Studies on the Control Mechanisms of Body Color Polymorphism and Prophenoloxidase Cascade in Insect

为明确昆虫幼虫颜色多态性的分子机制及其调控，研究了卷尾草（Agrius convolvuli）和家蚕（Bombyx mori）体色表达的相关因子。1)对绿、黑色幼虫的色素进行了分析。绿色幼虫表皮细胞中含有虫青素、AcINS（蓝色）和表皮类胡萝卜素结合蛋白eCBP（黄色）。另一方面，黑色幼虫表皮有色素，角质层有黑色素。2)研究了黑色素的前体及其转运。酪氨酸、左旋多巴、多巴胺是家蚕和旋花家蚕黑色素的前体。然而，β -丙氨酸只与棕色黑色素结合。3)对AcINS-a和-b进行了纯化和表征。AcINS基因的cDNA序列与Manduca ins具有高度的相似性。黑色幼虫表皮的AcINS含量约为绿色幼虫表皮的1/30。4)从旋花青幼虫中纯化出eCBP。eCBP的载脂蛋白约为27,500 Da，仅检测到叶黄素。ecbp的cDNA和n端氨基酸序列与雄性曼都卡（Manduca sexta） 29 k Da的核蛋白JP29具有高度的相似性。5)对家蚕颗粒型酚氧化酶（gPO）进行了纯化和鉴定。pPOs以原酶的形式合成。pro-gPO的分子量约为78,000 Da。左旋多巴和多巴胺gPO的Km值分别为1.67 mM和0.313 mM。纯化的pro-gPO未被酚氧化酶原活化酶活化。6)从柞蚕（Antherea pernyi）、柞蚕（A. yamamai）和柞蚕（Samia cynthiaricini）中分离到多巴脱羧酶（dopa decar羧酶，DDC）基因。这些DDC基因序列具有较高的相似性。7)从家蚕血淋巴中纯化并鉴定了模式识别蛋白。8)研究了酚氧化酶原从血淋巴到角质层的跨上皮转运机制。

To clear the molecular mechanisms and its control of larval color polymorphism in insect, the factors participating in body color expression in Agrius convolvuli and Bombyx mori were studied.1) The pigments of green and black larvae of A. convoluvli were analyzed. The green larvae have insecticyanin, AcINS (blue) and epidermal carotenoid-binding protein, eCBP (yellow) in epidermal cells. On the other hand, the black larvae have ommochrome in epidermis and melanin in cuticle.2) The precursors of melanin and its transports were examined. Tyrosine, L-dopa, dopamine were precursors of melanin of B. mori and A. convolvuli. However, beta-alanine was only incorporated in brown melanin.3) AcINS-a and -b were purified and characterized. The cDNA sequence of AcINS gene has high similarities with Manduca INSs. AcINS in the epidermis of the black larva was about 1/30 in the green larva.4) The eCBP were purified from the green larva of A. convolvuli. The apo-protein of eCBP was about 27,500 Da, and only lutein was detected in eCBP. The cDNA and N-terminal amino acids sequences of eCBPs showed high similarity with JP29, a 29 k Da nuclear protein of Manduca sexta.5) The granular phenoloxidase (gPO) of B. mori was purified and characterized. The pPOs were synthesized as proenzymes. The molecular mass of pro-gPO was about 78,000 Da. The Km values of gPO were 1.67 mM for L-dopa and 0.313 mM for dopamine. The purified pro-gPO was not activated by prophenoloxidase activating enzyme.6) The dopa decarboxylase (DDC) gene was isolated from Antherea pernyi, A. yamamai, and Samia cynthiaricini. The sequences of these DDC genes showed high similarity with each other.7) The pattern-recognition protein was purified and characterized from hemolymph of B. mori.8) The mechanisms of trans-epithelial transport of the prophenoloxidase from hemolymph to cuticle were investigated.