Mining of temperature stress functional genes in Gymnocypris eckloni based on transcriptome sequencing

【Objective】 The purpose of this study was to explore the temperature stress functional genes of Gymnocypris eckloni based on transcriptome sequencing, which could provide theoretical reference for exploring the adaptability and temperature response mechanism of Gymnocypris eckloni under heat stress. 【Method】 Sixty healthy Gymnocypris eckloni were selected and randomly divided into a control group and a heat stress group, which were reared in 10 and 28 ℃ water environments respectively. After the rearing, the liver tissues were obtained after MS-222 anesthesia. The liver tissues were ground with liquid nitrogen and then the total RNA was extracted by TRIzol method. The high-throughput sequencing was performed using the Illumina NovaSeq 6000 platform. The obtained unigenes were compared with the NR, NT, KO, SWISS-PROT, Pfam, GO and KOG databases for functional annotation analysis. Based on the transcript expression level, differentially expressed genes（DEGs） were screened by DESeq2, and KEGG signaling pathway enrichment analysis was performed. Six DEGs were randomly selected for real-time fluorescence quantitative PCR detection to verify the accuracy of the transcriptome data. 【Result】 GO functional annotation analysis results showed that a total of 23464 unigenes were successfully annotated in the GO database, involving 3 functional categories: biological processes, cellular components and molecular functions. KOG functional annotation analysis results showed that 16583 unigenes were successfully annotated in the KOG database, involving 26 KOG functional terms such as RNA processing and modification of chromosome structure and dynamics. KEGG functional annotation analysis results showed that unigenes annotated to the KEGG database involved in 5 major functional categories, among which the KEGG functional term with the largest number of annotated unigenes was signal transduction, followed by the immune system. DEGs analysis results showed that there were 2388 DEGs between the control group and the heat stress group, 1520 DEGs were up-regulated, and 868 DEGs were down-regulated. KEGG signaling pathway enrichment analysis results showed that the up-regulated DEGs were mainly enriched in signaling pathways such as antigen processing and presentation, endoplasmic reticulum protein processing, and estrogen. The down-regulated DEGs mainly enriched in metobolic pathways. The results of realtime fluorescence quantitative PCR showed that fragments per kilobase of exon model per million mapped fragments（FPKM） in DNAJA4, HSP90a, FKBP4, Caspase1, Caspase7 and Caspase8 transcriptomes were consistent with the gene expression variation trend, indicating that the transcriptome sequencing data had a high accuracy. 【Conclusion】 Under heat stress, the expression levels of immune-related DEGs of Gymnocypris eckloni are down-regulated, and the expression levels of heat shock protein are up-regulated. Heat stress may inhibit the immune function of Gymnocypris eckloni, and the heat shock protein family members may play an anti-heat stress role by stabilizing protein structure and maintaining cell homeostasis. Under heat stress, the expression levels of apoptosis-related DEGs Caspase7 and Caspase8 in Gymnocypris eckloni are significantly down-regulated, which may be related to the inhibitory effect of heat shock protein on apoptosis. Gymnocypris eckloni may reduce the risk of apoptosis by enhancing the activity of molecular chaperones under heat stress.