2018 Fiscal Year Research-status Report
Saving the frog: Understanding how Japanese frogs are resistant to a deadly worldwide fungal disease through in silico and in vitro assays of MHC and related immune genes
| Project/Area Number |
17K15053
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| Research Institution | The Graduate University for Advanced Studies |
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Principal Investigator |
QUINTIN LAU 総合研究大学院大学, 先導科学研究科, 特別研究員 (60794518)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Keywords | Rana / MHC / chytrid / frog |
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| Outline of Annual Research Achievements |
Study A- Expression of MHC class I and II in tadpoles during development: Completed qPCR experiments to measure MHC expression in Rana ornativentris tadpoles, and also conducted normal PCR to detect presence of MHC amplification from Xenopus tadpoles. Prepared and submitted manuscript.
Study B-Transcriptome of numerous Japanese frog species to study immune genes: isolated MHC class I and II sequences and validated using cloning/sequencing. Completed phylogenetic and supertyping analyses of the MHC sequences. Found that all Japanese frogs share some common physiochemical properties in MHC II based on supertyping anaylsis. Conducted preliminary computer-based prediction of binding affinity between frog MHC sequence and chytrid fungus sequence. Other immune genes were also isolated from transcriptome data including innate immune genes (Toll-like receptors and antimicrobial peptides).
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Study A: During the end of FY2018, we received heavy reviewer criticism related to our study of MHC expression in tadpoles, which drove us to the decision to continue this project with more robust and in-depth approach (i.e. transcriptome sequencing).
Study B: The isolation of MHC sequences from transcriptome data was complex. Since MHC is the most polymorphic gene in the genome, it was challenging to validate allele sequences based on transcriptome data alone (because each individual may have multiple MHC loci due to gene duplication). Thus, I had to conduct cloning and sequencing to confirm presence of true MHC alleles. This additional experimental work caused delay of subsequent analyses.
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| Strategy for Future Research Activity |
Study A- conduct cDNA library sequencing of Rana and Xenopus tadpole samples, isolate differentially expressed genes (DEGs) as well as expression levels of MHC class I and II. Update the former manuscript for submission
Study B- complete MHC supertyping analyses. Conduct further analyses of predicting MHC binding, utilizing more diverse sequences from the chytrid fungus after consulting with Australian collaborators.
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| Causes of Carryover |
Due to time constraints and delays in experimental work, the project has taken longer than expected to finish
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