| In addition to the improvement of culture conditions and the upgrading of feed nutrition formula,it is very important to study the genes related to the growth axis.In this study,PCR amplification,real-time fluorescence quantitative analysis,and enzyme cutting technology were used to clone and sequence the somatostatin(SS)gene sequence of Mauremys reevesii and construct a phylogenetic tree of SS gene.The effects of human growth hormone on the expression of SS mRNA,GHR mRNA,and IGF-1 mRNA of growth axis related genes in different genders were observed.To explore the effect of growth hormone changes on related genes and regulatory role of the tortoise,and provide a theoretical basis for further research on the molecular biology of tortoises and turtles and guidance of breeding production.(1)Molecular characteristics of SS c DNAThe core sequence of 294 bp c DNA was obtained by cloning and sequencing using the primers designed for the conserved region of SS in the closely related species of Mauremys reevesii.The phylogenetic tree showed that in reptiles,the homology of the SS gene of Mauremys reevesii was the highest(95.36%)with Terrapene carolina triunguis and Chrysemys picta.It has high homology with crocodile,is in the same branch,has the lowest homology with snake and lizard,and gradually decreases homology with birds and mammals.(2)Expression differences of SS,GHR,and IGF-1 in different tissuesThe differential expression of SS mRNA was detected by real-time fluorescence quantitative detection.The results showed that SS mRNA was expressed in the brain,pituitary,liver,stomach,and intestine.The highest expression was in the brain and the lowest in the liver.GHR and IGF-1 mRNA were expressed in the brain,pituitary,liver,kidney,pancreas,stomach,and intestine.The expression of GHR mRNA in the brain was the highest in males,followed by that in the liver.In females,the highest expression was in the liver,followed by that in the brain.Spleen expression was the lowest in both sexes.IGF-1 mRNA expression was the highest in the liver and the lowest in the stomach.(3)Difference of SS expression after injection of growth hormoneWithin one week after the injection,the changes of SS mRNA expression in the brain of female and male individuals were generally anti parabola trend,the male expression decreased after a small increase on the first day of injection,reached the lowest value on the fourth day,and then gradually increased;the female expression decreased rapidly after the injection,reached the lowest value on the second day,and then gradually increased,and was always lower than the control group.Within four weeks of continuous injection,the expression of SS mRNA in the male brain increased significantly in the second week(P<0.05)and reached the peak value,and decreased in the third and fourth weeks,which was lower than that of the control group in the fourth week;the expression of SS mRNA in the female brain decreased slightly in the first week,and increased signif icantly in the second week(P<0.05)and reached the peak value,then decreased gradually,which was slightly lower than that of the control group in the fourth week.In other tissues,the changing trend of the expression amount of female and male individuals is the same,which is down-regulation of the expression amount of pituitary,up-regulation of the expression amount of stomach and intestine.(4)Expression difference of GHR and IGF-1 after growth hormone injectionThe expression of GHR and IGF-1 mRNA in the livers of male and female individuals were similar in the four weeks after injection.GHR mRNA expression was significantly increased in the first week(P<0.05),decreased to the level of the control group in the second week,and then gradually increased.In the fourth week,the expression of males reached a significant level(P<0.05),in the third and fourth weeks,the expression of males and females reached a significant level(P<0.05),and there was no significant change in other tissues.IGF-1 mRNA expression was up-regulated in the first week,decreased to the level of the control group in the second week,and then gradually increased.In the male,it was up-regulated in the fourth week,while in the kidney,it was up-regulated(P<0.05),and in the spleen,it was down-regulated(P<0.05).However,in females,it was up-regulated to a significant level in the first,third,and fourth weeks(P<0.05),and there was no significant change in other tissues.After the injection of exogenous growth hormone,the expression of SS increased in the early stage and decreased gradually in the later stage,which indicates that somatostatin may have negative feedback regulation effect on growth hormone;however,the expression of somatostatin decreased in the later stage,indicating that the inhibition effect of somatostatin on growth hormone is not obvious.It is further speculated that the slow growth of Mauremys reevesii is not due to The high expression of somatostatin inhibited the growth hormone,but it was caused by the low expression of the self-growth hormone. |
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