Screening Of Differentially Expressed Genes And Expression Analysis Of Immune And Growth-related Genes Of Pomacea Canaliculata Before And After The Infection With Angiostrongylus Cantonensis

Objective:In this study to find and identify differentially expressed genes before and after Pomacea canaliculata infection with Angiostrongylus cantontensis,explore the spatial and temporal variations of these differentially expressed genes,a preliminary understanding of interaction relation between Angiostrongylus cantontensis and its intermediate host Pomacea canaliculata.Methods:1.Based on the early stage of transcriptome sequencing results and analysis of the Pomacea canaliculata infection with Angiostrongylus cantonlensis,screening differentially expressed genes associated with infection.Taken hepatopancreas and hemolymph samples after Pomacea canaliculata infection with Angiostrongylus cantontensis at 1d,10d,20d,after the extraction of total RNA transcription,is used for fluorescent quantitative PCR,the gene expression changes in the three period will be verified and further explored.2.Po,macea canaliculata raised in the laboratory was used as the experimental material,and infected by Angiostrongylus cantontensis.At Id,10d,20d and 30d after infection,hepatopancreas,cephalopods,kidneys,intestines and gills tissues were taken,and RNA was extracted and reverse-transcribed into cDNA.and blank control was set.Immune-related and cell growth-related gene were selected for bioinformatics analysis,and fluorescence quantitative PCR was used to investigate the expression distribution in Pomacea canaliculata tissues and the spatial and temporal expression differences after infection with Angiostrongylus cantontensis.3.Adult and juvenile Pomacea canaliculata raised in the laboratory was used as the experimental material.Hemolymph samples from juvenile and adult snails were taken at Id,10d,20d and 30d after infection with different concentrations of Angiostrongylus cantontensis(Three kinds of concentration:1500,2500 and 3500).RNA was extracted and reversely transcribed into cDNA,and blank control was set.Immune-related gene and cell growth-related gene were selected for bioinformatics analysis,and quantitative PCR method was used to investigate the expression differences in hemolymph.Results:1.After the results of transcriptome analysis of Pomacea canaliculata infected with Angiostrongylus cantontensis differentially expressed genes at 3 stages(Id,10d and 20d)after infection were screened out,20 and 18 genes were selected for quantitative fluorescence verification in liver tissue and hemolymph,respectively.The verified genes were mostly involved in cell growth and metabolism,signal transduction,immune response and stress response.The up-regulated genes include transforming growth factor gene,cellulase gene and ornithine decarboxylase gene.The down-regulated genes include glutathione S transferase gene,chitotriosidase gene,G lysozyme gene and a-tubulin gene.2.Spatiotemporal expression and bioinformatics of growth and immune-related genes in Pomacea canaliculata infected with Angiostrongylus cantontensis show the G type lysozyme cDNA of Pomacea canaliculata was 990bp,containing a 828bp complete open reading frame and 275 amino acids,which was the closest to Physella acuta in evolutionary relation.Quantitative results show that the G type lysozyme gene expressed in nonnal Pomacea canaliculata in hepatopancreas is highest.After infected with.Angiostrongylus cantontensis,all tissues are in decline.As the most suitable parasitic site of Angiostrongylus cantonensis,the gill tissue was most down-regulated,which was 1180.1 ±426.4,and the down-regulation of the kidney is the least,only 2.7±0.8.The downregulation of g-lys means that the anti-infection mechanism of Pomacea canaliculata is partially inhibited.a-tubulin cDNA,1452bp in total,was a complete ORF frame,encoding 483 amino acid sequences.In terms of evolutionary relationship,it was closed to scallops and mollusks as Crassostrea gigas,Mizuhopecten yessoensis.Results show that the a-tubulin gene expressed in normal in hepatopancreas is highest.After infected with Angiosrongylus cantontensis,it was also the most down-regulated,with a decrease multiple of 14.8 ± 1.4 compared with the control group.The decreased expression of alpha-tubulin gene is bound to affect the function of hepatopancreas detoxification,defense and metabolic hormones.3.Expression and bioinformatics analysis of immune and growth related genes in haemolymph of Pomacea canaliculata infected with different numbers of Angiostrongy^lus cantontensis show the total cDNA of the calreticulin protein gene is 1787bp,which contains a complete 1212bp open reading frame and encodes 403 amino acids,which is the closest to mollusks such as abalone in evolutionary relation.Quantitative expression results show compared with the blank control group,the calreticulin protein gene in each infection group increased to different degrees on day 1-20,and on day 30 of infection,the gene expression was the same as that in the blank control group,which may be related to the calreticulin protein gene's participation in the immune response in the early stage.In the later stage,the escape mechanism of Angiostrongylus cantonensis may be hidden in the internal environment of the body to avoid elimination,thus avoiding partial immune damage.The ornithine decarboxylase gene(1224bp)was translated into 407 amino acids for a complete open reading frame,which was the closest to Biomphalaria glabrata in evolutionary relation.The expression of ornithine decarbox)ylase gene was up-regulated in all stages in both low and medium concentration groups of adult snails.In the high-concentration infection group,gene expression remained at the same level as that in the control group,and the snail almost did not die.juvenile snail infection with low,medium and high concentration of Angiostrongylus cantontensis,the baby had different degrees of death,expression of ornithine decarboxylase gene were cut.The up-regulation of ornithine decarboxylase expression accelerates the synthesis of polyamines and is involved in the growth and repair of the body after damage.The decrease of ODC expression means the decrease of cell proliferation,differentiation and growth,leading to impaired repair function.Therefore,for later studies,ODC gene can be used as a potential molecular site for snail killing drugs.Conclusion:1.The expression changes of several genes in vivo after Pomacea canaliculata was infected with Angiostrongylus cantonensis,involving immune response,cell growth,signal transduction and other aspects.2.The expression of G lysozyme gene and a-tubulin was down-regulated in many tissues compared with the blank control group.3.Under different infection degrees of Angiostrongylus cantonensis,the expression of calreticulin protein gene in hemolymphatic cells was up-regulated at the early stage,which may be due to the involvement of calreticulin protein gene in immune response in Pomacea canaliculata.4.The expression of ornithine decarboxylase gene in hemolymph was different in adult and youth Pomacea canaliculata with different infection degrees,which may be related to the infection degree of Angiostrongylus cantonensis and growth status of Pomacea canaliculata.