抗冻蛋白基因LD4提高鱼类抗寒性能的分子机制研究

Fish antifreeze proteins are produced by fishes inhabiting the polar marine environments, serving as biological antifreezes that lower the freezing points of the blood and body fluids. Although diverse in compsoition and structure, all known antifreeze proteins function by a mechanism termed as ‘absorption-inhibition’, by which the antifreze molecules bind with ice and inhibit ice-growth. Thus, in the conventional sense, antifreze proteins protect the fishes from freezing demage by a physical interaction with the ice, rather any biochemical functions in cellular physiology. Recent years however, accumulating evidence indicated that some antifreeze protein types could confer cold protection functions to transgenic plants or animals even in temperatures above the freezing point when no ice could exist. In past few years, we identified a new type of antifreeze protein LD4, which showed stronger cold protection activities than other fish antifreeze proteins. In this study, we will conduct comparative studies between LD4 and other fish antifreeze proteins in the attempt to elucidate the mechanism of the superior cold protection function of the LD4. We will focus on identification of the interacting partners of LD4 and the mechanism by which it inhibits cold induced apoptosis, so to elucidate the transduction pathways of the LD4 function. This project will broaden our knowledges on the biochemical and physiological functions of antifreeze proteins, which provide new genes and approaches for anti-frost breeding of aquaculturely important species.

鱼类的抗冻蛋白是由生活于极地海洋的鱼类产生的能降低血液和体液冰点的蛋白。虽然种类和起源多样，它们都通过一个特殊的表面吸附于冰晶表面，抑制冰晶的生长。因此，传统意义上来说它们抗冻的功能是一种物理学的吸附和抑制功能，没有细胞生理学意义上的生物化学功能。近来，通过转基因的研究，越来越多的证据表明一些抗冻蛋白具有在没有冰晶存在的低温状态下保护细胞和个体不受低温胁迫伤害的能力。本实验室在过去的研究中获得鉴定了一种比其它鱼类抗冻蛋白具有更高抗低温胁迫功能的抗冻蛋白基因LD4。本项研究将利用这个蛋白基因与其它抗冻蛋白基因进行比较研究，揭示这个蛋白赋予动植物抵抗低温胁迫能力的分子机制。本项研究将从LD4抵抗低温引起的细胞凋亡，与其它蛋白或者生物分子间的相互作用，以及LD4引起的基因表达谱的改变等方面揭示LD4抵抗低温胁迫的信号通路。本项研究将拓展抗冻蛋白的生理学功能，为其在抗寒育种中的应用奠定理论基础。

先前认为鱼类抗冻蛋白的功能就是与冰晶结合从而抑制冰点状态的溶液发生冰冻。本项目的研究证实了来自于南极Lycodichthus dearborni有4个III型抗冻蛋白结构域组成的抗冻蛋白LD4不但有原先认识的对冰晶的抑制功能，还具有在非冰冻低温下保护细胞生理稳态的功能。我们发现，LD4可以与内质网的应激蛋白BIP相互作用，降低因低温引起的内质网应激反应和应激颗粒的形成以及胞质Ca2+的积累，从而大大降低低温下(4°C)细胞凋亡。项目通过转基因斑马鱼验证了LD4使原本不能在124°C低温刺激后正常发育的胚胎能完全正常地发育。项目构建了拟南芥和烟草的转LD4植株,发现这些植物在4°C长期胁迫后也能恢复生长。研究发现这种非冰冻温度下对动植物抗寒能力的提升与抗冻分子中所含的AFPIII结构域的数量呈正相关。本项目对LD4作用机制的阐明拓展了对抗冻蛋白功能的认识，为冰冻压力下分子进化提供新的视角，也为LD4基因在动植物抗寒育种和医学细胞保存中的应用奠定理论基础。