| Somatostatins(SSTs)is an upstream hormone which plays an important role in growth and development of vertebrates.It can not only directly inhibit the secretion of growth hormone(Gh),but also inhibit the growth of individuals by adjusting the GhIgf-1 axis.In the present study,four somatostatin genes(SST1,SST3,SST5,and SST6)were identified and characterized in S.argus;q RT-RCR was used to detect the expression levels of these four SST genes in 11 tissues(hypothalamus,pituitary,heart,liver,ovary,testis,kidney,spleen,stomach,intestine,muscle,gill).At the same time,q RT-PCR was used to examine the effect of SS-14 active peptides of the four SST genes on the expression of Igf-1 and Igf-2 in liver at different concentrations(0.1μM,1μM,10μM)and times(3h,6h)in vitro.The SS-14 active peptides of SST1 and SST3 were injected into S.argus.The transcriptome of pituitary and liver tissues were sequenced to screen and identify the differentially expressed genes related to the growth of S.argus,and the role of SST on the growth regulating axis was preliminary analyzed mechanism.The main results are as follows: 1.Identification and sequence analysis of SST geneFour types of somatostatin genes(SST1,SST3,SST5,and SST6)were successfully identified and characterized in the spotted scat.The full-lengths of c DNAs were 658,790,748 and 752 bp.Among them,the ORFs of these genes are 372,384,321 and 333 bp,encoding 123,127,106 and 110 amino acids respectively.Besides,SST1 and SST3 are located on the LG4 chromosome while SST5 and SST6 are located on the LG23 and LG8 chromosomes.The SST1 and SST3 amino acid sequences shared the highest sequence identities with their grouper(Epinephelus coioides)orthologs,which were 93.5% and 92.1%,respectively.The SST5 and SST6 shared the highest sequence identities with tilapia(Oreochromis.niloticus)and grouper(E.coioides),which were 77.9% and 96.1%,respectively.2.The temporal and spatial expression of S.argusOverall,the four genes are distributed and expressed differently in different tissues.It was observed that all 4 SSTs examined were abundantly expressed in the hypothalamus.SST1 and SST3 were equally expressed in the liver and muscle,but the expression of SST3 in the hypothalamus was significantly higher than that of SST1.In addition,SST3 was clearly expressed in the ovary,muscle,heart and liver.The expression of SST5 was gender-specific,being highly expressed in the ovaries of females and hypothalamus of males.The expression level of SST5 in the ovaries was much higher than was observed in the testis.Finally,SST6 exhibited the highest expression in the stomach,followed by the hypothalamus and intestine.3.Effects of SSTs on the expression of Igf-1 and Igf-2 in liver samples incubated with S.argus in vitroFour SS-14 active peptides were synthesized based on the amino acid sequences of four SST genes.The control group used 0.9% sodium chloride solution to incubate the liver fragments for 3h and 6h in vitro.Different types and different concentrations(0.1,1 and 10 μM)of SS-14 were used to incubate the liver fragments of S.argus in vitro for 3h and 6h.It was found that all SST except SST5 inhibited the expression of Igf-1 and Igf-2(P<0.05).4.Effect of S.argus SST on the expression of crucial genes of growth regulation in pituitary and liver.Different concentrations of SST1 and SST3 peptides(0.001 mg / kg,0.01 mg / kg and 0.1 mg / kg)were intraperitoneally injected into S.argus for 3 hours or 6 hours(control group was injected with 0.9% saline at the same dose).Transcriptome sequencing(RNA-seq)was performed on the pituitary and liver(a total of 21 samples).A total of 1,104,724,270 Clean Reads were obtained,and the Clean Read ratio reached 99.80%.46 differentially expressed genes(DEGs)were identified in the CP-vs-T1 P group in pituitary,including 24 up-regulated genes and 22 down-regulated genes;137 DEGs were identified in the CP-vs-T3 P group,including 116 up-regulated genes and 21 down-regulated genes.444 DEGs were identified in the CL3-vs-T1L3 group of S.argus liver,including 85 up-regulated genes and 359 down-regulated genes;1127 DEGs were identified in the CL6-vs-T3L6 group,including 703 up-regulated genes and 424 down-regulated genes.KEGG enrichment analysis found that in pituitary tissues,the DEGs were significantly enriched in Insulin secretion,MAPK signaling pathway,Metabolic pathways,etc.And in liver tissues,the DEGs were mainly enriched in Cell adhesion molecule,Focal adhesion and PI3K-Akt signaling pathway,etc.Whether the differentially expressed genes in the above pathway can directly affect the growth and development of S.argus requires further experimental verification.These results showed that SST has a significant inhibitory effect on Igf-1 and Igf-2 in the liver of S.argus.The transcriptome analysis of the pituitary and liver provides a theoretical basis for further research on the growth regulation mechanism and molecular characteristics of S.argus. |
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