实验海洋生物学重点实验室知识库

鲆鲽鱼类大多为较高纬度分布的重要海水养殖鱼类，冬季养殖都面临着较长的低温期。其中，大菱鲆Scophthalmus maximus是由欧洲引入我国的海水养殖鱼种，-1°C 到20°C 均可存活，但低于8℃时其生长速率显著降低。北方养殖中，每年要经历4个月的低温，使养殖场的生产效益显著降低。牙鲆Paralichthys olivaceus是中、韩、日三国的重要海水养殖鱼类。同样，冬季的低温海水影响其生长和存活，进而影响其养殖产业的产出。而有关鲆鲽鱼类的耐低温研究才刚刚开始，其耐低温机制、育种连锁标记等研究也几乎没有系统报道。本研究对雌核发育大菱鲆低温耐受组和敏感组进行转录组和miRNA高通量测序，筛选出低温胁迫下差异表达基因和miRNA，并进行关联分析，初步阐释低温下的miRNA-TF-mRNA调控网络及其耐低温的机制。同时，研究了低温下牙鲆能量代谢的关键调控因子AMPK及其相关基因表达变化，以及细胞水平上激活剂和抑制剂对该通路的调控。并在hsp70、hmgb1以及yb-1等耐低温相关基因中筛选了牙鲆低温相关的SNP。这些结果将为鲆鲽鱼类耐低温机制阐述与育种研究提供基础数据。具体研究结果如下：

1．雌核发育的大菱鲆幼鱼经0°C低温处理，获得低温耐受组（CT）和低温敏感组（CS）。对其进行RNA-seq和miRNA-seq高通量测序和分析。结果显示，相对于对照组，低温耐受组和敏感组对低温的应答在mRNA和miRNA水平上明显不同， mRNA很多上调，而miRNA的变化较少。进一步分析发现，miRNA-转录因子-mRNA网络参与了细胞周期、细胞膜组分、信号转导和昼夜节律通路的调控，从而调控大菱鲆对低温胁迫的应答。

2．对牙鲆雌核发育家系的鳃、肌肉及肝脏等组织在低温下（0℃）的组织形态变化进行了研究，发现低温对鳃的组织结构影响较大，肌肉组织结构几乎没有变化，但肝脏组织却有一定的损伤，呈现细胞凋亡的趋势。进一步对牙鲆AMPK及其相关基因低温胁迫下脑、肌肉和心脏组织中差异表达图式进行了分析，并在细胞水平探讨了其调控机制。结果表明，低温激活了脑组织中LKB1、CaMKKβ及AMPK基因， AMPK又激活了SITR1、FOXO1A和TFAM；在肌肉组织中，这些基因表达呈现出先降后升的趋势，对低温的应答存在滞后现象。在细胞水平，低温和激活剂AICAR均激活了AMPK及相关基因，抑制剂CC抑制了这些基因的表达。由此可以初步推断低温下AMPK的调控机制：首先低温胁迫通过Ca²⁺ 和AMP/ATP激活了LKB1 和CaMKKβ，进而激活了AMPK；AMPK又激活了FOXO1A和SIRT1，从而加强糖酵解和线粒体的生物合成，产生更多的能量，并增强抗氧化能力；活化的AMPK同时抑制了HMGCR，使胆固醇生物合成受阻。总的来说，在低温胁迫中，AMPK不但参与了能量调控还参与了对低温的应答。

3. 对hsp70、hmgb1及yb-1基因部分序列在牙鲆低温耐受组和敏感组进行SNP分子标记筛选。分别从hsp70、hmgb1及yb-1 获得了9、21和10个SNP位点。其中，hsp70和hmgb1各有3和2个SNP在低温耐受组和敏感组之间存在显著性差异；hsp70基因SNP 8的等位基因G仅出现在耐受组；hmgb1 SNP 7的基因型TT和等位基因T与耐低温相关，而基因型CC和等位基因C与低温敏感相关。单倍型分析发现，hsp70 基因的单倍型TTG与牙鲆的耐低温相关，而hmgb1基因的单倍型ATG和TCT分别与低温耐受和低温敏感相关联。

Most flatfish belong to important cultural fish at high latitude, and face a long period of low temperature in winter. Among them, turbot Scophthalmus maximus introduced from Europe has been becoming an important maricultural fish species in north China. It could live at temperature of -1 to 20°C but its growth rate would be significantly decreased when temperature below 8°C. There are about four months one year with lower temperature which makes fish growth decrease, and causes serious economic losses to farmers or enterprises. Olive flounder Paralichthys olivaceus is also a major mariculture flatfish species in China, Korea and Japan. Similarly, low temperature in winter influences its survival and growth, and then affects the output of its aquaculture industry. The research on flatfish cold tolerance is just beginning, and there few reports on the mechanism of cold tolerance and marker-assisted selective breeding. Therefore, it is necessary to strengthen relevant research. In this study, RNA-seq and miRNA-seq were performed under the cold condition in turbot with high-throughput sequencing, and differential genes and miRNAs under cold stress were screened. The related miRNAs and TFs to the cold tolerance were analyzed in this fish species, and the role of miRNA-TF-mRNA network was also revealed. In the meantime, the expression changes of AMPK, the key regulators of energy metabolism, and its upstream and downstream gene expression were analyzed in flounder under low temperature. And the regulation of activator and inhibitor on AMPK pathway in the cellular level was also analyzed. The SNPs of hsp70, yb-1 and hmgb1 associated with cold-tolerance were screened. These results will provide basic data for studying the mechanism of cold tolerance and breeding research in flatfish. The main research results are as follows:

1. Meio-gynogenetic diploid turbots were treated at 0°C to distinguish the cold-tolerant (CT) and cold-sensitive (CS) groups. The transcriptional reactions of them were analyzed by using RNA-seq and microRNA(miRNA)-seq. The results showed that there were quite different responses at both mRNA and miRNA levels, with more up-regulated mRNAs (1,069 vs. 194) and less down-regulated miRNAs (4 vs. 1) in CT versus CS relative to the control group. The network of miRNA- transcription factor- mRNA, regulating turbot different response to cold stress, was constructed, which involved in cell cycle, component of cell membrane, signal transduction and circadian rhythm pathways.

2. The gill, muscle and liver histological structure changes of gynogenetic flounder juveniles treated under 0°C were studied, and the results indicated that the gill tissue was obviously damaged and more serious lesions in some of the epithelial cell layers were observed in the CS group. There was almost no changing in the muscle tissue, while there is apoptosis in liver tissue with certain injure. The expression of flounder AMPK and its upstream and downstream genes in brain, muscle and heart tissues under cold stress were analyzed, and the regulation mechanism was explored at the cellular level. The results showed that low temperature activated LKB1, CaMKKβ and AMPK genes in the brain tissue, and the activated AMPK induced SITR1, FOXO1A and TFAM. In muscle tissue, the expression patterns of these genes showed a trend of decreasing firstly and then increasing, and there was a delay in response to low temperatures. At the cellular level, both cold stress and activator- AICAR activated AMPK pathway with a hysteresis, and inhibitor-CC inhibited these genes’ expression in the cells. Thus, the regulation mechanism of AMPK under cold stress was preliminarily analyzed. Cold stress activated LKB1 and CaMKKβ through AMP and Ca²⁺, led to AMPK to be activated, activated FOXO1A and SIRT1, which enhanced glycolysis and mitochondrial biosynthesis and produced more energy, resulting in enhancing ability of antioxidant stress. In the meanwhile, the activated AMPK inhibited HMGCR expression, and caused the inhibition of cholesterol biosynthesis. In general, AMPK is involved not only in energy regulation under low temperature but also in the responses to low temperature.

3. The correlation analyses between single nucleotide polymorphism locus (SNP) in flounder hsp70, yb-1 and hmgb1 and the trait of cold tolerance were performed. Nine, twenty-one and ten SNPs were screened from these genes’ partial sequences, respectively. Among them, 3 SNPs in hsp70 and 2 SNPs in hmgb1 showed significant difference in the two groups. Allele G of the SNP 8 (locus 1797) in hsp70 was only observed in the CT group. In the SNP 7 (locus 725) of hmgb1, genotype TT and allele T, and genotype CC and allele C were associated with cold tolerance and cold sensitive, respectively. Furthermore, one haplotype (TTG) generated from SNPs of hsp70 gene among CT and CS individuals showed significant relationship with cold tolerance. Two haplotypes (ATG) and (TCT) generated from SNPs of hmgb1 were significantly associated with resistance and sensitive in cold treatment, respectively.