6 research outputs found
Introduksi dan Ekspresi Gen GFP (Green Fluorescent Protein) dengan Promoter Berbeda pada Embrio Somatik Rumput Laut Kappaphycus alvarezii
No full textTransgenesis is genetic manipulation through gene transfer to introduce the gene coding a protein that encodes a character such as growth, disease resistance, and quality of fish meat or carrageenan content in the seaweed. As an early stage in order to produce transgenic seaweed, this study aims to test the promoter activity and the success of gene transfer of GFP as a marker. Construction of GFP gene with different promoters : CMV (cytomegalovirus), CaMV (cauliflower mosaicvirus), β-actin and keratin was transferred to somatic embryo seaweed by electroporation using a gene pulsher method (BIO RAD) with voltage: 300 V/cm, the pulse length : 0.5 millimeter/second, the pulse number : 4 time, the pulse interval: 0.1 second. Promoter activity was determined by analyzing the gene expression level of GFP using fluorescent microscope. The callus induction of Kappaphycus alvarezii for production of somatic embryos cell by different ratio of growth regulators and agar media concentrations. Callus induction was conducted at 0.8-1.0% agar medium containing IAA : kinetin = 1.0 : 1.0 ppm. Somatic embryos were cultivated by Conway medium (liquid culture). The results showed that CMV promoter drives expresses the number of fluorescent cells on average 34.10±1.49% with moderate and strong intensity levels of luminescence, the CaMV promoter drives luminescence intensity showed strong and percentage fluorescent cells on average 10.48±0.25%. β-actin promoter showed the intensity of luminescence in moderat level and the number of fluorescent cells was 8.85±2.31%, while keratin promoter drives weak luminescence intensity and the average number of fluorescent cells was 4.79± 0.26%. Thus, CMV is the best promoter and production of transgenic seaweed K. alvarezii could be conducted by electroporation method.Peningkatan produksi budidaya rumput laut dapat dilakukan melalui ekstensifikasi dan penggunaan bibit unggul (tumbuh cepat, tahan penyakit dan perubahan kondisi lingkungan). Permasalahan dihadapi dalam mencapai target produksi adalah rasio tumbuh yang semakin kecil akibat serangan penyakit dan lingkungan yang tidak mendukung. Selain itu, faktor penyakit dan lingkungan mempengaruhi mutu kandungan karaginan. Aplikasi teknologi transfer gen (transgenesis) dengan tujuan seperti peningkatan pertumbuhan, resistensi penyakit dan daya tahan terhadap kondisi lingkungan ekstrim, telah banyak diteliti pada berbagai spesies ikan, tetapi pada rumput laut masih sangat jarang dilakukan. Oleh karena itu perlu dilakukan perbaikan mutu genetik dalam rangka mendukung peningkatan produksi budidaya rumput laut. Salah satu penentu keberhasilan transgenesis adalah kemampuan promoter yang digunakan untuk mengendalikan ekspresi gen yang diintroduksi. Berbagai jenis promoter telah diuji aktivitasnya pada ikan seperti CMV (cytomegalovirus), β-aktin, keratin, sedangkan CaMV (cauliflower mosaicvirus) adalah promoter yang juga dapat aktif baik pada tanaman maupun hewan. Promoter yang telah diisolasi masih sangat terbatas, oleh karena itu perlu dilakukan uji promoter yang ada untuk mengetahui promoter yang sesuai dengan inang yang akan dibuat menjadi transgenik. Pada penelitian ini, sebagai langkah awal produksi bibit rumput laut bermutu tinggi menggunakan teknologi transgenesis, dilakukan introduksi gen GFP sebagai penanda untuk mengetahui aktivitas promoter CMV, CaMV, β-aktin dari ikan medaka, dan keratin dari ikan flounder Jepang. Transfer gen GFP dilakukan menggunakan metode elektroporasi pada embrio somatik K. alvarezii
Akselerasi moulting larva udang Vaname (Litopenaeus vannamei) dengan pemberian kalsium hidroksida Ca(OH)2
Get PDFKualitas dan pertumbuhan larva udang vaname sangat dipengaruhi oleh proses dan frekuensi molting, dimana pertumbuhan erat kaitannya dengan proses pergantian cangkang. Kalsium hidroksida Ca(OH)2 merupakan salah satu mineral kalsium yang berhubungan dengan kadar kalsium kulit dan kadar kalsium lingkungan seiring pertukaran kalsium secara terus-menerus antara tubuh dan lingkungan. Tujuan penelitian ini adalah mengevaluasi akselerasi dan frekuensi molting larva udang vaname serta pengaruh lanjut Kalsium Hidroksida Ca(OH)2 terhadap laju pertumbuhan larva udang vaname. Desain penelitian adalah eksperimental menggunakan Rancangan Acak Lengkap dengan 4 taraf dosis Ca(OH)2 : A. Tanpa penambahan Ca(OH)2 (kontrol), B. 2 mg/ L air, C. 4 mg/ L air, dan D. 6 mg/L air. Hasil penelitian menunjukkan bahwa dosis Ca(OH)2 sebanyak 4 mg/L air menunjukkan intensitas/persentase molting sebesar 67%, laju pertumbuhan berat mutlak sebesar 2.35 mg, pertumbuhan panjang mutlak sebesar 4,93 mm, dan tingkat kelulushidupan udang (SR) sebesar 49%
Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii
Get PDFPromoter regulates expression level of foreign gene in transgenic organism. This study was performed to select a
suitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Green
fl uorescent protein (GFP) gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycus
alvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP), caulifl ower mosaic virus (pCaMV-GFP),
medaka β-actin (pmBA-GFP) and Japanese fl ounder keratin (pJfKer-GFP) promoters were introduced by electroporation
method. Electroporation was performed using a gene pulser (BIORAD) with voltage of 300 V, pulse length of 0.5 ms,
pulse numbers of 4, and pulse interval of 0.1 s. Promoter activity was determined by analyzing GFP gene expression
level using a fl uorescent microscope. The results showed that CMV regulated highest number of fi lament callus
(34.10%±1.49) expressing GFP at medium to strong fl uorescence levels. CaMV promoter had relatively similar activity
with CMV, but lower number of fi lament callus expressing GFP (10.48%±0.25). mBA promoter drove GFP expression
at medium level and similar number of fi lament callus (8.85%±2.31) expressing GFP with CaMV, while JfKer promoter
had lowest activity by means in number of fi lament callus expressing GFP (4.79%±0.26) and GFP expression level. PCR
analysis for transgenic confi rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressing
fi lament callus in the same size (about 0.6 kb) with positive control of plasmid. Thus, CMV and CaMV promoters was
an appropriate promoter and foreign gene could be transferred to fi lament callus by electroporation method. Combining
this achievement with developing a culture method of fi lament callus to be thallus, stable transgenic breeding in K.
alvarezii can be feasible
Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii
Get PDFPromoter regulates expression level of foreign gene in transgenic organism. This study was performed to select asuitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Greenfl uorescent protein (GFP) gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycusalvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP), caulifl ower mosaic virus (pCaMV-GFP),medaka β-actin (pmBA-GFP) and Japanese fl ounder keratin (pJfKer-GFP) promoters were introduced by electroporationmethod. Electroporation was performed using a gene pulser (BIORAD) with voltage of 300 V, pulse length of 0.5 ms,pulse numbers of 4, and pulse interval of 0.1 s. Promoter activity was determined by analyzing GFP gene expressionlevel using a fl uorescent microscope. The results showed that CMV regulated highest number of fi lament callus(34.10%±1.49) expressing GFP at medium to strong fl uorescence levels. CaMV promoter had relatively similar activitywith CMV, but lower number of fi lament callus expressing GFP (10.48%±0.25). mBA promoter drove GFP expressionat medium level and similar number of fi lament callus (8.85%±2.31) expressing GFP with CaMV, while JfKer promoterhad lowest activity by means in number of fi lament callus expressing GFP (4.79%±0.26) and GFP expression level. PCRanalysis for transgenic confi rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressingfi lament callus in the same size (about 0.6 kb) with positive control of plasmid. Thus, CMV and CaMV promoters wasan appropriate promoter and foreign gene could be transferred to fi lament callus by electroporation method. Combiningthis achievement with developing a culture method of fi lament callus to be thallus, stable transgenic breeding in K.alvarezii can be feasible
BINARY VECTOR CONSTRUCTION OF KAPPA(κ)-CARRAGEENASE GENE AND TRANSFORMATION TO Agrobacterium tumefaciens AS MEDIATOR FOR SEAWEED TRANSGENIC GENERATION
Get PDFIncreasing of kappa (κ)-carrageenan content in Kappaphycus alvarezii seaweed is potentially be achieved by applying transgenesis technology. This study was performed to obtain a construction of κ-Carrageenase gene and Agrobacterium tumefaciens to carry those construction genes. The κ-Carrageenase (κ-Car) gene was involved in κ-carrageenan biosynthesis. The κ-Car gene sequence was ligated between the 35S CaMV promoter and tNos terminator sequences to generate pMSH/κ-Car expression vector. Transformation of pMSH/κ-Car plasmid to Escherichia coli was performed by heat-shock method, and to Agrobacterium tumefaciens by tri-parental mating method. The results showed that several colonies of E. coli and A. tumefaciens grew in the selective culture mediums containing antibiotic. PCR analysis using primers 35S-Forward and tNos-Reverse with DNA template from those bacterial colonies resulted DNA fragment of about 2,000 bp, the same as the total length of 35S CaMV promoter, κ-Car gene and tNos terminator sequences. Therefore, the construction of pMSH/κ-Car gene was succeeded and a colony of A. tumefaciens transformant carrying pMSH/κ-Car plasmid was successfully produced. Keywords: Agrobacterium tumefaciens, kappa(κ)-Carrageenase gene, transgenesis, vecto
Activity of Natural Compound Pothos tener Wall on Aeromonas hydrophila Infection to Prevent of Antibiotics
Get PDFThis study aims to discover how influential Photos tener Wall is as an anti-bacterial treatment for Aeromonas hydrophila. Methods: Cyprinus Carpio were reared for 28 days, and on the 29th and 30th days before Aeromonas hydrophila infection, the fish were adequately fasted. On the 31st day, they were intramuscularly challenged with A. hydrophila (105 CFU/mL) (the first day in A. hydrophila infection). The treatments given were (I) immersed trial with fresh (live) P tener Wall: (H1) Immersed with 15 g of P. tener Wall plant, (H2) Immersed with 30 g of P. tener Wall plant, and (H3) Immersed with 60 g of P. tener; (II) feeding trials in which the treatments given were (P1) 2% of P. tener Wall powder mixed with 1 kg commercial diet and (P2) 4% of P. tener Wall powder mixed with 1 kg commercial diet; Experiment III combined the best results from experiment I (H2) and experiment II (P2) and Oxytetracycline 5 g/kg feed as a control antibiotic. The result obtained was that the treatment of 30 g of fresh P. tener Wall or adding 4% simplicial P. tener Wall in the diet could increase koi fish's immune response and resistance to A. Hydrophila has a survival rate that reaches 100%. This treatment has the same effect as using antibiotic Oxytetracycline 5 g/kg of feed. An important aspect for further research is that P. tener wall can be tested on other fish diseases caused by bacteria or fungi
