Center for Integrated Animal Genomics

1. Research Experience in Molecular Biotechnology & Genomics • Summer 2008 Center for Integrated Animal Genomics Tierra Kershaw, Danielle Bowen and Max Rothschild Department of Animal Science Characterizing the HIF-1α transcriptional factor in Litopenaeus vannamei • A 691 base pair fragment was sequenced • Fragment was aligned with HIF-1α gene of: • P. pugio, Grass shrimp, (63% homology) • C. magister, Blue crab, (76% homology) • LDL receptor protein of P. monodon, Tiger prawn (96% homology) • Intron located at bases 391-493. • LDL receptor gene is likely mislabeled due to homology with HIF-1α gene in other species and lack of homology to other LDL receptor genes. • 9 SNPs identified, one exonic SNP selected for genotyping. • Calculated p-value of 0.3885 from linear regression model •  Mean weight for genotypes: AA=0.237g, AG=0.346g, GG=0.444g • Based on these results, there is a trend towards each copy of the G allele resulting in faster growth • Genotypes appeared to be within the Hardy Weinberg equilibrium • More research needs to be done using individuals exposed to hypoxic conditions Abstract The Hypoxia Inducible Factor (HIF-1α) is a vital transcriptional factor that regulates cellular responses such as glycolysis, respiration, and erythropoiesis during periods of hypoxic stress. This factor is necessary for animals in aquatic environments where the dissolved oxygen levels are often variable. Aquaculture could be greatly benefitted if animals are developed that can withstand hypoxic stress. It would also be important to know if this gene has any relationship with growth, to determine whether normal growth is affected during prolonged periods of hypoxia. HIF-1α has been widely studied in various mammalian and fish species. The only shrimp species that is known to have the gene is Grass shrimp (Palaemonetes pugio). In this study we determined that HIF-1α exists in pacific white shrimp (Litopenaeus vannamei) by using standard PCR technology and sequencing. A total of 9 SNPs were identified and one was used for genotyping. Based on the genotypes of a single population,there appeared to be a trend toward the G allele resulting in faster growth, though the trend was not statistically significant. However, more research can be done to analyze the remaining 8 SNPs that have not been studied. Results and Discussion Objective The purposes of this study were to partially sequence the HIF-1α gene in pacific white shrimp (Litopenaeus vannamei) and determine if the gene has an effect on growth. Figure 2: Tierra Kershaw displaying Litopenaeus vannamei individual • Methods and Materials • Primer design and optimization • Nucleotide blasts (http://ncbi.nlm.nih.gov/blast) were used to design • primer sets for conserved regions in the gene • PCR was performed and the products were electrophoresed on • 1.5% agarose gel • Bands were stained by ethidium bromide and visualized using an alpha imager (Innotech) • DNA sequencing and sequence analysis • ExoSAPit used to prepare PCR products for sequencing • Sequences were analyzed using Sequencher 4.0 software. • SNPs were identified using individual DNA samples. • Genotyping 76 individuals from L. vannamei population • One SNP was selected for genotyping • PCR performed to prepare for restriction enzyme digestion • Digested samples with BsrI via incubation at 65°C for 7 • hours • The product was electrophoresed on a 3% agarose gel • Performed linear regression of animal weight vs. genotype using the PROC GLM in SAS 9 software • Hardy Weinberg equilibrium was confirmed Background • Aquatic hypoxia in coastal water is increasing on a world wide scale (Diaz and Rosenberg, 1995). • Pacific white shrimp (Litopenaeus vannamei) are constantly exposed to changes in oxygen concentrations (Diaz and Rosenberg, 1995). • HIF-1α is studied primarily in mammalian and fish species • No significant research has been published about HIF-1α in L. vannamei on a molecular level • No studies have been completed about the effect of HIF-1α on body growth • Aquaculture Importance • L. vannamei is the most common species of shrimp farmed in the western hemisphere (FAO, 2008) • Low salinity requirement makes them easier and less expensive to farm (FAO, 2008) • Hypoxia is a major cause of production loss (Boyd and Watten, 1989) • Development of shrimp that can be maintained in both low DO levels and low salinity would be a benefit to shrimp farmers Figure 5:3 SNPs found within the gene Conclusions HYPOXIA NORMOXIA It has been determined that L. vannamei does possess the gene sequence for HIF-1α within its genetic code. The exact location within the genome remains unknown, since there is no map of the L. vannamei genome currently available. The sequenced fragment codes for only a small portion of the PAS domain within HIF-1α based on comparison to the gene in many other species, including P. pugio. More research must be done to amplify larger fragments in order to map out the entire gene for L. vannamei. Though a trend of significance was discovered between growth and genotype, more work needs to be completed. bHLH-PAS hydrolases HIF-1α OH | N OH | P HIF-1β CTAD VHL Ub | P300/CBP proteasome References HIF-1α Degraded! Target Genes Transcribed! Boyd, C.E. and Watten, B.J. 1989. Aeration systems in aquaculture. Reviews in Aquatic Science. 1: 425–472. Diaz, R. J. and Rosenberg, R. 1995. Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna. Oceanography and Marine Biology Annual Review. 33: 245–303. FAO. 2006. Cultured Aquatic Species Information Programme - Penaeus vannamei. Text by Briggs, M.In FAO Fisheries and Aquaculture Department [online]. Rome. Updated 17 Aug 2006. [Cited 10 Jul 2008]. <http://www.fao.org/fishery/culturedspecies/Litopenaeus_vannamei>. Figure 1:HIF pathway Acknowledgements Tierra Kershaw would like to think all of the members of Dr. Max Rothschild’s laboratory for assistance and support throughout this research experience. Figure 4:Mean weights for each genotype with standard error bars Figure 3: Genotypes of 5 individuals. From left to right: AG, AG, AG, GG, AG Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371