| Hirudo nipponia Whitman is an animal of Arhynchobdellida Blanchard,Hirudiniformes Caballero,Hirudinidae Whitman and Hirudo Linnaeus.It is one of the original animals of leech material in Chinese pharmacopoeia(2015 Edition).The dry and intact body of H.nipponia can be used as medicine.H.nipponia used as medicine is harvested in summer and autumn and dried in the sun or at low temperature after killing by boiled water.It has the effect of helping blood circulation and eliminating stasis.It has been used to treat impassable blood stasis syndrome,stroke hemiplegia,and injuries from falls,fractures,contusions and strains,and shown to be particularly effective in preventing and treating cardiovascular and cerebrovascular diseases.H.nipponia is widely distributed in China,north from the northeast provinces and Neimenggu,west to Sichuan,Gansu and south to Taiwan,Guangdong and Guangxi.It was also discovered in foreign countries including Japan,South Korea,Russia and Mongolia.H.nipponia mainly inhabits paddy fields,ditches,ponds,marshes and other fields with perennial water,feeding on blood of humans,livestock and amphibians.In order to further ascertain blood-feeding strategy of leeches,transcriptome sequencing on salivary glands of hematophagous leeches has been applied previously.Transcripts of anticoagulant and anti-thrombotic substance that resist host’s hemostatic system,such as hirudin,eglin C,antistasin,saratin,decorsin,ornatin,leech antiplatelet protein(LAPP)and destabilase,have been screened out and identified.Nevertheless,studies on the salivary gland of H.nipponia are fairly uncommon at present.In this study,H.nipponia was fed blood with different experimental treatment,to explore the blood-feeding strategy of salivary glands.Salivary glands of unfed H.nipponia were categorized as the control group(UFG).Salivary glands of H.nipponia that was fed for 20 minutes were categorized as the feeding group(FIG).Salivary glands of H.nipponia that were fully fed 24 h ago were categorized as the fed group(FG).Transcriptome sequencing on the salivary glands was applied,and transcripts of anticoagulant and anti-thrombotic substance were analyzed and identified.Then,the expression profiles of the salivary glands in the three groups were sequenced,and the blood-feeding strategy genes of the salivary glands of leech were analyzed and identified.The genes screened out were analyzed with bioinformatic methods,and their expression patterns were verified by qRT-PCR technology.Gene sequences with high integrity and reliable annotation were selected for prokaryotic expression and their biological functions were further analyzed to provide a theoretical basis for understanding the blood-feeding strategy of the salivary gland of leeches.The main conclusions are as follows:(1)Transcriptome sequencing data(6.5 Gb)of the salivary glands of the hematophagous leech Hirudo nipponia were obtained by using the BGIseq-500 platform.Three cDNA libraries corresponding to three blood meal stages were constructed.After assembly and removal of redundancy,a total of 41 895 unigenes were obtained.The values for total length,average length,N50 and GC content obtained were 46 785 456 bp,1 116 bp,1 968 bp and 42.12%,respectively.The transcriptome data were submitted to the NCBI Sequence Read Archive under accession number SRP151118.Then,the unigenes were compared to seven functional databases for annotation.Finally,24 723 unigenes(NR:59.01%),7 542 unigenes(NT:18.00%),20 526 unigenes(SwissProt:48.99%),19 303 unigenes(KOG:46.07%),19 862 unigenes(KEGG:47.41%),7 549 unigenes(GO:18.02%)and 19 707 unigenes(InterPro:47.04%)unigenes received annotation.When transcripts of unigenes related to anticoagulant and anti-thrombotic functions were screened out and gathered,bioinformatic methods were employed to assess integrity of the transcripts and reliability of the functional annotation.Finally,transcripts of pharmacological substance such as thrombin inhibitors,platelet aggregation inhibitors,factor Xa inhibitors and thrombolytic agents were obtained.(2)When transcripts that possessed potential antithrombin activity was being screened from the transcriptome database mentioned above,transcript unigene5370 was found to be annotated to hirudin HV3 from Hirudo medicinalis with an e-value of le-29 and named hirudin-HN.This transcript is a new gene encoding a thrombin inhibitor,belonging to protease inhibitor 114(hirudin)family.Hirudin-HN,with a 270-bp cDNA,encodes an 89-aa protein containing a 20-aa signal peptide.A mature Hirudin-HN protein shared typical structural characteristics with hirudin,including three conserved disulfide bonds,the PKP and DFxxIP motifs.Proteins(Hir and M-Hir)were obtained via prokaryotic expression,and the mature hirudin-HN protein was shown to have anticoagulant activity and thrombin affinity by using the chromogenic substrate S2238 and surface plasmon resonance(SPR)interaction analysis,respectively.The expression level of mRNA was studied by qPCR and Western blotting,and it was found that the mRNA expression level of hirudin-HN in the salivary gland of starving H.nipponia(UFG)was much higher than that in those feeding or fed leeches.These results provided a foundation for further research on the structure-function relationship of hirudin-HN with thrombin.(3)Transcript Unigene3091 was screened out from the transcriptome database mentioned above.The FPKM value of Unigene3091 is 12283.46,and it was annotated to Hirudin-HM1(Poecilobdella manillensis)with an e-value of 2e-14.Unigene3091,with a 237-bp cDNA,encodes a 77-aa protein containing a 20-aa signal peptide.Amino acid sequence alignment revealed that Unigene3091 shared a conserved structure of three disulfide bonds with hirudin.This typical structure leads to the formation of a stable core structure by the N-ternimal peptide chain.Nevertheless,other typical structures that exist in hirudin were not observed in Unigene3091,such as the PKP and DFxxIP motifs.A phylogenetic tree was constructed using the amino acid sequence corresponding to Unigene3091,and the sequence was closely related to hirudin-like factors(HLFs).Hence,this gene was named hirudin-like factor-HN(HLF-HN).The mature HLF-HN protein was obtained by prokaryotic expression.Using the chromogenic substrate S2238 revealed that the mature HLF-HN protein possessed antithrombin activity.Research results provided the theoretical basis for the study of hirudin-like factor gene in hematophagous leeches.(4)The salivary gland of H.nipponia produces and secretes a variety of pharmacologically active ingredients to effectively complete the blood-feeding process.Three cDNA libraries of the salivary glands corresponding to three feeding stages(UFG,FIG and FG)were established separately,and each library had three replications.A total of 216 161 299 raw reads were obtained by using BGISEQ-500 platform(BGI)to perform the sequence analysis of expression profiles of the cDNA libraries.Differentially expressed genes(DEGs)during three blood meal stages were screened out.In this study,specifically highly expressed genes in FIG group were defined as blood-feeding-related genes,and bioinformatic methods were used to analyze and evaluate blood-feeding strategy genes.The results showed that the expression of immune-related genes,vasodilator-related genes,digestion-related genes and development-related genes were involved in the blood-feeding strategy of the salivary gland of H.nipponia.(5)Transcript Unigene379 was screened out from differentially expressed genes in the salivary gland of H.nipponia during three blood meal stages.Unigene379,with a 411 bp cDNA,encodes a 136-aa protein containing a 21-aa signal peptide in its N-terminal region.Amino acid sequence alignment revealed that Unigene379 shared a conserved sequence of 14 cysteine,which formed 7 disulfide bonds,with lysozyme.A soluble fusion protein Trx-Des was obtained using Trx protein,a solubility enhancer.After separating Trx and Des proteins with thrombin,the activity of Des protein was detected.Des protein had the activity of lysozyme but not the activity of dissolved fibrin.In conclusion,a series of anticoagulation-related candidate genes were screened out by sequencing and analyzing the transcriptome of salivary glands of H.nipponia at three different feeding stages.Hirudin-HN and hirudin-like factor-HN proteins were obtained via prokaryotic expression,and both of them had been verified to have antithrombin activities.The relationship between hirudin-HN and thrombin was analyzed intensively through their 3D structure models.Screening of genes related to blood-feeding strategy of the salivary gland of H.nipponia through RNA-seq showed that the expression of immune-related genes,vasodilator-related genes,digestion-related genes and development-related genes were involved in the blood-feeding process of the salivary gland of H.nipponia.Prokaryotic expression was also employed to obtain destabilase-HN protein,of which lysozyme activity was verified.This study revealed that in order to take the blood meal successfully and safely,the salivary gland of H.nipponia probably expressed a series of blood-feeding strategy genes that possessed potential anticoagulant and bacteriostatic activities. |
PDF Full Text Request