Effects Of Feeding On Morphological Structure And Transcriptome Of Salivary Gland Cells In Hirudo Nipponia

Hirudo nipponia(Hirudo,Hirudinidae,Arhynchobdellae,Hirudinea)is widely distributed in China.The salivary gland with good market prospects is the main medicinal part of H.nipponia,due to a variety of bioactive substances.At present,the research on H.nipponia mainly focuses on artificial culture,biological active ingredients,and so on.There are few reports on salivary glands.The purpose of this study was to determine the effect of salivary glands of H.nipponia on feeding behavior,and to explore the culture system of salivary gland cells in vitro.The main results were as follows:(1)The morphological changes of salivary gland and its appendages of H.nipponia before and after feeding were observed,including various parts of salivary gland(cell body,extended tubule,opening area).The results showed that the changes of secretory granules in cells before and after feeding.(2)Six samples of salivary gland cells in H.nipponia before and after feeding were sequenced by Illumina HiseqTM,and eight differentially expressed genes were verified by RT-qPCR.1)After assembly and splicing,145981 unigenes were obtained,of which N50 was 1127 bp,with an average length of 675.04 bp.2)All unigenes were blasted in NR,NT,KOG,CDD,PFAM,Swissprot,TrEMBL,GO and KEGG databases.For the four major protein databases,NR,KEGG,Swissprot and KOG,55.64%,6.31%,41.98%and 27.62%unigenes were annotated,and 7594 unigenes were annotated respectively.3)The CDS obtained from the study mainly ranged from 200 to 300 bp.4)Compared with the control group,2650,6274,9032,3571 and 6939 differentially expressed genes were obtained at 0,1,3,5 and 10 days after feeding.13801 differentially expressed genes were clustered and analyzed by STEM software,and 22 sets of genes with similar expression patterns were obtained.In the GO database,translation,peptide biosynthesis and peptide metabolism were significantly enriched in biological processes.Ribosomes,ribosome subunits and cytosolic ribosomes were the terms of significant enrichment in cell composition.Ribosome structural components,structural and molecular activities and ribosomal RNA binding were significantly enriched in molecular functions.Ribosome pathway,oxidative phosphorylation pathway and citric acid cycle pathway was mainly enriched in KEGG pathway.5)The expression of eight differentially expressed genes were verified by RTqPCR,which was basically consistent with the results of RNA-Seq.(3)The salivary gland cells of H.nipponia cultured in vitro had got an initial exploration.1)By orthogonal test,the disinfection effect of antibiotic combinations with different concentrations and types was investigated.The best disinfection conditions were 100 U·mL-1-100 μg·mL-1 Penicillin-Streptomycin+160 μg·mL-1 Gentamicin+80μg·mL-1 Kanamycin+5 μg·mL-1 Amphotericin B.2)The best enzyme digestion method for H.nipponia was screened by CCK-8 cell activity test.The cell activity was best under the condition of 1 mg·mL-1 collagenase type Ⅱ for 30 min and 2 mg·mL-1 neutral protease for 45 min at 28 degrees.