| Sesarmops sinensis is a kind of omnivorous benthic crab,belonging to Crustacea,Decapoda,Brachyura,Grapsoidea and Sesarmidae.In China,it is widely distributed in the intertidal zone of Jiangsu,Shanghai,Zhejiang and other coastal areas.It lives in a complex environment.It detects changes in the water environment and water pollution.Due to the development of coastal areas and the emergence of illness in recent years,the ecological environment in which the S.sinensis is located has been severely damaged,its mortality rate has been increasing,and species resources have dropped sharply.In order to improve the environmental water quality of the habitat of S.sinensis,promote the economic development of coastal fisheries,and to prevent and control the occurrence of immune diseases,it is necessary to conduct research on immune related genes.High throughput sequencing technology provides convenience and foundation for analyzing transcripts and mining immune related genes.Based on the platform Illumina Hiseq 2000,to compare the transcriptome of hepatopancreas tissues of S.sinensis treated with phosphate buffer saline(PBS),peptidoglycan(PGN)and lipopolysaccharides(LPS),to analyze the differentially expressed genes of S.sinensis hepatopancreas tissues under pathogen PGN and LPS infection,and to reveal the immune related genes and immune related metabolic pathways in hepatopancreas of S.sinensis.The main results are as follows:1.In the hepatopancreas transcriptome stimulated by PBS and PGN,45,491,736 and 58,178,646 raw reads,44,414,140 and 56,834,314 clean reads were obtained from the two groups,respectively,with a total of 15.19 Gb.A total of 85,556 transcripts and39,039 unigenes were obtained.The average length of transcripts and unigenes were1,096 bp and 1,105 bp respectively.The value of Q30 was 91.40% or more.2.Gene function annotation was performed on seven databases,and finally 12,825(32.85%),3,729(9.55%),6,603(16.91%),9,602(24.59%),13,661(34.99%),13,661(34.99%)and 5,749(14.72%)unigene were respectively annotated in NR,NT,KO(KEGG),Swissprot,PFAM,GO and KOG databases.Of the 13,661 unigenes entries annotated to the GO database,125 are involved in “Immune system processes”and2,025 are involved in “Response to stimulus”.Of the 5,749 unigenes in the KOG database,42 unigenes were involved in the V classification “Defense mechanism”.In the KEGG database,6,603 unigenes were dispersed into 245 pathways,of which 247 unigenes were involved in the “Immune system”.3.Through unigene differential analysis,1,300 differentially expressed genes(DEGs)were screened out,of which 466 were significantly up-regulated genes and 834 were significantly down-regulated genes.Thirteen immune-related genes were screened from differentially expressed genes for qRT-PCR analysis,and the internal reference gene was actin gene β-actin.From the results,it can be concluded that the expression of these genes increased under PGN stimulation,which is the same as the result of transcriptome sequencing.These results indicating that the transcriptome data was of high quality and that the selected genes participate in the immune defense process of S.sinensis when faced with external stimuli.4.In hepatopancreas transcriptome of S.sinensis stimulated by PBS and LPS,there were 45,491,736 and 55,303,248 raw reads,44,418,190 and 53,694,142 clean reads in the two groups,respectively,and GC content was 49.14% and 49.87%.After Trinity assembly,94,485 transcripts and 42,421 unigenes were obtained,with average lengths of 1,124 bp and 1,098 bp,respectively,and total nucleotide numbers of106,225,673 and 46,577,934.5.Perform functional annotations on the obtained unigene and compare it with the seven public databases.The results in the Nr,Nt,KEGG(KO),Swiss Prot,PFAM,GO and KOG databases are 13,843(32.63%),4,189(9.87%),6,995(16.48%),10,415(24.55%),14,765(34.80%),14,765(34.80%),6,048(14.25%)unigenes were annotated.The 14,765 unigenes annotated in the GO database are divided into three categories: cell components,biological processes,and molecular functions,with a total of 55 subcategories.Among them,127 unigenes were classified as “Immune system processes” and 2,183 unigenes were annotated as “Responses to stimulus”.In the KEGG database,6,995 unigenes were dispersed into 245 pathways,of which 266 unigenes were involved in the “Immune system”.6.Through unigene differential analysis,1,950 differentially expressed genes(DEGs)were screened out,of which 1,144 DEGs showed significant up regulation and806 DEGs showed significant down regulation.KEGG enrichment analysis found that the most significant metabolic pathway for DEGs enrichment was “Regulation of actin cytoskeleton”.Using 18 S gene as internal reference gene,eight DEGs were screened from immune related genes and analysis by qRT-PCR.From the results,it can be concluded that the expression of these genes increased under LPS stimulation,which is the same as the result of transcriptome sequencing.These results proved that the transcriptome sequencing data was of high quality,and the selected genes participate in the immune defense process of S.sinensis.In conclusion,this study not only enriched the existing transcriptome data of S.sinensis,but also clarified the immune related pathways and immune defense mechanism of S.sinensis in response to external pathogens(PGN and LPS)infection,which provided a factual basis for the next study of immune function genes of S.sinensis,and helped us to make a deeper understanding of the immune defense mechanism of crustaceans. |
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