实验海洋生物学重点实验室知识库

     pH作为一种常见的环境因子，会对生物的生长发育、繁殖和免疫调节等多个生理调控过程产生重要影响。无论是陆地还是水生生物经常会经历pH的波动。海洋酸化，酸雨及其导致的水体和土壤的酸化都会导致生物体生存环境的pH降低。在本研究中，我们用模式动物秀丽线虫和海洋线虫作为动物模型，通过线虫响应不同pH环境的表型实验及转录组分析，为探究生物体感知和响应酸性pH胁迫的分子调控机制提供参考。主要研究结果如下：

1. 秀丽线虫对pH有宽广的耐受范围(pH 3.13~11.33)，但随着pH的下降，产卵时间有所延迟，在酸度很低的条件下(pH 2.93)，不能正常生长繁殖。我们以秀丽线虫正常NGM培养基pH 6.33为对照组，根据生长发育表型试验产生的差异选择pH 4.33，pH 3.13，pH 2.93作为酸度胁迫处理组。通过转录组分析，我们发现当pH下降到4.33时，秀丽线虫表皮合成和结构完整性所必需的基因col，dpy，nas和ptr的表达上调；当pH下降到3.13时细胞色素P450通路基因(cyp，gst，ugt和ABC转运蛋白基因)以及核激素受体基因nhr表达上调；热休克蛋白HSP70和HSP20家族基因表达下调。

2. 海洋线虫Litoditis marina相对于秀丽线虫酸性pH耐受范围较窄，当pH从5.92下降到5.33时(目前L. marina在实验室最佳生长条件的pH为5.92)，发育减慢，但可以产卵繁殖；当pH下降到4.33时，L. marina无法长成成体。通过转录组分析发现，当pH从5.92下降到4.33时，L. marina大量脂肪酸β-氧化通路基因表达上调；表皮相关基因nas和ptr，细胞色素P450通路基因，C型凝集素基因和HSP70家族基因表达显著升高。

3. 秀丽线虫和海洋线虫L. marina对酸性pH胁迫的响应既有区别又有相似之处。表皮合成与细胞色素P450通路基因的表达上调是秀丽线虫响应酸度胁迫的两个主要自我保护策略。L. marina则主要通过脂肪酸β-氧化，同时细胞色素P450通路基因，表皮相关基因等共同参与响应过程。秀丽线虫在pH 3.13时能够长成成体并且可以产卵繁殖，而L. marina在pH 4.33时无法长成成体，我们推测产生这种差异的关键在于，与秀丽线虫相比，L. marina基因组中的细胞色素P450通路基因发生了显著缩减，进而对酸性环境导致生理紊乱而积累在其体内的有毒物质的代谢能力减弱，所以表现的更不耐酸。细胞色素P450通路可能是线虫响应和适应极端酸性环境的关键通路。

    我们在线虫中发现的系统应答酸性pH胁迫调控模式，为理解生物能够在不断变化的pH环境中生存的分子机制提供了参考。我们的数据结果也会为筛选和识别生物有机体响应和适应酸性pH胁迫的关键基因的进一步研究奠定基础。

As an essential environmental factor, pH affects many life processes such as growth, development, metabolism, as well as immune regulation. Whether on land or in water, living organisms often experience pH fluctuations. Ocean acidification, acid rain and the resulting acidification of water and soil all lead to a decrease of living environmental pH, which results in a series of negative effects on organisms. At present, the molecular mechanism of how organisms perceive and respond to acidic pH stress is unclear. Therefore, using Caenorhabditis elegans and Marine nematode Litoditis marina as animal models, we find several regulatory mechanism underlying how nematodes response to acid pH stress, through phenotypic experiments and transcriptome analysis. The main discoveries are the following:

1. C. elegans has a wide tolerance range to pH (pH 3.13~11.33), but with the decrease of pH, its spawning time is delayed, and in the condition of very low acidity (pH 2.93), it cannot grow and reproduce normally. The pH of NGM is 6.33, thus, pH 6.33 is the control group, and pH 4.33, pH 3.13 and pH 2.93 are the acid environment treatment group according to the differences generated by the growth and development phenotype test. Through transcriptome analysis, we find that when the pH drops to 4.33, the expression levels of col, nas, dpy and ptr, which are essential for epidermal synthesis and structural integrity increase. When the pH drops to 3.13, the expression of the metabolism of xenobiotics by cytochrome P450 pathway genes (cyp, gst, ugt, and ABC transporters) and nuclear hormone receptor genes increase. And with the drop of pH, HSP70 and HSP20 genes of C. elegans are down-regulated.

2. L. marina has a relatively narrow tolerance range to acid pH stress compared to C. elegans. When the pH drops from 5.92 to 5.33 (the optimum growth condition of L. marina is 5.92 in our laboratory), the growth and development rate of L. marina slows down significantly, but it can still reproduce. However, when the pH drops to 4.33, L. marina cannot grow into adults. Transcriptome analysis shows that when pH decreases from 5.92 to 4.33, the fatty acid β-oxidation pathway genes and the unsaturated fatty acid synthesis genes in L. marina are up-regulated. The expression of cuticle related genes show different changes. The expression level of epidermal collagen genes col do not change, and the expression of 2 nas genes and 6 ptr genes are significantly up-regulated. What’s more, the expression levels of cytochrome P450 pathway related genes, lectin C genes and heat shock protein HSP70 family genes significantly increase.

3. In response to acid pH stress, C. elegans and L. marina have both differences and similarities. Cuticle synthesis and integrity, together with the reprogrammed xenobiotics metabolism by cytochrome P450 pathway are two major stress-responsive pathways for C. elegans in acidic stress environments. However, Marine nematode L. marina's response to the decrease of environmental pH is mainly through the metabolism of fatty acids. And the expression of drug metabolism by cytochrome P450 pathway genes, lectin C genes, cuticle related genes (only the ptr and the nas, no collagen genes) and heat shock protein HSP70 family genes increase in response to low pH environment. C. elegans is able to grow into an adult and reproduce eggs at pH 3.13, while L. marina is unable to grow into an adult at pH 4.33, showing more vulnerable to acid stress. We speculate that the key to this difference is that compared with C. elegans, the cytochrome P450 pathway genes in the genome of L. marina significantly reduce. Thus, the metabolic capacity of toxic substances accumulated in the body due to the physiological disorder caused by the acidic environment is reduced, the body is less acid resistant. The cytochrome P450 pathway may be a key pathway for nematodes to respond to and adapt to extremely acidic environments.

The systematic regulation pattern in response to acid pH stress found in nematodes provide a reference for understanding the molecular mechanisms by which organisms can survive in an environment with changing pH environments. Our data might lay the foundation to identify the master gene(s) responding and adaptation to acidic pH stress in further studies.

第1章 引言................................................................... 1

1.1 研究背景和意义.................................................................. 1

1.2 线虫生物学简介及相关研究进展...................................... 4

1.2.1 秀丽线虫生物学简介及相关研究进展 4

1.2.2 海洋线虫生物学简介及相关研究进展 4

第2章 秀丽线虫响应pH下降的转录组分析............ 7

2.1 材料与方法.......................................................................... 7

2.1.1品系的获得 7

2.1.2 线虫的同步化 7

2.1.3 秀丽线虫表型实验 8

2.1.4 RNA-seq建库准备及RNA-seq分析 8

2.1.5实时荧光定量PCR验证 9

2.2 实验结果............................................................................ 10

2.2.1秀丽线虫在不同pH环境下的产卵时间 10

2.2.2秀丽线虫在不同pH环境下的转录组分析 11

2.2.3表皮合成相关基因表达随pH的下降先升高后降低 12

2.2.4外源物质代谢细胞色素P450通路基因显著上调 14

2.2.5核激素受体基因表达上调 18

2.2.6热休克蛋白基因表达下调 18

2.2.7实时荧光定量PCR验证 18

2.3 讨论.................................................................................... 19

2.3.1表型实验 19

2.3.2表皮合成相关基因表达随pH下降先升高后下降 20

2.3.3外源物质代谢细胞色素P450通路基因显著上调 22

2.3.4热休克蛋白基因下调 24

2.4 本章小结............................................................................ 25

第3章 海洋线虫Litoditis marina响应pH下降的转录组分析............................................................................. 27

3.1 材料与方法........................................................................ 27

3.1.1品系的获得 27

3.1.2 海洋线虫的同步化 27

3.1.3 海洋线虫表型实验 28

3.1.4 RNA-seq建库准备及RNA-seq分析 28

3.2 结果与讨论........................................................................ 28

3.2.1海洋线虫L. marina在不同pH环境下的生长发育情况 28

3.2.2海洋线虫L. marina在不同pH条件下的转录组分析 29

3.2.3脂肪酸代谢相关基因表达随pH的下降而升高 31

3.2.4表皮相关基因表达差异变化 35

3.2.5细胞色素P450通路相关基因表达随pH的下降而升高 38

3.2.6热休克蛋白基因表达上调 39

3.2.7 C型凝集素基因表达随pH的下降而升高 40

3.3 本章小结............................................................................ 43

第4章 秀丽线虫和海洋线虫L. marina响应pH下降转录组变化的比较......................................................... 45

4.1 脂肪酸代谢通路基因的比较.............................................. 45

4.2 表皮相关基因的比较.......................................................... 45

4.3 细胞色素P450通路相关基因的比较................................ 45

4.4 C型凝集素基因的比较....................................................... 46

4.5热休克蛋白基因的比较....................................................... 46

第5章 结论与展望..................................................... 51

5.1 主要结论............................................................................ 51

5.2 不足与展望........................................................................ 51

参考文献......................................................................... 53

致 谢............................................................................. 65

作者简历及攻读学位期间发表的学术论文与研究成果 67