低氧胁迫下中华圆田螺的肝脏转录组学分析

Transcriptome analysis of liver tissue of Cipangopaludina cathayensis under hypoxic stress

中文关键词: 中华圆田螺  低氧胁迫  转录组  差异表达基因  热休克蛋白

英文关键词: Cipangopaludina cathayensis  Hypoxia  Transcriptome  Differentially expressed genes  Heat shock protein

中文摘要:

      为了探究低氧胁迫下中华圆田螺(Cipangopaludina cathayensis)肝脏组织基因的差异表达，本研究通过高通量测序技术，分析中华园田螺低氧胁迫组(2.5 mg/L)和常氧组(6.9 mg/L)某些基因的差异表达，并对差异基因进行生物信息学分析，进一步采用实时荧光定量PCR (RT-qPCR)对关键差异表达基因进行验证。结果显示，测序共获得232 379条基因(unigenes)，与对照组比较，低氧胁迫组筛到176个差异基因，包含64个上调基因和112个下调基因。GO功能注释分析显示，差异基因主要富集在生物学过程中的几丁质代谢过程和含氨基葡萄糖的复合代谢过程，细胞组分中的胶原三聚体成分，分子功能中的几丁质结合功能和糖衍生物结合功能。KEGG通路富集分析显示，差异基因主要集中于环境信息处理、遗传信息处理、代谢和生物系统这4大类通路。6个关键差异基因的RT-qPCR结果显示，热休克蛋白70B2和热休克蛋白β-6基因表达量上调，几丁质酶蛋白4、α-1胶原蛋白(XIV)、α-4胶原蛋白(XIV)、5-磷酸酶蛋白基因表达量下调，证实了转录组测序结果的可靠性。本研究发现，低氧胁迫激活了中华圆田螺适应缺氧的生理活动，并获得了低氧胁迫下中华圆田螺肝脏组织中相关功能基因的表达信息，为深入研究中华圆田螺响应低氧胁迫的调控机制提供了基础数据和理论依据。

英文摘要:

      Cipangopaludina cathayensis is a snail species unique to China. Domestic research on C. cathayensis has mainly focused on aquaculture technology, especially the paddy field breeding method, where water quality plays a critical role in the cultivation of C. cathayensis. Dissolved oxygen is one of the most important factors in the aquatic environment because it impacts a series of biological activities such as the growth and development, metabolism, reproduction, and breeding of aquatic animals. When the dissolution of oxygen in water is less than 2 mg/L, the water is in a low oxygen or anoxic state. Hypoxia can slow the growth and development of aquatic animals, reduce their disease resistance and reproductive ability, and, in serious cases, can lead to their death. In recent years, research on C. cathayensis has mainly focused on the anti-tumor mechanism, immune response, nutritional value evaluation, and antibiotic resistance. However, there is currently no report on the regulation of response to hypoxia in C. cathayensis either domestically or internationally. The purpose of this study was to explore the differential expression of genes in the liver of C. cathayensis under hypoxic stress. In this study, healthy C. cathayensis without mechanical damage were cultured in a hypoxia stress group (2.5 mg/L) and a normoxia (control) group (6.9 mg/L), with 90 C. cathayensis in each group and 3 replicates. For the low oxygen stress treatment, dissolved oxygen was decreased from 6.9 mg/L to 2.5 mg/L within 1 h and maintained for 24 h. The liver tissue was taken as the experimental material in both the hypoxia stress group and normoxic group. The total RNA was extracted and an mRNA library was constructed. The liver tissue samples of C. cathayensis from both groups were sequenced and analyzed using an Illumina HiSeq-2500 technology platform, and unigenes were compared and annotated in GO, KOG, Nr, and KEGG databases. The differentially expressed genes were analyzed using DESeq. Bioinformatics analysis was performed on the function of GO and KEGG of differentially expressed genes, and the key differentially expressed genes were further validated by qPCR. Transcriptome analysis results showed that 500 584 transcripts were assembled from the original data and 23 379 unigenes were obtained by sequencing, with an average length of 686.65 bp and N50 of 1 127 bp. Among the unigenes, 26 636 were found to be homologous to genes in the Nr protein database. Additionally, 22 907 unigenes were annotated in the GO database, 13 290 in the KOG database, and at least 4 179 in the KEGG database. Compared with the control group, 176 differentially expressed genes were screened in the hypoxia stress group, among which 64 and 112 were up- and down-regulated, respectively. Further, GO functional enrichment analysis found that the differential genes were mainly enriched in chitin metabolic and glucosamine-containing compound metabolic processes in the biological process. Differential genes were also enriched in collagen trimer in the cellular component and chitin binding in the molecular function. The enrichment analysis results of the KEGG pathway mainly focused on four pathway categories, namely environmental information processing, genetic information processing, metabolism, and organismal systems. Finally, the qPCR results of six key differentially expressed genes were obtained by RT-qPCR. Among the up-regulated genes under hypoxic stress were the heat shock proteins 70B2 and beta-6, and the down-regulated genes were chitinase-like protein 4, collagen alpha-1 chain (XIV), collagen alpha-4 chain (XIV), and phosphatase-related protein type 5, which confirmed the reliability of the transcriptome sequencing results. Studies have shown that, through transcriptome sequencing, the expression information of relevant functional genes in C. cathayensis liver tissues under hypoxic stress can be obtained. Among them, the expression of heat shock protein genes were up-regulated, indicating that hypoxic stress activated the physiological activities of C. cathayensis to adapt to hypoxia and protected the body from hypoxic damage. In addition, the down-regulated expression of related genes in the metabolic pathway indicates that the growth of C. cathayensis is affected under hypoxic environments. In conclusion, the results of this study provide basic data and a theoretical basis for the in-depth study of the regulatory mechanism of C. cathayensis in response to hypoxic stress.