Pub Date : 2020-01-02DOI: 10.22146/ijbiotech.52170
A. Budiani, Imam Bagus Nugroho, D. A. Sari, Inez Palupi, R. Putranto
Oil palm plantation in Indonesia is significantly affected by basal stem rot disease caused by the pathogenic fungus Ganoderma boninense . Tolerant oil palm cultivars toward G. boninense have been developed through a breeding program accelerated by the implementation of the CRISPR/Cas9 technology. This study was conducted to perform a gene knockout (KO) of oil palm that confers a putative defense‐related trait toward G. boninense . A plasmid pCRISPR_EMLP containing modules, i.e., 35S‐CaMV‐promoter‐driven CRISPR/Cas9, U6‐promoter‐driven sgRNA to the target EgEMLP gene (EL695076), and hygromycin resistance gene as the selectable marker, was established for Agrobacterium‐mediated delivery into oil palm calli (OPC). The transformed OPCs were regenerated and screened in DF (de Fossard) media containing hygromycin. The working concentration of hygromycin was successfully optimized for selection at 20 ppm. Through PCR‐based selection using HYG primers, we succeeded in discerning positive transformed OPC clones. The sequenced PCR products of genomic DNA as the template amplified using EMLP1 primers showed a point mutation, causing a frameshift in the edited EgEMLP and premature stop codon. Furthermore, in silico modeling demonstrated that the mutation resulted in a change in the C‐terminal region, affecting the tertiary protein structure. Moreover, electrophoresis analysis of PCR products of cDNA as the template from transformed OPC clones showed several samples with faint or undetected bands. This indicated that the CRISPR/Cas9 module induced a mutation that could destabilize the transcribed mRNA, e.g., premature degradation. Altogether, this study has successfully implemented CRISPR/Cas9 gene editing in oil palm in a model gene that is responsible for putative defense‐related traits toward the pathogenic fungus G. boninense .
{"title":"CRISPR/Cas9‐mediated knockout of an oil palm defense‐related gene to the pathogenic fungus Ganoderma boninense","authors":"A. Budiani, Imam Bagus Nugroho, D. A. Sari, Inez Palupi, R. Putranto","doi":"10.22146/ijbiotech.52170","DOIUrl":"https://doi.org/10.22146/ijbiotech.52170","url":null,"abstract":"Oil palm plantation in Indonesia is significantly affected by basal stem rot disease caused by the pathogenic fungus Ganoderma boninense . Tolerant oil palm cultivars toward G. boninense have been developed through a breeding program accelerated by the implementation of the CRISPR/Cas9 technology. This study was conducted to perform a gene knockout (KO) of oil palm that confers a putative defense‐related trait toward G. boninense . A plasmid pCRISPR_EMLP containing modules, i.e., 35S‐CaMV‐promoter‐driven CRISPR/Cas9, U6‐promoter‐driven sgRNA to the target EgEMLP gene (EL695076), and hygromycin resistance gene as the selectable marker, was established for Agrobacterium‐mediated delivery into oil palm calli (OPC). The transformed OPCs were regenerated and screened in DF (de Fossard) media containing hygromycin. The working concentration of hygromycin was successfully optimized for selection at 20 ppm. Through PCR‐based selection using HYG primers, we succeeded in discerning positive transformed OPC clones. The sequenced PCR products of genomic DNA as the template amplified using EMLP1 primers showed a point mutation, causing a frameshift in the edited EgEMLP and premature stop codon. Furthermore, in silico modeling demonstrated that the mutation resulted in a change in the C‐terminal region, affecting the tertiary protein structure. Moreover, electrophoresis analysis of PCR products of cDNA as the template from transformed OPC clones showed several samples with faint or undetected bands. This indicated that the CRISPR/Cas9 module induced a mutation that could destabilize the transcribed mRNA, e.g., premature degradation. Altogether, this study has successfully implemented CRISPR/Cas9 gene editing in oil palm in a model gene that is responsible for putative defense‐related traits toward the pathogenic fungus G. boninense .","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47329407","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-01-02DOI: 10.22146/ijbiotech.51734
R. Rumiyati, S. Sismindari, E. Semiarti, Sitarina Widyarani, Dewi Tika Sari, Brilliant Kharisma Apritadila, Anami Riastri
Studies 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 H 2 O 2 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 investgate 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 fbroblast adult (HDFa) cells exposed to UV‐B light. Carrot and tomato callus aqueous extracts were obtained by the maceraton method using aqua bidistlled solvent. Results showed that both carrot and tomato callus aqueous extracts at 0.5 mg/mL exhibited the highest cytoprotectve 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 ant‐inflammatory cytokine IL‐10 in HDFa cells.
{"title":"Cytoprotective activity of carrot and tomato callus extracts and the ex‐ pression of cytokines in UV‐B irradiated fibroblast cells","authors":"R. Rumiyati, S. Sismindari, E. Semiarti, Sitarina Widyarani, Dewi Tika Sari, Brilliant Kharisma Apritadila, Anami Riastri","doi":"10.22146/ijbiotech.51734","DOIUrl":"https://doi.org/10.22146/ijbiotech.51734","url":null,"abstract":"Studies 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 H 2 O 2 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 investgate 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 fbroblast adult (HDFa) cells exposed to UV‐B light. Carrot and tomato callus aqueous extracts were obtained by the maceraton method using aqua bidistlled solvent. Results showed that both carrot and tomato callus aqueous extracts at 0.5 mg/mL exhibited the highest cytoprotectve 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 ant‐inflammatory cytokine IL‐10 in HDFa cells.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48865196","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-12-09DOI: 10.22146/ijbiotech.51718
F. Dwivany, R. Esyanti, V. Suendo, A. S. Pratiwi, A. A. Putri
Banana is an important crop that demands proper methods in postharvest handling. As a climacteric fruit, the banana fruit ripening process is affected by ethylene. Several methods have been developed to extend the shelf life of a banana, such as using ethylene scrubbers. In this study, ttanium dioxide (TiO 2 ), a photocatalyst, was used as an alternatve method to delay the fruit ripening process. The effect of TiO 2 on the ripening‐related gene MaACS1 was investgated. Banana fruits were placed in a TiO 2 ‐coated glass chamber and observed for ten days. Fruit ripening in the treated chamber was delayed for eight days compared to the control. Total RNA was extracted from control and TiO 2 ‐treated fruit pulp and synthesized into cDNA. Reverse transcripton PCR was performed to investgate the gene expression, which showed that MaACS 1 expression was relatvely lower than treated control. The fnding of these studies suggested that the TiO 2 chamber has the potental to extend the shelf life of banana by delaying its ripening process and decreasing the expression of MaACS1 . To the best of our knowledge, no previous study has investgated the effect of TiO 2 on the expression of genes related to banana fruit ripening.
{"title":"Analysis of ethylene biosynthesis gene expression profile during titanium dioxide (TiO2) treatment to develop a new banana postharvest technology","authors":"F. Dwivany, R. Esyanti, V. Suendo, A. S. Pratiwi, A. A. Putri","doi":"10.22146/ijbiotech.51718","DOIUrl":"https://doi.org/10.22146/ijbiotech.51718","url":null,"abstract":"Banana is an important crop that demands proper methods in postharvest handling. As a climacteric fruit, the banana fruit ripening process is affected by ethylene. Several methods have been developed to extend the shelf life of a banana, such as using ethylene scrubbers. In this study, ttanium dioxide (TiO 2 ), a photocatalyst, was used as an alternatve method to delay the fruit ripening process. The effect of TiO 2 on the ripening‐related gene MaACS1 was investgated. Banana fruits were placed in a TiO 2 ‐coated glass chamber and observed for ten days. Fruit ripening in the treated chamber was delayed for eight days compared to the control. Total RNA was extracted from control and TiO 2 ‐treated fruit pulp and synthesized into cDNA. Reverse transcripton PCR was performed to investgate the gene expression, which showed that MaACS 1 expression was relatvely lower than treated control. The fnding of these studies suggested that the TiO 2 chamber has the potental to extend the shelf life of banana by delaying its ripening process and decreasing the expression of MaACS1 . To the best of our knowledge, no previous study has investgated the effect of TiO 2 on the expression of genes related to banana fruit ripening.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45924907","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-12-05DOI: 10.22146/ijbiotech.49939
P. Swarnalatha, V. Venkataravanappa, C. Reddy, M. Kumar, M. K. Reddy
Cisplatn 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.
{"title":"Molecular characterization of ageratum enation virus and beta satellite associated with leaf curl disease of fenugreek in India","authors":"P. Swarnalatha, V. Venkataravanappa, C. Reddy, M. Kumar, M. K. Reddy","doi":"10.22146/ijbiotech.49939","DOIUrl":"https://doi.org/10.22146/ijbiotech.49939","url":null,"abstract":"Cisplatn 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.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43687465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-12-05DOI: 10.22146/ijbiotech.51726
A. Setiawan, A. Wibowo, C. Teo, S. Kikuchi, T. Koba
Repetitive 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.
{"title":"Repetitive DNA sequences accelerate molecular cytogenetic research in plants with small chromosomes","authors":"A. Setiawan, A. Wibowo, C. Teo, S. Kikuchi, T. Koba","doi":"10.22146/ijbiotech.51726","DOIUrl":"https://doi.org/10.22146/ijbiotech.51726","url":null,"abstract":"Repetitive 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.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46358937","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-12-04DOI: 10.22146/ijbiotech.48732
A. Hermawan, Herwandhani Putri
Cisplatn 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.
{"title":"Data mining analysis of miR-638 and key genes interaction in cisplatin resistant triple-negative breast cancer","authors":"A. Hermawan, Herwandhani Putri","doi":"10.22146/ijbiotech.48732","DOIUrl":"https://doi.org/10.22146/ijbiotech.48732","url":null,"abstract":"Cisplatn 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.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45926776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-06-27DOI: 10.22146/IJBIOTECH.45551
Natalia Tri Astuti, Nurmalasari Darsono, Suvia Widyaningrum, W. D. Sawitri, S. Astuti, W. Darmanto
Sugarcane 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.
{"title":"Expression and purification of recombinant coat protein of sugarcane mosaic virus from Indonesian isolate as an antigen for antibody production","authors":"Natalia Tri Astuti, Nurmalasari Darsono, Suvia Widyaningrum, W. D. Sawitri, S. Astuti, W. Darmanto","doi":"10.22146/IJBIOTECH.45551","DOIUrl":"https://doi.org/10.22146/IJBIOTECH.45551","url":null,"abstract":"Sugarcane 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. ","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42424722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-06-26DOI: 10.22146/IJBIOTECH.42095
Resti Rahmawati, S. Sumadi, T. Hartatik
The 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 < 0.05), and ewes with the GG genotype had a higher litter size than those with the GA genotype. Genotype distributions for all identified SNPs were in agreement with Hardy-Weinberg equilibrium. We highlight that SNP g.333G>A may be useful as a genetic marker for litter size in Garut sheep.
{"title":"Identification of single nucleotide polymorphisms in GDF9 gene associated with litter size in Garut sheep","authors":"Resti Rahmawati, S. Sumadi, T. Hartatik","doi":"10.22146/IJBIOTECH.42095","DOIUrl":"https://doi.org/10.22146/IJBIOTECH.42095","url":null,"abstract":"The 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 < 0.05), and ewes with the GG genotype had a higher litter size than those with the GA genotype. Genotype distributions for all identified SNPs were in agreement with Hardy-Weinberg equilibrium. We highlight that SNP g.333G>A may be useful as a genetic marker for litter size in Garut sheep.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46303368","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-06-18DOI: 10.22146/IJBIOTECH.43989
Dian Prasetyo Wibisono, N. Arfian, M. M. Romi, W. Setyaningsih, D. C. R. Sari
Ischemic/reperfusion injury (IRI) causes acute kidney injury that may lead to chronic kidney disease. We investigated the correlation between kidney interstitial cells expansion, hemoglobin level, and erythropoietin expression as the chronic effects of single and repeated kidney IRI in mice. We created an IRI model using male Swiss mice by clamping the bilateral renal pedicles. Subjects were divided into four groups that contained six mice each: control/sham operation, single acute IRI, single chronic IRI, and repeated IRI. Our results showed that the single chronic and repeated IRI groups significantly increased the tubular injury score, decreased the hemoglobin level, and increased erythropoietin expression compared with the control. Lower hemoglobin levels in all of the groups compared with the control was associated with erythropoietin resistance. In single chronic and repeated kidney IRI, there were decreased creatinine levels compared with the control. The decreased creatinine levels from the single acute IRI group to the single chronic IRI group, suggesting a repair phase of IRI starting on day 7 occurred in the single chronic IRI group. A macrophage marker, CD68, and an inflammatory mediator marker, MCP-1, significantly increased in all IR groups, indicating inflammation occurred due to IRI. In conclusion, chronic and repeated kidney IRI induced interstitial cells expansion and inflammation associated with anemia.
{"title":"Inverse correlation of kidney interstitial cells expansion with hemoglobin level and erythropoietin expression in single and repeated kidney ischemic/reperfusion injury in mice","authors":"Dian Prasetyo Wibisono, N. Arfian, M. M. Romi, W. Setyaningsih, D. C. R. Sari","doi":"10.22146/IJBIOTECH.43989","DOIUrl":"https://doi.org/10.22146/IJBIOTECH.43989","url":null,"abstract":"Ischemic/reperfusion injury (IRI) causes acute kidney injury that may lead to chronic kidney disease. We investigated the correlation between kidney interstitial cells expansion, hemoglobin level, and erythropoietin expression as the chronic effects of single and repeated kidney IRI in mice. We created an IRI model using male Swiss mice by clamping the bilateral renal pedicles. Subjects were divided into four groups that contained six mice each: control/sham operation, single acute IRI, single chronic IRI, and repeated IRI. Our results showed that the single chronic and repeated IRI groups significantly increased the tubular injury score, decreased the hemoglobin level, and increased erythropoietin expression compared with the control. Lower hemoglobin levels in all of the groups compared with the control was associated with erythropoietin resistance. In single chronic and repeated kidney IRI, there were decreased creatinine levels compared with the control. The decreased creatinine levels from the single acute IRI group to the single chronic IRI group, suggesting a repair phase of IRI starting on day 7 occurred in the single chronic IRI group. A macrophage marker, CD68, and an inflammatory mediator marker, MCP-1, significantly increased in all IR groups, indicating inflammation occurred due to IRI. In conclusion, chronic and repeated kidney IRI induced interstitial cells expansion and inflammation associated with anemia.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46878029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-06-18DOI: 10.22146/IJBIOTECH.42582
Sogandi Sogandi, A. Z. Mustopa, I. Artika
Lactobacillus plantarum is widely found in either anaerobic plant matter or fermented foods, and it has been recognized as producing antimicrobial bacteriocins. This study aimed to characterize the antimicrobial bacteriocins of L. plantarum and detect its genes that encode plantaricins. Samples were isolated from traditional fermented foods from Indonesia. Antimicrobial activity was evaluated using the agar diffusion assay procedure. The titration method applied the maximum amounts of lactic acid at 1054 mg/mL and hydrogen peroxide at 3.85 mg/mL. Based on the results, the supernatant of the L. plantarum strains appeared to have a broad spectrum of antimicrobial activity against pathogens, which would be active at pH 2.0–12.0 and stable temperature. In addition, almost all of the L. plantarum strains contained plantaricin-encoding genes (e.g. plnA, plnF,plnJK, and plnW), which were grouped into one cluster as indicated by phylogenetic analysis. Therefore, this study discovered clear evidence of the potential of some L. plantarum strains to act as antimicrobial agents.
{"title":"The characterization of bacteriocins produced by Lactobacillus plantarum strains isolated from traditional fermented foods in Indonesia and the detection of its plantaricin-encoding genes","authors":"Sogandi Sogandi, A. Z. Mustopa, I. Artika","doi":"10.22146/IJBIOTECH.42582","DOIUrl":"https://doi.org/10.22146/IJBIOTECH.42582","url":null,"abstract":"Lactobacillus plantarum is widely found in either anaerobic plant matter or fermented foods, and it has been recognized as producing antimicrobial bacteriocins. This study aimed to characterize the antimicrobial bacteriocins of L. plantarum and detect its genes that encode plantaricins. Samples were isolated from traditional fermented foods from Indonesia. Antimicrobial activity was evaluated using the agar diffusion assay procedure. The titration method applied the maximum amounts of lactic acid at 1054 mg/mL and hydrogen peroxide at 3.85 mg/mL. Based on the results, the supernatant of the L. plantarum strains appeared to have a broad spectrum of antimicrobial activity against pathogens, which would be active at pH 2.0–12.0 and stable temperature. In addition, almost all of the L. plantarum strains contained plantaricin-encoding genes (e.g. plnA, plnF,plnJK, and plnW), which were grouped into one cluster as indicated by phylogenetic analysis. Therefore, this study discovered clear evidence of the potential of some L. plantarum strains to act as antimicrobial agents.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49327994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：