Knowledge Management System Of Institute of Oceanology, Chinese Academy of Sciences
|Alternative Title||The role of heat shock transcription factor1 and heat shock proteins respond to thermal stress in oysters|
|Place of Conferral||中国科学院海洋研究所|
|Keyword||牡蛎 热激 热激转录因子1 热激蛋白 调控关系|
作为栖息在潮间带的重要经济物种，牡蛎经常受到强烈的温度波动胁迫。近年来，随着全球变暖趋势加剧，栖息在潮间带的生物的生存压力与日俱增。牡蛎的夏季大规模死亡现象对产业造成巨大经济影响，其被认为与高温胁迫有关，因此研究牡蛎热适应机制具有重要生态和产业意义。本研究选取具有明显的地理分布和热适应能力差异的巨蛎属两姊妹亚种—长牡蛎（Crassostrea gigas gigas）和福建牡蛎（C. gigas angulata），通过对HSF1在牡蛎热激过程中的作用及其与HSP之间的调控关系的了解来阐释牡蛎热耐受性差异的调控机制。我们首先克隆鉴定热激响应的HSF1不同亚型；而后通过转录组（HSF1干扰后的热应激）和染色质免疫沉淀后测序（ChIP-seq）研究HSF1在热激中作用及其下游调控基因；之后在两姊妹亚种（C. gigas gigas和C. gigas angulata）中比较了HSP基因序列差异以及其与HSF1调控关系；最后通过对一个在两亚种中具有表达差异的HSP70基因的启动子序列研究，来阐释牡蛎中复杂的热适应调节机制。
为了研究在热激状况下的响应基因以及受HSF1调控的基因的情况，本研究处理得到以下四组材料：对照组、35 ℃-2 h热激组、HSF1干扰组和干扰后热激组。将这四种材料进行转录组测序，转录组分析结果得到了150个热激响应差异基因，其中包括七个HSP基因。加权基因共表达网络分析（WGCNA）得到的17个HSP基因中有六个也通过差异基因富集得到与热激相关。此外，我们发现在热激中，被HSF1上调和下调的基因分别是48和47个。在上调的基因中，我们得到了一个可能受HSF1响应热激调控的HSP70基因（CGI_10002387）。通过结合位点预测和荧光素酶双报告基因实验初步证实了HSF1对富集到的HSP70的调控作用。基于差异表达的基因和WGCNA分析，在热激条件下富集到了低氧信号通路。此外，在热激中，HSP70和HSP20是最早响应的。
Oyster, a commercially important species inhabiting in the intertidal zone, often exposed to temperature fluctuations. More stress pressure was put on the organisms inhabiting the intertidal zone with the global warming process going on in recent years. Summer mortality, which lead to great loss of economy and was thought be related to thermal stress. Thus, it is meaningful to study the mechanism of thermal stress in oysters. The materials, the Pacific oyster (Crassostrea gigas gigas) and Fujian oyster (C. gigas angulata), which with significantly differences in geographical distribution and ability to adapt to thermal stress, were used to illustrate the thermal stress mechanisms in oyster by identifying the role of HSF1 and the regulatory relationship between HSPs under heat shock stress. Firstly, we cloned the HSF1 isoforms which play an important role in heat shock response in C. gigas gigas and C. gigas angulata. Then, we performed transcriptomic (thermal stress following HSF1 interference), chromatin immunoprecipitation followed by sequencing (ChIP-seq) in the Pacific oyster. We compared the sequences variation and regulatory relationship between HSF1 and HSP in two congener oyster species (C. gigas gigas and C. gigas angulata). Those studies were used to explain the complex thermal adaptation regulation mechanism in oysters.
1. Isoforms of HSF1 under heat shock conditions in oyster
To determine which isoforms of HSF1 play an important role response to thermal stress in Fujian oyster and the Pacific oyster, we cloned the sequences and quantified gene expression level of isoforms in the heat-shocked materials. We found that there are two mainly isoforms of HSF1 (HSF1a and HSF1d) responding to heat shock in two subspecies and they expression differently under various thermal stress. Then, subcellular localization of two subtypes was performed and the results show that that distribution of both subtypes transformed from the dispersed state to points gathering in the nucleus when exposed to thermal stress. Interaction experiments show that they can be formed aggregates. Those results show that HSF1 isoforms which response to heat shock could contribute regulation in heat shock in Fujian oyster and the Pacific oyster.
2. RNAi based transcriptome for HSF1 regulatory relationship study in the Pacific oyster under heat shock
To determine the response genes and the genes regulated by HSF1 under heat shock, the oysters was separated to four groups, the control group, the heat shock group, the HSF1 RNAi group and the heat shock following RNAi group. Four groups were used to do RNA sequencing; the results of transcriptome analysis identified 150 genes responsive to heat shock including seven HSP genes. Six of 17 HSP genes enriched in response to heat shock according to weighted gene co-expression network analysis (WGCNA). In addition, we found that 48 and 47 genes were up- regulated and down-regulated by HSF1 in response to heat shock, respectively. Among the up-regulated genes, we found a HSP70 (CGI_10002387), which may be regulated by HSF1 in response to heat shock. We identified and confirmed one HSP70 potentially regulated by HSF1 in response to heat shock by binding site predictions and a dual luciferase assay. Hypoxia signaling pathways were enriched under heat shock conditions by differentially expressed genes and WGCNA analysis. In addition, our results revealed that the expression of HSP70 and HSP20 was initially triggered after 2-h of heat shock.
3. Identify the HSF1 regulated genes in the Pacific oyster under thermal stress by ChIP-seq
To sequence the fragment of binding sites in chromatin level which related to genes regulation are easier to find the gene regulated by HSF1 under heat shock condition. In the study, we analyzed the genes from the ChIP-seq results. The genes were obtained by peaks (916 peaks corresponding to HSF1 binding sites) annotated. The results showed that there were four HSP70 genes, two HSP40 genes and one small HSP gene that showed binding to HSF1. In Gene Ontology analysis, HSF1 target genes were related to signal transduction, energy production, and response to biotic stimulus. One HSP70 (CGI_10003417) with a binding site in the promoter region was validated to be regulated by HSF1 under heat shock.
4. Sequences comparison of differentially expressed HSPs and the regulatory relationship with HSF1 in the Pacific oyster and Fujian oyster
To investigate the reasons that differential gene expression level in response to heat shock in the Pacific oyster and Fujian oyster, sequence of the HSPs (with significant differences in expression) and their regulatory relationship with HSF1 in two subspecies were studied. The results of HSP sequence analysis showed that the proteins encoded by the corresponding HSP genes were highly similar and the functional domain were similar in two subspecies. It indicates that the functions of these HSP genes are conserved in both subspecies. The differences were found in the promoter sequences comparation. The degree of difference of HSPs was calculated by comparing the sequences in the promoter region. Analysis of the expression patterns of the two genes with large differences found that the expression patterns in the two subspecies are similar and may have the same regulatory relationship. Then, the regulatory relationship between HSF1 and HSPs was studied by Dual luciferase assay. It showed that genes with the largest differences in promoter regions were activated the most. Moreover, the active of isoforms changes most between the two subspecies. The results indicate that the difference in the promoter region sequence of the gene (CGI_10002594) may contribute to the difference in expression levels of the two subspecies under heat shock.
5. The promoter region Sequence of HSP70 (CGI_10002594) Gene
The sequence of the promoter region was used to analyze the role of sequence differences in regulatory relationships. The sequence analysis showed that there existed the sequence variation including insertion, deletion (indel) and SNP. And the Fujian oyster with more sequences with indel than the Pacific oyster. Furthermore, the indels were AT-rich repeats and were similar in both subspecies. Site mutation (A/G) can increase the possibility of sequence bond by HSF1, and it is found that there is a correlation between the fragments and A/G mutation. The results of linkage disequilibrium analysis showed that a high linkage disequilibrium characteristic of the AT-rich sequences and A/G mutations existed in the two subspecies. Moreover, the A/G mutant genotypes showed significantly differences in the comparison of the two subspecies. The Dual-luciferase assay showed haplotype II are more capable of being activated. In summary, the A/G site mutation and indel which contains AT sequence may affect gene expression.
Overall, the results revealed a possible HSF1–HSP regulatory relationship in the oyster. The results show that a more complex mechanism of heat shock regulation may exist in oysters than model species. The study may provide valuable information on the mechanisms of thermal tolerance and may provide insights into marker assistant breeding for thermal tolerance to possibly avoid summer mass mortality in this commercially important species.
|MOST Discipline Catalogue||农学 ; 农学::水产|
|Table of Contents|
|刘优利. 热激转录因子1和热激蛋白在牡蛎热响应中的作用机制研究[D]. 中国科学院海洋研究所. 中国科学院大学,2020.|
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