TC016383
Fly Ortholog:
Dan and Danr
Student Name:
Amnjot Hothi, Trevor Gilbert, Jake Stoler
School Name:
Miami University
Year of Study:
2018
Gene Information:
Dan is important for the modification of the distal region of the antenna. The protein features are Homeo domain, and winged helix.
NCBI Link:
iBeetle Link:
Tribolium Transcript: 5' UTR:
CGTGTTCGTCCGTCGCCAGTATTGATCGCCTGGACAAAGGAACTGACCACCGGCTGGCCAACCTCGCAAGAAACTACTACTTGCCACTAGCCGGCCGTCGCGAGCGGATGTCCGCACACCCATCATACCCGGTGTTTGAGAAAAGCGCGCGCCTGCGTCACCCCCAACTGCATTCGTCAAAACTTGGCCGCAAGTTTCGTTTGTTTGTGTGAAGTTTTTGCCGGTGAGGTTTTTTACAACTGCGCACTCGGGCGCACTCCCATTGGCCCGTTACACGCTTCCACCACGTGCTCCTCTCGGTGGAGACG
Tribolium Transcript: CDS:
ATGTCCACGAAGAACGTCAAGCGCTCGTTGCGCACTATTAGCGCCCACGGAAAGCTGGAGGCCATCAGGAGGGTGCATGACGGCGAGAGCAAGGCCTCGGTCGCGAGGGACATCGGCGTGCCCGAGTCCACCCTCAGGGGATGGTGCAAGAACGAAAGCAAAATTTCCTATCTCTCTCGACAGTCCAGCCCCGACACGGACGAATCCATCGAACCCAAAGACAAGCGGCCGAAAATCGAAGATCCGATAGTACAGCCGTTCAACCTCAGCCTAAAATCGTCAAACATTTACACACCCACCAATTCCGAGTCTCTGTACACGCCGATGGACTACACTAAAAACGACATCGATTGCACGCCGAAAACGCCGCAAAATCTCAGCATCAAGACGGAAACTACCCCCAAGACAGCCTCCCAAATAACCGAACGCGAGCGAAACCGAGCGGAATTGGCAAGGTTGAGCGTCGAACTTGGCTTGAACCGACCCGAAATGTTCACCTCCTCGTCTTCAACCAACAACAACACCTCTAGTCTTGCCGATCTAGCCGCGAACATCAACCTCCTCAGCCAGTGGAACAACATTCTGATGCAACATCAACTCAACAAGAAAGTCACCCCCGACACGACAACTCCAAGCAGCTCAACCCTCAAACAACAGAACTCGATCAAACCGCCCAAAGAACAACAGTCGGTTGAAGATTACATCAACAGTTGGTTGAAAGCGCAAGAGGCGATGTTGAGTCACAGCCGATCGAATGGACTTCCCCCGGTCACGACAACAACAAATTCAACATCAAACATGCAAAGTTCGTGGTTTTGGAATTGGTATAAGCAGCTCTCGTATCAGCAGAGTGCCACACCTGCCGTATCCCCGGAAAAGCCGATTTTGTATCAACAATTGACAAAGGATTCATCCGATGCGGAAAATCTCTCGGTTGATAAGTCGAACAACAACAAAGTGAAGTCGGTCTTGGACAATTTGCTTCATATCAACAACAATAACGTGACGATGGATAAAAAGGATTGCTTGTCGTATAGTGAGGCCGTCGAGCACGGGGAGAAATTCTTGAAGTGGCTGGAATCTTGCAGTAACCCTTCGGTGACGGCAATGCAGATAATGCAGTTCAGGGCGTTGTTGAGTAACGTGAAGAACAGTGCGGACAGGAAGAATGGTGACCTGCAGAACAAAACCAAAGTTAAAAGAAAGTGA
Tribolium Transcript: 3' UTR:
ATTTACTTTCGTCTAGTGAAAAAAGGCTGTTTAGCTACCAAATCTTATTAGTTAAAATATTATGTAGTTTATGTAAAAAGTCTATACGGGTGTTGATTCGATGGATACGGCGTTGGTAGTAACCATTAAATTTACGCCCGTTCTTCCTTAAATGTTACCTTGACAAACCGTTTATTGACGCTTAAAATTTGTTTGTACATACGTTACTTTAGTCGAATTTAGATAAATTTAATTGTAAAGACTATTAATAGTGCCAAATGATTTTCGCATTATTTTTGCTCAAATATGTAC
Tribolium Protein:
MSTKNVKRSLRTISAHGKLEAIRRVHDGESKASVARDIGVPESTLRGWCKNESKISYLSRQSSPDTDESIEPKDKRPKIEDPIVQPFNLSLKSSNIYTPTNSESLYTPMDYTKNDIDCTPKTPQNLSIKTETTPKTASQITERERNRAELARLSVELGLNRPEMFTSSSSTNNNTSSLADLAANINLLSQWNNILMQHQLNKKVTPDTTTPSSSTLKQQNSIKPPKEQQSVEDYINSWLKAQEAMLSHSRSNGLPPVTTTTNSTSNMQSSWFWNWYKQLSYQQSATPAVSPEKPILYQQLTKDSSDAENLSVDKSNNNKVKSVLDNLLHINNNNVTMDKKDCLSYSEAVEHGEKFLKWLESCSNPSVTAMQIMQFRALLSNVKNSADRKNGDLQNKTKVKRK
Forward Primer 1:
ACACCCACCAATTCCGAGTC
Reverse Primer 1:
CGACTGTTGTTCTTTGGGCG
Forward Primer 2:
GTATCCCCGGAAAAGCCGAT
Reverse Primer 2:
TCCTGTCCGCACTGTTCTTC
Gel Picture:
Cloned Fragment:
ACACCCACCAATTCCGAGTCTCTGTACACGCCGATGGACTATACTAAAAACGACATCGATTGCACGCCGAAAACGCCGCAAAATCTCAGCATCAAGACGGAAACTACCCCCAAGACAGCCTCCCAAATAACCGAACGCGAGCGAAACCGAGCGGAATTGGCAAGGTTGAGCGTCGAACTTGGCTTGAACCGACCCGAAATGTTCACCTCCTCGTCTTCAACCAACAACAACACTTCTAGTCTTGCCGATCTAGCCGCGAACATCAACCTCCTCAGCCAGTGGAACAACATTCTGATGCAACATCAACTCAACAAGAAAGTCACCCCCGACACGACAACTCCAAGCAGCTCAACCCTCAAACAACAGAACTCGATCAAACCGCCCAAAGAACAACAGTCG (KD)
Injection concentration:
The final dsRNA concentrations used were 1ug/uL, 100 ng/uL, and 10 ng/uL.
Number Injected:
We injected 33 beetles total. Eleven beetles were injected with 1ug/uL, eleven beetles were injected with 10ng/uL, and eleven beetles were injected with 100ng/uL.
Number Survived to Adult:
26 of the 33 beetles injected survived until adulthood. For the 1 ug/uL injection, 8/11 survived. For the 100 ng/uL, 9/11 survived. For the 10 ng/uL, 9/11 survived.
Number w/ RNAi Phenotype:
Three of the 1ug/uL beetles showed the RNAi phenotype. One of the beetles eventually died that was injected at an earlier stage.
Percent Penetrance:
1ug/uL - 37.5% 10ng/uL - 0% 100ng/uL - 0%
Assay Description:
We performed a qualitative analysis by looking under the microscope to observe the percent penetrance of the beetles. They were thoroughly observed for any abnormalities and then pictures were taken straight from the microscope.
RNAi Phenotype Description:
The observed phenotype of the beetles was a fusion of the distal segments in the antennae for the highest concentration of dsRNA used (1ug/uL).
RNAi Phenotype/Assay Images:
Discussion:
Our experiment showed inconclusive results. In the highest dosage RNAi (1 ug/uL), 3 out of the 8 of the surviving beetles showed fused segments of the distal region of their antennae. Lower doses of RNAi injections (100 ng/uL and 10 ng/uL) did not display any abnormalities. The loss of function phenotype in Drosophila melanogaster for dan is the production of the leg in place of the distal region of the antenna. Based on the results of our study and the known function of dan in Drosophila melanogaster, it is possible that the function of dan might be conserved in Tribolium castaneum. However, since the results of our study are inconclusive, we cannot provide full support for this statement. Therefore according to our results, future research is needed to fully support or reject the statement that dan is conserved in Tribolium castaneum.
Notebook:
Annotated Genbank File:
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