Transmisi dan Ekspresi Gen Penyandi Hormon Pertumbuhan pada Ikan Cupang (Betta imbellis) Transgenik Generasi Kedua

Abstract

Betta fish (Betta imbellis) is one of the endemic freshwater animals spread in several regions in Indonesia. B. imbellis fish is an ornamental fish commodity that has the potential to be cultivated. Betta fish fans prefer the larger size (giant) with a body length of > 6 cm than the regular size (approximately 5 cm). Betta fish giant size is still a favorite fish in international contests, and it is still difficult to get a fish that size. Giant-sized betta fish have a high market value, ranging from IDR 900,000 to IDR 950,000 per fish. One alternative method that can be applied to produce a giant betta fish is the transgenic method. Transgenic is a genetic engineering technique that is done by inserting foreign genes into the host genome, these genes are then expressed in certain phenotypes, and passed on to the next generation. Most transgenesis in fish use growth hormone (GH) encoding genes with the aim of increasing growth so that the maintenance time to reach selling size is shorter. Generally, GH transgenesis is also carried out in consumption fish. In ornamental fish, generally transgenic production is designed for color engineering. The G1 transgenic broodstock were identified by polymerase chain reaction (PCR) method, then crossbreeding was carried out by the reciprocal method by crossing transgenic G1 broodstock and non-transgenic fish. DNA was extracted from the fin-clipped tail, then used in the analysis of gene transmission in the second generation (G2) using the PCR method. Total RNA was extracted from pituitary, muscle, liver and fins following the Tri-Reagent Kit procedure for gene expression analysis. Complementary DNA (cDNA) synthesis was carried out using the Transcriptor First Strand cDNA Synthesis Kit. Parameters of reproductive performance (fertilization rate and hatching rate), growth (absolute growth rate, specific growth rate), fish survival, percentage of transgene transmission and gene expression level were statistically analyzed by analysis of variance ANOVA using the SPSS software version 18.0. Further analysis was performed using Duncan's test at a 95% confidence interval. This study showed that fertilization and hatching rates varied among crosses, and survival was the same in all crosses. Transgenic crossbreed fish was more than 90% progenies carrying the PhGH transgene, identified with transgene transmission from G1 parent to G2 ranging from 90.0% to 96.6%. The growth performance of G2 transgenic betta fish was higher than that of non-transgenic fish, which was 1.4-1.9 times. The PhGH gene expression was only detected in transgenic fish, whereas BeGH gene expression was detected in transgenic and non-transgenic fish with the same level of expression. The IGF-1 gene expression level of G2 transgenic betta fish was higher than that of non-transgenic betta fish. The mean length and body weight of transgenic fish were 4.28±0.70 cm and 1.77 ± 0.05 g, while the non-transgenic fish were 3.40±0.20 cm and 1.40±0.04 g, respectively In conclusion, G2 offspring have inherited the percentage of PhGH transgene inherited to G2 progenies with a higher percentage of transgenic fish compared to their G1 generation. The PhGH gene was expressed in all G2 transgenic so that had a higher growth rate compared to non-transgenic fish.