PKG基因对粘虫迁飞行为分化的调控机制研究

The Formation and regulation of migratory behaviors in Mythimna separata (Walker) is always the hot issue in the field of insect migration research. Previous studies have shown that the moths vary greatly in migratory physiological characteristics because of different rearing densities (1 larva per jar and 10 larvae per jar). The moths developed from gregarious larvae (10 larvae per jar) are more prone to migration than the solitary ones (1 larva/jar). In addition, the feeding behaviors are also different between "gregaria" and "solitaria". The former eat leaves more actively than the latter. For this reason, the investigation on the mechanism of migratory behavior differentiation in M. separate at the molecular level by the regulation of feeding behaviors is an important complement to migration mechanisms. Other studies have recently shown that PKG gene plays a key role in the regulation of foraging behaviors in classical model animals. The previous studies of the applicant have indicated that PKG is differentially marked with higher expression level in the solitary larvae than in the gregarious ones. This project is to investigate the transformation of feeding behavior and migratory physiological characteristics by the methods of pharmacology and RNA reference, and to further expound on the possible regulatory mechanism of PKG on migratory behavior differentiation in M. separate.

粘虫(Mythimna separata (Walker)) 迁飞行为发生与调控机制的研究一直为研究领域的热点。已有研究表明不同饲养密度幼虫（1头/瓶、10头/瓶）羽化成的成虫迁飞特性存在差异。群居型(10头/瓶)较散居型（1头/瓶）更有易于迁飞。群居型(10头/瓶)和散居型（1头/瓶）幼虫取食行为也存在差异，群居型幼虫取食更为积极且食量更大。因此，从分子水平调控粘虫幼虫取食行为来研究粘虫迁飞行为分化机制是研究粘虫迁飞机制的一个重要补充。已有研究表明PKG基因在调控经典模式动物觅食行为上发挥着关键作用。申请者前期工作已证明粘虫PKG基因在散居型幼虫脑中表达水平显著成性高于群居型。本项目拟通过药理学手段和RNA干扰技术处理粘虫来测定其取食行为、食物利用率和迁飞特性变化，进而阐明PKG对粘虫迁飞行为分化的可能调控机制。

粘虫Mythimna separata迁飞行为分化调控机制的研究一直为研究领域的热点。已有研究表明PKG基因在调控经典模式动物觅食行为上发挥着关键作用。从分子水平调控粘虫幼虫取食行为来研究粘虫迁飞行为分化机制是研究粘虫迁飞机制的一个重要补充。本课题以已建立的粘虫迁飞行为分化模型（群居型(10头/瓶)较散居型（1头/瓶）更有易于迁飞）为基础，运用生物信息学、RNA干扰、Western blot、药理学和荧光免疫组织化学等方法与技术，对PKG基因对粘虫迁飞行为分化的调控机制进行了研究。首先，阐明了两种生活型粘虫脑中PKG表达模式及蛋白活性。粘虫PKG基因在4龄幼虫、5龄幼虫和成虫三个虫态的两种生活型的脑中表达呈显著性差异。在4龄和5龄散居型幼虫脑中的表达水平显著高于群居型，而群居性成虫脑中PKG表达量显著高于散居性成虫；5龄散居型幼虫脑中PKG蛋白表达量与活性均显著性高于群居型，而成虫群居型PKG活性显著性高于散居型。其次，运用RNAi技术粘虫PKG基因进行了沉默，结果沉默效率不显著。第三，进而运用药理学技术对PKG蛋白活性进行激动或抑制，结果发现提高粘虫PKG蛋白活性可以提高粘虫幼虫取食量和幼虫体重，由此推测PKG可能调节消化酶的分泌；为此观察了粘虫细胞消化道的细胞类型和消化酶活性，结果显示其消化道有柱状细胞、杯状细胞、分泌细胞和再生细胞4种细胞，群居型的胰蛋白酶、淀粉酶和脂肪酶活性均显著性高于散居型；PKG参与调控成虫飞行行为，受PKG抑制剂KT5823处理的粘虫平均飞行次数显著少于对照组，平均每次停飞时间显著长于对照组，其他飞行参数没有显著性变化。幼虫期受药物处理后，其发育成的成虫的活性并不与迁飞行为分化模式一致，因此可以断定PKG不负责调控粘虫迁飞行为分化；PKG在粘虫脑中的调控区被定位在幼虫的后脑和成虫前脑、中脑和视叶的交界处。