| The Hyriopsis cymingii is a special and important freshwater pearl mussel in China.The economic value of Hyriopsis cymingii depends on its pearl breeding cycle,pearl production and quality.In order to improve the economic benefits of the pearl,the process of artificially cultivating pearls has gradually matured under the joint research of technicians and researchers.Our laboratory improved the cultivation process by co-cultivating the free cells and the nucleus,called free-cell-culture method.However,how to improve the proliferation ability of free cells has always been a key problem in the development of free-cell-culture technology.In order to improve the breeding cycle of the pearl and improve the quality of the pearl,this study aims to understand cell cycle regulation mechanism of free cell culture in vitro from Hyriopsis cumingii mantle,cyclins were screened and its function was explored.In this study,10 cyclin genes were screened in the transcriptome and digital gene expression profiling(DGE).Cyclin A,B,D2 and E,which are most related to cell division,were identified by bioinformatics analysis and literature support.And the four cyclin genes were verified and cloned for the first time in Hyriopsis cumingii.The length of the CDS region of the cloned cyclin A was 413 bp,and the cDNA length of the cyclin B gene was 1024 bp,including the ORF 768 bp,encoding 255 amino acid residues,3'-UTR 197 bp,cyclin D2 has a CDS region of 368 bp,and cyclin E has a CDS region of 391 bp;the four cyclin genes are analyzed by gene sequence similarity analysis.They all have homology with shellfish such as oysters and scallops.The amino acid sequence analysis of cyclin A,B,D2 and E showed that the cyclin A,B,D2 had cyclin box.Through predicting the target protein of cyclin A,B,D2 and E,we found the cyclin A and cyclin B proteins have a targeting relationship with CDK(cyclin-dependent kinase)1,2,and cyclin D2 has a targeting relationship with CDK6 and CDK4 in cell cycle regulation.Cyclin E could interacts with CDK1,2,4.In summary,the cyclin gene exists in Hyriopsis cumingii and they all have the structural characteristics of cyclin.In this study,the tissue expression profiles of cyclin A,B,D2 and E genes in female and male of Hyriopsis cumingii were constructed.The results showed that the four cyclin gene expressions were tissue-specific.The expression levels of cyclin A,B,and E genes in the gonads were significantly higher than those of the adductor muscle,foot,mantle,heart,blood,visceral mass,and gill,and it had significant female differences(P<0.05).The high expression of genes was associated with germ cell maturation and gonadal cell division.The expression of cyclin D2 gene in gill was significantly higher than that in other tissues(P<0.05),there was no significant difference between male and female expression(P>0.05).It was speculate the rate of gill cell has higher proliferation and division ability.In the cell cycle of the various tissues of Hyriopsis cumingii,the proportion of cells in the S phase was higher than that in G1/G0 phase and G2/M phase,and the proportion of cells in the G1/G0 phase was different in each tissue,it could cause the rate of cell division is different.In this study,RNAi technology was used to silence cyclin B and cyclin D2.The expression of cyclin B was significantly decreased in gonad and gill after cyclin B interference fragment injection.Meanwhile the phase change of the cells was measured by flow cytometry,it showed that the proportion of cells in the G2/M phase decreased and the cells in the dividing phase were decreased.It is hypothesized that cyclin B can regulate gonad and sputum cell division in Hyriopsis cumingii,and cyclin B interference fragment has no interference effect on mantle.it is speculated that cyclin B small interfering RNA(siRNA)mediated RNAi in Hyriopsis cumingii had no systematic.The cyclin D2 interference fragment injection has no interference effect on the gonadal,gill and mantle tissue of Hyriopsis cumingii,and its regulation function on cell division needs to further discussion.In this study,the mantle cells of Hyriopsis cumingii were cultured in vitro,and the expression changes of cyclin B and cyclin D2 genes and cell cycle phase were detected at 24,60,84,108 and 120 h.The results showed that the expression of cyclin B and cyclinD2 was significantly increased with the increase of culture time(P<0.05).Combined with the phase changes of the cell cycle of mantle cells at different cell culture time,the results showed that the two cyclins could accumulate and express continuously,but they do not reach the threshold value that could bind interactional CDK,and thus cannot promote cell division.In the following studies,the regulation mechanism of cell cycle in cell culture can be further explored by using the cyclin gene overexpression method.In this study,the functional studies on the cell cycle proteins of Hyriopsis cumingii were carried out at the molecular and cellular levels,and the regulation mechanism of cyclin in the Hyriopsis cumingii was preliminarily discussed,which provided a new research idea for promoting the mantle cells division in vitro.It provides theoretical support for establishing mantle cell system of Hyriopsis cumingii in vitro.And it lays a foundation for scientific research to optimize the pearl breeding process and promote the vigorous development of Chinese pearl industry. |
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