| Molting steroids play an important role in regulating the growth,development and reproduction of arthropods.The periodic fluctuations in titer regulate the periodic molting of arthropods,of which the main effect is 20 E.Scylla paramamosain with high nutritional value,belongs to the Crustacea,the Scylla genus,is one of the most popular economically farmed crabs in China.In this thesis,Scylla paramamosain is used as the research object,and several important regulatory genes during the synthesis and degradation of 20 E are identified.The expression patterns of these genes in different developmental stages,different tissues,and different stages of the molting cycle are analyzed.The concentration control lays a certain foundation.The main findings are summarized as follows:1.Excavation and identification of genes related to 20 E metabolism pathwayThe genes related to 20 E metabolism pathway were identified initially from the transcriptome of Scylla paramamosain,including Nvd,CYP307 al,CYP302al,CYP315 al,and CYP18a1.We obtained c DNA sequences of 1 Nvd,1 CYP302a1,1CYP315a1 and 1 CYP18a1 by clone technology,and these genes were preliminarily excavated,verified and systematically analyzed by the means of bioinformatics.Sequence analysis showed that the predicted molecular weights of amino acids encoded by sp-nvd gene were 52.37 kda,and those encoded by CYP302a1,CYP315a1 and CYP18a1 genes were 61.14,62.41 and 60.35,respectively,basically consistent with the size of cytochrome P450(56-62kda)in other vertebrates and invertebrates.The theoretical isoelectric points are 8.39,8.76,6.74 and 7.96,respectively.In addition,Signal 5.0 Server and TMHMM analysis revealed that the amino acid sequences of Sp-Nvd,Sp-CYP302a1,Sp-CYP315a1 and Sp-CYP18a1 all had no signal-free peptide sequences,and only the amino acid sequences of Sp-Nvd and Sp-CYP18a1 each had one predicted transmembrane domain at the N-terminal.In addition,sequence analysis also found that sp-nvd contained two conserved structural domains involved in enzyme catalysis in Nvd: a Rieske [2Fe-2S] characteristic domain and a non-heme Fe(II)binding motif,and the amino acid sequences of Sp-CYP302a1,Sp-CYP315a1 andSp-CYP18a1 had 5 conserved P450 characteristic regions including helix-K,helix-C,helix-I,PERF and heme-binding,as well as the PG rich region at the N-terminal2.Expression analysis of genes related to 20 E metabolism pathway in tissuesThe basic function of 20 E is to induce ecdysis,and periodic ecdysis plays an important role in the growth,reproduction and morphogenesis of arthropods.The crustacean YO uses the cholesterol in the food to synthesize Ecd through the steroid hormone synthesis pathway,and then converts to 20 E,thus exercising its regulatory function.At present,the synthetic organ of molting hormone has been identified in many kinds of crustaceans,which lays a foundation for the regulation mechanism of molting hormone.In this study,real-time PCR was used to analyze the tissue expression patterns of several genes(Nvd,CYP302a1,CYP315a1 and CYP18a1)related to metabolism of20 E in several acupoint blue crabs.The results of q-pcr showed that Sp-Nvd,Sp-CYP302a1 and Sp-CYP315a1 were all expressed in all tissues detected in female and male crabs,and they all had the highest expression in YO,while the expression levels in other tissues were relatively low,this is consistent with that of molting hormone synthesis organ.Strangely,Sp-CYP18a1,which may participate in the degradation of 20 E,have the highest expression in YO,in addition,Sp-CYP18a1 has higher expressions in thoracic ganglia and cuticle and middle gut in female crabs and in the heart and middle gut in male crabs,It is worth to research that Sp-CYP18a1 has high expression in YO further.In addition,the expression levels of Sp-Nvd,Sp-CYP302a1,Sp-CYP 315a1 and Sp-CYP 18a1 were found to be significantly higher in females than in males,suggesting that the effects of ecdysis hormone titer may be different in male and female growth and development,and a similar phenomenon was found in drosophila melanogaster.At present,the significance of this difference is still unclear and further study is needed3.Expression analysis of genes related to 20 E metabolism pathway during development of larvae and molting cycleThe most typical role of molting hormone is to cause molting shell due to periodic fluctuations in the molting cycle.Therefore,it is of great significance to analyze the expression patterns of related genes in the molting cycle.We detected the expression levels of Sp-Nvd、 Sp-CYP302a1、 Sp-CYP315a1 and Sp-CYP18a1 genes in different molting periods and analyzed their expression regulation patterns results show that,in a molting cycle,Sp-Nvd gene has significant changes in this cycle,in addition,Sp-CYP302a1 and Sp-CYP315 al equally significant changes in this cycle,both in this early phase(D)with the highest expression,then began to decline,and its change trend are the same as the change trend of the individual demand of 20 e,suggests that 20 e metabolism plays an important role in the process.We also analyzed the Sp-CYP18 al inYO,and He,the changing law of its variation pattern consistent in the two groups in the molting cycle changes significantly,in this period(E)with the highest expression capacity,at this late stage B expression quantity is relatively low.All of those prompt Sp-CYP18 al in 20 E metabolism has a more important role in the process of oddly,In addition,Sp-CYP18 al in the expression level of YO in C and E stage is relatively high,and the expression pattern of YO in its tissues indicates that the expression level of YO in the synthetic organ of ecdysis hormone is high,which indicates that the function of YO may have evolved to some extent,which still needs further study.In addition,the changes of these genes in the development process of young crab were analyzed,and the results showed that the expressions of Sp-Nvd、 Sp-CYP302a1、Sp-CYP315a1 and Sp-CYP18a1 genes had significant changes during the development of larva,suggesting that the functions of these genes were closely related to the development of larva.Sp-CYP18a1 showed a peak expression before and after Z3 period.In the actual producting process,rotifera was used as the main feed in Z1 and Z2 periods,and halogen larvae were added as the biological feed since Z3 period.Whether there is a certain connection between these phenomena is worth further discussion.In summary,5genes of 7 genes that may be involved in ecdysis hormone metabolism,including Nvd,CYP307 al,CYP302al,CYP315 al and CYP18a1,were preliminarily identified in acupoint Scylla paramamosain,and the ORF sequences of 4of them were first generation sequenced and the preliminary analysis results showed the sequence characteristics the patterns of the tissues and the change of the expression during molting cycle of Sp-CYP302a1 and Sp-CYP315a1 genes are consistent with speculated changes and synthesis of 20 E,but Sp-Nvd and Sp-CYP18a1 inconsistent,it may be that the function of these genes have certain diversity,it is worth to further study.Overall consideration,Sp-CYP302a1 gene may act as the Indicator gene of titer change of 20 E,this paper can provide basic data for related research. |
PDF Full Text Request