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

为了阐明昆虫幼虫颜色多态的分子机制及其调控，本研究以卷叶姬松茸和家蚕为材料，研究了影响幼虫体色表达的因素：1)分析了卷曲姬蜂幼虫绿色和黑色幼虫的色素。绿色幼虫在表皮细胞中含有昆虫蓝素、AcINS(蓝色)和表皮类胡萝卜素结合蛋白eCBP(黄色)。2)对黑色素前体及其转运进行了观察。酪氨酸、L多巴、多巴胺是家蚕和卷叶猴黑色素的前体。3)纯化并鉴定了AcINS-a和AcINS-b。AcINS基因的cDNA序列与Manduca INSS有很高的相似性。黑色幼虫表皮中的乙酰胆碱含量约为绿色幼虫的1/30。4)从绿色幼虫中提纯出eCBP。ECBP的载脂蛋白大小约为27,500 Da，仅检测到叶黄素。ECBPs的基因序列和N端氨基酸序列与家蚕核蛋白JP29有很高的同源性。5)家蚕颗粒酚氧化酶(GPO)的纯化和鉴定。以酶原的形式合成了多聚磷酸酶。原-GPO的相对分子质量约为78000 Da。L多巴和多巴胺的Km值分别为1.67 mM和0.313 mM。纯化的前GPO不被酚氧化酶原活化酶激活。6)分离了大蚕蛾、天南星和野桑蚕的多巴脱羧酶基因。这些DDC基因的序列具有很高的相似性。7)从白僵菌的血淋巴中纯化了模式识别蛋白，并对其进行了鉴定。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.