| Cell cycle process is an important part of biological growth and development,which is accurately regulated by a variety of regulatory factors,and cyclin dependent kinases(CDKs)are the core regulatory factors involved in cell cycle process.CDKs are widely involved in biological physiological processes.More and more studies show that CDKs play an important role in reproductive regulation.In order to study the role of CDKs in the ovarian development of white shrimp,the full-length cDNA sequences of CDK2,CDK8 and CDK9 genes were cloned and studied its function by RNA interference and in situ hybridization.The results are as follows:1、Expression of the cyclin-dependent kinase 2 gene(CDK2)influences ovarian development in the ridgetail white prawn,Exopalaemon carinicaudaSynchronization of ovary development is the premise for realizing large-scale seed breeding of small-and medium-sized shrimps.In order to explore the regulatory mechanism affecting the ovarian development of the ridgetail white prawn Exopalaemon carinicauda,the full-length cDNA sequence of CDK2 gene was cloned.The length of the open reading frame(ORF)of CDK2 is 918 bp,the amino acids with a conserved cyclin binding motif S＿TKc.The phylogeny shows that the CDK2 gene of E.carinicauda with ridge tail was clustered into one branch with the CDK2 gene of Macrobrachium nipponense and Macrobrachium rosenbergii.The results of q RT-PCR showed that the expression of CDK2 gradually increased in embryos from fertilized eggs to anterior nodal larvae,where it reached the highest level.The expression showed a significant downward trend in the post nauplius stage(P < 0.05).CDK2 gene was expressed in eye stalk,stomach,gill,intestine,ovary,hepatopancreas and other tissues,but the expression was the highest in ovary with vigorous cell division;By exploring the expression characteristics of CDK2 in different stages of ovarian development,it was found that the expression of CDK2 gene was the highest in stage III,which was significantly different from that in other stages(P < 0.05).RNAi results showed that reducing the CDK2 gene expression level could significantly delay ovarian development.The results of in situ hybridization further revealed that CDK2 plays an important role in the proliferation of oocytes,and the synthesis and transportation of exogenous yolk material.2 、 Cloning and function of the cyclin-dependent kinase 8 gene(CDK8)in ovarian development of the ridgetail white prawn,Exopalaemon carinicaudaIn this study,the full-length cDNA sequence of CDK8 gene was cloned.The open reading frame of the gene is 1434 bp,encoding 478 amino acids and there is an S＿TKC protein domain.The phylogeny shows that CDK8 gene had the highest homology with CDK8 gene of American crayfish,Penaeus monodon and Penaeus japonicus.q RT-PCR showed that the expression of CDK8 gradually increased in embryos from fertilized eggs to pre nauplius stage,and reached the highest in pre nauplius stage.The expression showed a significant downward trend in the post nauplius stage(P < 0.05).CDK8 gene was expressed in 9 tissues: eyestalk,stomach,heart,gill,intestine,muscle,ovary,hepatopancreas and ventral cord nerve,and the expression was the highest in ovary;At different stages of ovarian development,the expression of CDK8 gene in phase I was significantly higher than that in other periods(P < 0.05).The results of RNAi and in situ hybridization further revealed that CDK8 gene played an important role in the proliferation of shrimp oocytes and the synthesis and transportation of exogenous yolk substances.3 、 Cloning and function of the cyclin-dependent kinase 9 gene(CDK9)in ovarian development of the ridgetail white prawn,Exopalaemon carinicaudaWe cloned the full-length cDNA of the CDK9 gene and discussed its role in ovarian development.Our results show that the gene has an open reading frame of 1155 bp,encoded 383 amino acids and it has a conserved cyclin binding motif S＿TKc.The open reading frame of CDK9 was expressed as a 43.96 Kda molecular weight protein.The phylogeny shows that the CDK9 gene has the highest homology with the CDK9 genes of Penaeus vannamei and Penaeus japonicus.The results of q RT-PCR show that the expression of CDK9 gradually increased in embryos from fertilized eggs to pre nauplius stage,and reached the highest in pre nauplius stage.The expression showed a significant downward trend in the post nauplius stage(P < 0.05).The CDK9 gene was also expressed in the eye stalk,stomach,heart,gill,intestine,muscle,ovary,hepatopancreas,and ventral cord.The expression level was highest in the ovary.In the five stages of ovarian development,the expression of CDK9 gene in phase I was significantly higher than that in other periods(P < 0.05).The RNA interference results show that the expression of the CDK9 gene significantly affect the expression of the Vg and Vg R genes.The in situ hybridization tests showed strong expression signals in the membrane,cytoplasm,and nucleus of oocytes and previtellogenicoocytes.Nevertheless,following the proliferation of oocytes,the expression signals became much stronger in the oocyte membrane and in the whole follicular cells located in the interstitial space.Our findings suggest that CDK9 plays an important role in the synthesis and transportation of vitellin.This thesis has 28 figures,6 tables and 170 references. |
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