2017 Fiscal Year Research-status Report
Molecular studies on nutrient regulation of reproduction and immune responses in a tick
| Project/Area Number |
16K08094
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| Research Institution | University of Tsukuba |
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Principal Investigator |
Taylor DeMar 筑波大学, 生命環境系, 教授 (50261772)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | soft tick / nutrient pathway / immune genes / vitellogenin / Target of Rapamycin / engorgement / Ornithodoros moubata |
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| Outline of Annual Research Achievements |
Focus of the research for the second year of the grant was to confirm the sequence of the gene fragment to be TOR and analyze the expression of the TOR gene in at different times after engorgement and different tissues of mated female ticks.In addition, preparations for measuring effects of TOR on the immune genes was accomplished with analysis of the relish gene expression. Real-time PCR was also used to confirm the expression of the TOR gene and Vitellogenin (Vg) gene in unfed, immediately after beginning engorgement, 2 h, 6 h, 12 h, 1, 2, 3, 4, 5 and 6 days after feeding. Results showed TOR expression peaks occur before the Vg gene peak indicating TOR may be important in regulating Vg expression. Expression of the TOR and Vg gene were analyzed the RT-PCR in the midgut, ovary and fat body on day 6 after engorgement, day of the Vg expression peak, showing the TOR gene is expressed on in 3 tissues, but Vg is expressed only in the midgut and fat body. Effects of rapamycin injection on the Vg gene expression has also been confirmed by qPCR of the Vg gene. Gene specific primers are now being tested to prepare for qPCR of the TOR gene expression and preparation of double stranded RNA to confirm the functions of the TOR gene in the regulation of Vg. As far as the immune genes, the expression of relish gene has been confirmed during the same times in females by qPCR. After an appropriate TOR sequence for qPCR of the TOR gene and doubled stranded RNA is completed the importance of TOR in the regulation of the immune regulatory genes and defensin genes will also be examined.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Difficulties with cloning and creation of plasmids for the TOR and relish genes has delayed the double stranded RNA experiments to confirm the functions of the genes by knockdown experiments. All of the genes have now been identified and we are now getting help from a colleague for the preparation of the double stranded RNA so they final year we hope to be able to see the effects of TOR knockdown on the Vg, receptor and immune genes during this final year.
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| Strategy for Future Research Activity |
Confirm the expression patterns of the TOR gene by qPCR, prepare double stranded RNA for TOR to knockdown the TOR kinase gene and determine the effects on the Vg, receptors and immune genes in mated Ornithodoros moubata female ticks.
Finish writing and submit paper on the effects of rapamycin injection on vitellogenesis.
Finish writing and submit paper on the identification and characterization of the rel and relish immune genes.
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