Transcriptome Analysis Of Low Temperature Stress And Validation Of GeneRelated To Low Temperature Tolerance In Fenneropenaeus Chinensis

Fenneropenaeus chinensis is an important Marine shrimp species in northern China.It has very high economic value.Due to the requirement of releasing temperature higher than 14℃ for Fenneropenaeus chinensis,its breeding area and breeding time are limited.This has affected the size of its launch and unit of production.It is very important to breed low temperature tolerant strains of Fenneropenaeus chinensis,which can expand the culture area and size.In this study,through treatment of low temperature stress and transcriptome analysis of the low temperature stress group and the normal temperature control group,genes related to low temperature tolerance traits were screened out,bioinformatics analysis was performed and prokaryotic expression verification was carried out,in order to identify the molecular markers of low temperature tolerance in Fenneropenaeus chinensis and lay a theoretical foundation for low temperature breeding of Fenneropenaeus chinensis.Fenneropenaeus chinensis was treated with low temperature stress,and the families with better low temperature tolerance were taken as samples,and sent to BGI for RNA-Seq.The sequencing results were verified by real time RT-PCR.The 12 samples at normal temperature and low-temperature stress from the 40-day-old wild group,the 40-day-old breeding group and the 10-day-old wild group.576.53 M of Raw Reads were measured,521.39 M of clean Reads were obtained after filtration,accounting for 90.43%,and 75,905 Unigenes were obtained after assembly and removal.A total of 41986 Unigene annotations were obtained,accounting for 55.31%of All Unigene annotations,including 35.77%of KEGG database annotations and 11.93%of GO database annotations.There were 14,618 differentially expressed genes in the 10-day-old wild group,of which 5,703 were up-regulated and 8,915 were down-regulated.There were 13,870 differentially expressed genes in the wild group at 40 days of age,among which 6,821 were up-regulated and 7,049 were down-regulated.There were 22,914 differentially expressed genes in the 40-day-old breeding group,of which 12,783 were up-regulated and 10,131 were down-regulated.Among the GO annotations of different genes,there were 20 annotations for biological processes,and the two most enriched ones were metabolic process and cellular process.According to the enrichment bubble diagram,mRNA processing and regulation of gene expression in cellular process were most enriched.There are 14 classification notes of cellular component,and the most enriched ones are membrane class,membrane part class,cell class and cell part class,and organelle class in turn.There are 8 annotation items in molecular function category,and the two categories with the most enrichment are binding category and catalytic activity category.According to the enrichment bubble diagram,the binding category has the most enrichment in chromatin binding and sequence-specific DNA binding,while the catalytic activity category has the most activity in DNA binding transcription factor activity.KEGG annotation of different genes is mainly concentrated in signal transduction pathway of environmental information processing pathway,metabolic pathway,immune system and endocrine system related pathways of organismal system.With the small subunit ribosomal protein gene as the reference gene,the real time RT-PCR verification of CL161.Contigl,CL2560.Contigl,CL2861.Contig5,CL5186.Contig2.The differentially expressed genes were obtained by comparing the low temperature group and the normal temperature group.There were 9 up-regulated differentially expressed genes in more than 7 comparison groups,including transcription factors containing BZIP,rRNA,RRM domain proteins,cytochrome B5-like proteins,troponin C,extracellular proteins,tubulin subtypes,and 1ncRNAs.Among them,rRNA,cytochrome B5-like protein and IncRNAs were upregulated under low temperature,which is a new discovery in the related expression in response to low temperature.The significantly upregulated differentially expressed gene--CL2861.Contig5,which was identified as cytochrome B5-like protein by bioinformatics analysis,was predicted by open reading frame and the protein sequence was analyzed.An open reading frame ORF2861 was obtained.The molecular weight of ORF2861 was predicted to be 16.7 kDa.The secondary structure of ORF2861 was predicted to be 21.33%helix,6%β-chain and 72.67%ring.Two recombinant vectors pET28a-2861 and pET28b-MBP-2861 were successfully constructed by homologous recombinant method,imported into the competent E.coli BL21,the expression strain was formed.When induced by IPTG at 16℃,pET29b-MBP-2861 were soluble expressions.By comparing the survival rate of the strains expressing recombinant protein at 0 ℃ and-20℃ for 12 hours and 0℃ for 0-17 days,the results showed that the expression of recombinant protein at low temperature significantly improved the survival rate of the strains.The purified recombinant protein could not tolerate low temperature in vitro.