRNA duplex and AGO was further assessed via molecular docking, interaction analysis, and dynamics, using PatchDock Server, PLIP, and VMD/NAMD, respectively. The models between miR-145, predicted target site of ARF6 mRNA, and AGO protein returned stable thermodynamic variables with negative free energy. Specifically, the RNA duplex had an energy of -19.80 kcal/mol, while the docking had -84.58 atomic contact energy supported by 70 hydrogen bonds and 14 hydrophobic interactions. However, the stability of the RMSD plot was still unclear due to limited computational resources. Nevertheless, these results computationally confirm favorable interaction of the three molecules, which can be utilized for further transcriptomics-based drugs or treatments
Identification of single nucleotide polymorphisms in GDF9 gene associated with litter size in Garut sheep
No full textThe growth differentiation factor 9 (GDF9) gene has been regarded as having major impacts on ovulation rate and litter size in sheep. The aim of this study was to identify the single nucleotide polymorphisms (SNPs) of the GDF9 gene and their association with litter size in Garut sheep. For this purpose, a total of 60 ewes of Garut sheep were included in this study. Based on the sheep GDF9 reference sequences (Genbank Acc. No. AF078545.2), one pair of primers (5’-CTGCTGTTTAACCTGGATCGTG-3 5’-GGAGAGCCATACCGATGTCC-3 as forward and reverse, respectively) was used for PCR amplification. The results revealed that four SNPs (g.54C>T, g.60G>A, g.304G>A, and g.333G>A) were found in Garut sheep by direct sequencing. For SNP g.54C>T, the sheep exhibited the highest frequency of allele C and genotype CC. On the other hand, SNPs g.60G>A, g.304G>A, and g.333G>A showed a higher frequency of allele G than allele A, and the GG genotype was predominant in the population. SNP g.333G>A had a significant effect on litter size (p A may be useful as a genetic marker for litter size in Garut sheep
Diagnosis and molecular characterization of Anaplasma platys in dog patients in Yogyakarta area, Indonesia
No full textAnaplasma platys is a tick-borne, Gram-negative bacterium that causes anaplasmosis, a companion vector-borne disease impacting dogs. Information on this disease remains limited in Indonesia. Its symptoms are not specific, so molecular analysis is required for a rapid and accurate diagnosis. GroEL is an essential gene commonly used for classification and species identification of many groups of bacteria, including Anaplasma spp. In this study, a molecular diagnosis of anaplasmosis based on the groEL gene sequence was conducted using PCR. In addition, the genetic diversity of Anaplasma platys in infected dogs was determined. Blood samples were collected from 51 dogs suspected of anaplasmosis from Prof. Dr. Soeparwi Animal Hospital, animal clinics, and pet shops in the Yogyakarta area, Indonesia, based on anamnesis, histories of tick infestations, and clinical symptom examinations. DNA extraction and PCR targeting the groEL gene were performed, followed by sequencing. Phylogenetic tree analysis and construction were carried out using the BLAST and MEGA programs. Positive PCR sample results (amplicon length of 624 bp) were found in 6 of 51 dogs. Samples A1 (KHJ/C2), A2 (KHJ/A2), A3 (KSK/L), A4 (KHJ/L), and A5 (KNP/M2) had close ties to Anaplasma platys (AF478129.1) from GenBank. Phylogenetic analysis showed a very high homology value (100%) and bootstrap value of 100%. It can be concluded that there was no genetic diversity in the Anaplasma platys found in infected dogs in the Yogyakarta area
Repetitive DNA sequences accelerate molecular cytogenetic research in plants with small chromosomes
No full textRepetitive DNA sequences are highly abundant in plant genomes and are favorable probes for chromosome identification in plants. However, it is difficult to conduct studies on the details of metaphase chromosome structures in plants with small chromosomes due to their highly condensed status. Therefore, identification of homologous chromosomes for karyotyping and analyzing chromosome structures is a challenging issue for cytogeneticists without specific probes and precise chromosome stages. In this study, five repetitive DNA probes, i.e., 5S and 45S ribosomal DNAs (rDNAs), melon centromeric sequence (Cmcent), cucumber subtelomeric sequence (Type I), and microsatellite (CT)10 repeats, were used to identify primary constrictions and homologous chromosomes for karyotyping. Four and two loci of 45S rDNA were respectively observed on metaphase and pachytene chromosomes of Abelia × grandiflora. Cmcent was detected on both primary constrictions of melon pachytene and metaphase chromosomes. Furthermore, one pair of 5S rDNA signals were hybridized on melon metaphase chromosomes. Eight and two loci of 45S and 5S rDNA were respectively detected on cucumber chromosomes. Type I and (CT)10 probes were specifically hybridized on subtelomeric and interstitial regions on the chromosomes, respectively. These results suggest that repetitive DNA sequences are versatile probes for chromosome identification in plants with small chromosomes, particularly for karyotyping analyses
Expression and purification of recombinant coat protein of sugarcane mosaic virus from Indonesian isolate as an antigen for antibody production
No full textSugarcane mosaic virus (SCMV, genus Potyvirus, family Potyviridae) is a prominent pathogen of sugarcane (Saccharum sp. hybrids). It can cause losses in susceptible varieties, in crop as well as sugar production, economically. Although it has been studied in major sugar-producing countries, research on the definement of SCMV from Indonesian isolates based on molecular study has been very limited. This study aimed to obtain a proper recombinant antigens emanating from coat protein of SCMV from Indonesian isolate in order to produce polyclonal antibodies that cann be used for immunodiagnosis assays in a subsequent study. A gene-encoding coat protein of SCMV (CP-SCMV) was amplified using RT-PCR and cloned into vector pJET1.2. The cDNA was inserted into 6X His-tag expression plasmid of pET28a(+) and over-expressed in Escherichia coli BL21(DE3) to produce a recombinant protein. The highest expression was found in 0.1M IPTG induction media for 5 h at 37oC. SDS-PAGE analysis clarified that the recombinant CP-SCMV remained as an insoluble fraction. Purifications was carried out by the affinity Ni-NTA resin, followed by electroelution to obtain a highly purified protein. To meet the quality requirements of a proper antigen, the highly purified protein was concentrated. A molecular weight of the rCP-SCMV (approximately 40 kDa) was clearly observed by 10% SDS-PAGE at the concentration of 16.184 mg/mL.
Data mining analysis of miR-638 and key genes interaction in cisplatin resistant triple-negative breast cancer
No full textCisplatn is one of the chemotherapy for the treatment of triple‐negatve breast cancer (TNBC), but its effectveness is limited because of the phenomenon of chemoresistance. miR‐638 was shown to regulate chemoresistance; however, it has never been validated in the cisplatn‐resistant tumor from patents. This present study aimed to identfy the key gene regulatory networks of miR‐638 and evaluate the potental role of the miR‐638 and its targets as potental prognosis biomarkers for cisplatn‐resistance triple‐negatve breast cancer patents. The miR‐638 target was obtained from the miRecords database while the mRNA of chemoresistance biomarker candidate was obtained from the GSE18864 of GEO database, which is mRNA of cisplatn‐resistance TNBC patents. CCND1 and FZD7 are potental candidates for cisplatn chemoresistance biomarkers in patents with TNBC. Moreover, a Kaplan‐Meier survival plot showed that breast cancer patents with low mRNA levels of FZD7 had signifcantly worse overall survival than those in higher mRNA expression group. Taken together, miR‐638 plays a role in cisplatn resistance mechanism through a mechanism involving its target gene CCND1 and FZD7. Overall, miR‐638, CCND1, and FZD7 are candidates for cisplatn biomarker resistance in TNBC