飞蝗横纹肌肌球蛋白的序列分析与功能比较

The migratory Locusts (Locusta migratoria manilensis) are important agricultural pests in China. The plague of Locusts is associated with locusts' strong ability in moving and flying, which depends on locust muscle system. Striated muscle myosin is the most important functional protein in muscle. Contrary to vertebrates, locust has only one striated muscle myosin (hereafter referred as myosin) gene. We have obtained the full sequence of locust myosin gene and identified several alternative splicing exons in it. In theory, locust myosin gene encodes 360 types of myosin. However, locust myosin sequence as well as its tissue and development distribution remains to be determined experimentally. Another key issue is how the alternative splicing affects myosin function. In the proposed study, we will take two approaches to address these questions. First, we will determine the sequence of myosin in locust muscles by cDNA cloning and DNA sequencing. Second, we will purify myosin from locust muscles and analyze its function using a number of assays, to provide the structure-function relationship of locust myosin. Specific aims are 1) determine the sequence and function of locust myosin in various muscles; 2) determine the sequence and function of locust myosin in muscle under various development stages; 3) compare the sequence and function of locust myosin of gregarious and solitary locust. This study will not only pave the way for research in the field of locust muscle physiology, but also will provide clue on the molecular mechanism of locust phase transition and migration.

东亚飞蝗是我国一种重要的农业害虫，蝗灾的爆发与飞蝗很强的运动和迁飞能力有关。飞蝗的运动依赖于肌肉系统，横纹肌肌球蛋白（简称肌球蛋白）是肌肉中最重要的功能蛋白。与脊椎动物不同，飞蝗只有一个肌球蛋白基因。目前，我们已得到了该基因的全顺序，发现该基因的多个外显子可以发生选择性剪切，理论上可以编码360种不同的肌球蛋白，但实际编码的序列和分布还不清楚。另一个重要问题是选择性剪切如何影响肌球蛋白的功能。本研究将通过cDNA克隆测序的方法确定飞蝗肌肉中肌球蛋白的序列和种类；通过生化提取的方法从肌肉中得到纯化的肌球蛋白，测定其活性，建立飞蝗肌球蛋白结构与功能的关系。本研究将确定1）不同肌肉组织中肌球蛋白的序列与功能；2）不同发育阶段肌肉组织中肌球蛋白的序列与功能；3）群居型与散居型飞蝗飞行肌中肌球蛋白的序列与功能差异。本研究将为深入研究飞蝗肌肉系统奠定基础，也将有助于解析飞蝗型变与迁飞的分子机制。

飞蝗是重要的农业害虫，也是研究昆虫肌肉生理的模式动物。飞蝗拥有种类繁多的肌肉，它们的收缩为飞蝗各种形式的运动提供动力。然而，对于飞蝗肌肉中的各种组分是怎样引起肌肉功能多样性的，我们还知之甚少。此外，飞蝗可以发生散居型和群居型转换，散居型向群居型的转换是引起农作物大规模损害的重要原因，群居型飞蝗具有强大的飞行能力。在本项目研究主要研究成果包括：1）鉴定了飞蝗横纹肌肌球蛋白重链的Mhc基因，发现该基因可以通过可变剪切编码360种不同类型的肌球蛋白，在三种典型横纹肌中鉴定出13种肌球蛋白。2）通过组织提取，获得了飞行肌(胸部背纵肌)和跳跃肌(后足胫节伸肌)中的肌球蛋白，生化活性分析显示，与飞行肌中的肌球蛋白不同，跳跃肌中的肌球蛋白具有酸激活特性，推测该特性与跳跃肌能量代谢有关；序列分析暗示我们发现的酸激活特性在其他昆虫中可能是保守的。3）利用RNA-seq分析，发现群居型和散居型飞蝗飞行肌的基因表达存在多个方面的差异，包括转录调控和蛋白质加工，能量代谢和激素调节等方面有显著的表达差异。上述研究成果为今后研究飞蝗肌肉分化机制，及飞蝗群散两型变化的机制研究提供了分子基础。