Identification Of Somatostatin Receptors In Grass Carp And Preliminary Study On The Growth-Promoting Effect Of Its Antagonist

Somatostatin?SS?is a hormone that plays important regulatory roles in the growth and development of vertebrates,it is also called somatotropin release-inhibiting factor?SRIF?.SS can regulate the expression and secretion of a variety of hormones including growth hormone?GH?,gastrin,insulin and glucagon through its specific G protein-coupled receptor—somatostatin receptors?SSTs?,and further regulates metabolism,growth and development.In particular,SS can inhibit GH synthesis and secretion at the pituitary level in teleost fish,and subsequently affect the growth of them eventually.In teleost fish,somatostatin receptors have multiple subtypes arising from whole genome duplication.Studies have revealed that the expression of these receptor subtypes are tissue-specific,indicating the functional variability of SS in different tissues.In the present study,the coding sequences of grass carp SST subtypes were isolated and identified,and their tissue distribution was also explored.the SST subtypes expressed in the pituitary with high levels were determined,and their specific antagonists were screened.At the same time,their roles in regulating the growth and development of grass carp were preliminarily investigated.Firstly,seven grass carp SSTs coding sequences were cloned by using cDNA mixture from different grass carp tissues,and named as SST_1a,SST_1b,SST_2a,SST_2b1,SST_2b2,SST₃and SST₅.These seven receptor coding sequences are 1017bp to 1452bp in length and encode 338 to 483 amino acids,and all are intronless.All seven grass carp SST coding sequences possess seven transmembrane domains and the SSTs superfamily characteristic sequence:YANSCANPI/VLY.Multiple sequence alignment showed that the amino acid sequence identity of the seven SSTs was 44.83%.It also showed that the grass carp SSTs have a close relationship with zebrafish and far from humans and mouse.RT-PCR results showed that all subtypes of grass carp SSTs were expressed in the pituitary,liver and brain,but their expression patterns were different.Grass carp SST_2a,SST₃ and SST₅ are highly expressed in the pituitary,while SST_2b1 is almost exclusively expressed in liver.The polyclonal antibodies for these three receptor subtypes were obtained from biotechnology company,and the high protein levels of these three receptors were demonstrated in pituitary by Western blot.In view of the important role of SS in inhibiting the expression and secretion of GH in the pituitary,we selected SST_2a,SST₃,and SST₅,which are highly expressed in the pituitary as the study models.The three-dimensional structure of grass carp SST_2a,SST₃and SST₅ proteins was predicted by using a bioinformatics software,and the binding efficiency between the ligands and receptors was calculated by Autodock.By using this method,some specific antagonists for SST_2a,SST₃ and SST₅ were screened.At the same time,the pituitary of the young grass carp proved to have the same submicroscopic structure as the mature grass carp by Hematoxylin-eosin staining.Finally,MK-4256,a specific antagonist for SST_2a was selected to study its growth-promoting effect on grass carp by intraperitoneal injection.It was found that the grass carp injected with the antagonist?100ng and 300ng/10g body weight?had a higher body weight growth rate compared with the control group,but there was no significant difference in the fullness between the groups.The mRNA levels of insulin-like growth factor 1 in the livers were also detected,and the results showed no significant difference between the experimental and the control groups.In addition,the hepticsomatic index and the serum levels of alanine aminotransferase and aspartate aminotransferase in all groups were not significantly different,indicating that injection of MK-4256 did not have toxic effects on the grass carp liver.In this thesis,all the grass carp SST subtypes were isolated and identified for the first time,and their tissue distribution characteristics and the effect of SSTs antagonist injection on young grass carp growth were also explored.These experimets established foundation to reveal the roles of SSTs in regulating the growth and development of grass carp.