This study was aimed to produce recombinant growth hormone (rGH) from giant grouper (Epinephelus lanceolatus), giant gouramy (Osphronemus gouramy) and common carp (Cyprinus carpio) and compare their bioactivity potential by means of inducing the growth hormone of juvenile Nile tilapia (Oreochromis niloticus) as the model. DNA fragment encoding mature GH protein of giant grouper (El-mGH), giant gouramy (Og-mGH) and common carp (Cc-mGH) was amplified by PCR method. The purified PCR products were ligated to pCold-1 to generate pCold/El-mGH, pCold/OgmGH, and pCold/Cc-mGH protein expression vector, respectively. Each of the expression vectors was transformed into the Escherichia coli BL21. E. coli BL21 was cultured using 2xYT medium and protein production was induced by cold shock at 15±1 o C for overnight. The inclusion bodies of E. coli transformants containing protein expression vector were isolated by sonication method, and rGH production was analyzed by SDS-PAGE. Juvenile of Nile tilapia of average body weight of 12.41±3.28 g was intramuscularly injected once a week for 4 weeks with 1 μg inclusion body containing rGH per gram fish body weight. The result showed that rGH in molecular weight of about 25 kDa was obtained. Fish injected with rGH of El-mGH, Cc-mGH and Og-mGH grew 20.94%, 18.09%, and 16.99% faster, respectively, compared with the control. This result indicated that the three rGH produced in E. coli possessed biological activity when tested on Nile tilapia and further research is needed to find its effect on the growth of other aquaculture fish species. KEYWORDS: bioactivity, recombinant protein, growth hormone, Nile tilapiaProduction and bioactivity potential of three recombinant ... (Alimuddin)

Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10-90% (v/v), and was tolerant to organic solvents whose log P(ow) (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.

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The Cyca-DAB1*05 allele of major histocompatibility complex class II genes is recently suggested to have a link with the European common carp strain resistant to koi herpesvirus (KHV). In this study, a set of specific primers for Cyca-DAB1*05 was designed and applied as a marker to identify broodstocks of majalaya common carp strain subsequently used as a candidate resistant to KHV infection. From a total of 23 broodstock subjected to PCR analysis, two female and male fish, both having (P) and no Cyca-DAB1*05 (N), were selected and then diallelly mated. Disease resistance of progenies from 10 crosses was determined by a survival analysis in pond rearing and a laboratory challenge-test using cohabitation method. The results have revealed that the average survivals of PxP progenies for pond rearing and KHV challenge test were 86% and 100% higher (P < 0.05) respectively compared to that of NxN fish. Survival rate of PxN/NxP progenies was significantly lower (P < 0.05) than that of PxP fish. Furthermore, PCR analysis showed that almost 91% progenies of PxP crosses seemed to have a KHV resistant gene marker. Thus, this study suggests that the marker is associated with the KHV resistance in majalaya common carp strain, and farming of PxP progenies can be useful to increase common carp production.

Promoter 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.

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Economically, cacao bean products are divided into fine flavor and bulk cocoa. Fine flavor cocoa has white color beans while bulk cocoa has purple color beans. Bean color of fine-flavor cocoa beans is determined by the presence of a double recessive gene which is inherited from Criollo cocoa type. Analysis of the genetic background in this study used maturase K (matK) gene to ascertain the identity of the genotypes which will be used as a parent in crossing of fine flavor cocoa plants. The study aimed to investigate the genetic background of the promising clones that will be used as a parent in breeding program on fine flavor cocoa based on maturase K (matK) gene in order to ensure the identity of the genotype that will be used in parent crossing and it had Criollo ancestor. DNA analysis was conducted at Agency for the Assessment and Application Technology (BPPT), Serpong, West Java. DNA analysis was conducted on eight genotypes consisting of four genotypes of fine flavor cocoa (ICCRI 02, DRC 16, PNT 16 and DR 2) and four genotypes of bulk cocoa (MCC 01, MCC 02, Sulawesi 1, and KW 617). The results showed that Maturase K (matK) was one of chloroplast gene which could be used to study phylogenetic and evolution on cocoa. Two primers Mac 02 and Mac 09 were used for amplification of matK gene on cocoa with a rate of homology 99-100% with position 872 bp for Mac 02 and 1153 bp for Mac 09. The results of the phylogenetic analysis showed that the cocoa genotypes would be used as parent crossing included DR 2, ICCRI 02, DRC 16, PNT 16, MCC 01, MCC 02, Sulawesi 1, KW 617 and HJ 2 tended to have ancestral Criollo as female parent.

This study aimed to produce recombinant growth hormone (rGH) protein of common carp (Cyprinus carpio) and evaluate its bioactivity. DNA fragment encoding mature GH protein of common carp (mCcGH) was amplified by polymerase chain reaction (PCR) method and PCR products were then ligated into pCold I to generate pCold I-mCcGH protein expression vector. Escherichia coli BL21 (DE3) harboring pCold I-mCcGH was cultured in the 2xYT medium at 15 °C for 24 hours and protein production was induced by isopropyl-beta-thio galactopyranoside (IPTG). The inclusion bodies containing rGH protein from E. coli transformants were isolated by sonication method and analyzed by SDS-PAGE. The result showed that rGH with molecular weight of about 25 kDa was obtained. Common carp juveniles with average body weight of 5.2±0.4 g were intramuscularly injected once a week for 4 weeks with rGH protein solution from 1 μg bacterial cells per gram fish body weight. The result showed that juveniles fish injected with rGH grew 106.56% higher than control. This result indicated that rGH produced in E. coli BL21 possessed biological activity and it may be useful to improve growth of aquaculture species.Key words: growth hormone, recombinant protein, common carp ABSTRAKPenelitian ini bertujuan menghasilkan protein rekombinan hormon pertumbuhan (growth hormone, GH) dari ikan mas (Cyprinus carpio) dan menguji bioaktivitasnya. Fragmen DNA penyandi protein matang (mature) GH ikan mas (mCcGH) diamplifikasi dengan menggunakan metode PCR dan hasilnya kemudian diligasi ke dalam pCold-I untuk menghasilkan konstruksi vektor ekspresi pCold-I-mCcGH. Plasmid pCold-I-mCcGH ditransformasi ke bakteri Escherichia coli BL21 (DE3), dikultur dalam media 2xYT cair pada suhu 15°C selama 24 jam dan produksi protein diinduksi dengan menggunakan isopropyl-beta-thio galactopyranoside (IPTG). Badan inklusi yang mengandung protein rekombinan GH (rGH) dari bakteri E. coli transforman diisolasi menggunakan metode sonikasi dan dianalisis dengan menggunakan SDS-PAGE. Hasil penelitian menunjukkan bahwa rGH dengan bobot molekul sekitar 25 kDa berhasil diproduksi. Benih ikan mas dengan bobot rata-rata 5,15±0,4 g diinjeksi secara intramuskular satu kali per minggu selama 4 minggu dengan larutan rGH hasil ekstraksi dari 1 µg pelet bakteri/g bobot ikan. Benih yang disuntik dengan rGH tumbuh sekitar 100% lebih cepat bila dibandingkan dengan kontrol yang tidak diinjeksi rGH. Hasil ini mengindikasikan bahwa rGH yang diproduksi dalam bakteri E. coli memiliki bioaktivitas dan dapat bermanfaat untuk memacu pertumbuhan spesies ikan-ikan budidaya. Teknologi transgenik bermanfaat untuk menyisipkan gen-gen yang berguna dan menghasilkan ikan budidaya dengan karakter unggul. Contoh penggunaan teknologi transgenik adalah penyisipan gen penyandi hormon pertumbuhan (growth hormone (GH)) yang dapat memacu pertumbuhan ikan menjadi beberapa kali lipat dari ikan normal