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Overexpression of Sugarcane Sucrose Transporter 1 (SoSUT1) Gene Increases Rice Yield
No full textThe growth and development of plants are determined by photosynthesis, which ultimately results in sucrose. Sucrose is synthesized in the source then translocated to all parts of the plant (sink). The translocation process of sucrose from source to sink is controlled by sucrose protein called sucrose transporter. SoSUT1 is a gene that encodes a sucrose transporter 1 (SUT1) protein in sugarcane. Rice transformation with the SoSUT1 causes overexpression SUT, which is expected to increase the translocation of sucrose into the seed of rice plants. This research was conducted by introducing SoSUT1 in rice plants Inpari 14 SS. Transformation using Agrobacterium tumefaciens vector in apical bud explant Indica rice cv. Inpari 14 SS results in 26 events positive rice contains genes SoSUT1. This study aims to elucidate the inheritance of transgene in the next generation and to characterize its effect on the morphology and the yields of a subsequent generation. The study is conducted by planting the seeds of T1 and T2 plants in media containing Hygromycin and using PCR analysis for further analysis. As the results, from 26 events on the T1 plant, only 3 events of T3 plants were confirmed on the T3 plant. The overexpression of the SoSUT1 gene could increase the number of tillers, the number of productive tillers, panicle length, and panicle exit length, also increasing the number of spherical grains, reducing the number of empty grains and increasing the weight of 1000 grains
Optimization of Expression Condition, Two Dimensional And Melting Point-Based Characterization of Recombinant Human Interferon Alpha-2a Fusion and Non Fusion Forms
No full text Recombinant Human Interferon Alpha-2a (rhIFNα-2a) is a therapeutic protein that used in hepatitis and cancer treatments. In our previous research, we developed higher molecular weight of the protein through human serum albumin fusion. The fusion and non fusion form of rhIFNα-2a were produced in Pichia pastoriswith 86 kDa and 19 kDa in size respectively. In previous research, protein yield was not reproducible due to unoptimized expression conditions. This reseach was aimed to optimize expression condition process and to characterize the fusion and non fusion forms of rhIFNα-2a. The parameters to observe in overproduction include nutrient (media and methanol concentration) and non nutrient (temperature andincubation period). Affinity and size exclusion cromatographicwere compared in protein purification. BCA assay was used to determine quantity of protein. Protein characterization was conducted using two-dimensional SDS PAGE and denaturation analyses. The optimal condition of expression was achieved using complex media with 1% of methanol for 3 day incubation period at 25°C. The protein yield was reproducible and higher comparing to previous research. Affinity chromatography resulted in higher purity of the proteins comparing to size exclusions. Characterization using two dimensional gel analysis revealed that isoelectric point of rhIFNα-2a is 6.5 for fusion form and 6.0 for non fusion form. The melting points of fusion protein were 56°C and 62°C whilst that of non fusion was 56°C
Non Invasive Detection of Dengue Viruses from Saliva: In vitro Study
No full text In the previous paper, we had succeeded in developing an early detection system of dengue viruses using Sugar liganded Gold Nano Particle (SGNP) only from 6 μL serum. It has been reported that dengue virus is also detected in the saliva and urine of the patient. The evidences lead to the possibility of developing non-invasive methods of dengue virus detection. In this in vitro study, we evaluated the utility of SGNP to capture and concentrate dengue virion in 10% saliva solution. The results showed that dengue virion was successfully detected in 10% of saliva solution. Analysis of virion stability during storage showed that virions in salivary samples were stable up to 3 days at temperature wherease the RNA has significantly degraded. Although still a preliminary study, the data obtained show the prospect of SGNP as a non-invasive dengue virus detection method, as well as the development of POC (Point of Care) method. Clinical trials using saliva from dengue viruses infected patients need to be done to prove the effectiveness of the SGNP method
Optimization of Somatic Embryogenesis Induction of Cassava (Manihot esculenta Crantz)
No full textThe embryogenesis (SE) has important role for genetic engineering of cassava (Manihot esculenta Crantz). However, the success of SE induction depend on plant growth regulator s (PGR)s and treatment enriched in induction media. This experiment tried to induce callus formation of cassava from several in vitro explants: immature leaf, apical bud, and internode; and to develop somatic embryogenesis of cassava in several media enriched with tyrosine and copper sulphate (CuSO4) added into media enrich with picloram as treatment. Different response of explants source to callus induction treatment from those three varieties in callus induction as well as friable callus formation were found in this experiment. The best medium to induce varied with variety; MS media supplemented 12 mg/L picloram + 0.5 mg/L CuSO4 was the best for “Adira 4” and half MS and half GD media supplemented 12 mg/L picloram + 100 mg/L tyrosine for “Malang 6”. All treatments resulted somatic embryo which developed indirectly and in morphologically normal somatic embryo
Agrobacterium-mediated Transformation of Banana Musa acuminata AA cv "“Mas Lampung” with hpt Gene Using Sterile Corm as Target Tissue
No full textThe protocol for Agrobacterium-mediated transformation of local banana plants cv “Mas Lampung” (AA) has been established. A selectable marker gene (hpt) has been used to study the transformation using in vitro corm slices as target tissues. Banana in vitro corm slices were co-cultivated with the EHA105 strain of Agrobacterium tumefaciens harbouring binary vector pCAMBIA 1301 containing hygromycin resistance gene (hpt) as a selectable marker and intron-containing β-Glucuronidase (gus-intron) gene as a reporter gene driven by CaMV 35S promoter. Polymerase Chain Reaction (PCR) were used to examine the existence of hpt gene in plants resulted from the transformation. Using primer pairs specific for hpt gene, our PCR analysis on leaves showed the presence of the hpt transgene in banana transgenic plants at first generation (T0) of transformation. To prove the existence of hpt gene in the fruits of transgenic banana plants, PCR analysis were also carried out. The data showed that the hpt gene could be amplified from banana fruits of tested samples. These result demonstrates that the Agrobacterium-mediated transformation method used in this experiment has been successful to transfer gene into banana plants. Thus, the transformation method reported here could be used as a standard protocol to transfer another useful genes into local banana plants cv. “Mas Lampung”. Furthermore, the presence of transgene in fruits of banana transgenic plants is important achievement especially for transgene that is expected to be expressed in the fruit including to introduce vaccine genes into banana fruits for edible vaccine. 
Construction of pY-Af Vector for Expression of Thermostable α-L-Arabinofuranosidase in Saccharomyces cerevisiae
No full textIn this research, construction of expression vector for thermostable α-L-arabinofuranosidase in Saccharomyces cerevisiae BJ1824 was conducted. The Escherichia coli/S. cerevisiae shuttle vector, pYES2 was used as parental vector in construction. The abfA gene encoding α-L-arabinofuranosidase from Geobacillus thermoleovorans IT-08 was amplified by PCR, in which the plasmid pTP510 was used as a template. The amplification product was treated with SacI and XhoI and then subcloned to the pYES2 vector, which was previously digested with SacI and XhoI. The recombinant plasmid was designated as pY-Af and propagated first in E. coli Top10, and then transformed into S. cerevisiae BJ1824. For α-Larabinofuranosidase (AbfA) production, the yeast transformants were grown in YNBG selective medium and YPG rich medium, using galactose as an inducer. The AbfA activity was assayed by measuring the amount of p-nitrophenol (pNP) released from p-nitrophenyl-α-L-arabinofuranoside (pNPA) substrate at pH 6.0 and 70oC for 30 min. The recombinant AbfA activity was detected in either of culture medium (0.98%), cellassociated (14.17%) and intracellular (84.85%) when recombinant yeast was grown in YPG rich medium
Identification of Differentially Expressed cDNA in Cassava under Drought Stress Using cDNA-RAPD Approach
No full textCassave is an important carbohydrat
Construction and Expression of Immunotoxin Anti EGFRvIII scFv-HPR Conjugate in Pichia pastoris as A Targeted Drug Candidate for Cancer Therapy
No full textEpidermal growth factor receptor variant III (EGFRvIII) is a mutant of EGFR lacking 267 amino acids (exon-2 through 7) within its extracellular domain, resulting in the formation of a new epitope as a tumor specific target. EGFRvIII is commonly found in GBM, breast, ovarian, prostate, and lung carcinomas. Antibodies or part of antibodies (e.g. single chain variable fragment or scFv) with specific activity against this receptor have been developed. These antibodies are internalized into the cell after receptor binding. Ribonucleases can be cytotoxic due to their inherent ability to degrade RNA, therefore causing cell death via inhibition of protein synthesis. The aim of this research is to construct, clone and express an immunotoxin-based conjugate combining an anti-EGFRvIII scFv with a HPRmut (human pancreatic ribonuclease mutant variant) in Pichia pastoris. The anti-EGFRvIII scFv gene was cloned into yeast expression vector pPICZαA and fused with EGFP gene as a marker. The HPRmut gene was subsequently cloned at the C-terminal of the scFv::EGFP fusion, resulting in the scFv::EGFP::HPR fusion construct. The fusion construct was successfully obtained and nucleotide sequence of plasmid was verified. This construct was used to transform P. pastoris SMD 1168H. The gene fusion was successfully transformed and expressed in P. pastoris with a transformation efficiency of 102 cfu/μg DNA. The transformed yeasts were screened on agar media containing up to 1000 μg/ml zeocin. Yeast transformants showed green fluorescent due to the expression of EGFP gene. The recombinant proteins have been expressed and secreted from P. pastoris as showed by immunoblotting and SDS-PAGE analyses, then purified by affinity chromatography method. The selected yeast transformant produced at least 15.85 mg of purified protein per liter of yeast culture.