| The razor clam(Sinonovacula constricta)is one of the four major farmed shellfish in our country,that is an important economical shellfish because of its rich meat and fresh flavor.In recent years,the artificial seedling technology has been broken through,but the spawning mechanism of the razor clam is still unclear.The spawning time of the razor clam was strictly limited at night and were ineffective when using heating,drying in the shade,running water stimulation,reducing salinity,and shading to induce labor at the daytime,and them spawning time was strictly limited at night,that was unlike other bivalve mollusks.It is speculated that the oviposition of razor clam may be tightly regulated by endogenous circadian clocks other than environmental factors.To explore the physiological and molecular mechanisms of nocturnal spawning of clam,the circadian changes of the feeding rate,heart rate and sex hormones by measuring of razor clams in our study,and that was preliminarily determined whether there were circadian rhythm changes in the lifestyle and sex hormones of clams.The circadian clock genes and rhythm genes related to spawning were determined by transcriptome sequencing.The key circadian clock genes Bmal1 and Period c DNA full-length were cloned,and the technology of q RT-PCR,Western blotting,immunofluorescence,si RNA was used to study and analyze the relationship between Bmal1 and Period genes and oviposition of clams.Our study purpose is to preliminarily explore the molecular mechanism of the circadian clock regulating the nocturnal spawning of clams through the correlation analysis of biological clock genes and physiological,biochemical indicators,which will have important application value and reference for other mollusca spawning molecular mechanism research and industrial seedling breeding significance.1.The 72 h heart rate changes of female and male clams were tracked and measured in real time using a non-invasive heartbeat infrared monitor.The heart rate of clams was higher than that in the daytime between 20:00-04:00 at night,existing a circadian rhythm,which speculated that the razor clam was a nocturnal animal.The number of particles of Chaetoceros musculi ingested by the clam during the day and night were counted by a hemocytometer,and the results showed that the feeding rate of clam was higher at 00:00-02:00 and 06:00-08:00 in the night than at 12:00-14:00 and18:00-20:00 in the day of 72 h with a significant difference(P < 0.05).The changes of sex hormone concentrations in the ovary/testis during the reproductive period of clams at different times(00:00,06:00,12:00 and 18:00)within 72 h were detected by using ELISA reagent test kit.The results showed sex hormone concentrations were the highest at 00:00 and the lowest at 18:00,displaying a diurnal variation pattern of first decreasing and then increasing(P < 0.05).In this experiment,the circadian rhythm changes of food intake and heart rate in razor clam,indicating that the biological clock might regulate many life activities of the clams.The results of sex hormones furtherly indicated that the biological clock might be involved in the nocturnal spawning of the razor clams.2.RNA-Seq high-throughput sequencing technology was used to perform light(12:00 and 18:00)and dark(00:00 and 06:00)transcriptome sequencing analysis of the ovarian tissue of clams during the reproductive period,and a total of 3379 DEGs of samples were screened.The GO enrichment and KEGG significant enrichment of bioinformatics methods were used to identify KEGG pathways and genes related to spawning,and the key biological clock genes Bmal1,Period,Timeless,etc.were screened in the transcriptome library.Besides,the ovarian circadian clock genes(ROR,Timeless)and sex steroid stimulation-related(EGR3,DHCR7,STS,cyp19,WNT,SRD5A)rhythm genes were furtherly screened for expression in 5 tissues(ovary,testis,gill,hepatopancreas,and siphon)and time(00:00 and 12:00)by using q RT-PCR technology.The results showed that the expression levels of ROR,EGR3 and STS were higher at 00:00 than at 12:00,the expression levels of Timeless,DHCR7,cyp19,WNT and SRD5A were all at 12:00 higher than 00:00(P<0.05).The key biological clock genes Bmal1 and Period were expressed in 72 h,the results showed that Bmal1 and Period had stable circadian rhythm expression.In this experiment,KEGG pathways,ovarian circadian clock genes and rhythm expression genes related to oviposition were found,indicating that the circadian clock exists in the ovarian tissue of clams,and participates in and regulates the nocturnal spawning activities of clams through circadian clock genes and related genes.3.The full-length c DNA sequences of Bmal1 and Period genes were obtained by RACE cloning technology.The full-length c DNA of Bmal1 was 1944 bp encoding 647 amino acids(aa),while Period gene was 3327 bp encoding 1108 aa.The expression level of Bmal1 gene in the five tissues(ovary,testis,gill,hepatopancreas,and siphon)of clam was basically the highest at 00:00-06:00 at night,and the lowest at 12:00-18:00during the day(the gill tissue showed the opposite trend);Period genes were the highest at 12:00-18:00 during the day,and the lowest at 00:00-06:00 at night,all of which had a certain circadian expression pattern.At the protein level,the changes of Bmal1 and Period protein content were delayed by 6 h-12 h compared with the m RNA level,and strong fluorescence signal were found in mature oocytes,sperm cells,hepatocytes cells in hepatopancreas,epithelium cells at the edge of gill filaments and epithelial cells at the edge of the end of the siphon.The results of si RNA interference showed that the interference efficiency of Bmal1 and Period genes reached the highest on the 5th day(P< 0.01),and their protein expression changes were basically consistent with m RNA levels.At the biochemical level,the contents of testosterone in testis and estradiol in ovary were measured after Bmal1 and Period gene si RNA interference,and the results showed that testosterone and estradiol contents were significantly decreased from 1d to9 d after the interference(P < 0.01).The molecular and protein results of this experiment indicated that the circadian clock genes Bmal1 and Period of the clams existed in multiple tissues with opposite expression patterns,indicating that the Bmal1 and Period genes of they might combine with other circadian clock genes to form a 24 h circadian rhythm.si RNA interference results indicated that the circadian clock gene involved in the nocturnal spawning of razor clam may be related to the secretion and synthesis of sex hormones. |
PDF Full Text Request