Cloning And Expression Of GH/IGF Axis Related Genes In Sexual Dimorphism And Molting Of Fenneropenaeus Chinensis

Fenneropenaeus chinensis,also known as the Oriental prawn,belong to Arthropoda,Crustacea,Decapoda,Natantia,Dendrobranchiata,Penaeidae,Fenneropenaeus,are the main econmic prawn in the Yellow Sea and Bohai Sea.The molting is one of the inevitable processes in the culture of Fenneropenaeus chinensis,which is a key factor affecting its growth and development.In this paper,we used the transcriptome sequencing data of Fenneropenaeus chinensis in our laboratory as the target of this study to screen for candidate genes related to the growth and molting of Fenneropenaeus chinensis.The full-length sequences of these genes were studied through validation and gene cloning.And Quantitative real-time PCR(quantitative real-time PCR,qRT-PCR)was used to analyze the expression levels of these genes in male and female Fenneropenaeus chinensis at different stages of molting.The main research contents were as follows:1.Cloning and sequence analysis of GH/IGF axis related genes from Fenneropenaeus chinensisIn this study,the reference sequences of GRTP1,GHITM,GHSR,IGFBP2,IGFBP4 and IGFBP-rP1 were obtained from the transcriptome of Fenneropenaeus chinensis.In this chapter,IGFBP4 and IGFBP-rP1 genes of Fenneropenaeus chinensis were first cloned successfully by RACE method.The IGFBP4 gene cDNA 1061 bp and ORF sequence 660 bp,encods 219 amino acids and contains an IGFBP domain;it has the highest homology with Penaeus monodon;it is also the closest relative to the Penaeus monodon.The total length of IGFBP-rP1 gene is 1657 bp,including an 816 bp ORF,encoding 271 amino acids,which spans the membrane once,with one transmembrane structural domain: TM1,an N-glycosylation site at position 81,and includs three structural domains,Ig,IGFBP and KZAZAL＿FS;it has the highest homology with Penaeus monodon;it is also the closest relative to the Penaeus monodon.The partial cDNA(complete ORF and partial UTR)sequences of the GRTP1,GHITM,GHSR and IGFBP2 genes were obtained by PCR amplification.The GRTP1 gene contains an ORF of 1017,encoding 338 amino acids,and has a conserved structural domain of the Rab GAP-TBC supergene family;it has the highest homology with Penaeus monodon;it is also the closest relative to the Penaeus monodon.The GHITM gene contains an ORF of 1023,encoding 340 amino acids,and has a conserved structural domain of the BI-1-like supergene family;it has the highest homology with Penaeus monodon;it is closely related to the Penaeus monodon,the Litopenaeus vannamei and the Penaeus japonicus.The GHSR gene contains an ORF of 1425,encoding 474 amino acids,with three N-glycosylation sites at position 65,356 and400,and a conserved domain of the 7 tm-GPCRs supergene family;it has the highest homology with Penaeus monodon;it is closely related to the Penaeus monodon,the Litopenaeus vannamei and the Penaeus japonicus.The IGFBP2 gene contains an ORF of 399,encoding 132 amino acids,with one N-glycosylation site at position 106,and a conserved domain of the IGFBP supergene family;it has the highest homology with Litopenaeus vannamei;it is also the closest relative to the Penaeus japonicus.2.Expression analysis of GH/IGF growth axis related genes in different tissues of F enneropenaeus chinensisIn this chapter,qRT-PCR was used to detect the expression of growth axis related genes in eight tissues of Fenneropenaeus chinensis,including eye stalk,gill,heart,stomach,hepatopancreas,intestine,muscle and blood.The results showed that the genes were expressed in all tissues,and the expression was the lowest in both heart and stomach.The tissues with highest expression levels of GRTP1,GHITM,IGFBP2 and IGFBP4 were hepatopancreas.The tissues with highest expression of GHSR gene was intestinal tract.The tissue with the highest expression of IGFBP-rP1 was blood.The gene expression significantly higher than all of the above tissues(P<0.05).3.Expression expression of GH/IGF growth axis related genes in sexual dimorphismof Fenneropenaeus chinensisIn this chapter,qRT-PCR was used to detect the expression levels of growth axis related genes in six tissues including eye stalk,gill,hepatopancreas,intestine,muscle and blood of male and female Fenneropenaeus chinensis.The results showed that the expression of GRTP1,IGFBP2,IGFBP4 and IGFBP-rP1 genes was significantly higher in male than in female in both sexes.The expression of GHITM and GHSR genes was significantly higher in female than in male in both sexes.The expression of IGFBP2 gene was higher in female than in male in the eye stalk.No significant differences were found in other genes.4.Expression expression of GH/IGF growth axis related genes in the molting cycle of Fenneropenaeus chinensisIn this chapter,qRT-PCR was used to detect the expression levels of growth axis related genes in six tissues including eye stalk,gill,hepatopancreas,intestine,muscle and blood of Fenneropenaeus chinensis at different stages of molting.The results showed that the expressions of GRTP1,GHITM and GHSR genes was higher in the middle molting period than in the late molting period than in the inter molting period and early molting period,and showed a trend of first increasing and then decreasing throughout the molting.The expression of IGFBP2 gene was higher in the early molting period than in the late molting period than in the middle molting period than in the inter molting period,and showed a trend of first increasing and then decreasing throughout the molting.The expression of IGFBP4 gene was higher in the early molting period than in the middle molting period and in the late molting period than in the inter molting period,and showed a trend of first increasing and then decreasing throughout the molting.The expression of IGFBP-rP1 gene was higher in the early molting period than in the middle molting period and in the molting period than in the inter molting period.