TC002602
Fly Ortholog:
Evi5
Student Name:
Danielle Owens, Jordan Brady
School Name:
Miami University
Year of Study:
2018
Gene Information:
Evi5 stands for “Ecotropic viral integration site 5 orthalog”. This gene has often been studied in drosophila for its role in border cell migration and interaction with specific members of Rab proteins. Evi5 acts as a regular membrane trafficking regulator and regulates border cell migration as well as acting as a gap protein for Rab11. It was found that a loss of function of Evi5 in drosophila blocked border cell migration by disruption the Rab11 dependent polarization of the guidance receptors. Border cells are a cluster of 6-8 migratory cells found in the ovary of drosophila and are important in the expression of the torso-like gene, which activates the receptor tyrosine kinases (Tor) at the terminal of the embryo. Tor then can function as a receptor for prothoracicotropic hormone (PTTH) that initiates metamorphosis. Torso-like gene also plays a role in developmental timing. These relationships are interesting insights into how Evi5 may indirectly impact the body size of organisms, though the only observed connection has been in tribolium. In our study, we would like to confirm the hypothesized relationship between Evi5 and body size in tribolium and explore what insights this relationship could have in the field of molecular biology.
iBeetle Link:
Tribolium Transcript: 5' UTR:
GCGGTGACAGTTCGATTTTGGACATTTGCGTGGTTAGGTATCGACGCGTGCGACGCTAGACACGTCAAATACGGCGATTACTGTTTTTATTTATCTCTTCGTTTTTAATTTATTTGGAAAGCTTTAATTTTGTGTGAGAACTGGGAGATTGTTAATGTGACGATATTGTAAAGTTTGATTTTATTTGTTTCGAATTCGTCAAGTGGTTGTTGGTGAAAGTGTGGGTGTTCTTTTATGGTAAACAAAAACATATTCTAGGGTTTTCGGATCATTTTAGTGGTGGTGGATCGTTGAGCTGTCTCGTGGTCGAGTCCGATGGAGGCTGCTACTCAGCA
Tribolium Transcript: CDS:
ATGCCTCCGACAGCCATGAGGCTCGAGAGCCCTCCTTGCAACAATAATAACAACGCATCTGGAGCAGAACTTGAACTTCTCGCCAAACTGGAGGAAGCAAACCGTCTTTACGAGTCCGACGCCAAGTCCCTCAATTCGCTGAGCGGGGCGTCGGGGCACTCGAGGAAAAGTTCCGACACTTCGCAAATATCGCTAACCTCGGGCACGAGCTCCACACAAGATCGAGCTGAGGATTGCGGTGAGGAGGATTTGTGGACCACTTGGGGGCGTATTGTCAATGATTGGGAGAATAATTGGAAGAAGCAGAATGCTCAAATCAGGGAATTGGTGCGGAAGGGCATTCCGTTACATTTCAGGGCGATAGTTTGGCAATTGCTGTGCAGTGCAGCCGAAGCTCCTGAAAAGAAACTCTACGCCGAATATATCAAGACAAAATCTCCGTGTGAAAAAGTCATCCGTCGAGACATCGCCCGCACTTACCCCGAGCACGACTTCTTCAAAGAAAAAGATGGTCTTGGCCAAGAATCTCTCTTCAACGTCATCAAAGCCTACTCCCTACACGATCGGGAAGTGGGTTATTGCCAGGGATCGGGTTTTATCGTCGGACTTTTACTTATGCAAATGCCAGAAGAGGAAGCTTTCGCTGTGCTCGTTAAAATTATGCAAGATTACCGCATGCGTGACATGTTCAAACCGAGTATGGCAGAATTGGGCCTTTGTATGTACCAGCTCGAGAACCTGGTCGCTGAATATTTGCCAGACCTCAACCAACATTTCCAGTCACAGAATTTCCACACTTCGATGTATGCTTCCAGTTGGTTTCTGACACTGTTTACCACCGCTTTGTCTCTGCCGCTAGCGTGCAGGATCATGGATGTTTTTTTATCAGAGGGAATGGAGATCATTTTCAAAGTGGCACTTGCCATGTTGACGTTGGGCAAAGACGAACTCATGTCGCTCGATATGGAGGGAATGTTGAAGTTTTTCCAAAAAGAACTTCCCGTCAAAGCCGAAGCTGACCCGGAAAACCTTATGCAAGTAGCCTACAATATGAAATACAATGCCAAGAAAATGAAAAAATTGGAAAAAGAATACAATGTGATAAAAACAAAGGAACAAGAGGAAATGGCTGAATTGAAACGCTTGAGAACCGAAAACAAGCAGTTGAAACATCGCTGCGAAATGTTGGAGGAAGAAAGCAAAGCTCTAGCTGATCGTTTGGTCCGCGGTCAAGTCAGCAGAGCTGAAGAAGAAGAAACTACTTTTGTAGTTCAACGTGAATTGGACGCTTTAAGACACACTCATTTGGAAACGACACACCAATTAGCTTTAGCGAACGAAAAAATTAGGTCATTGTCTTTGATGATGGAAGAAACTCACACTTCGAGACAATCTTCCATCGACGAAATTTCCCTCAAACAAGAGCAACTGCAACAAAGGGAAGAAATGATCGAATGCCTCCAAGAAGAACTGGTCAAAGTGCGATTACGTGAAGCTGAAAACGACGCTTTAATTCGCGACTTAAGAGCCAGAATACACGAGTTGGAAGAAGACAAAAAAACACTCCGTGAAATAACACCAGACAATAGTGTTGCCCACCTCCAGGAAGAGCTAATCGCTGTGAAGCTCCGCGAAGCTGAAGCTAACCTCTCTTTGAAGGATTTGCGCCAAAGAGTGTCAGAACTTAGCAATCAGTGGCAGCGCCACCTCCAGGAGCACAAACAGGAGCCGACTGCCTCCGGGGAACCACACAGCACTCCCAAGAAACTCCTCTTCTGGGAGAACCGAAACAATGAGACGCAGAAGTTGGAGGACGAGCTTATGACCACTAGGATACGAGAAATGGAGACTTTGACTGAAGTGAAGGAGTTGAGGCTTAAGGTGATGGAGCTTGAGACTCAGGTGCAGGTCAGTACGAATCAGTTGAGACGCCAGGATGAGGAAATTAAGAAGCAGAGAGATGAGTTGGAGAAGGCTGAGTTGAGGGATCGGGAAGCTGCGGCTCGTTTGTTGGAGGAGCATCGCCGGTATTCGGATCTTGAGAGTAAAATGCAAGATGAGTTGATGAAGGCGAGGATTAGCGATGCGGAGAAAACGCAAGCTATTGCCGAGCTCACGCAAAAGATCAGCCAGCTGGAGTTGAAGAGCCAGGAAATTGCCACCGAAGGCGAGCTTCGAATCCAGTCGGTCGACGATAGCGATCGAGTTAGAGAATTGCAAGATAAAGTGGCTGATCTTCAAGCCGAGGTCATGAGATTGGAATCGTTCAAGAGACGAATAATCGCCGGGGGTGGTCGTCTGCCGTCGGAGAGGTCTTGTTCCATCGACAGCACCGACGACGATCCCAAGATCAGATTGGACGATCCTTCCCTGAGATTAAACTTAGTCGAGTCTCCTACCTCCAAATCCGAGCTTACTTTTAGCGACATGTGTCAAAATCCTACTATAAAAAACGACAACAAAGTAGATCTAGATCCCAACATGAATACGCACACCACTATAGATGCTCACGAAGAGAAGTGTGCCCCAAAGGTCTGA
Tribolium Transcript: 3' UTR:
CAACACGACTGAAATTTTAGGAATAAGATTTTGTAGTATTTTTTGCCTATATATACTTTTCCAATGGTCAATATAACGGGATTTGTCATTATAGAGTACATACTACAAACGAATAGGAAATTTGGTGCATTTAAGTAAGTCTAGGTCGTCATAATTTATTGTTTATTTTGTATTAATATATTTTTAGATTTATGTACTTAACCGATTCTAGTGTGGAAATTTCGTGTATTTTGTTAATGTCTGGTATGTAATTTAAAATAGGGTAGAATGTTCTTGGGAATTCAACCAAGAGTAACTTATGAATTAGTGAGACAACTAGGAGATGTTAGATTAGATTTTCTAATATTCTATCTGTGTTACAATGGTAGTATTAAATTGAGAGGGCTGTTTGTTTATACTAAGAATATTTTGCTGCGCAAAGTCGTTGAAACAGTATTTTTCTTTAGAAGTGGACAAGTTGATGAACCAATCAACGGTGCTATTTTTATCAGTTGCAATAATTCAAAAAACATTCCAAACGTTAAACATCGAAAGTTTTATTTTTAGATAGAAATTAATAAATACTCTTAGAGAAACATATTGCACATT
Tribolium Protein:
MKRDKSFMHSMVLQKLVSSRYWYTRRPRKRGGGSLSCLVVESDGGCYSAMPPTAMRLESPPCNNNNNASGAELELLAKLEEANRLYESDAKSLNSLSGASGHSRKSSDTSQISLTSGTSSTQDRAEDCGEEDLWTTWGRIVNDWENNWKKQNAQIRELVRKGIPLHFRAIVWQLLCSAAEAPEKKLYAEYIKTKSPCEKVIRRDIARTYPEHDFFKEKDGLGQESLFNVIKAYSLHDREVGYCQGSGFIVGLLLMQMPEEEAFAVLVKIMQDYRMRDMFKPSMAELGLCMYQLENLVAEYLPDLNQHFQSQNFHTSMYASSWFLTLFTTALSLPLACRIMDVFLSEGMEIIFKVALAMLTLGKDELMSLDMEGMLKFFQKELPVKAEADPENLMQVAYNMKYNAKKMKKLEKEYNVIKTKEQEEMAELKRLRTENKQLKHRCEMLEEESKALADRLVRGQVSRAEEEETTFVVQRELDALRHTHLETTHQLALANEKIRSLSLMMEETHTSRQSSIDEISLKQEQLQQREEMIECLQEELVKVRLREAENDALIRDLRARIHELEEDKKTLREITPDNSVAHLQEELIAVKLREAEANLSLKDLRQRVSELSNQWQRHLQEHKQEPTASGEPHSTPKKLLFWENRNNETQKLEDELMTTRIREMETLTEVKELRLKVMELETQVQVSTNQLRRQDEEIKKQRDELEKAELRDREAAARLLEEHRRYSDLESKMQDELMKARISDAEKTQAIAELTQKISQLELKSQEIATEGELRIQSVDDSDRVRELQDKVADLQAEFPTPITSPDTEPWKWLES
Forward Primer 1:
ACCTCCAGGAGCACAAACAG
Reverse Primer 1:
CAAGATCCGAATACCGGCGA
Forward Primer 2:
TCGGGAAGTGGGTTATTGCC
Reverse Primer 2:
TTCGGCTTTGACGGGAAGTT
Gel Picture:
Cloned Fragment:
TCGGGAAGTGGGTTATTGCCAGGGATCGGGTTTTATCGTCGGACTTTTACTTATGCAAATGCCAGAAGAGGAAGCTTTCGCTGTGCTCGTTAAAATTATGCAAGATTACCGCATGCGTGACATGTTCAAACCGAGTATGGCAGAATTGGGCCTTTGTATGTACCAGCTCGAGAACCTGGTCGCTGAATATTTGCCAGACCTCAACCAACATTTCCAGTCACAGAATTTCCACACTTCGATGTATGCTTCCAGTTGGTTTCTGACACTGTTTACCACCGCTTTGTCTCTGCCGCTAGCGTGCAGGATCATGGATGTTTTTTTATCAGAGGGAATGGAGATCATTTTCAAAGTGGCACTTGCCATGTTGACGTTGGGCAAAGACGAACTCATGTCGCTCGATATGGAGGGAATGTTGAAGTTTTTCCAAAAAGAACTTCCCGTCAAAGCCGAA
Injection concentration:
1 µg/µl
Number Injected:
18
Number Survived to Adult:
17
Number w/ RNAi Phenotype:
0
Percent Penetrance:
0
RNAi Phenotype Description:
There was no observable change in the body size of our beetles, but they appeared to be healthy.
RNAi Phenotype/Assay Images:
Discussion:
Evi5 is a highly conserved gene present in both drosophila and tribolium species. In a previous study reported on iBeetle, RNAi was done on the Evi5 gene in tribolium and the resulting phenology was an overall smaller body size, with few early fatalities. It is not known exactly why one gene may have such a large effect on the organism or if it is really Evi5 alone that caused that effect, as it has not been extensively studied. This gene has often been studied in drosophila for its role in border cell migration and interaction with specific members of Rab proteins. Evi5 acts as a regular membrane trafficking regulator and regulates border cell migration as well as acting as a gap protein for Rab11. It was found that a loss of function of Evi5 in drosophila blocked border cell migration by disruption the Rab11 dependent polarization of the guidance receptors.
In our study, when RNAi was performed, no observable difference in phenotype occurred in the beetles. Seventeen out of eighteen of the beetles survived into adulthood and they all appeared healthy. The lack of phenotypic change could have been due to the fact that in the previous study another factor could have influenced the resulting reduced body size. It is also possible that in the previous study, the beetles were injected at an earlier state, allowing the gene to play a larger role in development. Lastly the primers used in this experiment may not have allowed a successful RNAi, meaning that the beetles developed normally. Further study on this gene could allow a deeper insight into its function and if it plays a role in the development of body size in tribolium castaneum.
Notebook:
Annotated Genbank File:
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