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ISOLASI DAN PENGKLONAN FRAGMEN cDNA DARI GEN PENYANDI MULTIDRUG RESISTANCE ASSOCIATED PROTEIN DARI Melastoma affine Suharsono, Suharsono; Firdaus, Syarifin; Suharsono, Utut Widyastuti
Makara Journal of Science Vol. 12, No. 2
Publisher : UI Scholars Hub

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Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP) encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE

ISOLASI DAN PENGKLONAN FRAGMEN cDNA DARI GEN PENYANDI MULTIDRUG RESISTANCE ASSOCIATED PROTEIN DARI Melastoma affine Suharsono, Suharsono; Firdaus, Syarifin; Suharsono, Utut Widyastuti
Makara Journal of Science Vol. 11, No. 2
Publisher : UI Scholars Hub

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Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP) encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE

ISOLASI FRAGMEN cDNA DARI GEN PENYANDI AKTIN DARI Melastoma malabathricum Hannum, Saleha; Akashi, Kinya; Suharsono, Utut Widyastuti; Hartana, Alex
Makara Journal of Science Vol. 14, No. 2
Publisher : UI Scholars Hub

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Isolation of cDNA Fragment of Gene Encoding for Actin from Melastoma malabthricum. M. malabathricum grows well in acidic soil with high Al solubility, thereby it can be used as a model plant for tolerance to aluminum and acid stresses. Actin is housekeeping gene used as an internal control for gene expression analysis. The objective of this research was to isolate and clone the cDNA fragments of MmACT encoding for actin of M. malabathricum. Total RNA was isolated and used as the template for cDNA synthesis by reverse transcription. Four cDNA fragments of MmACT, called MmACT1, MmACT2, MmACT3, and MmACT4, had been isolated and inserted into pGEM-T Easy plasmid. Nucleotide sequence analysis showed that the size of MmACT1 and MmACT2 is 617 bp, whereas MmACT3 and MmACT4 is 735 bp. The similarity among these four MmACT is about 78%-99% based on nucleotide sequence and about 98%-100% based on amino acid sequence. Phylogenetic analysis based on amino acid sequence showed that at 1% dissimilarity, the MmACT1, MmACT2, MmACT3 and the ACT5 Populus trichocarpha are clustered in one group, while the MmACT4 is grouped with ACT9 P. trichocarpa and ACT1 Gossypium hirsutum, and these two groups are separated from actin group of monocotyledonous plants. The sequence of MmACT fragments were registered in GenBank/EMBL/DDBJ database with accession numbers AB500686, AB500687, AB500688, and AB500689.

ISOLASI DAN PENGKLONAN FRAGMEN cDNA DARI GEN PENYANDI MULTIDRUG RESISTANCE ASSOCIATED PROTEIN DARI Melastoma affine Suharsono, Suharsono; Firdaus, Syarifin; Suharsono, Utut Widyastuti
Makara Journal of Science Vol. 12, No. 2
Publisher : UI Scholars Hub

Show Abstract | Download Original | Original Source | Check in Google Scholar

Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP) encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE

Isolasi dan Pengklonan Fragmen cDNA Gen Penyandi H+-ATPase Membran Plasma dari Melastoma malabathricum L. Muzuni, ,; Sopandie, Didy; Suharsono, Utut Widyastuti; Suharsono, ,
Jurnal Agronomi Indonesia (Indonesian Journal of Agronomy) Vol. 38 No. 1 (2010): Jurnal Agronomi Indonesia
Publisher : Indonesia Society of Agronomy (PERAGI) and Department of Agronomy and Horticulture, Faculty of Agriculture, IPB University, Bogor, Indonesia

Melastoma malabathricum L. grows well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying acid and aluminum stress is a plasma membrane H+ -ATPase protein encoded by PMA gene. The objective of this research was to isolate and clone the cDNA fragment of MmPMA encoding plasma membrane H+ -ATPase from M. malabathricum L. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of MmPMA cDNA had been successfully isolated by PCR by using total cDNA as template and PMA primer designed from conserved region for corresponding gene. This fragment had been successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5". Nucleotide sequence analysis showed that the length of MmPMA fragment is 806 bp encoding 268 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MmPMA fragment was 81% identical to part of PMA of Sesbania rostrata, Juglans regia, and Prunus persica. Based on deduced amino acid sequence, MmPMA was 94% identical to part of PMA of Juglans regia; 93% to PMA of S. rostrata, and Arabidopsis thaliana. MmPMA fragment has phosphorylation intermediate domain (DKTGT) and ATP binding domain (KGAP, DPPR, MITGD, and GDGVN). Keywords: isolation, Melastoma malabathricum L., MmPMA fragment, sequencing

RNAi dari Fragmen 3’UTR Gen Penyandi H+ -ATPase Membran Plasma Melastoma malabathricum L. dapat Menghambat Pertumbuhan Tanaman Tersebut Muzuni, ,; Sopandie, Didy; Suharsono, Utut Widyastuti; Suharsono, ,
Jurnal Agronomi Indonesia (Indonesian Journal of Agronomy) Vol. 41 No. 2 (2013): Jurnal Agronomi Indonesia
Publisher : Indonesia Society of Agronomy (PERAGI) and Department of Agronomy and Horticulture, Faculty of Agriculture, IPB University, Bogor, Indonesia

The RNA silencing technique is an effective tool to examine the biological function of the target mRNA in plants. The recent development of GATEWAYTM cloning technology makes it easy to construct the RNAi vectors with trigger sequences and to analyze the function of a target gene. The objective of this research was to construct RNAi including the 3’UTR fragment of the gene coding plasma membrane H+-ATPase from Melastoma malabathricumL., 3’UTRMmpma. RNAi vector had been successfully constructed using GATEWAYTM cloning technology with the 3’UTRMmpma was used as double-stranded RNA (dsRNA) trigger sequence, pENTRTM/D-TOPO®as entry vector, and pANDA plasmid as destination vector. RNAi had been successfully introduced into M. malabathricumL. mediated by A. tumefaciensEHA101 to analyze the function of Mmpma gene in the detoxifying Al stress. Based on the test of transgenic plants tolerance to Al stress showed that in the nutrient solution including 3.2 mM Al (AlCl3.6H2O), the transgenic plants underwent growth suppression especially roots and leaves, whereas non-transgenic plants underwent growth normally. It showed that suppression of Mmpmagene expression by RNAi to M. malabathricumL. caused the plant became sensitive to Al.Keywords: 3’UTRMmpma, A. tumefaciens, Al stress, RNAi vector

Isolasi dan Pengklonan Gen Penyandi H+-ATPase Membran Plasma dari Melastoma malabathricum L. ., Muzuni; Sopandie, Didy; Suharsono, Utut Widyastuti; ., Suharsono
Jurnal Agronomi Indonesia (Indonesian Journal of Agronomy) Vol. 42 No. 1 (2014): Jurnal Agronomi Indonesia
Publisher : Indonesia Society of Agronomy (PERAGI) and Department of Agronomy and Horticulture, Faculty of Agriculture, IPB University, Bogor, Indonesia

ABSTRACT Melastoma malabathricum L. is an Al-accumulating plant that grows well in acidic soils with high level of soluble aluminum in the tropics. One of the important proteins in the detoxifying Al stress is a plasma membrane H+-ATPase, a most abundant protein on the plasma membrane, encoded by PMA gene. The objective of this research was to isolate and characterize the gene encoding plasma membrane H+-ATPase from M. malabathricum L. Full length cDNA of MmPMA had been successfully isolated through a gradual isolation of the gene. The 5’ end and middle part of the MmPMA gene had been successfully isolated by PCR by using total cDNA as template and pma primers designed from some plants, while the 3’ end of Mmpma had been isolated by 3’ RACE. The parts of the gene had been successfully joined by PCR. The joining product was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the length of MmPMA coding sequence was 2,871 bp encoding 956 amino acids with molecular weight of 105.29 kDa and a predicted pI value of 6.84. Local alignment analysis based on nucleotide of mRNA showed that MmPMA is 82% identical to pma Vitis vinifera; 81% to pma Juglans regia, pma Populus trichocarpa, pma Sesbania rostrata, and pma Prunus persica and 80% to pma Lycopersicon esculentum. Based on deduced amino acid sequence, MmPMA is 94% identical to PMA Vitis vinifera and PMA Juglans regia; 93% to PMA Populus trichocarpa; 92% to PMA Vicia faba, Lycopersicon esculentum, and Arabidopsis thaliana, AHA4. MmPMA has 10 transmembrane domains, 4 cytoplasm loops, 6 functional domains and 3 autoregulatory domains.Keywords: aluminum, cDNA, MmPMA, PCR, RACE

Isolasi, Identifikasi, dan Produksi Miselia Rhizopus sp. Berkadar Asam Nukleat Rendah untuk Pengembangan Mikoprotein Firmansyah, Riza; Sukarno, Nampiah; Suharsono, Utut Widyastuti; Sukarno, Sukarno; Fadillah, Wendi Nurul
Jurnal Ilmu Pertanian Indonesia Vol. 29 No. 2 (2024): Jurnal Ilmu Pertanian Indonesia
Publisher : Institut Pertanian Bogor

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.18343/jipi.29.2.179

Fungi, including Rhizopus sp., are food sources that commonly contain high nucleic acid levels. Therefore, the nucleic acid content must be reduced to achieve health standard requirements. This study aimed to isolate, identify, and produce Rhizopus sp. mycelium containing low nucleic acid. The Rhizopus spp. were isolated from tempeh collected from 12 different areas in Indonesia. Fungal identification was conducted based on morphological characteristics. The fungal isolates were selected based on mycelial growth and spore production on PDA. Biomass production of mycelium was carried out in potato extract and soybean extract media obtained from 200 g/L and 333.3 g/L, respectively. In each medium, 6 sugar levels were added, namely 0, 2, 3, 4, 5, and 6 g/L. Mycelium nucleic acid content reduction was achieved by heat treatment at 50°C and 60°C for 15 minutes and measured by a spectrophotometer at 260 nm. Fifty-eight isolates that were identified into 3 species were obtained in this experiment: R. oryzae, R. stolonifera, and R. microsporus. R. Microsporus had higher mycelium biomass and lower spore number than the other species. R. Microsporus produced a higher mycelium biomass in the soybean extract medium with 5 g/L additional sugar. The nucleic acid content of the 50°C heat-treated mycelium was 1.82% and 1.73% at 65°C. These values fulfilled the standard of mycelial nucleic acid content permitted in food by the USDA. Keywords: morphology, Rhizopus microspores, spore, soybean extract, tempeh

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