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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
Abstract
Objective
Results and Discussion
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.
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.
•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 HIF1α 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
Methods and Materials
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
HYPOXIA
NORMOXIA
bHLH-PAS
hydrolases
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
VHL
HIF-1β
P300/CBP
proteasome
•ExoSAPit used to prepare PCR products for sequencing
•Sequences were analyzed using Sequencher 4.0 software.
•SNPs were identified using individual DNA samples.
• 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
HIF-1α
Degraded!
Target Genes
Transcribed!
Figure 5: 3 SNPs found within the gene
Genotyping 76 individuals from L. vannamei population
HIF-1α
CTAD
Figure 2: Tierra Kershaw displaying
Litopenaeus vannamei individual
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•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
Weight
References
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.
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Aquaculture Importance
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.
Boyd, C.E. and Watten, B.J. 1989. Aeration systems in aquaculture. Reviews in
Aquatic Science. 1: 425–472.
Mean Weight
Figure 1: HIF pathway
Conclusions
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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>.
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AA
AG
GG
Genotype
Figure 3: Genotypes of 5
individuals. From left to
right: AG, AG, AG, GG, AG
Figure 4:Mean weights
for each genotype with
standard error bars
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.
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371