庆网蛱蝶高温适应分子机制及其在同域蝴蝶中趋同演化的研究

Temperature exhibits overall and deep impacts on organism, and organism may adapt local temperature through genetic variation. The Glanville fritillary butterfly (Melitaea cinxia) lives in both Xinjiang of China and Åland Islands of Finland. The ambient temperature is significantly different between these two areas. In June when adults fly in the field, the daily highest temperature is about 25℃ in Åland, whereas it is above 35℃ in Xinjiang. The Glanville fritillary butterflies living in Xinjiang should possess higher adaptation to high temperature than those in Åland. In our previous study, it was found that phosphoglucose isomerase (PGI) showed higher thermal stability from Chinese population than Finnish population with different PGI allozymes. In this project, we will focus on molecular mechnism of adaptation to high temperature in the Glanville fritillary butterfly through the following studies. First, we will require recombinant PGI, and then detect their thermal stability, so that we may understand the contribution of each amino acid substitution to thermal stability of PGI. Second, we will clone the genes involved in adaptation to high temperature from the other species of butterfly living in the same region, and detect whether there are same amino acid substitutions in these species, i.e. whether convergent evolution occurred under selection of high temperature. These studies will be helpful for us to understand the molecular mechanism of adaptation to high temperature in the Glanville fritillary butterfly, and provide basic information for discovering the general rules of adaptation in organisms. In the meantime, the results also would be helpful for understanding the adaptation potential to global warming in the Glaville fritillary butterfly, which may provide scientific evidence for conservation.

温度对生物有着全面而深刻的影响，而生物可通过遗传变异来适应当地的温度。庆网蛱蝶在中国新疆和芬兰Åland岛均有分布，每年六月，Åland岛白天最高气温为25℃，而在新疆则在35℃以上，可见生活于中国的庆网蛱蝶对高温环境具有更强的适应性。本组前期研究发现，中国种群的葡萄糖磷酸异构酶（PGI）比芬兰种群具有更高的热稳定性。本项目将通过如下研究来探讨庆网蛱蝶高温适应的分子机理：首先，体外表达定点突变的重组PGI，通过酶活测定来分析氨基酸替换对蛋白质热稳定性的贡献；然后，在新疆地区其它种蝴蝶中克隆高温适应相关基因，检测其是否具有和庆网蛱蝶一样的氨基酸替换模式（即趋同演化）。这些研究将有助于我们更加全面深入地了解庆网蛱蝶高温适应的分子机制，为揭示生物高温适应的基本规律提供数据支撑。

温度对生物有着全面而深刻的影响，而生物可通过遗传变异来适应当地的温度。庆网蛱蝶在中国新疆和芬兰Åland岛均有分布，每年六月是其成虫期，新疆气温显著高于Åland岛。我们采集了两地的庆网蛱蝶，蛋白提取液高温处理后再进行酶活测定，结果表明中国种群的葡萄糖磷酸异构酶（PGI）比芬兰种群具有更高的热稳定性。通过对pgi基因的分析发现，发现芬兰种群共发现5个pgi等位基因，优势等位基因为pgi-d；中国种群有2个等位基因，优势等位基因为pgi-g3。原核表达主要的PGI酶型，纯化后测定其热稳定性和催化反应动力学参数，结果表明，G3-PGI比D-PGI具有更高的热稳定性，但催化效率比D-PGI低。序列比对结果表明，G3-PGI和D-PGI之间氨基酸差异数量最多，共存在5个位点的氨基酸替换；通过定点突变逐个替换氨基酸，再测定其热稳定性，结果表明，Q35H、T49M和V64I这三个氨基酸替换是引起热稳定性改变的根本原因。通过对PGI蛋白三维结构的模拟，可以分析得到这三个位点基本处于两个单体的交界面处，氨基酸的替换影响了它们与周围其它氨基酸的相互作用，使得两个单体的稳定性发生变化。对中国新疆和庆网蛱蝶同域分布蝴蝶（铂蛱蝶、荨麻蛱蝶、豆粉蝶和露娅小蚬蝶）的PGI氨基酸序列进行分析，结果表明，铂蛱蝶、荨麻蛱蝶、豆粉蝶和露娅小蚬蝶的PGI序列在35、49和64位均表现出与新疆庆网蛱蝶G3-PGI的氨基酸类型的高度一致。这表明，共同生活在新疆高温环境下的蝴蝶在分子水平上采用了相同的氨基酸替换规律以提高PGI蛋白的热稳定性，从而适应高温的环境，表现为一种对高温环境适应的趋同演化现象。这一结果有助于我们更好地理解全球气候变暖下生物的适应机制。