Analisis DNA Genomik Heat Shock Protein 70 sebagai Kandidat Marka Cekaman Suhu pada Ikan Gurami Osphronemus goramy

Abstract

Temperature stress often causes high mortality in giant gourami. One strategy to prevent this threat is to use giant gourami that are adaptive to temperature stress. Adaptive fish can be produced by molecular marker-based selection that utilizes genes associated with giant gourami resilience traits. However, the availability of gene sequence information and molecular markers for selection has not been widely done. This research was conducted as an initial stage for the development of molecular marker-based selection of gourami adaptive to temperature stress through gene expression analysis and gene identification. This research was carried out in three stages. The first stage aims to analyze gene expression levels related to gourami resilience. The second stage aims to identify markers in the gourami genome related to resilience. The third stage aims to select gourami using the Tetra primer amplification refractory mutation system-polymerase chain (Tetra-ARM PCR) method and evaluate its correlation with fish resilience. The study began with raising 200 fish to be conditioned in culture containers. For gene expression analysis, 20 giant gourami were transferred to a 55 cm × 45 cm × 45 cm aquarium with a water level of 35 cm. The fish were kept for fourteen days at 28 - 30°C and fed commercial feed with a protein content of 39% by mouth. Water changes of 30% of the aquarium volume were done once a day. The temperature stress test was conducted in two aquarium containers measuring 55 cm × 45 cm × 45 cm with a water height of 35 cm. Each aquarium had its water temperature regulated with the first aquarium at a hot temperature of 32-34 °C and the second aquarium at a cold temperature of 18-20 °C. The fish were transferred directly from the rearing container to the first aquarium for one hour, then the fish were transferred to the second aquarium for one hour. Furthermore, fish behavior was observed and differentiated into two groups of fish, namely adaptive fish and susceptible fish. Adaptive fish are fish that can still swim well, while vulnerable fish are fish that have collapsed at the bottom of the aquarium. A total of five adaptive fish and five vulnerable fish were taken for total RNA extraction. Expression analysis was performed on three resilience-related genes, namely heat shock protein 70 (HSP70), Cu-superoxide dismutase (CuSOD), dan Mn-superoxide dismutase (MnSOD) using the qPCR method. The results showed that adaptive fish had a higher gene expression level than the susceptible fish for all the genes tested. The results indicate that there is an important role of these genes in the response of giant gourami to temperature stress. Therefore, these genes can be used as a potential gene as a molecular marker for giant gourami selection. The second phase of the study aims to identify single nucleotide polymorphisms (SNPs) in the HSP70 genome sequence and evaluate fish survival after temperature stress testing. The preparation of adaptive and susceptible fish giant gourami samples for HSP70 genome sequence analysis was carried out in stages, with three replicates. A total of 40 giant gourami were used at each stage. Temperature stress treatment, determination of adaptive and susceptible fish was carried out the same as in the preparation of samples for HSP70 gene expression analysis. A total of 45 adaptive and 45 susceptible fish were obtained from the three stages of the stress test. The caudal fin of the fish was cut for DNA extraction. A total of 13 fish each from adaptive and susceptible fish were randomly taken for DNA extraction. Furthermore, the samples were sent to a sequencing service provider company, namely 1st Base Laboratory. The sequences obtained were then analyzed to find SNPSs. The position and variation of SNPs obtained were used as the basis for designing candidate HSP70 gene SNPs primers for the tetra-ARMs PCR test. SNPS analysis showed that 76.9% of adaptive fish had genotype C, while 92.3% of susceptible fish had genotype T at the 788th nucleotide base of the HSP70 genome (SNPS-3). Then, the evaluation of TKH was carried out by raising adaptive and susceptible fish from the temperature stress treatment for the preparation of DNA samples separately in aquarium measuring 55 cm × 45 cm × 45 cm with a water level of 35 cm and a density of 2 fish/L. Fish were reared using the same method used in the rearing of fish for the preparation of HSP70 gene expression analysis samples. Fish survival was calculated after 40 days of rearing. The results showed that the adaptive fish had a TKH of 86.6% while the susceptible fish was 31.1%. These results indicate that adaptive fish have better resilience than vulnerable fish. The third phase of the study aimed to select gourami using the Tetra-ARM PCR method and evaluate its correlation with fish resilience. A total of 50 fish that had been conditioned in the culture container were used for the SNPs marker confirmation test. Fish fins were taken for DNA extraction and SNPs marker confirmation tests were carried out using the tetra-ARM PCR method. The amplification results showed that there were three types of DNA bands with sizes of 300 bp, 400 bp, and 700 bp. These results indicated that there were three different genotypes in the identified fish. Fish were grouped based on their genotype, then subjected to temperature stress as described above. Furthermore, the fish were reared using the same method in the survival analysis after the above stress test. The number of living and dead fish was counted based on their genotypes. Furthermore, a correlation analysis was conducted by correlating the fish genotypes and post-rearing TKH. The results of tetra-ARM PCR analysis showed that there were 22% CC, 32% TT, and 46% CT genotyped fish. Based on correlation analysis using the chi-square method, the results showed that there was no significant difference between the genotype differences and the level of resilience of gourami (p>0.05). Based on the overall results in this study, it can be concluded that adaptive giant gourami have higher gene expression than susceptible fish for all the genes tested. Then candidate adaptive fish molecular markers can be found in the 788th HSP70 genome sequence (SNPs-3) with the CC genotype. However, further research is still needed on larger fish populations so that the analysis of the relationship between the genotypes obtained and fish resilience can have a better correlation.