Indonesian Journal of Biotechnology
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Regression Analysis for the Identification of RAPD Markers Linked To Drought Tolerance in Sorghum
No full textSorghum (Sorghum bicolor) can actually withstand in dry or drought condition better than other crops,therefore it can be grown at different agroclimatic conditions and its product can be used for different purposessuch as food, feed and industrial raw material. However at severe condition, the productivity will also dropdrastically. The aim of this research was to identify RAPD marker linked to the drought tolerance. In thisresearch, varieties of sorghum used as research materials were Durra, Zhengzu, the mutants of Durra andZhengzu (from 300 Gy gamma radiation) B-100 and Zh-30, and the F2 seeds from Zh-30 x B-100 and B-100 xZh-30. Drought screening was carried out using 0.3 % KI during sorghum vegetative stage. DNA extractionwas done using a modified CTAB method. PCR was carried out for RAPD analysis. PCR amplification productswere scored and analyzed using SAS program. The result showed that potassium iodide can be used fordrought screening during the vegetative stage and regression analysis using the logistic method can be usedto identify RAPD markers that is linked to drought tolerance in sorghum. The logistic analysis showed thatband A8-480 was linked to drought tolerance in sorghum
16s rRNA Identification of Pediococcus spp. from Broiler and Studies of Adherence Ability on Immobilized Mucus
No full textThe objectives of this research were to study taxonomical status of lactic acid bacteria (LAB) isolated from broiler and adherence ability on mucus in vitro. Molecular analysis was performed by analyzing 16S rRNA gene using universal primer. The adherence assay on mucus was carried out using microplate method with total plate count (TPC), absorbance (A550) and confirmed by scanning electron microscopy (SEM). The results of this studies revealed that three of LAB isolates have closed relation to Pediococcus acidilactici (99.9%) species.Three isolates of P. acidilactici have adherence ability on broiler mucus higher than that on porcine mucin with an adherence percentage of 55.5% versus 50.8% and absorbance A550 of 0.061 versus 0.051, respectively. The highest adherence ability showed by P. acidilactici R02 with adherence percentage was 59.3% and absorbance A550 = 0.068. Adherence on mucus were affected by the addition of 3 g/l of gastric juice and 0.3% (b/v) of bile salt. Adherence analysis using SEM also showed that the adherence on broiler mucus was higher than the adherence on porcine mucin. Altogether this adherence studies, suggest that three isolates of P. acidilactici LAB were capable of colonizing host intestinal mucus in vitro as important property to be promising probiotic bacteria for broiler.Key words : adherence, broiler, Pediococcus, mucus, 16S rRN
Identification of Protease Producing Halophilic Bacteria from Bledug KuwuMud Volcano
No full textThe objective of this research was to isolate and identify the halophilic bacteria from Bledug Kuwu-mudvolacano having an ability to produce proteolytic enzyme. From this work, 6 bacterial isolates were obtainedfrom mud and water samples using artificial sea water media after incubation at room temperature. Threeout of the 6 isolates (BKL-3, BKL-5, and BKA-1) were selected for further analysis. BKL-3, BKL-5 and BKA-1exhibited an ability to grow at salt concentration greater than 10%. BKL-3 could grow on media supplementedwith 15% of salt, meanwhile BKL-5 and BKA-1 could grow at 20% of salt, respectively. Furthermore, thoseisolates also exhibited proteolytic activity when they were grown on casein media. The phylogenetic analysisbased on the 16SrRNA gene sequences showed that the BKL-3 belong to the group of Bacillaceae, whilst BKL-5and BKA-1 isolates were relatively distance from the group of Halomonadaceae. Therefore, BKL-5 and BKA-1could be considered as the allegedly new species that were separated from Halomonadacea
Genetic Variation Analysis of Mold (Magnaporthe oryzae B.Couch) Using Random Amplified Polymorphic DNA
No full textMagnaporthe oryzae B.Couch is a host-specific fungi, certain strain only infect certain host plant species. Genetic variety among M. oryzae isolates was explained by dendogram which was constructed using similarity data of Random Amplified Polymorphic DNA (RAPD). Dendogram construction was achieved by computer software, Numerical Taxonomy System (NTSYS). The aim of the research were to study the genetic variation among M. Oryzae using RAPD and to construct a dendogram of genetic similarities among the ten isolates from green foxtail (Setaria viridis L.), finger millet (Eleusine coracana L.) and rice (Oryza sativa L.).RAPD was performed in 30 cycles using 5 primers (OPA-02, OPA-03, OPA-04, OPA-05, OPA-07). Polymorphism data was used to constructed dendogram using Dice index and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) in NTSYS software. There were 68 polymorphism fragments from 74 amplified fragments.Three clusters were formed in the dendrogram, based on host pathotype: foxtail millet type, finger millet type and rice type. There were two subclusters in foxtail millet type based on mating type, MAT1-1 dan MAT1-2. Thus, RAPD could be used as a method for genetic variation analysis of Magnaporthe oryzae to show host-specific specificity.Key words: Magnaporthe oryzae, RAPD, mating typ
Functional Analysis of OsKANADI1, A Florigen Hd3a Interacting Protein in Rice (Oryza sativa L.)
No full textOsKANADI1 is considered as a florigen Hd3a interacting protein. To study the function of OsKANADI1, the expression pattern of OsKANADI1 was performed by semiquantitative RT-PCR with various wild-type tissues in the floral transition stage. The results demonstrated that OsKANADI1 was expressed in all organs of wild-type plants, but was highest in roots and leaves. We hypothesize that OsKANADI1 is a transcription factor in rice because it contains a GARP domain and posses a nuclear localization signal. To determine whether OsKANADI1 encodes a nuclear protein, full-length OsKANADI1 fused to GFP was introduced into onion epidermis cells by particle bombardment. The result revealed that OsKANADI1 was localized in the nucleus, suggesting that OsKANADI1 may be a transcription factor. Functional analysis was carried out using a reverse genetics approach to generate gain of function mutant (overexpression) and knockdown mutant (RNAi). The results showed that suppression of OsKANADI1 by RNAi displayed branching and increasing tiller number in several lines. This phenotype resembles to the Hd3a overexpressed plants indicating they possibly function in similar pathway.Key words : OsKANADI1, Transcription factor, Hd3a interacting protein, Ric
Gynura procumbens Prevents Chemoresistance through Inhibition MDR1 Expression on MCF-7 Breast Cancer Cell Line and Sensitizes the Cells to Doxorubicin
No full textThe long-term exposure of doxorubicin (Dox) causes enhancement in MDR1 expression that leads tobreast cancer cell resistance. This protein become a serious problem in cancer treatment and also well-knownas negative prognostic factor in breast cancer malignancies. The new approach using natural chemopreventivesubstance was developed to inhibit this resistance progress. This study was aimed to investigate whether ethylacetate fraction of Gynura procumnens (FEG) can prevent chemoresistance through suppressing the MDR1 proteinexpression. MCF-7 cell was used as chemoresistance cell model. The MCF-7 cells were maintained with 100nM Dox-contained medium for five weeks. The chemoprevention effect of FEG was investigated by treatedMCF-7/Dox with sub-toxic concentration of FEG. The cytotoxic properties of MCF-7 cells were determinedusing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Immunofluorescenceand western blotting analysis was performed to detect the MDR1 expression. MCF-7/Dox cells need higherconcentration for inhibiting cell growth, were compared with MCF-7, shown by IC50value. The MDR1 proteinlevel elevated after Dox exposure in time dependent manner. The FEG treatment decreased MDR-1 proteinlevel with dose dependent manner. FEG in combination with DOX potentiates the DOX effect on breast cancercell growth inhibition. The FEG prevents the chemoresistance development in breast cancer cell line, MCF-7induced by Dox through inhibiting MDR1 expression. The additional of FEG enhances Dox effect on cell deathinduction. Thus, FEG could be developed as co-chemotherapy agent for reverse multidrug resistanc
Isolation and Purifi cation of Chitinase Bacillus sp. D2 Isolated from Potato Rhizosfer
No full textPotato Cyst Nematodes (Globodera rostochiensis) is one of the important potato’s pests and caused economic looses up to 70% in the several centrals of potato plantations in Indonesia. Potato Cyst Nematodes (PCN) shell component of egg shell containing chitin (inner layer) and vitelline/protein (outer layer), so the purpose of research was to fi nd out of chitin degrading bacteria for controlling of egg’s PCN by cutting of their life cycle. The results showed that Bacillus sp. D2 isolated from potato rhizosphere could produce extra cellular chitinase in the medium containing of 0.20% colloidal chitin and fermented for 72 hours. Result of chitinase purifi cation using ammonium sulphate precipitation and DEAE-Cellulose ion-exchange chromatography showed a specifi c activity 2691,052 U/mg and analyzing using SDS-PAGE 12.5% resulted in molecular weight 30 kDa. The apparent Km and Vmax of chitinase towards colloidal chitin were 2 mg/ml and 2.2 μg/h, respectively.
Molecular Marker Confirmation for Member of Anopheles barbirostris Van Der Wulp 1884 in Different Localities
No full textVector and non-vector forms of Anopheles barbirostris have been recognized in Indonesia. However, because of their similarity in morphology, they were considered to be a single species. This information has led to the hypothesis that Anopheles barbirostris is a complex of species, which are morphologically indistinguishable from each other by ordinary methods. Objectives of the research was to identify the member of Anopheles barbirostris by PCR Assay. Samples were taken from two localities in Java, two in Sulawesi, two in Flores Indonesia, one from Thailand, one from China. The study was to develop a PCR-based technique of rDNA ITS2 region. Results showed that there are at least four species within the Anopheles barbirostris population studied, namely Anopheles barbirostris species DW, DX, DY and DZ. The length of the sequence amplified for species W, species X, species Y, and species Z were 339bps, 247bps, 165bps. and 157bps, respectively. Verification of the method was carried out with 270 mosquitoes from eight different field-collection sites using various sampling methods. Samples collected from Singaraja-Flores were identified as species W and X. All specimens collected from human bite outdoors were identified as species X; this species showed to be predominant among indoor light trap, indoor human bite and indoor resting collections Samples from Reo-Flores were identified as species W and X. All specimens from Manado and Palopo in Sulawesiwere identified as species Z. Similarly only species Y was found in samples from Thailand, while specimens from Salaman and Jambu in Java were identified as species W or species X. These species-specific molecular markers for the Anopheles barbirostris, complex appear to be reliable over a wide geographical area. However, larger number of samples is still needed from throughout the range of this species.Key words: Anopheles barbirostris, ITS2, PCR, Specific primer diagnosti
Selection of Yeast Strains for Ethanol Fermentation of Glucose-FructoseSucrose Mixture
No full textThis study was aimed to compare the ability of some yeast strains to consume sugars (sucrose, glucoseand fructose) and to convert them into ethanol during fermentation. The results of this comparison will be thebasis of considerations in choosing the right strain to be used as a mixed culture to increase the productionof ethanol from substrate containing a mixture of sucrose, glucose and fructose, such as juice of cane andsweet sorghum. The study was conducted using fermentation in substrate consisting of glucose, fructose,and sucrose separately, glucose-fructose mixture, and glucose-fructose-sucrose mixture using some yeaststrains: FNCC3012, OUT7009, OUT7027, OUT7055, OUT7080, OUT7096, OUT7903, OUT7913, and OUT7921.Following the fermentation, analysis of the produced ethanol and the remaining sugar was conducted. Theresults of study indicated that the strains with the highest substrate consumption were OUT7921, OUT7096,OUT7055, OUT7027, and OUT7913 for glucose, fructose, glucose-fructose mixture, sucrose, and glucosefructose-sucrose mixture, respectively. Strains that produced highest concentration ethanol were OUT7096 inglucose and sucrose substrates, OUT7921 in substrate of glucose-fructose mixture and sucrose, OUT7913 insubstrate of glucose-fructose-sucrose mixture. Upon consideration of each strain capacity, both in consumingsugar and producing ethanol, the recommended strains for use in mixed culture in bioethanol fermentationusing mixed substrate of glucose, fructose and sucrose are OUT7096, OUT7913, and OUT7921
Cloning and Expression of ORF124 Koi Herpesvirus as a Vaccine
No full textKoi herpesvirus (KHV) which also known as Cyprinid herpesvirus 3 (CyHV-3), Koi herpes-like virus,and carp interstitial nephritis gill necrosis virus (CNGV), caused signifi cant morbidity and mortality in koiand common carp (Cyprinus carpio). The case fatality rate of this disease is 80–100%. Glycoprotein has beenused for vaccine development as sub unit vaccine against viruses. The aim of this research was to clone andexpress membrane glycoprotein ORF124 KHV as a candidate of recombinant vaccine. ORF124 KHV gene wassuccessfully cloned into pBSKS and sequenced. Result showed that ORF124 KHV (isolate from Indonesia) had100 % similarity with Cyprinid herpesvirus 3 strain TUMST1 (from Japan), 99% similarity with Koi herpesvirus strain KHV-U (from USA) and Koi herpesvirus strain KHV-I (from Israel). Prediction analysis of T and B cellepitopes showed that ORF124 KHV protein had 14 and 11 T cell epitopes (IAd, Rothbard/Taylor pattern),and had 10 B cell epitopes, suggested that the protein can be used as a vaccine candidate. ORF124 gene hasbeen expressed in Escherichia coli under pET32-a(+)vector