| Pelteobagrus fulvidraco is also known as Yellow catfish.With the increasing exorbitant fishing and the deterioration of water ecological environment,it is urgent to study the nest selection and utilization during the artificial propagation of Pelteobagrus fulvidraco.Pelteobagrus fulvidraco is a kind of benthic fish.The artificial nest used for spawning can provide a good environment for the effective reproduction of parents and the growth and development of individuals.As one of the downstream molecules of clock gene,Period 2 expression products gather in the cytoplasm and reach the peak value,then transfer to the nucleus,form oligomer with CRY,and then inhibit its transcription expression and regulate the reproductive activity of fish.Circadian rhythm gene,as a regulator of many biological processes,has become an important factor affecting its growth and reproduction.Therefore,it is necessary to study the exact role of clock gene Period 2 in the reproduction of bony benthic fish.In this study,we explored the oviposition preference of several species of Pelteobagrus fulvidraco in the breeding period,cloned and analyzed the full length of Period 2 gene c DNA sequence of Pelteobagrus fulvidraco.At the same time,we monitored the expression of Period 2in the blood during parental reproduction and during the ontogeny of the parents of Pelteobagrus fulvidraco,and located the protein during the early ontogeny.The following achievements have been achieved:1.To explore the preferences of yellow catfish(Pelteobagrus fulvidraco)for artificial fish nests during the breeding Period,preferences for different nest colors and materials were determined under different water temperatures in June-July 2018.The experimental spawning fish nests were hemispherical(25 cm in diameter and 12 cm deep).Gravel palm and plastic fish nests(4-6 pieces)were placed in the same specification.Additionally,6-layer nylon fishing nets(30 cm×40 cm)and palm pieces of fish nests(30 cm×40 cm)were folded.In the cement tank(290 cm×120 cm×100 cm),the egg-laying preference experiment was carried out under two temperature intervals(24-26 ? and 28-32 ?).The ratio of males to females in the experimental compartment was 1:1.The body length and weight of females was(167.8±19.0)mm and(99.9±21.7)g,respectfully,whilst for males the body length and weight was235.1±18.8 mm and 188.5±38.1 g,respectively.The results showed that the average number of eggs in the hemispherical brown palm(no stone)fish nest,under a water temperature of 24-26 ?,was 628.49 grains/m2 and the average egg attachment rate per unit area was 2.77%.The average egg attachment in the hemispherical palm(no stones)fish nest,under a water temperature of 28-32 ?,was 225.48 grains/m2 and the average egg attachment rate per unit area was 1.40%.After analyzing the experimental results,it was concluded that 24-26 ? is the optimal water temperature range for yellow catfish spawning.The analysis also showed that yellow catfish have a significant preference for spawning in brown fish nests(P<0.05),compared to gray,pink,yellow,and green nests.Furthermore,the sticking effect of eggs on the hemispherical palm(no stone)fish nest was significant(P<0.05).The diminishing preference order for other fish nests was:hemispherical palm(with stones)>flat palmettos>hanging palms>hemispherical plastics>cement pool.For sticking eggs,the top preference is the brown hemispherical palm fish nest under the water temperature environment of 24-26 ?.The results of this study have important significance for guiding the artificial reproduction of yellow catfish,spawning field repair,and resource proliferation of sticky egg fish.2.Sequence analysis of Pelteobagrus fulvidraco showed that the full-length c DNA of Period 2 gene was 4593 bp,including 357 bp 5 '-untranscribed region(5'UTR),216 bp 3'-untranscribedl region(3'UTR)and 4020 bp open reading frame(ORF),encoding1339 amino acids.The homology and phylogenetic analysis showed that Period 2 of Pelteobagrus fulvidraco had the highest homology with Period 2 of channel catfish,and the evolutionary relationship was relatively close.Multiple sequence comparison and phylogenetic analysis showed that the sequence similarity between Pelteobagrus fulvidraco and demersus fulvidraco was higher than that of human,rodent and microorganism.3.The expression results of Period 2 gene in blood tissue of the yellow catfish show that the overall female and male changes are consistent.During the short period before and after spawning of female parents,the expression of Period 2 was lower than that of non-induced spawning,and a small peak appeared during spawning.The maleparent returns to the level of the uninduced period after a peak occurs when the female parent lays eggs.Compared with males,females had lower Period 2 expression levels before and after spawning.Period 2 may accumulate in the ovary when the female lays eggs.The maturation of the ovary may accelerate the expression of the Period 2 gene in the ovary.4.The expression of Period 2 gene in the tissues of Pelteobagrus fulvidraco showed that the expression of Period 2 was significantly higher in the early stage of embryo development.With the development of embryo and the absorption of yolk nutrition,Period 2 showed a continuous downward trend.It is suggested that the generation of Period 2 is directly caused by the fact that the fertilized egg may be exposed to light and affected by light since leaving the mother.However,the decreased expression of Period 2 m RNA is caused by light.In this process,SCN receives light signals,and then pineal gland responds and secretes Period 2 protein.In addition,the expression level of Period 2 was significantly increased during the period of eating and conversion,indicating that the expression level of Period 2 was affected by other external environment,such as nutritional intake factors and digestive factors.The generation of Period 2 is accelerated by continuous light and exogenous nutrients.This study can provide new ideas for the study of the mechanism of gene progression and degradation in the external environment.5.The circadian cycle protein of Pelteobagrus fulvidraco is partly affected by SCN.Since it became fertilized egg,Period 2 protein is affected by the mother,which can be considered as a type of circadian rhythm.Until the development of receptive cells in the retina of fertilized eggs,the environment has a greater impact on individual growth and development.In the initial stage,the effects of photoreceptor cell development and feeding on the expression of Period 2 protein are different.With the growth of individuals exposed to external environment,especially the accumulation of light effect,the rhythmic oscillation is more obvious.6.The results of immunohistochemical studies on Pelteobagrus fulvidraco show that Period 2 protein imprinting was detected in yolk of Pelteobagrus fulvidraco.Those show that Pelteobagrus fulvidraco carried Period 2 protein from the mother.Strong signals of Period 2 protein were detected in simple columnar epithelial cells of brain,liver,body surface and intestine,and Period 2 protein was mainly expressed in brain area.Period 2 can regulate food intake.IHC results showed that the positive results ofindividuals were weak from day 4 to day 13.However,the signal increased from the13 th to the 19 th day after membrane rupture.In addition,on the 16 th and 19 th day,there was a small positive peak signal.The distribution of per2 in the brain is stable and regular.The results were highly consistent with the quantitative m RNA expression pattern.The results showed that the initial stage of ingestion was closely related to Period 2 protein,and the expression of Period 2 and protein signal intensity might be used as indicators of early stages of development of Pelteobagrus fulvidraco. |
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