Diapause, Stress Tolerance and Molecular Chaperones in Artemia franciscana

甲壳类动物Artemia franciscana的胚胎发育成游动的幼虫或被囊并进入滞育，这是一种以发育停滞和新陈代谢大大减少为特征的生理状态。被包裹的胚胎（囊肿）具有很强的耐受性，在极端温度、干燥和无氧环境下存活下来。因此，A. franciscana囊肿代表了地球上最耐压力的多细胞生命形式之一。在以滞育为目标的发育过程中，franciscana胚胎表现出差异的基因调控，产生滞育起始、维持和终止所需的蛋白质，并维持最大的耐受性时期。本研究的总体目标是阐明金银花调控滞育特异性发育的细胞、分子和生化机制，并确定胚胎如何在严重的生理应激下存活。热休克因子1 （HSF1）通常激活生理应激期间表达的基因，将进行测试，以确定它是否调节滞育期间的基因表达。特别令人感兴趣的是一个丰富的，滞育特异性分子伴侣，p26，它在缺乏能量供应的情况下起作用。P26被认为与滞育胚胎中的变性蛋白结合，保护它们免受不可逆变性。因此，p26的底物蛋白将被鉴定。一些分子伴侣如Hsp40、Hsp70和Hsp90需要能量才能活动。这些蛋白质的合成将在滞育胚胎发育过程中被研究，以及它们在能量有限时的保护功能，如滞育过程中发生的。调节滞育终止的蛋白质也将被确定。因此，拟议的研究解决的一个核心问题是，a . franciscana的胚胎如何在严重的压力下存活下来，这种压力会很快杀死大多数其他生物。分子伴侣是否负责压力耐受性，如果是，它们是如何促成这一过程的？回答这些问题将为滞育和基本细胞蛋白（如分子伴侣蛋白）的功能提供重要的基础见解。在应用层面上，拟议的研究可能有助于在水产养殖中使用A. franciscana，该生物被用来喂养商业上重要的鱼类和水生无脊椎动物的幼虫。例如，Hsp70在a. franciscana对细菌感染的抗性中起作用，这表明所提出的工作将导致a. franciscana培养的改进。此外，更好地了解滞育对农业、林业和医学都有影响，因为它可能使人们能够发展出控制能够在冬季生存的害虫的方法，并试图在滞育期间消灭它们，这对加拿大有直接的好处。

Embryos of the crustacean, Artemia franciscana, develop into swimming larvae or they encyst and enter diapause, a physiological state characterized by developmental arrest and greatly reduced metabolism. Encysted embryos (cysts) are very stress tolerant, surviving extreme temperature, desiccation and years without oxygen when hydrated at ambient temperature. A. franciscana cysts therefore represent one of the most stress tolerant multicellular life forms on Earth. A. franciscana embryos exhibit differential gene regulation during diapause-destined development, producing proteins required for the initiation, maintenance and termination of diapause, with maintenance the period of greatest stress tolerance. The global objectives of the proposed research are to elucidate cell, molecular and biochemical mechanisms by which A. franciscana regulate diapause-specific development and determine how embryos survive severe physiological stress. Heat shock factor 1 (HSF1) which normally activates genes expressed during physiological stress will be tested to determine if it regulates gene expression during diapause. Of particular interest is an abundant, diapause-specific molecular chaperone, p26, which functions in the absence of an energy supply. p26 is thought to bind denaturing proteins in diapause embryos and protect them from irreversible denaturation. Consequently, substrate proteins of p26 will be identified. Several molecular chaperones such as Hsp40, Hsp70 and Hsp90 require energy for activity. The synthesis of these proteins will be investigated during diapause-destined embryo development, as will their protective function when energy is limiting, as occurs in diapause. Proteins that regulate diapause termination will also be identified. Thus, a central question addressed by the proposed research is how embryos of A. franciscana survive severe stressors which kill most other organisms rather quickly. Are molecular chaperones responsible for stress tolerance and, if so, how do they contribute to this process? Answering these questions will provide significant fundamental insights into diapause and the functions of essential cellular proteins such as the molecular chaperones. On an applied level, the proposed research may facilitate the use of A. franciscana in aquaculture where the organism is used to feed the larvae of commercially important fish and aquatic invertebrates. Hsp70, for example, plays a role in the resistance of A. franciscana to bacterial infection, indicating that the proposed work will lead to improvements in the culture of A. franciscana. Moreover, a better understanding of diapause has consequences for agriculture, forestry and medicine as it may allow for the development of methods for the control of insect pests able to survive winter and attempts at their eradication when in diapause, this of direct benefit to Canada.