| Pelodiscus sinensis is a kind of widespread breeding medium and small species,belonging Testudines,turtle family.Male turtle growing faster than female ones,have higher gum-content and rich in nutrients,are more welcomed by market.Therefore,hatching a higher proportion of male turtle were important on aquaculture industry.But the molecular mechanism of sex determination in P.sinensis has been a long-standing mystery.Steroid hormone plays an important role in sex differentiation and development of vertebrates.In vertebrate animals,steroidogenesis is regulated by a series of steroidogenic enzymes.Steroidogenic acute regulatory protein(StAR)transports cholesterol from outer to the inner membranes of mitochondria,and it well know that StAR as the rate-limiting factor in the biosynthesis of most steroid hormones.However,there is no evidence about the roles of StAR in steroidogenesis during sex differentiation and gonadal development in P.sinensis,it still needs more further exploration.In this study,the sequence full-length of StAR,different temperature expression at different development stages of this gene,the influence of Letrozole on the StAR gene expression and StAR expression in gonad had been analyzed by clone,q RT-PCR technique separately in order to clarify the molecalor mechanisms of StAR in P.sinensis.At the same time,the differently expression of the circular RNA between the testis and ovarium was studied.The main results of this thesis are as follows:1.The StAR full-length c DNA of P.sinensis is 2377 bp,which contains 903 bp of open reading frame(ORF)and encodes 300 amino acids.The results of amino acid multi-comparison analysis showed that the turtle is the highest similarity,followed by bird and fish.The results of q RT-PCR showed that StAR was mainly expressed in the testis of P.sinensis,and also expressed in ovary,heart and brain.The results showed that the expression of StAR gene was in a dynamic state,and there were differences in different temperature.2.In this experiment,the antigenic fragment of StAR amino acid sequence was cloned and the recombinant plasmid was constructed with p ET-32a vector.Bamh I and Hind III were selected as restriction endonuclease sites,the recombinant plasmid Pet32a-star was introduced into BL21(DE3)receptive state and induced by IPTG to obtain polypeptide protein.SDS-PAGE electrophoresis showed that the size of the StAR peptide protein was 35.6 Ku.The serum was purified by Nickel column and immunized by dialysis.The titer of antibody detected by ELISA was 8.1×10~5,which reached the following experimental standard.The expression of StAR protein in the gonad of soft-shelled Turtle was detected by immunohistochemistry using the prepared mouse polyclonal antibody as the first antibody.The results showed that StAR was expressed in leydig cells of ovary and in leydig cells,spermatogonia and cytoplasm of testis.The expression of StAR in testis,ovary and brain of P.sinensis was detected by western blot.The results showed that StAR was highest express in testis,followed by ovary and brain.3.In this study,the aromatase inhibitor Letrozole was used to treat male P.sinensis.The q RT-PCR results showed that the expression of StAR gene in testis at the 24th hour was significantly increased compared with the control group.The expression of StAR gene showed a trend of decreasing first and then increasing with time.After seven injections,the expression of StAR gene in the testis of soft-shelled Turtle treated with low concentration was significantly higher than that of control group,while the expression of StAR gene in the testis of high concentration treatment group was not significantly different from that of control group.However,there was no significant difference in the expression of StAR gene between the two concentration groups at 24h and 48h,and the difference was significant between the two concentration groups at 96h,over time,the expression of StAR gene decreased in the low concentration group,but increased in the high concentration group.After seven injections,the expression of StAR gene in the two treatment groups was significantly different from that in the control group.4.The number of differently expressed circular RNAs in testis and ovary of P.sinensis was1468,of which 169 were down-regulated in females and 541 were up-regulated in females.The results of GO analysis showed that these different circular RNAs were mainly enriched in GO terms such as immunesystem process,extracellular region,nucleic acid binding transcripition factor activity.The results of COG analysis showed that these differential circular RNAs’coding genes were mainly clustered in Transcription,Replication,Recombination and repair,Singal transduction mechanisms.These results suggest that StAR may be involved in the gonad development and spermatogenesis of P.sinensis and may play a role in the maintenance of testis function. |
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