| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2017: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2015: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
|
|---|
| Outline of Final Research Achievements |
Sperm motility-initiating substance (SMIS) is a key protein for the internal fertilization of Cynops pyrrhogaster. The deduced amino acid sequence of SMIS cDNA included 14 cysteine residues, and 6 of them construct the cysteine knot (CK) motif. CK motif is thought to mediate protein polymerization and stabilize protein conformation. 1) We performed genome editing using CRISPR/Cas9 system for targeting to 4th cysteine residues of the CK motif. Western blotting using the anti-SMIS antibodies revealed that the lack of the 4th cysteine resulted in the change of polymerization and/or conformation status of SMIS in correlation with the physiological activity. 2) SMIS genes of Cynops ensicauda and Hynobius lichenatus were identified by RNAseq and found the presence of the CK motif in the deduced amino acid sequences. 3) By the Western blotting, differences in the molecular sizes and the possible polymerization status were suggested among species that undergo distinct mode of reproduction.
|
