TC002498
Fly Ortholog:
Dual Oxidase (duox)
Student Name:
Clayton Deible & Kailee Tateman
School Name:
Miami University
Year of Study:
2017
Gene Information:
Duox, or Dual Oxidase, is a gene that has not been studied in depth in many species. The studies that have been done were used to mainly show the function of the Duox gene rather than looking at the conservation of that gene in similar species. The main focus of this study are not only evaluate potential conservation of function of the gene through similar species as Drosophila, but also, the divergence in function duox has in Tribolium. Duox is a gene that codes for an important protein that takes part in the electron transport chain. The physiological impacts that these proteins have, have been shown to cause changes in pathogen protection in a species but the main focus of this study is the function it has in wing morphology. This gene works with another gene to create the stability needed for wing structures, specifically with the cuticles in the wing. When the Duox gene was knocked down or overexpressed, the wing curved either down or up respectively.
iBeetle Link:
Tribolium Transcript: 5' UTR:
ACTTTCTCTTCACTGTTGATGGAGAGGTTTTGTTTAAGCAATCTGTTAGTGTGAATGAAGTACAGTGTAGATATGCGTGAGTGTGTGTGCTGTCTCTTCGCAAAATCCTCAACAGTGACTATGTCGTCCACTTAATTTATTTTAATCGCGAGTGCGGGATGTTAAACGCTTGATTTATACACGGTTTTTTATCGTTCTTCGAAC
Tribolium Transcript: CDS:
ATGGTTTCTTTAACATCTCTTTTATTTCACGTACTCACTTTCTGTGTCATTGTCCTCGTTGCCATTTCGACAGTACGACCTGAAGTCCGGACGGAGAAGCAACGCTATGACGGATGGTTCAACAATTTGGCTCATCCAGACTGGGGCTCTGTGGGGTCACACTTGATTCGGAGGGCCCCCTCGGCCTACTCCGACGGGGTGTACATGCTAGCGGGGCAAAACCGGCCCAGCCCCCGCAAGCTGAGCCGCCTATTCATGCGGGGCCTCGACGGCCTGGGCTCCATGAACAACCGCACGGCCCTTTTGGCCTTCTTCGGCCAAATGGTCACCTCTGAGGTGATGATGGCCTCCGAGAGCGGCTGTCCGATCGAAATGCACCACATCGAGATCGAGAAATGCGACGATGTGTACGATAAGGAGTGTCGCGGGGACAAGTACATACCGTTCCACAGGGCCGGGTACGACAGGAAGACGGGGCAGAGCCCCAACAGCCCCCGGGAGCAGTTGAATCAAGTGACGAGTTGGATCGATGGGAGTTTTATTTATAGTACCAGTGAGCCCTGGGTTAATGCAATGCGGTCGTTTCGTAATGGCACTTTTTTGACGGATGCCACGGGCAAAATGCCGGTGCGGAATTCGATGAGAGTGCCGCTTTTTAATAACCCTGTGCCACATGTCATGAAAATGTTAAGTACTGAGAGGCTATTTTTACTCGGCGACCCCCGAACCAACCAAAACCCCGCCCTCCTCACCATCAGTATTCTGTTTTTCCGATGGCATAATGTCATCGCCGAACGCGTCCAAAAAGAGCACCCGGACTGGTCCGACGAGGACGTCTTCCAACGCACGAGACGCATCGTCGTCGCCACCGTCCAAAACATCATCGCTTATGAATACATCCCAGCATTCCTAGGCCAACCACTCCCGGAGTACTCCGGGTACAAACAAGACGTCCATCCCGGAGTCACACACGTATTTCAAAGCGCGGCTTTTCGTTACGGCCATTCCCTGATCCCTCCCGGACTTTATCGAAGAGACGGTGAATGCAACTTCGAGGAGTCTCCGATGGGATTAAGGCTCTGCGCCACGTGGTGGGACTCCAATGATGTCTTAATTAGCCACTCTTTGGAGAAGCTCTTAATGGGAATGGCATCCCAACTGGCTGAACGTGAAGACTCAGTTTTGTGTTCAGACGTCAGAGACAAGTTATTCGGTCCGATGGAGTTCTCCAGACGAGATTTGGGAGCTTTGAACATAATGCGTGGCAGAGACAGCGGCCTCCCTGATTACAACACGATCCGGGCCTATTACGGCTTACCAAAAATAAAAAAATGGGCCGACATCAACCCGAAATTATTCGAAGAAAAACCAGAATTGTTGCGAGCACTTGTCTCTGCTTACGCCAATAACATCAACAACATTGACGTTTATGTTGGGGGCATGTTGGAGAGTTATGGGGGACCTGGGGAACTCTTCACCACCGTCATCAAGGAGCAGTTTGCGCGGCTACGAGACTCGGATAGGTTTTGGTTCGAGAACGAGGGCAATGGGATTTTTACCAGAGAGGAGATTGCGAGTTTGAGGAAAGTTACGCTGTGGGATGTTATTGTGAATAGTACGAACATCGCGCCGAATCATATCCAGAAAAATGTGTTTTTCTGGATGAGTGGGGATCCCTGCCCGCAGCCGATGCAGCTGAATACGTCCCTGATGGAGCCGTGTAAAATTCTGAATGGATACGATTATTTCGAGGGCAACGAACTAGTCTACATCTACGCTTGTGTCTTCCTGGCCTTCGTGCCAATCCTCTGCGCTGGCGCCGGCTACGGCGTCGTCAAACTCCAAAACCGGCGCCGTCGCCGCCTCAAAATCAAACAAGAGGAACTCCGCAACTGCGTCGAGGGCAAACTCCCCGTGGACACAATGATCGTCCAAGAGTGGCTCCACGCCAACCACAAACGCCTCGTCAAAATCAAATTCGGCCCCGAAATCGCCATCCACATCATGGGCCGAAAAGGCGAAAAACTCCGAACCGTCAACTTCAAAAACACCGAAACGGTCTACGTCGAAATCTCCGTCGAACTGACCAATCAAAAGAAACCAATGCTCCTGATCCGAGTCCCACGTGACCACGACCTCGTCATCGAACTAGATTCGATCGGATCCCGGAAAAAGTTCCTCACAAAACTGGAACTCTTCCTAACTTCGAATAAAAAATCCCTGATTATCACAGAAGTCGTGCGCGAAATCATGCTGGCGAAGGCGGAGACGAGCGAGCGGAGACAGAAAAAGCTGGAGCACTTCTTCAGAGAGGCGTACGCTTTGACCTTCGGGCTGAGACCCGGGGAGCGGCGTCGCAGGTCCGACGCCAGCGTCGACGGCGAGGTGGTGACCGTGATGCGGACCTCGCTCACCAAAAGCGAGTTCGCCAGCGCCCTGGGGATGAAGCATGACGCGGTCTTCGTGAAGAAGATGTTCAATATTGTGGATAAAGACGGCGATGGGCGCATCTCCTTCCAGGAGTTTCTGGACACTGTGGTGTTGTTTTCCAGGGGAAAGACCGACGATAAGTTGAGGATCATCTTCGACATGTGCGATAACGACGGGAATGGGGTTATCGACAAGGAGGAGTTGTCGGAGATGTTGAGGTCGTTGGTGGAGATTGCGAGGACGACGAGTTTGAGCGACGATCACGTCACGGAGTTGATTGAGGGGATGTTCCAGGACGCGGGGTTGCAGCATAAGCATTTTTTGACGTATAATGACTTCAAGGAGATGATGAAGGAGTATAAAGGGGAGTTCGTCGCGATTGGGTTGGATTGTAAGGGGGCCAAACAGAACTTTTTAGACACTTCCACCAATGTTGCAAGGATGACTAGTTTTCACATAGAACCGACCCTCGACTCTGAAAAAAACTACCTCGAACTGAAATGGAACTCCCTCACCACATTTCTGGAAGAAAACCGCCAAAACATCTTCTATTTATTTATTTTCTACGTGATAACGATCGCTCTGTTCGTCGAACGCTTCATACACTACTCATTTATGGCCGAACATACCGACTTGCGCCACATAATGGGCGTCGGAATTGCGATAACTCGCGGCTCTGCCGCCTCTTTATCTTTCTGTTACAGCATTTTGTTGCTAACAATGTCCCGGAACCTTCTTACAAAACTGAAAGAATTCTCCATCCAACAATACATTCCGCTCGACACTCACATCCAATTTCACAAAATCGCAGCTTGCACAGCACTGTTTTTTTCACTGTTACACACCGTTGGCCACATTGTCAACTTTTACCACGTCTCGACTCAACCGCTCGAAAATTTGAGATGTCTCACAAACGAGGTGCGATTTCCCTCGGATTATAAACCAGGAATCACATTTTGGTTGTTCCAAACAATAACAGGTCTTACAGGAGTCTTCTTGTTTGTGATAATGAGCATCATTATAGTATTCGCTCACCCGACAATCAGGAAAAAGGCCTACAACTTTTTTTGGGCCACACATAATCTCTATATAATTTTGTACATCCTGTGTCTTATTCACGGGCTTGCGAGGCTCACAGGGGCCCCCAGATTTTGGCTATTTTTCATCGGCCCTGGAACTATTTACATTCTTGATAAAGTCGTGAGCTTACGCACCAAATACATTCCCTTGGATGTGATCGAAACCGAGCTTTTACCATCCGACGTGATAAAAATAAAGTTTTACAGGCCACCGAATTTGAAGTATTTGTCAGGACAATGGGTCCGTTTAGCTTGTACTGCCTTCAAAAGTCACGAATTTCACTCGTTTACACTGACTTCGGCACCTCATGAAAACTTTCTCTCGTGTCATATTAAAGCCCAAGGTCCTTGGACGTGGAAACTCCGAAACTACTTCGACCCCTCAAACTACAACCCGGAAGACCAGCCAAAAATCCTCCTCGAAGGCCCCTTCGGTGGAGGCAACCAGGACTGGTACAAATTCGAGGTCGCTGTTATGGTTGGCGGCGGAATCGGGGTAACCCCCTACGCCTCCATCCTAAATGACCTAGTTTTCGGCACTTCCACAAACCGATACTCGGGAGTAGCGTGCAAAAAAGTTTACTTTTTATGGATTTGTCCGTCACACAAACACTTCGAATGGTTCATTGACGTGTTGCGCGATGTTGAGAAAAAAGATGTCACCAATGTCTTAGAGATTCACATTTTCATTACGCAATTCTTCCATAAGTTTGATTTGAGAACAACAATGTTGTACATTTGCGAAAATCACTTCCAACGGCTTTCGAAAACCTCGATCTTTACCGGGCTGAAGGCAGTCAATCACTTTGGACGCCCCGACATGACTTCGTTTCTCAAATTCGTCCAAAAGAGGCACAGTTATGTGAGCAAAATCGGGGTTTTCAGTTGCGGCCCGCGTCCCTTGACCAAGAGCGTGATGTCGGCTTGCGACGACGTCAACAAGGGCCGAAAACTGCCCTACTTCATCCACCACTTCGAAAACTTCGGCTAG
Tribolium Transcript: 3' UTR:
No 3' UTR
Tribolium Protein:
MVSLTSLLFHVLTFCVIVLVAISTVRPEVRTEKQRYDGWFNNLAHPDWGSVGSHLIRRAPSAYSDGVYMLAGQNRPSPRKLSRLFMRGLDGLGSMNNRTALLAFFGQMVTSEVMMASESGCPIEMHHIEIEKCDDVYDKECRGDKYIPFHRAGYDRKTGQSPNSPREQLNQVTSWIDGSFIYSTSEPWVNAMRSFRNGTFLTDATGKMPVRNSMRVPLFNNPVPHVMKMLSTERLFLLGDPRTNQNPALLTISILFFRWHNVIAERVQKEHPDWSDEDVFQRTRRIVVATVQNIIAYEYIPAFLGQPLPEYSGYKQDVHPGVTHVFQSAAFRYGHSLIPPGLYRRDGECNFEESPMGLRLCATWWDSNDVLISHSLEKLLMGMASQLAEREDSVLCSDVRDKLFGPMEFSRRDLGALNIMRGRDSGLPDYNTIRAYYGLPKIKKWADINPKLFEEKPELLRALVSAYANNINNIDVYVGGMLESYGGPGELFTTVIKEQFARLRDSDRFWFENEGNGIFTREEIASLRKVTLWDVIVNSTNIAPNHIQKNVFFWMSGDPCPQPMQLNTSLMEPCKILNGYDYFEGNELVYIYACVFLAFVPILCAGAGYGVVKLQNRRRRRLKIKQEELRNCVEGKLPVDTMIVQEWLHANHKRLVKIKFGPEIAIHIMGRKGEKLRTVNFKNTETVYVEISVELTNQKKPMLLIRVPRDHDLVIELDSIGSRKKFLTKLELFLTSNKKSLIITEVVREIMLAKAETSERRQKKLEHFFREAYALTFGLRPGERRRRSDASVDGEVVTVMRTSLTKSEFASALGMKHDAVFVKKMFNIVDKDGDGRISFQEFLDTVVLFSRGKTDDKLRIIFDMCDNDGNGVIDKEELSEMLRSLVEIARTTSLSDDHVTELIEGMFQDAGLQHKHFLTYNDFKEMMKEYKGEFVAIGLDCKGAKQNFLDTSTNVARMTSFHIEPTLDSEKNYLELKWNSLTTFLEENRQNIFYLFIFYVITIALFVERFIHYSFMAEHTDLRHIMGVGIAITRGSAASLSFCYSILLLTMSRNLLTKLKEFSIQQYIPLDTHIQFHKIAACTALFFSLLHTVGHIVNFYHVSTQPLENLRCLTNEVRFPSDYKPGITFWLFQTITGLTGVFLFVIMSIIIVFAHPTIRKKAYNFFWATHNLYIILYILCLIHGLARLTGAPRFWLFFIGPGTIYILDKVVSLRTKYIPLDVIETELLPSDVIKIKFYRPPNLKYLSGQWVRLACTAFKSHEFHSFTLTSAPHENFLSCHIKAQGPWTWKLRNYFDPSNYNPEDQPKILLEGPFGGGNQDWYKFEVAVMVGGGIGVTPYASILNDLVFGTSTNRYSGVACKKVYFLWICPSHKHFEWFIDVLRDVEKKDVTNVLEIHIFITQFFHKFDLRTTMLYICENHFQRLSKTSIFTGLKAVNHFGRPDMTSFLKFVQKRHSYVSKIGVFSCGPRPLTKSVMSACDDVNKGRKLPYFIHHFENFG
Forward Primer 1:
5'-AGTGAGCCCTGGGTTAATGC-3'
Reverse Primer 1:
5'-TCCGGGTGCTCTTTTTGGAC-3'
Forward Primer 2:
5'-TGCAGCTGAATACGTCCCTG-3'
Reverse Primer 2:
5'-TTTTCGGCCCATGATGTGGA-3'
Gel Picture:
Cloned Fragment:
TGCAGCTGAATACGTCCCTGATGGAGCCGTGTAAAATTCTGAATGGATACGATTATTTCGAGGGCAACGAACTAGTCTACATCTACGCTTGTGTCTTCCTGGCCTTCGTGCCAATCCTCTGCGCTGGCGCCGGCTACGGCGTCGTCAAACTCCAAAACCGGCGCCGTCGCCGCCTCAAAATCAAACAAGAGGAACTCCGCAACTGCGTCGAGGGCAAACTCCCCGTGGACACAATGATCGTCCAAGAGTGGCTCCACGCCAACCACAAACGCCTCGTCAAAATCAAATTCGGCCCCGAAATCGCCATCCACATCATGGGCCGAAAA
Injection concentration:
0.6 ug/ul
Number Injected:
15 for the 0.6 ug/ul concentration and 10 for the 1.0 ug/ul concentration
Number Survived to Adult:
10 survived to adults in the 0.6 ug/ul concentration and 3 survived to adults in the 1.0 ug/ul concentration
Number w/ RNAi Phenotype:
None for either concentration
Percent Penetrance:
0%
Assay Description:
No assay was performed. Only observational analysis of the beetle phenotype.
RNAi Phenotype Description:
Not observed in Tribolium. We hypothesized that the duox gene had gained a new function that facilitated the evolution of unique wing structure in Coleoptera. We predicted to see downward curvature in the wing and possibly the elytra as well.
RNAi Phenotype/Assay Images:
Assay Data:
There were abnormalities in the sampling we performed RNAi on. One pupi from the lower concentration sample displayed a mass on its ventral cavity. This pupi did not survive past this developmental stage. Two adults in the lower concentration had wing abnormalities that we can not attribute to our RNAi due to low occurrence across samples. One adult had a crinkled left wing. The other adult had a hole in the same spot in the wing and the elytra.
Discussion:
After observational analysis of the beetles we injected with our specific RNAi sequence, we did not detect the phenotype we had predicted to observe. Most all the beetles appeared to have completely normal wing and elytra structure. Two of the 25 had differing wing structure, however, we do not believe this is associated with the RNAi. We injected at two different concentrations, 0.6 ug/ul and 1 ug/ul. In the lower concentration sample we began with 15 injected beetle larva. Of those 15, two of the larva and one of the pupi died. Of the twelve remaining, two adults and one pupi showed differing phenotypes from the rest of the sample. One pupi had an abnormal protrusion on the ventral cavity. Two adults showed odd wing structure. One had a crinkled left wing, the other had a hole in the same spot on the elytra and wing.
There are some possible explanations for why we did not see our predicted phenotypes. One such explanation is that our RNAi did not form correctly and so did not bind to the DNA segment of our gene. Another reason why we may not have seen our predicted phenotype is because we chose the wrong paralog in C. tribolium. There were 3 different isoforms of our Dual Oxidase gene, however these isoforms should not have been different enough that we needed to create different RNAi. In addition to the isoforms, there was another paralog that we could have chosen which may have been the correct gene to use. Another reason why we may not have seen any phenotypic changes to the wing is because of genetic redundancy. Because there are isoforms and paralogs, it is possible that there are more genes besides our own that that are used in cuticle structure in the wing, in which case we would not only need to knock our gene of interest out but also find out what other genes are playing a role and knock them out as well.
In addition to this study, there are more routes that we can study. This includes testing to see what the isoforms of Dual Oxidase do along with the function of our paralog as well. Also, our Dual Oxidase gene has a variety of functions in different species such as the generation of reactive Oxygen species and epithelial cells in humans. The reason why knowing the function of Dual Oxidase is important is because it is a highly conserved gene throughout different species. Since it is this highly conserved, it can be thought that this conservation is an indication of how important the gene is to development.
Notebook:
Annotated Genbank File:
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