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Rough Periwinkles at Emersion Presence Or Absence of Response in Gene Expression of Aspartate Aminotransferase?

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Rough Periwinkles at Emersion Presence Or Absence of Response in Gene Expression of Aspartate Aminotransferase? Rough periwinkles at emersion Presence or absence of response in gene expression of aspartate aminotransferase? CH-14 Cecilia Helmerson Degree project for Master of Science (Two Years) in Marine Sciences and Biology Degree course in Marine ecology 45 hec Spring and Autumn 2014 Department of Biological and Environmental Sciences University of Gothenburg Examiner: Kerstin Johannesson Department of Biological and Environmental Sciences University of Gothenburg Supervisors: Marina Panova and Olga Ortega Martinez Department of Biological and Environmental Sciences University of Gothenburg Illustration: Cecilia Helmerson 2014 Index ABSTRACT ............................................................................................................................................................ 4 1. INTRODUCTION ............................................................................................................................................. 5 2. MATERIALS AND METHODS ................................................................................................................... 7 2.1 SAMPLING AND ACCLIMATION ....................................................................................................................................... 7 2.2 EMERSION EXPERIMENT .................................................................................................................................................. 8 2.3 DISSECTION AND EXTRACTION ................................................................................................................................... 10 2.4 QUALITY CHECK AND REVERSE TRANSCRIPTION .................................................................................................... 10 2.5 QUANTITATIVE PCR ...................................................................................................................................................... 10 2.6 DATA ANALYSIS ............................................................................................................................................................... 13 3. RESULTS ......................................................................................................................................................... 14 3.1 EFFECT OF EMERSION .................................................................................................................................................. 14 3.2 CORRELATIONS AND COMPARISONS BETWEEN TARGETS ..................................................................................... 16 4. DISCUSSION ................................................................................................................................................... 18 4.1 MAIN RESULTS ................................................................................................................................................................ 18 4.2 HUMIDITY ........................................................................................................................................................................ 18 4.3 REGULATION OF GENE EXPRESSION ......................................................................................................................... 18 4.4 CORRELATION AND COMPARISONS BETWEEN TARGETS ....................................................................................... 19 4.5 INDIVIDUAL VARIATION ................................................................................................................................................ 20 4.6 TECHNICAL ASPECTS ..................................................................................................................................................... 20 4.7 ONE GENE OF MANY ...................................................................................................................................................... 20 5. CONCLUSION ................................................................................................................................................ 21 6. ACKNOWLEDGEMENTS .......................................................................................................................... 21 7. REFERENCES ................................................................................................................................................ 21 7. SUPPLEMENTARY MATERIAL .............................................................................................................. 24 S1- EXTRACTION PROTOCOL .............................................................................................................................................. 24 S2 – MOPS GEL AND BUFFERS ......................................................................................................................................... 24 S2.1 Agarose gel for RNA ................................................................................................................................................ 24 S2.2 Running buffert MOPS ........................................................................................................................................... 24 * S3 - CDNA SYNTHESIS ........................................................................................................................................................ 24 S4 – PLATE DESIGN ............................................................................................................................................................... 25 S5 – STANDARD CURVES ...................................................................................................................................................... 26 S6 NORMALITY TESTS ........................................................................................................................................................... 28 S. 6.1 Between treatments ................................................................................................................................................ 28 S. 6.2 Between targets ....................................................................................................................................................... 29 S7 TEST ASSESSING HOMOGENEITY OF VARIANCES ...................................................................................................... 30 S. 7.1 Between treatments ................................................................................................................................................ 30 S8 KRUSKAL WALLIS ANOVA .............................................................................................................................................. 31 S9 GEOMETRICAL MEAN ..................................................................................................................................................... 32 Rough periwinkles at emersion Presence or absence of response in gene expression of aspartate aminotransferase? Cecilia Helmerson Supervisors: Marina Panova1 and Olga Ortega-Martinez2 Degree Project for a Master of Science in Marine Sciences and Biology, 45 hec Department of Biology and Environmental Sciences, Sven Lovén Centre of Marine Sciences - 1Tjärnö/2Kristineberg, University of Gothenburg E-mail address: [email protected] or [email protected] Phone: 0046-739804988 Abstract Many selective forces shape evolution in rocky shore organisms. In the intertidal snail Littorina saxatilis desiccation at emersion is thought to be an important selective force. One approach to study responses to desiccation is through measuring gene expression of genes putatively involved in coping with the exposure. In the exposed ecotype of L. saxatilis a candidate gene for studying response to desiccation is aspartate aminotransferase (Aat). Aat is an enzyme initially active in anaerobic energy production that may be used at emersion. There are two allozyme alleles Aat120 and Aat100 and the allele frequencies vary with shore height. The activity of the enzyme varies depending on the genotype. This study investigated if there was an up-regulation of the enzyme at transcript level during emersion. Snails were sampled at an exposed atidal shore in the Aat hybrid zone and acclimated to laboratory conditions. An emersion event was simulated and gene expression quantified at different time points with quantitative PCR. The study found no significant response in gene expression for cytosolic Aat or mitochondrial Aat, but found a significant correlation between the two. The hypothesis of up-regulation in response to desiccation however cannot be rejected. Humidity throughout the experimental emersion event was low, but higher than dry air conditions. Snails might therefore not utilise Aat at the humidity and timeframe of the experimental setup. Based on results, the directions for further studies will be testing extreme stress, difference between genotypes and investigating expression difference at tissue level. The measuring of gene expression and enzyme activity together also allows testing an alternative hypothesis - that regulation is not at transcript level. Simulated emersion and other experimental approaches also should be compared to natural emersion events. Keywords desiccation, anaerobic energy production, glutamate-oxaloacetate transaminase, gastropod 4 Degree Project for a Master of Science in Marine Sciences and Biology Cecilia Helmerson University of Gothenburg 1. Introduction Rocky shore
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