温度渐变和骤变下凡纳滨对虾ddit4l基因的表达分析

Expression analysis of the ddit4l gene in Litopenaeus vannamei under sudden and gradual change of temperature

中文关键词: 凡纳滨对虾  高温  Lv-ddit4l  基因表达

英文关键词: Litopenaeus vannamei  High temperature stress  Lv-ddit4l  Gene expression

中文摘要:

      为探索凡纳滨对虾(Litopenaeus vannamei) DNA损伤诱导因子-4 (DNA damage inducible transcript 4-like, ddit4l)的结构及功能，以转录组测序获得的unigene序列为基础，采用直接PCR扩增方法，获得ddit4l完整的ORF序列(Lv-ddit4l)。该基因的ORF长495 bp，编码164个氨基酸，分子量为18.51 kDa，理论等电点为5.35，包含1个RTP801C超基因家族功能结构域。多序列比对结果和系统进化树显示，Lv-ddit4l与北太平洋雪蟹(Chionoecetes opilio)的同源性最高，为59.76%，且与北太平洋雪蟹先聚为一支。组织表达结果显示，Lv-ddit4l在眼柄、鳃、肝胰腺、神经、肌肉5种组织中均表达，且表达量基本相同。在温度渐变和温度骤变条件下，Lv-ddit4l在不同组织中表达变化模式有较大差别。与26℃水温(对照组)相比，水温渐变至32℃时，眼柄中Lv-ddit4l表达量显著上调(P<0.05)。之后水温缓慢升至最高温38℃再以同样的速度回温。回温至32℃时，鳃、肝胰腺中Lv-ddit4l表达量显著上调(P<0.05)。在环境水温从26℃骤变至36℃后10 min时，鳃、肝胰腺、神经中Lv-ddit4l表达量显著上调(P<0.05)；24 h时，肌肉中Lv-ddit4l表达量显著上调(P<0.05)。当环境温度分别从38℃、36℃恢复回26℃后，凡纳滨对虾体内原先上调表达的Lv-ddit4l表达量呈下调恢复趋势。研究表明，Lv-ddit4l可能与凡纳滨对虾响应高温胁迫过程相关。

英文摘要:

      Temperature is an important environmental factor that affects the growth, development, and metabolism of shrimp. High temperatures tend to cause a decrease in the immunity and production performance of shrimp, resulting in a significant increase in shrimp disease and mortality. Based on transcriptome sequencing data, we screened for the involvement of DNA damage-inducible transcript 4 like (ddit4l) in the temperature stress process of Litopenaeus vannamei. Ddit4l (also known as Redd2 and Rtp801L) is involved in regulating cell survival, proliferation, and apoptosis by affecting DNA damage repair factors, hypoxia-inducible factors, and other signaling pathways when stimulated by DNA damage, hypoxia, ischemia, and oxidative stress, among others. Ddit4l may be an important transducer of pathological stress in autophagy through mammalian target of rapamycin (mTOR) signaling. Autophagy is a process in which cells use lysosomes to degrade their own damaged organelles and macromolecules, and the process plays an important role in maintaining the balance of protein metabolism and the stability of the intracellular environment, promoting cell growth and development. An important regulator of autophagy is the mTOR, which responds to various stimuli, such as growth factors, cellular energy status, oxygen concentrations, and stress, to regulate cell metabolism and growth. Ddit4l and its homolog ddit4 are upstream mTOR inhibitors in several tissues and cell models. We set up sudden temperature change and gradual temperature change experiments to induce a stress response in L. vannamei. In the gradual change experiment, the water temperature was increased from 26℃ to 38℃ at a rate of 4℃ per day, and cooled back to 26℃ at the same rate. Two individuals were randomly selected from each of the three parallel experiments in the experimental group at each time point. The eyestalk, gill, hepatopancreas, nerve, and muscle were used for subsequent RNA extraction and gene expression quantification. RNA quality and concentration were also examined. The partial ddit4l cDNA (complete ORF and partial UTR) of L. vannamei was obtained directly by PCR amplification to evaluate the structure and function of the Lv-ddit4l gene. The Lv-ddit4l sequence contained a 495 bp open reading frame, encoding 164 amino acids, with a molecular weight of 18.51 kDa, and an isoelectric point of 5.35. The instability index was 65.83, which classifies the protein as unstable. Lv-ddit4l contains a highly conserved RTP801-C domain. BLAST alignment and phylogenetic tree analysis showed that the Lv-ddit4l gene had 59.76% similarity with the Chionoecetes opilio ddit4l gene, and first clustered with C. opilio. A real-time RT-PCR confirmatory experiment considered three parallel groups for each sample with 18S rRNA as the reference gene. Relative gene expression was analyzed using the 2–ΔΔCt method. Quantitative analysis of gene expression levels showed that Lv-ddit4l was widely expressed in all of the examined tissues, with similar expression levels at 26℃. When the temperature was increased gradually from 26℃ to 32℃, the Lv-ddit4l gene in the eyestalk was significantly increased (P<0.05), and was 10.37 times higher than that in the control (A-26℃). Therefore, this gene is hypothesized to respond to temperature changes in the eyestalk of L. vannamei. The expression of Lv-ddit4l in the eyestalk, gills, hepatopancreas, nerves, and muscles did not change significantly when the water temperature was gradually increased to the highest temperature of 38℃; it is speculated that this gene may play a role in pre-heat stress. When the temperature was gradually cooled from 38℃ to 32℃, the expression of Lv-ddit4l increased significantly in the gill and hepatopancreas (P<0.05), which were respectively 3.37 and 2.03 times higher than that of the control group (A-26℃). There was no significant difference in gene expression under different levels of gradual changes in temperature stress in the nerve and muscle. The expression in the gill, hepatopancreas, and nerve was significantly up-regulated (P<0.05) at a sudden change in water temperature to 36℃ for 10 min, and the change in expression in the hepatopancreas was 24.06 times higher than that in the control group (A-26℃). Gene expression in the gills and nerves was 1.22-fold and 3.67-fold higher than that in the control (A-26℃), respectively. When the water temperature was increased to 36℃ suddenly for 24 h, Lv-ddit4l gene expression increased significantly in the muscle (P<0.05). Lv-ddit4l showed significant up-regulation of expression in the gill, hepatopancreas, nerve, and muscle with sudden changes in temperature, suggesting that this gene may play a role in the stress induced by sudden increases in temperature. When the temperature was restored from 38℃ and 36℃ to 26℃, the expression of the Lv-ddit4l gene was downregulated in L. vannamei. The above results indicate that Lv-ddit4l is associated with the response to high-temperature stress.