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饮食营养调控线虫生长发育的机制研究
曹绪文
Subtype博士
Thesis Advisor张留所
2022-05-20
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士学位
Degree Discipline海洋生物学
Keyword秀丽线虫 海洋线虫Litoditis marina CeMM饮食 生长发育 脂肪酸
Abstract

饮食和营养调控生长发育、繁殖和寿命等重要生命过程,然而饮食摄入和营养成分如何调控动物生长发育的确切分子机制仍不清楚。本论文利用模式生物秀丽线虫Caenorhabditis elegans和潜在的海洋模式动物海洋线虫Litoditis marina为研究材料,比较研究了在化学成分明确的CeMMC. elegans maintenance medium营养环境下,线虫生长发育调控的分子机制。研究结果将为营养环境如何调控动物生长发育的分子机制提供新认知,也将为研究饮食如何调控人类健康,和全球气候变化背景下海陆动物如何应对变化的营养环境及海洋生态系统保护奠定坚实的理论基础。主要研究结果如下:

1. 通过比较转录组分析发现,在CeMM营养环境下,快速生长的eat-2tmc-1突变体中,数个脂肪酸合成和延长基因的表达显著上调,而多个脂肪酸β-氧化基因表达显著下调。饮食添加脂肪酸实验表明,C17ISO、棕榈酸和硬脂酸均可以显著加快秀丽线虫在CeMM营养环境下的生长发育。CRISPR基因敲除和转基因营救实验表明,eat-2突变体的快速生长依赖于负责合成C17ISO的延长酶基因elo-5elo-6,并且通过组织特异性营救实验表明elo-6主要在肠道中发挥调控生长发育的作用。此外,比较转录组分析发现,elo-6C17ISO可能通过控制表皮合成和hedgehog信号通路基因的表达调控秀丽线虫的生长发育。另外,基于代谢组分析,本研究发现elo-6C17ISO可能通过增加氨基酸、氨基酸衍生物和维生素的生物合成以促进秀丽线虫生长发育。

2. 与秀丽线虫相似,饮食添加C17ISO同样能够促进海洋线虫L. marinaCeMM营养环境下的生长发育,显示了C17ISO在秀丽线虫和L. marina之间发育调控作用的保守性。比较转录组分析发现:在秀丽线虫中,饮食添加C17ISO可能主要通过上调表皮合成及hedgehog通路相关基因的表达促进生长发育;在海洋线虫L. marina中,饮食添加C17ISO则可能通过上调氨酰-tRNA生物合成等蛋白质合成相关基因和跨膜转运蛋白相关基因的表达,以及下调神经信号相关基因的表达来促进生长发育。

3. 与秀丽线虫一致,饮食添加硬脂酸同样也能够促进海洋线虫L. marinaCeMM营养环境下的生长发育。比较转录组分析表明,硬脂酸既可能通过相似的机制,如上调跨膜转运蛋白相关基因的表达促进秀丽线虫和L. marina的生长发育;也可能通过两种线虫中特异的调控机制促进生长发育。比如在秀丽线虫中,表皮合成基因的表达显著上调;而在L. marina中,氨酰-tRNA生物合成、翻译起始和延伸及核糖体生物合成相关基因的表达显著增加,神经信号相关基因的表达被显著抑制。

4. 和饮食添加棕榈酸促进秀丽线虫的生长发育相反,棕榈酸的添加导致海洋线虫L. marinaCeMM营养环境下的大量死亡。转录组分析表明,饮食添加棕榈酸后,秀丽线虫中hedgehog信号通路和表皮合成相关基因的表达显著上调,而L. marinahedgehog信号通路和表皮合成相关基因的表达则显著下调。此外,在L. marina中,添加棕榈酸也可能通过抑制核糖体生物合成基因的表达和增加应激激素合成基因的表达而威胁L. marina的生存。

Other Abstract

Diet and nutrition regulate important life processes such as growth and development, reproduction, and longevity, yet the exact molecular mechanisms underlying how dietary intake and nutrient composition regulate animal growth and development remain unclear. In this thesis, the model organism Caenorhabditis elegans and the potential marine model animal Litoditis marina were used as research materials to comparatively investigate the molecular mechanisms of nematode growth and development regulation in the chemically defined CeMM (C. elegans maintenance medium) nutrient environment. The results of the study will provide new insights into the molecular mechanisms underpinning how the nutritional environment regulates animal growth and development, and will also lay a solid theoretical foundation for studying how diet regulates human health, as well as how marine and terrestrial animals respond to the changing nutritional environment and marine ecosystem conservation in the context of global climate change. The main findings are as follows.

1. Comparative transcriptome analysis revealed that the expression of several fatty acid synthesis and elongation genes was significantly up-regulated, while the expression of many fatty acid β-oxidation genes was significantly down-regulated in fast-growing eat-2 and tmc-1 mutants in CeMM nutrient environment. Experiments of dietary fatty acid supplementation showed that C17ISO, palmitic acid and stearic acid could significantly accelerate the growth and development of C. elegans in CeMM nutrient environment. CRISPR knockout and transgenic rescue experiments showed that the rapid growth of eat-2 mutant depended on the elongase genes elo-5 and elo-6, which are responsible for synthesizing C17ISO, and tissue-specific rescue experiments demonstrated that elo-6 functioned mainly in the intestine to regulate growth and development. Furthermore, comparative transcriptome analysis revealed that elo-6 and C17ISO might regulate the growth and development of C. elegans through controlling the expression of cuticle synthesis and hedgehog signaling pathway genes. In addition, based on metabolomic analysis, this study found that elo-6 and C17ISO might promote the growth and development of C. elegans by increasing the biosynthesis of amino acids, amino acid derivatives and vitamins.

2. Similar to C. elegans, dietary supplementation of C17ISO could also promote the growth and development of marine nematode L. marina in CeMM nutrient environment, showing the conserved developmental regulatory role of C17ISO between C. elegans and L. marina. Comparative transcriptome analysis revealed that dietary supplementation with C17ISO might promote growth and development of C. elegans mainly by up-regulating the expression of cuticle synthesis and hedgehog pathway related genes, while it might promote growth and development of the marine nematode L. marina by up-regulating the expression of protein synthesis related genes, such as aminoacyl-tRNA biosynthesis and transmembrane transporter related genes, and down-regulating the expression of neuronal signaling related genes.

3. In line with C. elegans, dietary supplementation of stearic acid could also promote the growth and development of the marine nematode L. marina in CeMM nutrient environment. Comparative transcriptome analysis indicated that stearic acid might promote the growth and development of C. elegans and L. marina through similar mechanism, such as up-regulating the expression of transmembrane transporter related genes; it might also promote growth and development through specific regulatory mechanisms in two nematodes. For example, the expression of cuticle synthesis genes was significantly up-regulated in C. elegans; while the expression of aminoacyl-tRNA biosynthesis, translation initiation and elongation, and ribosomal biosynthesis related genes was significantly increased, and the expression of neuronal signaling related genes was significantly suppressed in L. marina.

4. In contrast to that dietary supplementation of palmitic acid promoting the growth and development of C. elegans, palmitic acid supplementation led to massive mortality of the marine nematode L. marina in CeMM nutrient environment. Transcriptome analysis showed that the expression of hedgehog signaling pathway and cuticle synthesis related genes was significantly up-regulated in C. elegans, while the expression of hedgehog signaling pathway and cuticle synthesis related genes was significantly down-regulated in L. marina after dietary supplementation of palmitic acid. Moreover, in L. marina, palmitic acid supplementation might also threaten the survival of L. marina by repressing the expression of ribosome biosynthesis genes and increasing the expression of stress hormone synthesis genes.

Subject Area生物学
MOST Discipline Catalogue理学::海洋科学
Language中文
Table of Contents

第1章 绪论... 1

1.1 乙酰胆碱信号调控动物饮食与健康的研究进展... 1

1.2 脂肪酸调控动物发育与健康的研究进展... 3

1.2.1 单甲基支链脂肪酸C17ISO.. 4

1.2.2 硬脂酸... 6

1.2.3 棕榈酸... 7

1.3 饮食和营养调控线虫发育的研究进展... 8

1.4 海洋线虫Litoditis marina. 11

1.5 本研究的目的和意义... 12

1.6 技术路线... 12

第2章 饮食和营养调控秀丽线虫生长发育的分子机制... 14

2.1 研究背景... 14

2.2 材料方法... 14

2.2.1 秀丽线虫品系和培养方法... 14

2.2.2 CeMM配制... 15

2.2.3 秀丽线虫的同步化... 16

2.2.4 转录组测序和分析... 16

2.2.5 非靶代谢组学分析... 18

2.2.6 实时定量PCR(qPCR)... 18

2.2.7 饮食添加脂肪酸... 19

2.2.8 质粒制备... 19

2.2.9 CRISPR/Cas9基因编辑... 20

2.2.10 转基因品系... 23

2.2.11 统计分析... 23

2.3 结果... 23

2.3.1 乙酰胆碱受体基因eat-2延缓秀丽线虫在CeMM上的生长发育... 23

2.3.2 eat-2突变可能通过调控脂肪酸代谢基因的表达促进秀丽线虫发育... 25

2.3.3 tmc-1突变可能也是通过调控脂肪酸代谢通路基因的表达促进秀丽线虫发育... 30

2.3.4 饮食添加脂肪酸加速了秀丽线虫在CeMM营养环境下的生长发育... 35

2.3.5 eat-2突变体在CeMM营养环境下的快速生长依赖于elo-5和elo-6. 39

2.3.6 elo-6通过其在肠道中的表达调节秀丽线虫的生长发育... 42

2.3.7 elo-6功能缺失可能通过下调表皮合成和hedgehog信号减慢秀丽线虫的生长发育... 43

2.3.8 饮食添加C17ISO可能通过上调表皮合成和hedgehog信号促进秀丽线虫生长... 47

2.3.9 氨基酸、氨基酸衍生物和维生素可能促进秀丽线虫在CeMM上的发育... 48

2.4 讨论... 51

2.5 本章小结... 59

第3章 脂肪酸调控海洋线虫生长发育的转录组特征... 60

3.1 研究背景... 60

3.2 材料和方法... 60

3.2.1 海洋线虫L. marina. 60

3.2.2 海盐-CeMM配制... 60

3.2.3 饮食添加脂肪酸... 61

3.2.4 L. marina的同步化培养... 61

3.2.5 转录组测序分析... 61

3.2.6 实时定量PCR分析... 62

3.3 实验结果... 62

3.3.1 C17ISO、棕榈酸和硬脂酸在CeMM饮食环境下调控海洋线虫L. marina生长和发育... 62

3.3.2 饮食添加C17ISO、硬脂酸和棕榈酸的转录组分析... 64

3.3.3 氨酰-tRNA生物合成通路基因表达在添加C17ISO和添加硬脂酸后显著上调... 70

3.3.4 翻译起始、延伸和核糖体生物合成相关基因的表达在添加C17ISO和硬脂酸后显著上调... 73

3.3.5 跨膜转运蛋白相关基因表达在添加C17ISO和添加硬脂酸后显著上调... 74

3.3.6 神经信号基因的表达在添加C17ISO和添加硬脂酸后显著下调... 75

3.3.7 添加棕榈酸抑制了核糖体生物合成、hedgehog通路和表皮合成相关基因的表达... 78

3.3.8 添加棕榈酸显著上调了acs-7和acox-1.1基因的表达... 79

3.3.9 实时定量PCR(qPCR)验证... 80

3.4 讨论... 82

3.4.1 C17ISO和硬脂酸可能通过上调蛋白质合成相关基因的表达促进L. marina的生长发育... 82

3.4.2 C17ISO和硬脂酸可能通过上调跨膜转运蛋白相关基因的表达促进L. marina的生长发育... 84

3.4.3 C17ISO和硬脂酸可能通过抑制神经信号传导促进L. marina的生长发育... 85

3.4.4 棕榈酸可能通过抑制核糖体生物合成、hedgehog通路和表皮合成相关基因影响L. marina的生存... 87

3.4.5 棕榈酸可能通过上调应激激素合成影响L. marina的生存... 87

3.5 本章小结... 87

第4章 脂肪酸调控秀丽线虫和海洋线虫L. marina生长发育的异同... 89

4.1 饮食添加C17ISO调控秀丽线虫和海洋线虫发育表型和转录组表达特征的异同... 89

4.2 饮食添加硬脂酸调控秀丽线虫和海洋线虫发育表型和转录组表达特征的异同... 92

4.3 饮食添加棕榈酸调控秀丽线虫和海洋线虫发育表型和转录组表达特征的异同... 99

第5章 结论... 105

5.1 主要结论... 105

5.2 创新点... 106

5.3 研究展望... 106

参考文献... 107

致 谢... 130

作者简历及攻读学位期间发表的学术论文与研究成果... 131

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/178395
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
曹绪文. 饮食营养调控线虫生长发育的机制研究[D]. 中国科学院海洋研究所. 中国科学院大学,2022.
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