| Hirudinaria manillensis(Hirudinea:Arhynchobdellida:Hirudinea:Hirudinaria)is a type of blood-sucking leech widely distributed in many countries.Due to the variety of effective anticoagulant bioactive proteins it secretes in blood sucking,H.manillensis has been extensively applied as a medical animal resource in therapeutic treatment in recent years.This species has threrfore attracted rising attention.However,the current research on H.manillensis is limited to phylogency,artificial breeding,the extraction and identification of bioactive components,and etc.The absence of genic and genetic research has restricted the understanding of this important medicinal animal and its'medicinal research process seriously.In this study,we analyzed the differentially expressed genes(DEGs)by using the obtained transcriptomes of H.manillensis under different treatment conditions,which provided new evidences that reveal the mechanism of the blood-sucking habit of H.manillensis,and subsequently used them to explore new genes of anticoagulation.In this study,we generated transcriptome sequencing of several H.manillensis individuals under two different treatment conditions,starving and after blood sucking.We later analyzed the changes of gene expression and related metabolic pathways.Further,functional genes related to blood-sucking habits were identified accordingly.These results were not only explained the mechanism of blood-sucking,but also provided a reliable molecular basis for pharmaceutical development of H.manillensis.Furthermore,11 DEGs were verified by qRT-PCR to support our results.At last,all produces result and main conclusions are collected and decribed as follows:1.We established two cDNA libraries of H.manillensis under two different treatment conditions,then obtained a total of 14.06 Gb of Clean data by sequencing.After de novo assembly and splicing,a total of 55,998 Unigenes were obtained with a total length of 37,208,146 bp,the GC value of 43.70%,N50 at 1,391 bp,and average length of 664.45 bp.2.After blast with the databases of NR,KEGG,Pfam,COG,KOG,eggNOG,Swiss-prot,and GO,a total of 26,469 Unigene were annotated.Among them,the number of annotated Unigene in NR database was the highest,accounting for 75.58%of the total number of annotated Unigene.Among these Unigene,the homologous sequences with Helodella robusta were dominant.3.A total of 40,603 CDS were obtained.Among them,35,009 CDS ranged from 100 to 1,000 bp occupied the most mount,accounting for 84.16%.While the 1,623 CDS with the length over 2,000 bp were the least,accounting for 2.74%.4.The transcriptome data were standardized by RPKM,and the screening criteria were strictly controlled(FDR?O.01 and FC?2).Then 2,371 DEGs were detected in H.manillensis after blood sucking compare with starving,of which 892 had the up-regulated expression and the expression level of 1,479 genes were down-regulated.Among these DEGs,485 were functionally annotated by GO database,with the most genes related to energy metabolism,protein synthesis processes,stress responses and immune processes.The KEGG pathway enrichment of DEGs showed that a total of 535 DEGs were annotated and involved 174 metabolic pathways.Significantly enriched KEGG pathways include oxidative phosphorylation,ribosome and MAPKs signal pathway,and etc.Further analysis of these KEGG pathways showed that in the oxidative phosphorylation pathway,most DEGs associated with the electron transport chain were down-regulated and the metabolic process was reduced.At the same time,expression levels of most DEGs related to the ribosome metabolism pathway were up-regulated.These results indicate that the metabolic process was weakened and the protein synthesis was significantly enhanced thusly.5.According to the characteristics of blood-sucking animals,we selected the functional groups of the genes related to the blood sucking process,including anticoagulant-related genes,genes involved in defense response and response to oxidative stress.The results showed that the expression level of DEGs was significantly increased after the blood sucking in H.manillensis,which indicated that the anticoagulant ability,defense ability and stress response of H.manillensis were significantly enhanced.These processes ensured that H.manillensis can absorb blood successfully,meanwhile,resist the invasion of bacteria and the oxidative stress of hemoglobin in the process of blood sucking.6.qRT-PCR was used to test the expression levels of the 11 anticoagulants' coding genes:Hirudin,Saratin,Antistasin,Guamerin,Apyrase,Destabilase,Lectin C,Eglin C and Ficolin.The results showed that the qRT-PCR results were consistent with RNA-Seq results and confirmed the reliability of the transcriptome analysis. |
PDF Full Text Request