超活性AFP在白蜡虫抗冻中的关键作用及其分子机理

The Chinese white wax scale insect, Ericerus pela, is a traditional resource insect, and has strong adaptability to extremely low temperatures by lowering their supercooling point (SCP) below the temperature of environment. This characteristic has great effect on the division and expansion of new white wax producing regions. However, the molecular mechanism underlying the strong supercooling ability of E. pela remains unclear. It is assumed that E. pela contains the hyperactive antifreeze protein (AFP), which plays a key role in the antifreeze process by significantly lowering the freezing point. To unclose the molecular mechanism underlying the antifreeze process of E. pela, this study focus on the scientific problem that hyperactive AFP plays key role in the antifreeze process of E. pela. The AFP will be isolated by ice affinity method combined with cold reduced differently expressed genes from the overwintering E. pela adults collected from Changchun City, and from the cold induced before-overwintering E. pela adults collected from Kunming City. The thermal hysteresis activity will be measured through heterologous protein expression in vitro, then the hyperactive AFP will be identified. The role of the hyperactive AFP in regulation of SCP will be analyzed emphatically. Finally, RNAi will be employed to analyze effect of the hyperactive AFP on the SCP and the survival rate of E. pela under low temperature. Moreover, the function of hyperactive AFP in antifreeze process of E. pela will be validated. The expected results will illuminate the molecular mechanism underlying the adaptability of E. pela to low temperatures.

白蜡虫是我国传统资源昆虫，对极端低温环境具有很强的适应能力，该特点对区划和扩大新的白蜡产区具有重要影响，但白蜡虫适应极端低温环境的分子机理尚不清楚。白蜡虫过冷却点（supercooling point, SCP）低于环境温度，由于抗冻蛋白（antifreeze protein, AFP）可以显著降低冰点，超活性AFP在白蜡虫抗冻过程可能起到了关键作用。为揭示白蜡虫抗冻的分子机制，本项目围绕超活性AFP形成了白蜡虫超强的过冷却能力这一科学问题，拟采用冰吸附的方法，并结合低温诱导差异表达基因分析，从长春越冬白蜡虫和昆明低温诱导白蜡虫中分离AFP，然后通过蛋白的体外表达进行热滞活性测定，鉴定出超活性AFP，着重分析超活性AFP对溶液过冷却点的降低作用，最后通过RNA干涉研究超活性afp基因对白蜡虫SCP和低温存活的影响，明确其对白蜡虫抗冻的关键作用，从而揭示白蜡虫适应极端低温环境的分子机理。