Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
|Place of Conferral||中国科学院海洋研究所|
|Keyword||皱纹盘鲍（Haliotis discus hannai Ino） 耐热性 代谢生理 代谢组 转录组|
皱纹盘鲍（Haliotis discus hannai Ino）属于原始腹足目、鲍科、鲍属，是我国具有高经济价值的养殖贝类之一。近年来，夏季大规模死亡事件频繁发生，已成为制约产业发展的瓶颈问题，其原因及解决办法尚无深入认识。该物种自然分布于我国北部沿海、朝鲜沿岸及日本东北部，为低温种，适宜温度范围为15-22°C。21世纪以来，随着人工育苗与养殖技术的飞速发展，我国福建、广东沿海已与辽东、山东半岛一样形成商品化规模，成为皱纹盘鲍的主要产区。国内鲍养殖企业大多采用“南北调养”的养殖模式，即每年的11月初，将壳长2-3cm鲍苗运往南方越冬，次年5月运回北方。在这个过程中部分鲍留在南方继续进行全年养殖，相当于经历了暖驯化的过程，该过程是否对鲍的适温性提高产生作用，是否对鲍遗传育种具有利用价值，还有待研究。同时，鲍应对热胁迫的响应机制方面，尤其是在生理生化响应及分子生物学机制方面的认知仍远远不足。这些问题的解决对选育优良品种、优化养殖策略具有重要意义。本研究以实验室驯化的皱纹盘鲍幼鲍为研究对象，从表型、生理特征、代谢产物和转录水平四个层面就皱纹盘鲍一龄幼鲍对温度胁迫的响应及耐热性机制进行研究，旨在为鲍产业可持续发展提供数据支撑。主要研究结果如下：
经过62天的低温（10°C）和高温（30°C）驯化养殖，低温驯化群体（L 组）没有发生一例死亡现象，保持了100% 的存活率，高温驯化群体（H 组）也有接近80% 的存活率；经过24小时连续监测，后者平均耗氧率是前者的1.98倍，差异显著（t = -5.912、P = 0.000），表明H群体具有更高的有氧代谢率；L 群体幼鲍的平均壳长增长率、壳宽增长率和增重率分别是 H 群体相应指标的5.10、25.17和168倍。表明环境温度对幼鲍生长、增重具有极大影响，与低温相比，过高的培养温度会增加幼鲍代谢率并抑制其生长和增重。然而高温驯化后的幼鲍表现出对高温胁迫的耐受性。将 L 组和 H 组各45只幼鲍缓慢升 / 降至20°C，稳定1周后置于31°C海水中进行热激，L 组24小时后全部死亡，而H组在48小时的观测期内仅1例死亡。
H 群体幼鲍血糖含量显著高于L 群体（t = -2.615、P = 0.031），表明高温环境下幼鲍对能量的需求更高；H群体的丙氨酸氨基转移酶（ALT）和天门冬氨酸氨基转移酶（AST）活力较高，血蓝蛋白含量较L群体略低，但这三项指标两群体间差异不显著（P > 0.05）；通过对腹足肌肉、鳃、外套膜和肝胰腺相关生理生化指标的分析发现，H 群体较L 群体幼鲍组织内含有更多的游离葡萄糖（GLU），两个主要无氧代谢酶——乳酸脱氢酶（LDH）和亚牛磺酸脱氢酶（TDH）活力也有不同程度的提高，表明高温下无氧代谢也参与了细胞能量供应；另外，H 组幼鲍各组织SOD活力普遍高于 L 组，但CAT活力在除腹足肌肉外的其他3个组织（鳃、外套膜和肝胰腺）却明显低于 L 组，且鳃和外套膜组织中脂质过氧化标志物MDA含量有显著增加（P < 0.05），而维持高酶活和修复过氧化损伤会增加H 组鲍细胞对能量的需求。
将L和H两个群体于20°C水温下稳定一周后，于31°C下进行3小时的急性热激，分别标记为LH 和HH 组。L 和 H两个群体幼鲍热胁迫前后分别共筛选到了1815和1314个显著差异代谢物，L 组差异代谢物排在前三位的是羧酸及其衍生物类（17.65%）、二元胺类（8.82%）和酰基脂肪酸类（8.82%），而H 组则为有机氧合化合物类（18.92%）、羧酸及其衍生物类（16.22%）和二元胺类（5.41%）。两组鲍在热激前后的差异代谢物种类上表现出明显差异。
以Fold Change > 1.5 或 < 0.6 且VIP > 1.5为标准从两组分别筛选出42和100个与热胁迫响应相关的代谢标志物，其中有28个代谢物重叠。在两组共同的代谢标志物中， 2-甲基丁酰肉碱（2-Methylbutyroyl-carnitine）含量显著增加（P < 0.05），该物质在正常细胞中检测不到，它的增加与一种缬氨酸分解酶异丁基辅酶a脱氢酶功能障碍或缺失有关。犬尿素（L-Kynurenine）和吲哚-3-乳酸（DL-Indole-3-lactic acid）在热胁迫后的细胞内也有显著积累（P < 0.05），这两种物质是色氨酸分解代谢过程的中间产物，在正常细胞中的含量处于动态平衡状态，过量将引发一系列恶性反应，如神经功能紊乱和细胞凋亡。犬尿素的大量积累与犬尿素-3-单加氧酶功能障碍有关。热胁迫还导致酪氨酸代谢中产物N-乙酰-L-酪氨酸（N-Acetyl-L-tyrosine）含量的显著增加，而一种细胞质酶芳香族-L-氨基酸脱羧酶的功能失常可以引起该物质在细胞中大量积累。戊二酸（Glutaric acid）只在于L 组高温胁迫后的组织中有大量的积累，该物质及其衍生物具代谢毒性，会导致神经功能紊乱和肌肉痉挛。戊二酸含量的大幅增加至少与三种蛋白功能障碍有关，分别是戊二酰辅酶a脱氢酶、电子转移黄素蛋白和电子转移黄素蛋白-泛素氧化还原酶，前者参与赖氨酸、色氨酸和羟赖氨酸的线粒体氧化代谢，后两在蛋白质和脂质分解中起重要作用，并协助产生能量。
此外，与脂肪酸氧化障碍（FAOD）相关的代谢标志物如硬脂酰肉碱（Stearoylcarnitine）含量在热激后的细胞内也显著增加（P < 0.05），这一现象被证明与脂酰肉碱移位酶Ⅱ功能缺失有关，它是一种线粒体内膜酶，它的功能障碍导致长链脂酰基肉碱无法释放肉碱分子变回脂酰辅酶a进行b-氧化，而是在细胞内蓄积，因此，我们同时在LH与HH组也检测到了L-棕榈酰肉碱（L-Palmitoylcarnitine）水平的显著提高（P < 0.05），在LH组检测到了肉碱（L-Carnitine）含量的显著降低（P < 0.05），而长链脂酰肉碱分子具有细胞毒性，可以影响细胞膜的分子动力学特性及引发细胞凋亡。辛二酸（Suberic acid）含量的显著增加也在两个热激组（LH与HH）中被检测到（P < 0.05），该物质的累积至少与三种脂肪酸氧化代谢相关酶的功能障碍有关，它们分别是：中链酰基辅酶a脱氢酶、肉碱/脂酰肉碱转位蛋白和丙二酰辅酶a脱羧酶。而与中链酰基辅酶a脱氢酶功能异常有关的另一种代谢标志物——C10脂酰肉碱（Decanoy-L-carnitine）在LH组被检测到含量显著上调（P < 0.05）。
另外，一些有毒代谢物质如戊二酸（Glutaric acid）、癸酰肉碱（Decanoy-L-carnitine）、古洛糖内酯（L-Gulonic gamma-lactone）和b-丙氨酸（beta-Alanine）只在LH组内检测到显著的增加（P < 0.05）；而一些稳定细胞内环境的有益物质如葡萄糖-6-磷酸（D-Glucose-6-phosphate）、4-羟基肉桂酸（4-Hydroxycinnamic acid）、反式-2-羟基肉桂酸（trans-2-Hydroxycinnamic acid）和N-乙酰-葡萄糖胺（N-Acetyl-D-glucosamine）和巯丙甘氨酸（Tiopronin）只在HH组内含量显著增加（P < 0.05）。与促进、稳定代谢相关的代谢物如核黄素（Riboflavin）、泛酸（Pantothenate）和胆碱（Choline）以及与稳定和修复生物膜、神经保护有关的CDP-胆碱（CDP-choline），HH组的含量显著高于LH组（P < 0.05）。两个驯化及热激组的代谢物差异表明，经历高温驯化的幼鲍在促进稳定代谢、修复细胞损伤和保护神经组织方面拥有明显的优势，这可以从代谢层面帮助解释其耐热性高的原因。
以Fold Change ≥2 且FDR < 0.01为标准对各比较组差异表达基因进行筛选，L和H 群体热激前后分别有478和709个基因出现差异表达，前者包含315个上调基因，163个下调基因，后者包含562个上调基因和147个下调基因。L vs LH 与 H vs HH两个组存在246个共同差异基因，各自分别有232和463个特有的差异基因。通过KEGG富集分析发现，两组的共同差异基因在内质网蛋白加工途径具有最显著的富集，相关基因多数为分子伴侣蛋白。对皱纹盘鲍5个热激蛋白家族基因与其他物种进行比较分析，结果表明，在基因数量方面，皱纹盘鲍除HSP 90家族基因数量较多外，但其他家族基因数量较长牡蛎少。
对参与内质网蛋白加工途径的分子伴侣蛋白、泛素化过程相关蛋白以及涉及自噬、凋亡相关的17个基因进行qPCR验证，基因表达趋势均与转录组差异分析结果一致。蛋白质识别加工途径上的基因包括 DnaJC 3（属于Hsp 40家族）、CCT7（也被称为TRiC，参与蛋白质折叠）以及2个NEF基因（HYOU1和SIL1）的mRNA含量在两个群体热激后显著积累（P < 0.05），HSPA5（也被称为Bip 或者 GRP 78，属于HSP 70家族）基因在热胁迫后虽然表达量有所上升，但没有显著变化（P > 0.05）；内质网相关的蛋白质降解途径上有5个伴侣蛋白基因（HSPA1S、HSP110、HtpG、CRYAB和BAG3）和1个泛素蛋白Ubiqutin基因表达水平也在两组热激后均有显著上调（P < 0.05）。尤其是基因CRYAB（属于HSP 20家族）在热激后的转录水平升高万倍以上，表明Alpha-crystallin蛋白在皱纹盘鲍幼鲍热胁迫响应调控方面起着重要的作用。与泛素化相关的其他基因中，E1蛋白基因在高温组（H和HH）中的表达水平显著高于低温组（L和LH），但是热激前后的表达量没有显著变化（P > 0.05）；E2蛋白基因热激前后表达无显著差异， 但是H和HH组总体比L和LH组高，且HH显著高于L和LH组；E3蛋白基因在HH组的表达量显著高于其他3组（P < 0.05）。此外，与蛋白重新折叠和降解有关的CRYAA蛋白、与有毒聚集物清除有关的p62蛋白以及凋亡抑制蛋白cIAP1的基因也是HH组的表达量显著高于其他3组（P < 0.05）。表明高温驯化群体经热激后在错误折叠蛋白的降解方面存在一定的优势，这可以从分子层面帮助解释其具有更高耐热性能的原因。
Pacific abalone Haliotis discus hannai Ino is one of the cultured vetigastropoda with high economic value in China. In recent years, frequently occurred massive summer mortality has restricted the industry development, while few is known on the causes. Pacific abalone, with the preference temperature range 15-22°C, naturally inhabit along northern Yellow Sea to the coast of Korea and northern east Japan. Since the 21st century, with the rapid development of artificial nursery and aquaculture technology, Fujian and Guangdong have become major production areas of Pacific abalone. Most aquaculture companies applied "north-south relay" strategy to culture Pacific abalone. In early November, abalones with shell length of 2-3cm are transported to the south for overwintering, and then returned to the north in May of next year for estivating. Some abalones are retained at south in summer and thus experience warm acclimation during this process. It is still unknown whetehr warm acclimation benefit for high temperature tolerance. At the same time, understanding the mechanism of abalone thermal stress response is urgently need, especially from the perspectives of metabolism and molecular level, to provide theoretical support for new aquaculture mode development and genetic improving of abalones. In this study, we use H. discus hannai juvenile to analyze the thermal response and tolerance mechanism from four aspects: phenotype, physiological characteristics, metabolites and mRNA level. Main results are as follows:
1. Phenotypic adaptability of temperature in Haliotis discus hannai juvenile
At the end of the 62-day culture process, there was no death in the low-temperature acclimated population (group L), which maintained a 100% survival rate, while the high-temperature acclimated population (group H) had a survival rate of nearly 80%. We calculated average oxygen consumption rate (OCR) via 24-hour continuously monitoring the dissolved oxygen change. Rusult showed that OCR of individuls from group H was 1.98-fold as many as that of ones from group L (t = -5.912, P = 0.000). High OCR means high aerobic metabolic rate and more ATP output of oxidative phosphorylation (OXPHOS). Nevertheless, this increased energy flux didn’t be used for the accumulation of body composition because of lower values of the average shell growth rate, shell width and bady weight growth rates of abalone from group H (0.196, 0.040 and 0.006 of corresponding values of ones frome group L, respectively) indicating environmental temperature had a great influence on the growth performance of juvenile abalone, namely, excessive high temperature inhibits abalone’s growth and weight gain. Although individuals in group H sacrificed theirs rapid growth performance, they had higher heat tolerance. After being matained at same temperature (20°C) for one week, 45 abalones from each group were exposed to 31°C immediately, the mortality rate of L group was 100% while the number of H group was only 2.2% during 48-hour observation period.
2. Metabolic adaptability of temperature in Haliotis discus hannai juvenile
The hemolymph glucose (GLU) content of juvenile abalone in H group was significantly higher than that in L group (t = -2.615, P = 0.031), indicating that abalone under high temperature environment had higher demand for energy substance. Higher activity of ALT and AST together with lower content of hemocyanin was observed in hemolymph of ones from group H, but there were no significant difference between the two groups in these three parameters (P > 0.05). The physiological and biochemical analysis results of the foot muscle, gill, mantle and hepatopancreas showed that GLU concentrations and the activities of two major anaerobic metabolic enzymes of abalone--lactate dehydrogenase (LDH) and tauropine dehydrogenase (TDH) in these four tissues from H group were increased compared with those from L group indicating that anaerobic metabolism took part in fueling the cellular energy demand. Otherwise, the SOD activities in four tissues from H group were generally higher than that from group L whereas the CAT activities in gill, mantle and hepatopancreas were lower than that of L group. The content of lipid peroxidation biomarker--malonaldehyde (MDA) in gill and mantle were significantly higher in group H, this is one of the reason why organisms require more energy under heat stress.
3. Analysis of heat stress response and heat resistance of Haliotis discus hannai based on metabolome
Abalones from two groups were kept at 20°C for one week in order to homogenization then put into 31°C immediately and incubated for 3 hours, labeled as LH and HH respectively. The hepatopancreas from group L, H, LH and HH were sampled for metabolome analysis. In total, 1815 and 1314 significantly changed metabolites were found in LH and HH group compared with L and H group, respectively. Most of these metabolites in L vs LH were carboxylic acids and derivatives (17.65%), diazines (8.82%) and fatty acyls (8.82%), while the top three classes of differential metabolites in H vs HH were organooxygen compounds (18.92%), carboxylic acids and derivatives (16.22%) and diazines (5.41%) indicating the differential metabolites undergoing heat shock between the two groups were obviously unlike.
A number of 42 and 100 signifcantly different metabolite biomarkers (MBs) were identifed in L vs LH and H vs HH, respectively, with parameter setting is FC > 1.5 or < 0.6 and VIP value > 1.5. Among the 28 same MBs of these two group, associated with partly break up of amino acids in mitochondria were significantly accumulated. Such as 2-methylbutyroylcarnitine (P < 0.05), usually not detected in normal individuals, the elevation of 2-methylbutyrylcarnitine suggests a deficiency of isobutyryl-CoA dehydrogenase, which helps break down the amino acid valine. The L-kynurenine and DL-Indole-3-lactic acid which are partly break down metabolites of the amino acid tryptophan accumulated significantly (P < 0.05) in cells after heat stress. Kynurenine-3-monooxygenase (KMO) deficiency contributes to an accumulation of kynurenine and high level of this metabolite is proved to be associated with neurotransmitter metabolic disorders. DL-Indole-3-lactic acid induces caspase-8 and caspase-9, which results in caspase-3 activation and then apoptosis. Heat stress also led to a significant increase in the content of N-Acetyl-L-tyrosine (P < 0.05), a intermediate product of tyrosine metabolism and associated with aromatic L-amino acid decarboxylase deficiency. Significant high glutaric acid only be detected in LH group compared with group L (P < 0.05). Chronically high levels of glutaric acid are associated with at least three enzymes dysfunction including glutaryl-CoA dehydrogenase, electron transfer flavoprotein (ETF) and electron transfer flavoprotein dehydrogenase (ETF-QO). The glutaryl-CoA dehydrogenase deficiency in which the cell is unable to completely break down the amino acids lysine, hydroxylysine, and tryptophan. When one of ETF and ETF-QO is defective or missing, the mitochondria cannot function normally, partially broken-down proteins and fats accumulate in the cells and damage them.
Parallelly, cellular contents of MBs associated with fatty acid oxidation disorder (FAOD) were observed of a large increment in ones expoesed to heat stress (P < 0.05). Such as stearoylcarnitine and L-palmitoylcarnitine which are found in significantly greater amounts of cells with carnitine palmitoyltransferase (CPT) II deficiency that is a metabolic disorder characterized by this enzymatic defect that prevents long-chain fatty acids from being transported into the mitochondria for utilization as an energy source. The long chain acylcarnitines have cytotoxicity, which can affect the molecular dynamics of cell membrane and induce apoptosis. Large amount change of suberic acid was detected in both LH and HH group (P < 0.05). Elevated levels of this unstaruated dicarboxylic acid are found in individuals with medium-chain acyl-CoA dehydrogenase deficiency (MCAD). Suberic acid is also found to be associated with carnitine-acylcarnitine translocase (CACT) deficiency, malonyl-CoA decarboxylase deficiency. Other metabolic biomarker, the decanoy-L-carnitine was detected significantly accumulated in LH group (P < 0.05). In a word, high temperature can result in incomplete decomposition of amino acids and fatty acids in hepatopancreas cells of juvenile abalone, illustrating that the pathway of oxidativing these two energy substances to yield energy was dysfunction. This phenomenon is first observed in marine mollusc.
When organisms suffering heat stress, demand of energy greatly increased. Due to the fact that mitochondria are energy factories of cells, the stability of mitochondrial structure and function plays an important role in heat stress tolerance of Haliotis discus hannai. Some metabolic toxic substances such as glutaric acid, decanoy-L-carnitine, L-gulonic gamma-lactone and beta-alanine were found significantly increased only in the LH group (P < 0.05). While some beneficial substances such as D-glucose-6-phosphate, 4-hydroxycinnamic acid, trans-2-hydroxycinnamic acid, N-acetyl-D-glucosamine and tiopronin were able to accumulate rapidly only in the individual of group HH (P < 0.05). In addition, metabolites related to promoting and stabilizing metabolism such as riboflavin, pantothenate, choline as well as CDP-choline which is related to stabilizing and repairing biomembrane and neuroprotection in hepatopancreas of group HH was significantly higher than that of group LH (P < 0.05). In a word, individuals of group H had obvious advantages in promoting, stabilizing metabolism, repairing molecular damage and protecting nerve, which can help explain the high heat tolerance of abalones from this group.
4. Analysis of heat stress response and heat resistance of Haliotis discus hannai based on transcriptome
A total of 12 mRNA samples form L, LH, H and HH group (4 group and 3 repetitions in every group) were sequenced then the clean data were mapped to the reference genome. Based on the mapped results, 24,464 of novel genes were functionally annotated. Gene expression analysis was performed and differentially expressed genes were identified based on their expression levels in different samples. There were 478 differential expression genes in group L vs LH including 315 up-regulated genes and 163 down-regulated genes while 709 genes in group H vs HH among which 562 up-regulated and 147 down-regulated. KEGG pathway enrichment analysis of shared differentially expressed genes of L vs LH and H vs HH was executed and results showed that the most significant pathway is protein processing in endoplasmic reticulum and genes enriched in this pathway were mostly molecular chaperone. The gene expression of 17 genes including molecular chaperone, ubiquitination-related proteins together with proteins involved in autophagy and apoptosis were estimated by qPCR assay. The gene expression pattern were consistent with the transcriptome results.Data showed that mRNA levels of HYOU1 and SIL1, DnaJC 3 (belonging to HSP 40 family) and CCT7 these genes involved in protein recognition and processing were incresased significantly in the hepatopancreas of two populations after heat shock (P < 0.05). Although the transcriptional level of gene encoding HSPA5 (also known as Bip or GRP 78) increased after heat stress, there was no significant change (P > 0.05). The mRNA contents of five chaperone proteins (HSPA1S、HSP110、HtpG、CRYAB and BAG3) in ER-related protein degradation pathway as well as Ubiqutin, were significantly up-regulated after heat shock in both groups (P < 0.05). It is noteworth that the transcription difference of CRYAB coding gene was more than 10,000 times pointing that alpha-crystallin protein may play an important role in the regulation of heat stress response in juvenile abalone Haliotis discus hannai. The expression levels of E1 and E2 protein gene did not change significantly before and after heat shock (P > 0.05). The expression level of E3 protein encoding gene, which regulates cell cycle, apoptosis, transcription, protein quantity control and signal transduction, increased significantly only in group HH (P < 0.05). In addition, the mRNA concentrations of the CRYAA protein which is involved in protein refolding and degradation, the p62 protein which is associated with toxic aggregate clearance were only significantly increased in the HH (P < 0.05). The gene expression of apoptosis inhibitor cIAP1 was increased significantly in LH and HH compared with L and H, respectively (P < 0.05) and value of HH was even higher than that of LH (P < 0.05). This indicates that from the aspect of regulation of cell fate, group H has a certain degree of advantage, which is one of the reasons for its strong heat resistance.
|Subject Area||海洋科学 ; 海洋生物学 ; 生物学 ; 生物化学 ; 脂类生物化学 ; 新陈代谢与营养生理学 ; 分子生物学|
|MOST Discipline Catalogue||理学 ; 理学::海洋科学 ; 理学::生物学|
|Funding Project||National Natural Science Foundation of China ; National Natural Science Foundation of China ; National Natural Science Foundation of China ; National Natural Science Foundation of China|
|Table of Contents|
|高婷婷. 高温与低温驯化下皱纹盘鲍幼鲍耐热性的代谢组学与转录组学比较分析[D]. 中国科学院海洋研究所. 中国科学院大学,2019.|
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