【作者】 刘芳；

【导师】 邹成钢；

【作者基本信息】 云南大学 ， 生物化学与分子生物学， 2017， 博士

【摘要】 低温环境会对生物体的生理活动产生很重要的影响,尤其对变温动物的影响更为深远。生物体为了应对冷应激,获得更大的生存机会,已经形成了不同的抗冷机制。目前关于变温动物应对冷应激的机制,主要集中在通过离子梯度的修复、代谢产物的产生和通过细胞膜结构的调整和膜成分的改变获得冷耐受,但是参与其中的信号机制还不是很清楚。在树蛙和昆虫类等物种中已有研究报道,在低温刺激下蛋白激酶A(PKA)会被激活,而在本研究中的实验结果表明低温刺激会使cAMP升高,这提示了变温动物的冷耐受与cAMP/PKA信号通路有相关性。在本研究中,利用模式生物秀丽隐杆线虫(Caenorhabditis elegans)易于遗传操作的特性,通过遗传学手段直接证明了秀丽隐杆线虫的冷耐受需要cAMP/PKA信号通路的参与。研究发现,在冷应激下,秀丽隐杆线虫(C.elegans)的cAMP/PKA信号通路会被激活;此外,cAMP/PKA信号通路失活,会增加线虫对冷应激的敏感性。同时发现,在冷应激下,秀丽隐杆线虫体内的脂肪会发生水解。通过筛选29个与线虫脂代谢相关的基因,发现只有激素敏感脂肪酶HOSL-1参与调控脂肪的水解,并且其作用于cAMP/PKA信号通路的下游参与线虫对冷的抵抗。进一步研究发现,在低温环境中,线虫体内的水分会丢失,且随着冷暴露时间的延长,水分丢失越显著。脂肪水解产生的甘油有利于秀丽隐杆线虫的冷抵抗。当加入外源的甘油不仅能恢复冷敏感型线虫对冷的耐受,还会延长野生型线虫在冷下的生存时间。此外,本研究还发现,秀丽隐杆线虫的冷抵抗,需要KIN-1/PKA调控的水通道蛋白AQP-1、AQP-3和AQP-7的参与,沉默其中任一基因后,都会使线虫对冷更为敏感。最后,本研究的实验结果表明,在冷应激下,KIN-1/PKA调控线虫的冷耐受具有组织特异性,沉默肠道或神经元中的kin-1,都会导致线虫在冷下的生存时间缩短。综上所述,秀丽隐杆线虫在抵抗低温的过程中,肠道中的KIN-1/PKA以细胞自主调控的方式诱导肠道中的激素敏感脂肪酶HOSL-1表达;神经元中的KIN-1/PKA以细胞非自主的方式上调肠道、表皮和肌肉中的水通道蛋白AQP-1、AQP-3和AQP-7,促进甘油的渗透,从而保护线虫免受由于低温环境而导致的高渗压力的影响。本研究揭示了一个在进化上保守的抗冷机制,该机制普遍存在于变温动物和恒温动物中,这将为生物体抗冷防冻,提供一个理论依据。更多还原

【Abstract】 Low environmental temperature can have a significant impact on physiological and behavioural processes in a variety of living organisms,especially for poikilotherms.The mechanisms of cold resistance were reported by previously studies mostly focus on the rebuilt of ion gradients,the production of metabolite and the modification of membrane lipids.However which signalling pathway play a role in poikilothermic to cold stress has not been illuminated.Several studies have shown that the activity of the PKA is increased in wood frogs and some insect at low temperatures.In this study,we also observed the increase of cAMP levels in C.elegans under cold stress.These researchs implicated that the cAMP/PKA pathway may involve in the cold tolerance of poikilotherms.In this research,using a genetically tractable metazoan animal,by genetic means,we found that cAMP-PKA pathway involved in cold stress of C.elegans,which is required for resistance to cold.Our result showed that cold stress activates the cAMP/PKA pathway as well,genetic inactivation of the core components in the cAMP-PKA pathway enhanced the susceptibility of C.elegans to cold stress.Under cold conditions,The lipid content was eventually reduced.To identify which gene(s)is involved in fat mobilization,we screened 29 genes in lipid metabolism pathway,found that only HOSL-1 is involved in fat hydrolysis during cold stress.Meanwhile,cAMP-PKA pathway promotes fat mobilization via up-regulating hosl-1 to resist cold stress.Furthermore,we show that cold stress led to a decrease in water contents in WT worms.And the time of cold treatment is longer,the lost in water contents are more.Another key finding is that the glycerol produced by lipid hydrolysis is beneficial to cold resistance in C.elegans.Exogenous application of glycerol was not only sufficient to restore the resistance to cold stress in kin-1(ok338)mutant or hosl-1 RNAi worms,but also extended the lifespan of WT worms under cold conditions.Our result also proved that in C.elegans,aquaporins are required for response to resistance cold stress in a PKA-dependent manner in C.elegans.Knockdown of aqp-1,aqp-3,and aqp-7,but not other aquaporins,by RNAi led to enhanced sensitivity to cold stress.Finally,under cold condition,PKA in neurons and intestine regulates cold stress.we found that both intestinal-specific knockdown of kin-1 or neuronal RNAi of kin-1 resulted in a decrease in the survival of worms during cold stress.Taken together,the mechanism of cold tolerance in C.elegans is that KIN-1 in the intestine induces the expression of a ligase gene hosl-1,which in turn mediates lipid hydrolysis to produce glycerol.Meanwhile,KIN-1 in the neurons up-regulates the expression of three aquaporins in the intestine,epidermis,and muscles for glycerol transport in a cell-nonautonomous manner,thereby protecting worms against hyperosmotic stress induced by water loss.Our findings provided an example of an evolutionarily conserved mechanism for cold tolerance that has persisted in both poikilothermic and homoeothermic animals.Meanwhile,our work provided a theory for how organisms adapt cold environment and resistance to cold stress.更多还原