| Cyclina sinensis is a great economic value of marine animals.In recent years,due to environmental degradation and the drop resistance,a large area of clam mortality occurs repeatedly,causing enormous economic losses,so to get disease resistance genes of clam have great significance for the health of sustainable development of clam aquaculture in China.There is significant value to transcriptome studies,further study of the immune response mechanism of the clam and the identification of the immune-related genes will provide molecular biology basic to reveal the adaptability of the species.Most shellfish EST data in GenBank focused on scallops,oysters,mussels,abalone and other bivalves,combined with the results of the literature search and registration of the GenBank EST Clams,it should be said the study of gene function in clam has just started at home and abroad,so build massive transcriptome sequencing library in clam is necessary.Clam gene sequences by biological analysis,can effectively provide information on the molecular structure of the gene.The research provides reliable guarantee for study the function of immune-related genes in clam.The main contents include building clam transcriptome library and sequenceing,cDNA sequence advanced analysis and bioinformatics analysis,providing an important foundation for elucidating the mechanism of genes related to immune and signal transduction pathways of immune molecule.In this study,under the stimulate of micrococcus luteus and vibrio anguillarum respectively,the blood of clam were extracted total RNA.purified mRNA products added fragmentation buffer will break into short segments,reverse transcribed using random primers were synthesized cDNA.The purified double-stranded cDNA and then by the end of the repair,A-tailing,plus sequencing joints,purification and PCR amplification to complete the entire library preparation.The library sample of a single bacteria stimulate(6h and 12h)were mixed.The clam transcriptome library was stimulated by micrococcus luteus which peak at 552 bp,was stimulated by vibrio anguillarum in 556bp.Using second-generation sequencing technology MiSeq sequencing mode to complete the clam transcriptome sequencing by pair end double-ended(PE),the sequencing raw data of the sample stimulated by micrococcus luteus(sample T)was 3.22G,getting 12916040 Raw reads,then remove the connector information,low quality bases,not measured base,getting 10966322 Clean Reads;the sequencing raw data of the sample stimulated by vibrio anguillarum(sample M)was 3.98G,getting 15930210 Raw reads and 13389388 Clean Reads.High quality sequencing and low error rate made library sequencing successful.Transcriptome sequences were assembly spliced by bioinformatics tools.In sample T,the total number of transcripts is 137940 and the total number of genes is 57877,average contig is 644.32bp.In sample M,the total number of transcripts is 148238 and the total number of genes is 62152,average contig is 618.50bp.The ORF of Unigene was predicted and analyzed.To identify the all unigene sequences,we performed Blast alignments againstm NR protein database,Uniprot/Swissprot protein database and the Unigene eggNOG.GO annotation information of Unigenes were got by BlastGO Software.By KEGG pathway,functional annotation and pathway of the unigenes were found and predicted.Differentially expression of Unigenes were analyzed.According to the homology comparison of sequence,functional annotation of genes and pathway analysis,the results show that A total of 893 genes annotated to 15 immune related pathway,some important immunity related genes were found in the data including heat shock protein,toll-like recepter and so on.This result provides feasibility for studying the expression of these disease resistance factor genes and protein function analysis,it will provide molecular biology basic to analyze the immune response mechanism and immune defense system in shellfish.Finally,the Toll 2 gene sequence and MyD88 signal molecule of pathway were analyzed and discussed. |
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