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Tim (Timeless) is the second core circadian clock gene identified after the discovery of Per, which plays an important role in the regulation of animal reproduction. In order to explore the molecular mechanisms of Tim to regulate spawning in Sinonovacula constricta, the cDNA sequence of Tim was cloned and analyzed, the circadian expression patterns of Tim gene/protein in different tissues were analyzed by qRT-PCR and immunofluorescence technology, and the regulatory effects of Tim on sex hormone secretion were investigated by siRNA technology. The ORF sequence of Tim was 3 090 bp, encoding 1 029 amino acids and containing three conserved domains (TIMELESS superfamily、TIMELESS_C and FIil). Tim was expressed in test tissues, and showed higher expression levels in the hepatopancreas, siphon, ovary and testis. The expression levels of Tim were higher at 12:00–18:00 in the daytime and lower at 00:00–06:00 in the night time, which exhibited circadian rhythmic expression patterns in the ovary and testis. Tim protein was mainly expressed in hepatocytes of the hepatopancreas, epithelial cells of siphons, mature oocytes of the ovary, and spermatids of the testis. And protein fluorescence signals of Tim were slightly stronger at 12:00 during the day than at 00:00 during the night in the ovary and testis. After siRNA injection, the expression levels of Tim significantly decreased from the 3rd to 7th day in the siRNA treatment groups of the ovary and testis (P < 0.05). Meanwhile, the contents of testosterone in the testis and estradiol in the ovary decreased significantly from the 1st to 9th day after siRNA injection (P < 0.01). Consequently, it could be concluded that Tim gene/protein exhibited circadian rhythmic expression patterns in the ovary and testis, and the contents of sex hormones significantly decreased after Tim interference, which suggested that Tim might regulate the nocturnal spawning by mediating the secretion of sex hormones in S. constricta. These findings preliminarily explored the regulatory effects of circadian clock gene Tim on nocturnal spawning of S. constricta, which will provide important application value for in-depth analysis of spawning mechanism and optimizing industrialized seedling breeding technology in mollusks.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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