中国科学院海洋研究所机构知识库

盐度环境影响动物的生长发育、繁殖和生存等重要生命过程，但无脊椎动物如何应答盐度变化的分子机制仍不清楚。本论文以模式生物秀丽线虫（Caenorhabditis elegans）和海洋模式种海洋线虫（Litoditis marina）为实验材料，比较研究了线虫应答盐度变化的分子机制。本研究结果将为无脊椎动物如何应答盐度胁迫和发育调控机制提供新认知，也将为研究全球气候变化背景下海-陆动物如何应对盐度环境变化以及生命从海洋到陆地或相反方向演化过程中盐度适应的分子机制奠定坚实的理论基础。主要研究结果分为以下几点：

1、通过Illumina RNA测序分析，发现与野生型线虫相比，脂肪酸代谢和细胞色素P450代谢等通路基因在具有高盐抗性的osm-11突变体中显著上调，而钙信号传导等通路基因的表达在osm-11突变体中受到显著抑制。进一步通过CRISPR基因编辑技术制备基因敲除突变体，并与osm-11突变体杂交获得双突变体，发现在osm-11突变体中上调表达的脂肪酸代谢相关基因acdh-12和acs-17，以及P450代谢相关基因ugt-15是osm-11突变体高盐抗性所必需的。进一步通过比较转录组分析，本研究发现相较于osm-11单突变体，氧化磷酸化等通路相关基因在osm-11;acdh-12双突变体中显著下调，而自噬和线粒体自噬等通路相关的基因表达在高盐敏感的双突变体中显著上调。鉴于高盐抗性下降可能和线粒体功能相关，本研究进一步的实验发现线虫在高盐条件下的死亡与线粒体碎片化相关。另外，本研究观察到在高盐环境中，与osm-11突变体相比，野生型线虫的脂滴大小显著增大，这可能与其脂肪酸氧化基因dhs-28和daf-22的表达显著下调相关，而在osm-11突变体中敲除这两个基因导致其高盐抗性显著降低。此外，本研究发现，高盐抗性可能与铁死亡呈负相关，osm-11突变体饮食添加二十碳四烯酸（DGLA，Dihomo-γ-Linolenic Acid）导致其高盐抗性显著降低，并且与野生型动物相比，高盐抗性osm-11突变体中铁死亡抑制相关基因如ftn-1显著上调，而在osm-11;acdh-12双突变体中ftn-1和ftn-2均显著下调。

2、通过ONT长读长测序（lrRNA-seq），本研究在3‰、30‰和60‰盐度环境下共发现了6464个新的海洋线虫转录本。通过长读长测序发现的海洋线虫不同盐度间的差异表达基因（DEGs，differentially expressed genes）与本研究之前报道的Illumina短读长RNA测序数据呈高度正相关。相较于3‰盐度环境，ONT lrRNA-seq和Illumina数据在30‰盐度均富集到氧化还原酶活性、阳离子跨膜转运和离子跨膜转运等GO条目。类似地，与30‰盐度相比，在60‰盐度中，海洋线虫细胞外间隙、角质层结构成分、特异通道活性、离子传递、特异跨膜转运子活性等GO条目在ONT和Illumina数据中均显著富集。此外，本研究发现随着盐度增加，79个基因的表达显著上调，而119个基因表达显著下调。通过对30‰与3‰盐度相比较时获得的214个含有差异可变剪切（DAS，differential alternative splicing）事件的基因进行GO富集分析，发现细胞组分组装和辅酶生物合成等GO条目显著富集。相似的，与30‰盐度相比，60‰盐度下获得含有DAS事件的基因的GO分析发现，细胞组分组装和辅酶生物合成等GO条目也显著富集。同时，本研究发现在30‰与3‰、60‰与30‰以及60‰与3‰的比较中，分别有86、125和81个含有DAS的基因是DEGs。进一步，本研究阐释了海洋线虫在不同盐度条件下的可变多聚腺苷酸（APA，alternative polyadenylation）的使用特征。

3、通过ONT lrRNA-seq，本研究在3‰、9‰、15‰和30‰盐度环境下共发现26423个秀丽线虫转录本，包括6280个新转录本。其中野生型N2中发现4127个新转录本，而在osm-11突变体中发现了4060个新转录本。通过GO富集分析，发现相较于N2，在30‰高盐环境下，osm-11突变体中上调DEGs的GO分析显著富集的条目包括蜕皮周期、细胞外区域和氧化还原酶活性等，而下调DEGs富集的GO条目为UDP-糖基转移酶活性和细胞内膜结合细胞器等。通过对30‰盐度下osm-11 vs. N2比较中的DEGs与海洋线虫L. marina 30‰ vs. 3‰ 的DEGs进行LOA分析，本研究发现两物种方向一致的重叠DEGs中，上调DEGs 61个，下调DEGs 31个；方向不一致的重叠DEGs中，上调DEGs 38个，下调DEGs 59个；在秀丽线虫中特异上调的DEGs 492个，下调DEGs 278个；而L. marina高盐特异上调的DEGs 2570个，下调DEGs 2481个。

4、通过ONT lrRNA-seq，本研究在海洋线虫L. marina 30‰ vs. 3‰盐度对比中发现了259个差异可变剪切（DAS）事件，属于214个基因，其中86个基因为DEGs；在秀丽线虫30‰盐度下，osm-11 vs. N2 对比中发现了120个DAS事件，涉及107个基因，其中8个为DEGs。通过分析秀丽线虫野生型N2 30‰ vs. 3‰对比组差异可变剪切，发现相较于低盐，高盐条件导致了381个DAS事件，属于327个基因，其中170个是DEGs；而osm-11突变体在30‰ vs. 3‰比较中发现，相较于低盐，高盐环境导致194个DAS事件发生，涉及171个基因，其中52个为DEGs。在L. marina 30‰ vs. 3‰ 比较中发现的214个发生DAS事件的基因，与各秀丽线虫对比组中的DAS事件相关基因比较时发现，与30‰ osm-11 vs. N2相比包括2个基因重叠（CC8.2和tos-1），与osm-11突变体 30‰ vs. 3‰相比包括5个基因重叠（F02E11.7、mak-1、tos-1、rml-1和Y39E4B.6），而与野生型N2 30‰ vs. 3‰相比含9个基因重叠（CC8.2、ceh-13、F02E11.7、iglr-3、mdt-26、mod-1、rml-1、T12D8.9和Y39E4B.6）。

5、通过ONT lrRNA-seq，本研究发现在30‰盐度下，秀丽线虫osm-11突变体具有比野生型N2更长的3' UTR，海洋线虫L. marina的3' UTR显著长于osm-11突变体。在3‰盐度下，3' UTR具有30‰盐度下相似的变化趋势。与3‰盐度相比，在30‰盐度下，N2和L. marina 的3' UTR均显著变长，而osm-11突变体含有不同PAS位点的3' UTR变化趋势不一致。另外，本研究发现与低盐3‰相比，在30‰高盐环境下，海洋线虫中17个转录本选择远端PAS位点，而38个转录本利用近端PAS位点；在30‰盐度下，相较于秀丽线虫野生型N2，osm-11突变体中6个转录本选择远端PAS位点，6个利用近端PAS位点；与3‰相比，在30‰高盐环境下，osm-11突变体有26个转录本选择远端PAS位点，而12个利用近端PAS位点；相较于与3‰，在30‰盐度下，野生型N2线虫中23个转录本选择远端PAS位点，而21个转录本利用近端PAS位点。

本论文研究显示秀丽线虫osm-11突变体的高盐抗性依赖于脂肪酸代谢相关基因acdh-12和acs-17以及P450通路基因ugt-15，acdh-12基因突变通过下调线粒体能量合成，上调线粒体自噬等通路基因降低osm-11突变体的高盐抗性。首次在海洋线虫L. marina中进行了不同盐度环境下的长读长转录组分析(ONT lrRNA-seq)，阐释了L. marina不同盐度下，差异表达基因(DEGs)、差异可变剪切(DAS)、3' UTR 和APA等变化特征。通过ONT lrRNA-seq，首次比较研究了秀丽线虫osm-11突变体、野生型N2和海洋线虫L. marina在不同盐度下DEGs、DAS、3' UTR和APA变化特征的异同。
 

Salinity affects critical life processes such as growth, development, reproduction, and survival in animals. However, the molecular mechanisms underlying how invertebrates respond to salinity changes remain incompletely understood. In this study, we used the model organisms Caenorhabditis elegans and marine nematode Litoditis marina to comparatively investigate the molecular mechanisms underpinning nematodes' response to salinity environments. The findings of this project will provide novel insights into how invertebrates respond to different salinity conditions, and will also lay a solid theoretical foundation for investigating how marine and terrestrial animals respond to the changing salinity environment in the context of global climate change, as well as the mechanisms underlying salinity adaptation during animal transition from ocean to land or vice versa. The main results are summarized as follows:

1. Through Illumina RNA sequencing, we found that the expression of genes in certain pathways such as fatty acid metabolism and cytochrome P450 metabolism were significantly increased in osm-11 mutant compared with wild-type animals, while genes in calcium signaling pathway were significantly repressed in hypersaline resistant osm-11 mutants. Next, through generating gene knock-out in osm-11 mutant to obtain double mutants, we found that fatty acid metabolism related genes such as acdh-12 and acs-17, as well as P450 metabolism related gene ugt-15 are required for hypersaline resistance in osm-11 mutants. Further, through RNA sequencing, we found that genes belonging to pathways such as oxidative phosphorylation were significantly downregulated in osm-11;acdh-12 double mutant in comparison to osm-11 single mutant, while the expression of genes in pathways such as autophagy and mitophagy was significantly upregulated in the hypersaline sensitive double mutant. Moreover, we observed that mitochondrial fragmentation might contribute to the death of wild-type nematodes under hypersaline conditions. In addition, we observed that the size of lipid droplets were significantly enlarged in wild-type worms compared to osm-11 mutant under hypersaline environment, which might correlated with decreased expression of β oxidation genes dhs-28 and daf-22 in wild-type animals, and knock out either genes in osm-11 mutants impaired their hypersaline resistance. Additionally, we found that hypersaline resistance might be negatively correlated to ferroptosis as revealed by supplementation of DGLA (Dihomo-γ-Linolenic Acid) reducing hypersaline resistance in osm-11 mutant, and ferroptosis inhibition related genes such as ftn-1 was significantly upregulated in osm-11 mutant compared with wild-type animals, and both ftn-1 and ftn-2 were significantly downregulated in osm-11;acdh-12 double mutant in comparision to osm-11 single mutant.

2. Through ONT lrRNA sequencing under 3‰, 30‰ and 60‰ salinity environments, we found 6464 novel transcripts in marine nematode L. marina. DEGs obtained from the current lrRNA-seq exhibited highly correlation with those identified in our previous reported Illumina short-read RNA sequencing data. When compared 30‰ to 3‰ salinity condition, we found that GO terms such as oxidoreductase activity, cation transmembrane transport and ion transmembrane transport were shared between ONT lrRNA-seq and Illumina data. Similarly, GO terms including extracellular space, structural constituent of cuticle, substrate-specific channel activity, ion transport, substrate-specific transmembrane transporter activity were shared between ONT and Illumina data under 60‰ compared with 30‰ salinity. In addition, we found 79 genes were significantly increased, while 119 genes were significantly decreased, when salinity increased. Furthermore, through GO enrichment analysis of 214 genes containing DAS (differential alternative splicing) events in 30‰ compared with 3‰ salinity, we found that GO terms such as cellular component assembly and coenzyme biosynthetic process were enriched. Additionally, we observed that GO terms such as cellular component assembly and coenzyme biosynthetic process were also enriched in 60‰, when compared with 30‰ salinity condition. Moreover, we found that there were 86, 125, and 81 genes contained DAS were DEGs, for comparisons between 30‰ vs. 3‰, 60‰ vs. 30‰, and 60‰ vs. 3‰, respectively. In addition, we demonstrated the alternative polyadenylation APA usage in marine nematode under different salinity conditions.

3. Through ONT lrRNA-seq under 3‰, 9‰, 15‰, and 30‰ salinity environments, we revealed 26423 transcripts in C. elegans, of which 6280 were novel transcripts. Among these novel transcripts, 4127 were found in the wild-type N2 animals, while 4060 were identified in osm-11 mutants. When compared osm-11 mutant to N2 under 30‰ salinity condition, we found that GO terms such as molting cycle, extracellular region, and oxidoreductase activity were significantly enriched for the upregulated DEGs, while the downregulated DEGs were enriched in GO terms such as UDP-glycosyltransferase activity and intracellular membrane-bounded organelle. Through LOA analysis of significant DEGs between osm-11 vs. N2 under 30‰ salinity conditions and 30‰ vs. 3‰ conditions for the marine nematode L. marina, we identified the overlapping DEGs with the same direction between both species, including 61 upregulated DEGs and 31 downregulated DEGs. While, the overlapping DEGs with opposite direction included 38 upregulated DEGs and 59 downregulated DEGs. In C. elegans, 492 genes were specifically upregulated and 278 genes were specifically downregulated, whereas in L. marina, 2570 genes were specifically upregulated and 2481 genes were specifically downregulated under hypersaline conditions.

4. Through ONT lrRNA-seq, we found 259 differential alternative splicing (DAS) events in the marine nematode L. marina under 30‰, when compared with 3‰ salinity condition, which belonged to 214 genes, of which 86 were DEGs. In C. elegans, under 30‰ salinity conditions, the osm-11 vs. N2 comparison revealed 120 DAS events in 107 genes, of which 8 were DEGs. Analysis of differential alternative splicing in C. elegans wild-type N2 under 30‰ vs. 3‰ condition, we found 381 DAS events in 327 genes, of which 170 were DEGs under hypersaline conditions. Similarly, in the osm-11 mutants, 194 DAS events belonging to 171 genes were observed under 30‰ compared with 3‰ condition, of which 52 were DEGs. When comparing the genes containg DAS in L. marina under 30‰ in comparision to 3‰ salinity, with those containing DAS events in the above mentioned C. elegans comparisons, we found that there were 2 overlapping genes (CC8.2 and tos-1) when compared with osm-11 vs. N2 under 30‰ salinity; there were 5 overlapping genes (F02E11.7, mak-1, tos-1, rml-1, and Y39E4B.6) when compared with osm-11 mutant 30‰ vs. 3‰, and there were 9 overlapping genes (CC8.2, ceh-13, F02E11.7, iglr-3, mdt-26, mod-1, rml-1, T12D8.9, and Y39E4B.6) when compared to wild-type N2 30‰ vs. 3‰.

5. Through ONT lrRNA-seq, we found that in 30‰ salinity, the average length of 3' UTR of C. elegans osm-11 mutants was longer than that of wild-type animals, while 3' UTR of marine nematode L. marina was longer than osm-11 mutants. Similar trends in 30‰ salinity as mentioned above were observed in 3‰ salinity condition. Both C. elegans wild-type N2 and marine nematode L. marina exhibited longer 3' UTRs under 30‰ than those in 3‰ salinity, whereas the osm-11 mutant showed distinct 3' UTR length changes for different PAS motif usage. Furthermore, we found that compared to hyposaline condition (3‰), 17 transcripts in L. marina chosed distal PAS sites, while 38 transcripts utilized proximal PAS sites under hypersaline condition (30‰). Under 30‰ salinity condition, 6 transcripts in the osm-11 mutant selected distal PAS sites, and 6 utilized proximal PAS sites when compared to the wild-type N2. Under 30‰ salinity condition, the osm-11 mutant had 26 transcripts choosing distal PAS sites, while 12 utilized proximal PAS sites when compared to 3‰ salinity condition. While, under 30‰ salinity condition, the wild-type N2 had 23 transcripts choosed distal PAS sites, while 21 transcripts utilized proximal PAS sites when compared to 3‰ salinity condition.

This study demonstrated that the hypersaline resistance of osm-11 mutant was dependent on the fatty acid metabolism-related genes such as acdh-12 and acs-17, as well as the P450 pathway gene ugt-15. acdh-12 mutation repressed the expression of genes in mitochondrial energy synthesis, and increased the expression of genes in mitophagy, which might reduce the hypersaline resistance in osm-11 mutant. For the first time, long-read transcriptome analysis (ONT lrRNA-seq) was applied to the marine nematode L. marina under different salinity conditions. The characteristics of DEGs, DAS, 3' UTR, and APA changes in L. marina across varying salinities were elucidated. Using ONT lrRNA-seq, the DEGs, DAS, 3' UTR, and APA changes between osm-11 mutant, wild-type N2, and L. marina under different salinity conditions were compared for the first time, highlighting their similarities and differences.
 

第1章 绪论 1

1.1 海洋无脊椎动物应答盐度环境变化的研究进展 1

1.2 模式生物秀丽线虫盐度胁迫响应与适应的研究进展 3

1.3 Notch 信号通路 4

1.4 Oxford Nanopore Technologies (ONT) 长读长测序技术简介 5

1.5 本研究的目的和意义 6

1.6 技术路线 7

第2章 秀丽线虫应答高盐胁迫的分子机制 9

2.1 研究背景 9

2.2 材料与方法 9

2.2.1 秀丽线虫品系 9

2.2.2 盐度梯度NGM板制备 11

2.2.3 盐度耐受实验 11

2.2.4 CRISPR/Cas9基因编辑和转基因营救 11

2.2.5 转录组样品准备 13

2.2.6 转录组样品测序 13

2.2.7 qPCR验证 14

2.2.8 Luperchio重叠分析（Luperchio Overlap Analysis，LOA） 14

2.2.9 WormExp 15

2.2.10 ChiP-atlas 15

2.2.11 显微观察 15

2.2.12 碘化丙啶染色实验 15

2.2.13 数据统计 16

2.3 结果 16

2.3.1 osm-11 突变体促进秀丽线虫的高盐抗性 16

2.3.2 osm-11 突变体与野生型之间的比较转录组分析 20

2.3.3 osm-11突变体促进高盐抗性依赖于acdh-12 27

2.3.4 osm-11单突变体与osm-11;acdh-12双突变体间的比较转录组特征 37

2.3.5 acdh-12可能通过dhs-28和daf-22调控osm-11突变体的高盐抗性 42

2.3.6 在高盐环境中对acdh-12依赖和非依赖性的转录组特征 46

2.3.7 线粒体碎片化可能参与了高盐胁迫 51

2.3.8 osm-11突变可能通过经典的Notch信号通路促进高盐抗性 55

2.3.9 osm-11突变体的高盐抗性依赖于甘油合成酶基因 60

2.3.10 胰岛素信号通路基因akt-1突变促进osm-11突变体高盐抗性 62

2.3.11 osm-11突变体可能通过抑制铁死亡促进对高盐抗性 64

2.4 讨论 66

2.5 小结 69

第3章 海洋线虫响应不同盐度环境的转录组特征 71

3.1 研究背景 71

3.2 材料与方法 71

3.2.1 线虫品系 71

3.2.2 样品收集与ONT lrRNA测序文库制备 71

3.2.3 ONT的转录组构建 72

3.2.4 基因和转录本丰度预测 72

3.2.5 差异表达分析 72

3.2.6 可变剪切分析 73

3.2.7 可变多聚腺苷酸化分析 73

3.2.8 3' UTR 分析 73

3.2.9 基因集和通路富集分析 73

3.3 结果 74

3.3.1 不同盐度条件的ONT lrRNA 测序结果概述 74

3.3.2 DEGs分析 74

3.3.3 差异可变剪切（DAS）和DEGs的分析 79

3.3.4 3' UTRs 概述 82

3.3.5 APA 分析 84

3.4 讨论 86

3.4.1 ONT lrRNA-seq的DEGs与Illumina数据具有高度相似性 86

3.4.2 通过lrRNA-seq揭示了随着盐度增加而上调和下调的DEGs 89

3.4.3 不同盐度条件下的DAS鉴定 93

3.4.4 海洋线虫在不同盐度条件下显示出明显的可变多聚腺苷酸位点使用差异 94

3.5 小结 95

第4章 海陆近缘种线虫高盐应答的比较研究 97

4.1 研究背景 97

4.2 材料与方法 97

4.2.1 线虫品系 97

4.2.2 样品收集与ONT lrRNA测序文库制备 97

4.2.3 ONT的转录组构建 97

4.2.4 基因和转录本丰度评估 97

4.2.5 差异表达分析 97

4.2.6 可变剪切分析 97

4.2.7 可变多聚腺苷酸化分析 97

4.2.8 3' UTR 分析 97

4.2.9 基因集和通路富集分析 98

4.3 秀丽线虫野生型N2和osm-11突变体应答不同盐度环境的转录组特征 98

4.3.1 不同盐度条件的ONT lrRNA测序结果概述 98

4.3.2 DEGs分析 98

4.3.3 差异可变剪切（DAS）分析 105

4.3.4 3' UTRs 概述 107

4.3.5 APA 分析 110

4.4 秀丽线虫和海洋线虫L. marina的盐度响应ONT 长读长比较转录组分析 112

4.4.1 两物种DEGs 比较 112

4.4.2 两物种DAS 比较 121

4.4.3 两物种3' UTR 比较 126

4.4.4 两物种APA 比较 128

4.5 小结 131

第5章 结论 133

5.1 主要结论 133

5.2 创新点 134

5.3 研究展望 135

参考文献 137

致 谢 149