| Hematopoiesis is the process of hematopoietic stem cells into mature blood cells and lymphocytes,and it plays an important role in the survival of animals.Cyclin-dependent kinases(CDKs)are core regulators in the process of cell cycle regulation,and participate in cell proliferation,cell differentiation and gametogenesis.In vertebrate,the interaction between CDKs and hematopoietic system are widely studied.Currently,it is found that CDKs also play an important role in invertebrates.However,the research on the regulation of hematopoiesis in crustaceans is still limited.In this work,the cDNA sequence of EsCDK2 was cloned,and its tissue and cell distribution characteristics and response patterns to hematopoietic stimulation were analyzed.In addition,we conducted a preliminary study on the hematopoietic regulation mechanism of EsCDK2by using the methods of biochemistry,cell biology,molecular biology and other means in the Chinese mitten crab(Eriocheir sinensis).A CDK2(designed as EsCDK2)with a serine/Threonine protein kinase catalytic domain was identified from Chinese mitten crab(Eriocheir sinensis).The full-length cDNA sequence of EsCDK2 was of 2405 bp with an open reading frame(ORF)of 909bp.EsCDK2 shared 66%-81%sequence similarities with previously identified CDK2s.It was clustered with the CDK2 from Penaeus monodon in the invertebrate branch of the phylogenetic tree.The mRNA transcripts of EsCDK2 were highly expressed in hematopoietic tissue(HPT)and gonad,while lower in hemocytes,heart,gills and muscle.Immunohistochemistry was used to detect subcellular localization,and EsCDK2 protein distributed in both cytoplasm and nucleus of HPT cells.The expression of EsCDK2 mRNA in HPT of E.sinensis was significantly up-regulated and reached a peak level at 3 h post the stimulations with Aeromonas hydrophila(2.31-fold,p<0.05)and Lipopolysaccharide(LPS)(2.02-fold,p<0.05).After an exsanguination stimulation,the total hemocyte counts(THC)decreased significantly to 0.42×10~7/ml(0.39-fold,p<0.05)at 0.5 h,then returned to a normal level at 6 h,and the mRNA expression of EsCDK2 in HPT cells was up-regulated at the early phase from 0.5 h to 6 h.In addition,the effect of EsCDK2 on the proliferation of hematopoietic tissue cells was investigated using RNA interference technique.After EsCDK2-dsRNA injection,the mRNA expression level of EsCDK2 in HPT decreased significantly(0.53-fold,p<0.01)at 24 h post the injection of EsCDK2-dsRNA,and the percentage of new-born hemocytes in HPT also decreased significantly from 37.7%to 16.3%(0.43-fold,p<0.01).After interference of EsCDK2 mRNA expression,the percentage of G0-G1 phase cells significantly increased to 85.01%(1.26-fold,p<0.01),while S phase and G2-M phase cells significantly decreased to 7.92%(0.46-fold,p<0.01)and 7.07%(0.43-fold,p<0.01)respectively,indicating that the cell cycle of HPT cells was arrested at G1phase after the inhibition of Es CDK2 expression.In conclusion,EsCDK2 is similar to the CDK2s of vertebrate in structure,and is mainly expressed in hematopoietic tissue cells and gonad cells.After immune stimulation and exsanguination,THC decreased significantly while the relative expression of EsCDK2 mRNA in hematopoietic tissues increased significantly.After the interference of EsCDK2,the THC and the percentage of new-born hemocytes in hematopoietic tissues decreased significantly.Besides,the cell cycle of hematopoietic tissue cells is blocked in G1 phase,indicating EsCDK2 has been involved in regulating the hematopoiesis of Chinese mitten crab by regulating the cell cycle of hematopoietic tissue cells from G1 to S phase.These results are of great significance for the further study of the composition of the immune system and the regulation mechanism of hematopoiesis. |
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