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毛蚶耐高温品系选育及其应对高温胁迫的响应机制
Alternative TitleBreeding of a heat-resistant strain of Scapharca subcrenata and its response mechanisms to thermal stresses
邹德生
Subtype硕士
Thesis Advisor王春德
2022-05-12
Training institution中国科学院烟台海岸带研究所
Degree Grantor中国科学院大学
Place of Conferral中国科学院烟台海岸带研究所
Degree Name工程硕士
Degree Discipline生物工程
Keyword毛蚶,高温胁迫 生理生化指标 多组学联合分析 响应机制 Scapharca subcrenata Heat-stress Physiological and biochemical indexes Multi-omics analysis Response mechanism
Abstract毛蚶(Scapharca subcrenata)是我国重要的经济贝类种类,在中国贝类养殖业中占有重要的商业地位。近年来,由于夏季持续高温加上弧菌病害频发,常导致毛蚶等滩涂贝类出现大规模死亡。本文针对当前养殖毛蚶耐高温性能差、夏季死亡率高、良种缺乏等问题,收集山东本地的毛蚶野生群体,采用高温连续胁迫的方法选育耐高温的毛蚶品系,并探索毛蚶应对高温胁迫的响应机制,为毛蚶耐高温品系的分子辅助育种打下基础,推动我国毛蚶养殖业的健康、可持续发展。 (1)耐高温毛蚶品系的选育 收集东营海区的毛蚶野生群体,首先通过温度耐受性实验确定本实验毛蚶的96 h高温半致死温度为30.2 ℃。然后采用高温胁迫种贝和高温培养浮游幼虫的方法,选育出第一代本地毛蚶耐高温新品系。 (2)毛蚶响应高温胁迫的比较转录组学分析 为揭示毛蚶耐热的分子机制,本研究首先进行了热应激(30 ℃)下毛蚶血细胞的转录组分析。RNA-Seq和实时荧光定量PCR的结果表明,热休克蛋白(HSPs)、凋亡(TRAF6、GRP78)等可能是调控热应激的关键差异表达基因(DEGs)。GO和KEGG富集分析显示,DEGs主要与凋亡、NF-kappa B信号通路、TNF信号通路和RIG-I-like受体信号通路有关。在高温胁迫下,参与蛋白质生物合成和水解的DEGs的表达显著升高,推测毛蚶可能通过调节蛋白质代谢、DNA复制和抗凋亡系统来适应短期热应激;然而随着高温胁迫时间延长,组织缺氧和代谢失调将对毛蚶造成不可逆的损伤。 (3)毛蚶应对快速和缓慢升温胁迫下的生理和转录响应 为了解毛蚶应对自然升温和快速升温的机制,本研究测定了该实验毛蚶的96小时高温半致死温度,然后研究了它们对快速和缓慢升温(32 ℃)胁迫下的生理和转录反应。快速升温处理组(AHT)的累积死亡率(96小时死亡率52%)明显高于缓慢升温处理组(CHT)(7天死亡率22%)。在两种高温胁迫模式下,血细胞的凋亡率和坏死率呈时间依赖性显著增加。AHT处理组毛蚶抗氧化酶(SOD和CAT)活性在短时间内急剧上升,随后迅速下降,12 h内达到较低水平,而CHT处理组抗氧化酶活在较长时间内维持较高水平。无论AHT还是CHT处理组,丙二醛含量均呈现先升高后逐渐恢复的变化趋势。此外,快速和缓慢升温胁迫均可诱导毛蚶热休克蛋白(HSPs)、凋亡(TRAF6、GRP78和Casp-3)和抗氧化反应(GST和MRP)相关基因表达显著上调,RGN、MT和PRX的表达显著下调。但在CHT处理组中,抗氧化酶的活性和大多数基因的表达可恢复到初始水平,抗逆的可塑性更高。推测抗凋亡系统、抗氧化防御系统和热休克蛋白在毛蚶响应高温胁迫中发挥重要作用。 (4)耐高温毛蚶与野生毛蚶对高温胁迫响应机制 为了解耐高温组(NTR)和野生组(WT)对高温胁迫的响应机制,本研究对NTR和WT毛蚶进行了高温(32 ℃)胁迫,分别在胁迫后24 h和96 h取鳃组织进行转录组学、蛋白质组学和代谢组学关联分析。结果表明,与WT-C相比,WT-H24在差异表达基因(DEGs)、差异表达蛋白(DEPs)和差异表达代谢物(DEMs)GO和KEGG主要富集在膜及其成分、NOD-like受体信号通路、癌症通路和生物素代谢途径;随着时间的延长,WT-H96差异主要富集在核苷酸结合、MAPK信号通路、凋亡通路;而NTR-c在差异主要富集在催化活性、NOD-like受体信号通路、MAPK信号通路和核酸切除修复;NTR-h24主要富集在钙离子结合、PI3K-Akt信号通路;NTR-h96与NTR-h24富集程度相同,但是随着时间的延长不饱和脂肪酸的生物合成途径富集量增加。这些结果表明,相比于毛蚶野生群体,毛蚶耐高温群体本身已经激活抗性(NOD-like受体信号通路)和修复通路(核酸切除修复),再受到高温胁迫时,具有更强的耐热性来抵御外力损伤,但是在长时间的高温胁迫下毛蚶的鳃组织也会逐渐出现细胞损伤现象。 本研究揭示了毛蚶响应高温胁迫的分子机制,表明适当地利用缓慢升温胁迫方法有助于选育毛蚶耐高温新品种,研究结果可为毛蚶耐高温新品系的分子标记辅助育种奠定基础。
Other AbstractThe ark shell, Scapharca subcrenata, is an economically important shellfish in China. In recent years, high temperature, frequently combined with the occurrence of Vibrio diseases in summers often leads to massive mortality in mudflat shellfish. Aiming at these problems, this study focused on the selection of heat resistant strain from local populations from Shandong Province and the exploration of the response mechanisms of ark shell to thermal stress. Our results may provide essential basis for molecular assisted breeding of heat-resistant strains of ark shell, and promote healthy and sustainable development of ark shell aquaculture industry in China. (1) Selection of a heat-resistant strains of ark shellIn this study, semi-lethal upper temperature of ark shell was determined as 30.2 °C by temperature tolerance test. The first generation of new heat-resistant strains of ark shell was reproduced from the brook stocks selected by thermal stresses. (2) Comparative transcriptome analyses of ark shell in response to thermal stresses To reveal the underlying molecular mechanisms of heat resistance (32 ℃) in the ark shells, transcriptomic analyses were carried out with haemocytes sampled from ark shells exposed to thermal stress. RNA-seq and quantitative real-time PCR showed that heat shock proteins (HSPs) and apoptosis (TRAF6, GRP78) may be the key differentially expressed genes (DEGs) regulating heat stress. GO and KEGG enrichment analyses showed that the DEGs were mainly associated with apoptosis, NF-kappa B signaling pathway, TNF signaling pathway and RIG-I-like receptor signaling pathway. Noteworthily, the expression of DEGs involved in protein biosynthesis and proteolysis was significantly elevated in ark shells under heat stress. It is speculated that the ark shell may adapt to short-term heat stress by regulating protein metabolism, DNA replication and anti-apoptotic system. However, with the aggravation of high temperature stress, tissue hypoxia and metabolic disorder will cause irreversible damage to the S. subcrenata. (3) Physiological and transcriptional responses to acute and chronic thermal stress in ark shell In order to understand the mechanism of response to acute or chronic stress rise of the ark shell, this study first measured the 96 h median lethal temperature of the ark shell, and then studied their physiological and transcriptional responses to the stress of rapid and slow temperature (32 ℃) rise. A significantly higher cumulative mortality (52% in 96 h) was observed in the acute heating treatment (AHT) group than that (22% in 7 days) in the chronic heating treatment (CHT) group. The apoptosis and necrosis rates of haemocytes were increased significantly in a time-dependent manner under both thermal stress strategies. Activities of antioxidant enzymes (SOD and CAT) increased dramatically in a short time followed by a quick decline and reached to a lower level within 12 h in the AHT group, but maintain relatively high levels over a long period in the CHT group. The contents of MDA were increased significantly firstly and restored to the original later in both acute and chronic thermal stress. Moreover, expression of the genes related to heat shock proteins (HSPs), apoptosis (TRAF6, GRP78, and Casp-3) and antioxidant responses (GST and MRP) could be induced and up-regulated significantly by thermal stress, expression of RGN, MT and PRX was down-regulated dramatically under the two heating treatments. However, the activity of antioxidant enzymes and the expression of most genes could be restored to the initial level in the CHT, so the plasticity of stress resistance was higher. These results suggested that anti-apoptotic system, antioxidant defense system and heat shock proteins could play important roles in thermal tolerance of ark shells. (4) Response mechanism of heat-resistant and wild ark shell strains to thermal stress In order to understand the response mechanism of heat-resistant strain (NTR) and wild strain (WT) to thermal stress, transcriptomic, proteomics and metabolomics association analyses were performed with gills sampled from animals from the NTR and the WT strain after thermal stress (32 ℃). The results showed that compared with the WT that were not exposed to thermal stress, GO and KEGG in differentially expressed genes (DEGs), differentially expressed proteins (DEPs) and differentially expressed metabolites (DEMs) of the WT-H24 were mainly enriched in membrane and its components, NOD-like receptor signaling pathway, cancer pathway and biotin metabolism pathway. With the extension of time, the differences in the WT-H96 were mainly enriched in nucleotide binding, MAPK signaling pathway and apoptosis pathway. However, in the NTR that were not exposed to thermal stress, the differences were mainly enriched in catalytic activity, NOD-like receptor signaling pathway, MAPK signaling pathway and nucleic acid resection repair. The NTR-h24 was mainly enriched in calcium ion binding and PI3K-Akt signaling pathway. The enrichment of DEGs, DEPs and DEMs in the NTR-h96 group was similar to that of the NTR-h24, but the unsaturated fatty acid biosynthesis pathway was gradually enriched with the extension of time. These results indicate that compared with the WT-C, the resistance (NOD-like receptor signaling pathway) and repair pathway (nucleic acid resection repair) have been activated in the NTR-C group, which exhibited a stronger heat resistance to resist external damage when subjected to thermal stress again. However, under the long-term high temperature stress, cellular damage will gradually become apparent in the gills of the ark shell. The results herein may reveal the molecular mechanism of response to high temperature stress of the ark shells and suggest that the method of slow heating stress could be used in the breeding of a heat-resistant strain of S. subcrenata. The results in this study may also lay the groundwork for marker-assisted selection of heat-resistant strains in S. subcrenata.
Pages106
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/34399
Collection中国科学院烟台海岸带研究所知识产出_学位论文
Recommended Citation
GB/T 7714
邹德生. 毛蚶耐高温品系选育及其应对高温胁迫的响应机制[D]. 中国科学院烟台海岸带研究所. 中国科学院大学,2022.
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