TC003913
Fly Ortholog:
TweedleD
Student Name:
Valerine Rajathi and Anna Osatuke
School Name:
Miami University
Year of Study:
2017
Gene Information:
The gene that we are primarily interested in is the TweedleD gene, belonging to the Tweedle family of genes most of which are involved in cuticular protein expression. Previous research (Guan et al., 2006) has shown that mutation of the TweedleD gene in Drosophila has given rise to offspring with short and stout larval and pupal stages.
iBeetle Link:
None
Tribolium Transcript: 5' UTR:
AGGTATACGTCCTTGTTATAAGGATGTTTAGAAAAGTGGGGAACAATCTTATAAATAAAATTAAATGACTTTATCGCACCAATTCAATTAAA
Tribolium Transcript: CDS:
ATGAACCTTCCACTGCTAATTGCAGCCCTCGCTCCTTTGGTCGCTTGCGACGTAAGCCATCTGGGGTACCAGTACCACGCCCCAGCGACCAGTTACGACGTCCCTTTTGGCTCCATTGGAAGCCTGAAGCACGACAGGCAGTACTACCATGTCAACAAACCCGGGATTTTGAGTGGGGTTGTTGGCACCACCCCTGCCTCCAACCTACAAACCCCCATACACGAGTTCTCCGCCTTCAGAGGAGGCGAAACCTTCAACTCCTTTCTCCACCAACCAGCGGACTTCGGAGTTACGGTTAAACCAATCCTCAACACTGTAAGTCCAACCGTTGGACCGGTTTTCGGTTCGGTTAACAACGTAGTTCCACAATCGGACTTTGGAGTTACGGTTAAACCGATCCTCAATACTGTAAGTCCAACCGTTGGACAGGCTTTCGGAGTTAGCACAGTGAGTTCAACTGCAGCCCCGGTTATCGGTTCGGTTAACCCACAATCGGACTTCGGAGTTACGGTTAAACCGTTTATTGACACCGTGAGCACAACTGTTCGGCCGATTTTCGACAATGTTGTTCCACAATCGGACTTCGGCGTTACGGTTAAACCGGTTGGACCGCTTTTAGGGTTAGCTCCACAATTGGACTTCGGCGTTAAATCGGTTTTCAACACCGTAAGTCCAACGGTGGGGCCGGCTTTCGGTTCGGTTGACAATTTCGGAGTTACGGTTAAACCGGTTCTTAACACTGAGGGAGCAGTCCAATCTTCGGGTGTGGAAGTCCAAAAGCATTTGTACTTCTTTTCCGCTCCGGACGATGAGCCTGAGGAAGTTAAACCCCGGATTATTTTTCCCCAAGCTCCGGTCAAGAAAAATCTCAAAGTATTGTTTATCAAAGCTCCGACGTTCCGTACTTCCCCGGTTCAAATCCCAGCGTTGCCGCAAAACGAAGAAAAGACTCGTGTCTACGTTTTGGTGAAGAAACCGGAAGAAGCGACGATTATTCTTCCGACTCAAGCGCCAACACAGCCAACTAAACCTGAGGTTTATTTCATCAAATACAAGACGCAGATGGAGGCTGAAGAAGCGATTTCGCAAGTGCAAGGTGGGGCAGGAGGTGATGTTTCCATTAGTGCGAATTCGGTTGATCATGATCATGATTTTCTGAGTGGGATCCAAGACAGTGATTTGGCTCAGAAATTAGGTTCTGGTCTTGTCTCAGGTTCCACAGAAGGTTCGATTTTAGGGTTTAATAACCAGCAAGGTTTTGGTGGGTTTGTTGGGGTGAAGGATTCGTTTGTGTCCACAACTCCTGCACCCATTGAGCTGAACCAAGGTTTTGGTGGGTTTGGGGGAAAAATCGAGTCGTTTTCAACGACTTTGAGCCCTTTGGGGGTGGTCTCCACCACGCCGACGATTTTGCAAGGAGGTGGCATTCATAAGGTTTATGGGCCGCCACAGAAACGCTCTTATTAA
Tribolium Transcript: 3' UTR:
TAAAGAAATTTTGTGATAGTA
Tribolium Protein:
MNLPLLIAALAPLVACDVSHLGYQYHAPATSYDVPFGSIGSLKHDRQYYHVNKPGILSGVVGTTPASNLQTPIHEFSAFRGGETFNSFLHQPADFGVTVKPILNTVSPTVGPVFGSVNNVVPQSDFGVTVKPILNTVSPTVGQAFGVSTVSSTAAPVIGSVNPQSDFGVTVKPFIDTVSTTVRPIFDNVVPQSDFGVTVKPVGPLLGLAPQLDFGVKSVFNTVSPTVGPAFGSVDNFGVTVKPVLNTEGAVQSSGVEVQKHLYFFSAPDDEPEEVKPRIIFPQAPVKKNLKVLFIKAPTFRTSPVQIPALPQNEEKTRVYVLVKKPEEATIILPTQAPTQPTKPEVYFIKYKTQMEAEEAISQVQGGAGGDVSISANSVDHDHDFLSGIQDSDLAQKLGSGLVSGSTEGSILGFNNQQGFGGFVGVKDSFVSTTPAPIELNQGFGGFGGKIESFSTTLSPLGVVSTTPTILQGGGIHKVYGPPQKRSY
Forward Primer 1:
5’- (TTGACACCGTGAGCACAACT)
Reverse Primer 1:
5’- (CACCCGAAGATTGGACTGCT)
Forward Primer 2:
5’- (CTTCCCCGGTTCAAATCCCA)
Reverse Primer 2:
5’- (CCTTCACCCCAACAAACCCA)
Gel Picture:
Cloned Fragment:
ttgacaccgtgagcacaactgttcggccgattttcgacaatgttgttccacaatcggacttcggcgttacggttaaaccggttggaccgcttttagggttagctccacaattggacttcggcgttaaatcggttttcaacaccgtaagtccaacggtggggccggctttcggttcggttgacaatttcggagttacggttaaaccggttcttaacactgagggagcagtccaatcttcgggtg
Injection concentration:
1.8µg/µL
Number Injected:
15
Number Survived to Adult:
14
Number w/ RNAi Phenotype:
0
Percent Penetrance:
0%
RNAi Phenotype Description:
The beetles appeared to exhibit no change in the axial ration as shown in the Drosophila. However, after two days of incubation in ethanol, they exhibited a breakdown in their cuticle and showed an unravelled musculature.
RNAi Phenotype/Assay Images:
Discussion:
Our hypothesis that RNAi of the TweedleD ortholog would cause a change in the axial ratio of Tribolium was not supported.
This could probably be because the TweedleD gene has 27 other paralogs which could have resulted in functional redundancy over the course of evolution. Also, the concentration of dsRNA injected could not have been sufficient enough to elicit an appropriate response in the beetles.
However, an alternate unexpected phenotype, where the beetles looked like they had "exploded" out of their cuticle was observed. The primary research paper about the TweedleD gene in Drosophila mentioned that this gene is instrumental in maintaining protein cross-linking for cuticle integrity. This could be related to the phenotype where the disintegration of the cuticle was observed in Tribolium after soaking in Ethanol for 48 hours.
Here it looks like a case of divergence of gene function though at one point of time it might have evolved from a common ancestor sharing the same function.
Notebook:
Annotated Genbank File:
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