实验海洋生物学重点实验室知识库

饮食调控动物和人类重要的生命过程如发育、繁殖和寿命。已经有报道表明化学成分明晰的CeMM饮食延缓秀丽线虫的生长发育并延长其寿命，但CeMM饮食是否调控其他线虫的生长发育和寿命，以及潜在的分子调控机制尚不清楚。本研究通过CRISPR基因编辑技术获得了slo-1与ace-4突变体，发现slo-1与ace-4基因突变均促进秀丽线虫在CeMM的生长发育，进一步RNA-seq对slo-1(lzq49)与ace-4(lzq46)突变体进行了转录组测序和分析，初步揭示了ace-4与slo-1基因突变体在CeMM饮食环境下的发育调控转录组特征。另外，我们发现CeMM饮食延缓潜在的海洋模式线虫Litoditis marina的生长发育但不延长其寿命，通过转录组分析，我们探究了秀丽线虫和海洋线虫在CeMM和OP50饮食环境下的全基因组转录特征，为深入了解饮食调控动物发育和寿命的分子机制提供了新认知。主要研究结果如下：

1. 在CeMM饮食条件下，我们发现秀丽线虫slo-1 与ace-4基因突变促进线虫在CeMM营养环境下的生长速率，进一步通过转录组分析，我们发现相较于野生型，slo-1与ace-4突变所导致的转录组变化特征既有相同之处，也有差异。相较于野生型，slo-1(lzq49)与ace-4(lzq46)突变体中，与免疫和防御相关的几类基因如pals-，clec-的表达显著上调，表明slo-1(lzq49)与ace-4(lzq46)突变体可能通过上调免疫和防御相关基因的表达以适应CeMM营养胁迫并表现为快速生长表型。通过GO富集分析，发现在CeMM饮食环境下，相较于野生型，slo-1突变体的abu/pgn-、 ptr-基因发生了显著下调。相较于野生型，ace-4突变体的abu/pgn-家族基因， ptr-家族基因同样也发生显著下调。另外，在slo-1(lzq49)与ace-4(lzq46)上调的差异基因中，62个基因是共有的，45个基因是ace-4(lzq46)中特有，106个基因是slo-1(lzq49)中特有的；在下调的差异基因中，399个基因是共有的，260个基因是ace-4(lzq46)中特有，81个基因是slo-1(lzq49)中特有的。

2. 课题组前期报道了CeMM饮食延缓秀丽线虫的发育并延长其寿命，本论文发现CeMM饮食延缓海洋线虫L. marina的生长发育，但不能延长其寿命。通过KEGG富集分析，我们发现在秀丽线虫中，许多FOXO DAF-16信号通路、溶酶体和外源物质P450解毒系统相关基因的表达在CeMM饮食环境下显著上调，这可能导致了秀丽线虫寿命的延长。值得注意的是，我们发现L. marina的溶酶体和外源物质P450解毒系统基因的表达显著下调，这可能是CeMM饮食不能延长L. marina的寿命原因之一。此外，秀丽线虫在CeMM饮食环境下RNA转录和蛋白质生成及相关过程基因明显受到抑制，这些基因不仅可能涉及秀丽线虫寿命的延长，还可能延缓其在CeMM上的发育，但是上述蛋白合成相关通路并未在L. marina得到富集。我们发现CeMM饮食可能通过抑制不饱和脂肪酸合成基因的表达延缓L. marina生长发育。

本研究为确定长寿和发育所需的关键基因提供新认知，进一步通过基因编辑和转基因过表达等技术和方法对本研究发现的调控生长发育与长寿的关键候选基因进行功能验证，将为深入解析海-陆近亲物种应答全球气候变化背景下的营养环境变化的分子机制提供新思路。

Diet regulates the development, reproduction and longevity of animals and humans. A chemically defined CeMM diet was reported to delay the development but extend the lifespan of Caenorhabditis elegans. However, whether CeMM diet regulates that of other nematodes and the underlying molecular mechanism remains unclear. In this study, we obtained slo-1 and ace-4 mutants via CRISPR gene editing, and found that loss function of both genes promoted the development and growth of C. elegans on CeMM.  Transcriptome analysis of slo-1(lzq49) and ace-4(lzq46) was further performed by RNA-seq technique to explore the transcriptome characteristics of these two mutants in the CeMM nutrition environment. In addition, we found that CeMM slowed down the growth but did not extend the lifespan of the marine nematode L. marina. Through RNA-seq, we further analyzed the genome-wide transcriptional characteristics of C. elegans and L. marina in CeMM and OP50 diets, respectively, which will provide new insights into the molecular mechanisms underlying how diet regulates the development and longevity of animals. The main results are as follows:

1. We found that the mutations in slo-1 and ace-4 accelerated the growth of C. elegans in the CeMM nutritional environment. Through transcriptome analysis, we found that there were both similarities and differences in transcriptome changes in slo-1 and ace-4 mutants compared with wild-type worms. In slo-1(lzq49) and ace-4 (lzq46) mutants, several pals- and clec- genes, which were related to defense against external factors, were up-regulated. These results suggested that slo-1(lzq49) and ace-4 (lzq46) mutants might be better adapted to nutritional stress and accelerate the growth by up-regulating the expression of immune and defense-related genes on CeMM. We found that abu-/pqn- and ptr- family genes were significantly down-regulated in slo-1 mutants on the CeMM diet compared with the wild type. Similarly, we found that abu-/pqn- family genes and ptr- family genes were also significantly down-regulated in ace-4 mutants with CeMM diet compared with the wild type. Additionally, we found that, among the up-regulated DEGs, 62 genes were significantly increased in both slo-1(lzq49) and ace-4(lzq46) mutants, 45 genes were only up-regulated in ace-4(lzq46) mutant, while 106 genes were only up-regulated in slo-1(lzq49) mutant. Among the down-regulated DEGs, 399 genes were significantly decreased in both two mutants, 260 genes were only down-regulated in ace-4(lzq46) mutant, whereas 81 genes were only down-regulated in slo-1(lzq49) mutant.

2. It was previously reported that the CeMM diet not only slows down the development but extends the lifespan of C. elegans, in this study, we found that CeMM diet delayed the development of L. marina, but did not prolong its lifespan. In C. elegans, we found that many genes associated with FOXO/DAF-16 signaling pathways, lysosomes and exogenous metabolism were significantly up-regulated on CeMM, which may contribute to the longevity of C. elegans. Notably, we found that the gene expression of the lysosomal and xenobiotic metabolism pathway was significantly down-regulated under the CeMM feeding conditions, which might explain why the CeMM diet could not promote the lifespan of L. marina compared to bacterial feeding. Additionally, down-regulation of several RNA transcription and protein generation and related processes genes in C. elegans on CeMM might not only be involved in extending longevity but also contribute to attenuating the development of C. elegans on CeMM, however, the RNA transcription and protein synthesis related pathways were not enriched in L. marina. We found that unsaturated fatty acids synthesis genes were significantly down-regulated in L. marina on the CeMM food environment, which might contribute to slowing down the growth of L. marina.

This study provides new insights into the identification of key genes which involved in longevity and development regulation. Further research on the functions of key candidate genes through gene editing will provide novel insights into the physiological changes of marine and terrestrial organisms under the dynamics of nutrient environment in the context of climate changing.

第1章 引言... 1

1.1 研究背景和意义... 1

1.2 饮食调控线虫生长发育的研究进展... 2

1.3 饮食调控线虫寿命的研究进展... 5

第2章 slo-1与ace-4调控秀丽线虫生长发育的转录组分析... 9

2.1 材料与方法... 9

2.1.1 品系获得以及培养... …9

2.1.2 线虫的同步化... …9

2.1.3 CeMM准备... ..10

2.1.4 CRISPR/Cas9介导的基因编辑... ..10

2.1.5 线虫CeMM实验... ..11

2.1.6 RNA-seq建库准备及RNA-seq分析... ..11

2.1.7 实时荧光定量PCR验证... ..12

2.2 实验结果... 13

2.2.1 slo-1与ace-4功能缺失促进秀丽线虫在CeMM上的生长发育... .13

2.2.2 秀丽线虫slo-1 与ace-4突变体在CeMM饮食环境下的转录组分析... ..15

2.2.3 slo-1突变体的转录组特征分析... ..28

2.2.4 ace-4突变体的转录组特征分析... ..31

2.2.5 实时荧光定量PCR验证... ..33

2.3 讨论... 34

2.3.1 ace-4 与slo-1突变促进线虫在CeMM饮食环境下的生长发育... ……………………………………………………………………..34

2.3.2 ace-4 与slo-1突变体可能通过上调免疫与防御通路基因的表达促进线虫生长发育... ..34

2.3.3 ace-4 与slo-1突变体可能通过调控未折叠蛋白反应通路基因促进线虫生长发育... ..35

2.3.4 ace-4 与slo-1突变体间转录组特征的异同... ..35

2.4 本章小结... 38

第3章 饮食调控秀丽线虫和海洋线虫发育和寿命的转录组分析... 40

3.1 材料方法... 40

3.1.1 品系的获得... ..40

3.1.2 海洋线虫的同步化... ..40

3.1.3 海洋线虫表型实验... ..41

3.1.4 15‰海盐的CeMM准备... ..41

3.1.5 RNA-seq建库准备及RNA-seq分析... ..41

3.1.6 daf-16靶基因的确定... ..42

3.1.7 实时定量PCR (qPCR)验证... ..42

3.2 结果... 43

3.2.1 CeMM饮食延缓L. marina的生长发育，但不延长其寿命... ..43

3.2.2 不同饮食环境下秀丽线虫和海洋线虫的转录组分析 ..44

3.2.3 C. elegans 和 L. marina的共同转录组特征... ..47

3.2.4 在CeMM饮食条件下秀丽线虫独特的转录组特征... ..51

3.2.5 在CeMM饮食条件下L. marina独特的转录组特征 ..55

3.2.6 实时荧光定量PCR（qPCR）验证... ..57

3.3 讨论... 58

3.3.1 CeMM延长秀丽线虫的寿命，但不能延长海洋线虫L. marina的寿命... ..59

3.3.2 CeMM饮食延迟秀丽线虫和海洋线虫的生长发育... ..64

3.4 本章小结... 66

第4章 结论与展望... 67

4.1 主要结论... 67

4.2 不足与展望... 68

参考文献... 69

致 谢... 82

作者简历及攻读学位期间发表的学术论文与研究成果 84