The Study of Evolutionary Origin of the Antifreeze Gene in Rhagium
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Roskilde Universitet / Roskilde University Den Naturvidenskabelige Bacheloruddannelse / The Natural Science Bachelor Programme _____________________________________________ The study of evolutionary origin of the antifreeze gene in Rhagium mordax. __________________________ ____________ _ ___________________ Group members: Saudat Alishayeva Nicolaj Stelzner Grønvall Alexander Varnich Hansen Philip Kruse Nina Štrancar Project supervisor: Peter Kamp Busk Semester: 5th Semester Date: 18/12/2018 Abstract This project report details the attempt to isolate and sequence candidate sequences for the evolution of the antifreeze protein gene present in Rhagium mordax. Larvae of the R.mordax species were collected, their DNA extracted and underwent PCR amplification with the use of specially designed primers which targeted sequences similar to ones coding for antifreeze genes. In addition, to validate the species we have applied DNA barcoding method. PCR amplifications were analysed with gel electrophoresis. Several PCR products were observed and sent for direct Sanger sequencing. Due to a problem with sequencing, we have been unable to identify the sequences and draw the conclusions of the origin of the antifreeze gene. Although the sequencing failed, results from the previous steps indicate that the methods utilized in this project were feasible for the study of R. mordax genetics. 1 Table of contents Abstract 1 Table of contents 2 Introduction 4 1.1 Hypothesis 5 2. Theory 6 2.1 Historical Overview of the Discovery of the Antifreeze Proteins 6 2.2 Convergent Evolution and R. mordax 6 2.3 Mutations - Gene Duplication 8 2.4 Antifreeze Proteins and Their Function 10 2.5 Degenerate primer design 10 3. Experimental methods 15 3.1 Сollection of R. mordax 15 3.2 DNA barcoding and CO1 gene 16 3.3 DNA Extraction and Purification 16 3.4 Analysis of the DNA Quality and Concentration 17 3.5 Primers Design 18 3.5.1 Forward Primer 18 3.5.2 First Reverse Primer 19 3.5.3 Second Reverse Primer 19 3.5.4 Primer Modification 20 3.4.5 CO1 primers 21 3.6 Polymerase Chain Reaction 22 3.7 Gel Electrophoresis 22 3.8 Gel Samples Extraction and Purification 22 3.9 Sequencing 24 3.10 Bioinformatics analysis 25 4. Results 26 4.1 The first agarose gel 27 4.2 The second agarose gel 28 4.3 The third agarose gel 30 2 4.4 Sequencing 31 4.5 Bioinformatics analysis 32 5. Discussion 36 5.1 Purification of the larvae DNA with QIAamp mini kit 36 5.2 PCR settings 36 5.3 Gel-1 examination 37 5.4 Primers specificity 37 5.5 Gel-2 examination 38 5.6 Gel DNA extraction 38 5.7 Sequencing 39 5.8 Interpretation of bioinformatics analysis 40 6. Conclusion 41 7. Perspective 41 8. References 44 9. Appendix 47 3 1.Introduction Organisms around the world have evolved mechanisms to optimise their chances of survival to the different climate. One of such examples are organisms found in colder climates where there is a chance of freezing, which may cause damage to an organism. A discovery in the late 1960s by Arthur L. DeVries and his team shed light on how some Antarctic fish species preserve their blood in the liquid state despite being in ice-cold water (DeVries et al., 1970). The team has found glycoproteins which were able to depress the freezing point of water. This revelation was just one of the later discovered proteins, which came to be commonly known as Antifreeze Proteins (AFPs), also labeled ice structuring proteins (ISPs) or more generally, ice-binding proteins (IBPs). In this project, we refer to them as AFPs (Davies, 2014). The proteins are not exclusive to fish but have been identified in organisms such as insects, vertebrates, bacteria, plants, and fungi (Bar Dolev et al., 2016). While they share a common function, their structure exhibits great variation, which hints they have evolved independently in different species during evolution (Davies, 2014). The mechanism behind the AFPs' function is their binding to ice crystals. The adsorption restricts crystal growth, and that causes the freezing point to be depressed (Bar Dolev et al., 2016). The research of AFPs is not limited to their structure or function, but also extends to analysis of their core elements, such as AFP-coding DNA sequences. By obtaining the DNA sequence which codes an AFP, it is possible to analyse possible evolutionary origins between species that exhibit AFPs, observing effects on their function if mutations are implemented, or produce modified AFPs for utilisation in industry. 4 In this project, we designed primers based on AFP protein amino acid sequences originating from Rhagium mordax beetle and performed PCR followed by direct sequencing. 1.1 Hypothesis Our study hypothesizes that the gene responsible for the antifreeze protein has appeared in Rhagium mordax after a gene duplication event, as was previously shown to be the case for the arctic fish Notothenia where similar antifreeze genes evolved by gene duplication. If a gene duplication event has taken place, then we expect to see a similar gene to rmAFP in the genome of R. mordax. The goal is thus to find sequences similar to the gene coding for AFPs, which could provide an insight from which sequences the rmAFP originated. 5 2. Theory 2.1 Historical Overview of the Discovery of the Antifreeze Proteins The antifreeze activity was first observed in the 1950s when a Norwegian scientist P.F. Scholander set out an expedition to reveal the mystery of the mechanism by which the Arctic fish Notothenia can survive in water with colder temperature than the freezing point of their blood. He concluded from his experiments that there was “antifreeze” in the blood of Arctic fish. But only later, in the late 1960s, was the actual antifreeze protein was isolated from the Antarctic fish by Arthur DeVries. These proteins were later named antifreeze glycoproteins (AFGPs) or antifreeze glycopeptides to emphasize the difference with newly discovered non-glycoprotein biological antifreeze agents (AFPs). There are several possible explanations as to what led to the evolution of antifreeze genes. One of the hypotheses (Hudait, 2018) states that the changes in climate during the glacial period could be a possible source of the selective forces that drove convergent evolution of the novel mechanisms as the means of adaptation to the cold conditions. 2.2 Convergent Evolution and R. mordax Convergent evolution is the phenomenon of species developing same traits without transfer of genes. This can occur by emergencies of a similar character (in our case freezing of water) and leads to various molecular mechanisms that compensate for this specific environmental hazard. Only mechanisms that are alike are referred to as convergent evolution. According to (C. Deng et al. 2010) the antifreeze gene that was discovered in arctic fish evolved from a mutated copy of gene that codes for sialic acid synthase (SAS) enzyme. The sequence alignment of the genes evolved in Rhagium mordax and 6 Notothenia fish shows no significant similarities. Therefore it’s fair to assume that the antifreeze gene in R. mordax has evolved convergently with the Notothenia gene. Carl Linnaeus has identified all beetles as Coleoptera Order in 1758. (Linnaeus, 1758, Systema Naturae) According to (Duman et al. 2004) antifreeze genes evolved convergently within Coleoptera Order in 14 species: Figure 1: Antifreeze proteins of Coleoptera order (Duman et al. 2004). The publication does not consider Rhagium mordax., so on total there are about 15 species of beetles with the adaptation to similar condition. In case of Rhagium mordax and inquisitor there is an ancestral heredity of the gene. Even though their amino acid sequences coding for the antifreeze proteins are 30% diverse (Kristiansen, 2012), they share enough similarity to say that their most recent common ancestor had this gene. The Coleoptera order is the largest of all orders and contributes to 400 000 species of beetles. Since only 15 of species that belong to unrelated suborders have evolved to have the antifreeze gene it is convenient to assume that most of them have acquired this gene independently from each other. The structure of all antifreeze proteins is made of repetitive domains, forming because of very high content of Tyrosine amino acids. Moreover, the convergent evolution of those genes allowed some species to have more efficient antifreeze proteins than others hence to survive in even harsher conditions. For example, the beetle of our primary interest, R. mordax can survive temperatures that are down to 7 -20°C, while an interesting beetle from Alaska Cucujus clavipes puniceus is able to survive in −58°C and its larvae is capable of living under −100°C (Sformo et al, 2010). In most cases, evolution led not only to adaptation through the emergence of convergent antifreeze genes in the beetles, but also to alternative mechanisms that suppress the melting point, such as having high high sugar and glycerol content in blood. In the case of previously mentioned Cucujus clavipes puniceus, the species achieves prevention of ice crystal formation with combination of deliberate dehydration and antifreeze activity. In conclusion, the adaptation to frost environment is a convergent process of evolution that appeared to favour the antifreeze proteins together with combinatorial effects of several alternative factors. 2.3 Mutations - Gene Duplication Mutations are changes in the genetic code in an individual cell or in an entire organism. Mutations come in many different variants. One variant, the spontaneous mutation can be caused by errors in DNA replication as well as spontaneous lesions, among other things. Spontaneous mutations are also categorized. An example would be tautomerism, in which the isomer of one base is changed via. the repositioning of a hydrogen atom, which results in the base bonding differently, leading to replication errors due to incorrect base pairing (Griffiths et al, 2000).