Molecular neuro-physiological study of insect diapause mechanism

以家蚕脑中提取的c DNA为模板，用聚合酶链式反应(PCR)扩增出复眼视蛋白基因。我们扩增了一条约200个碱基的条带，然后亚克隆了一个编码视蛋白样蛋白基因的210个碱基的DNA片段(70个氨基酸)。在此DNA序列的基础上，我们进行了3‘-5’-Race方法来确定ORF区域的全长。克隆的DNA序列长约1600个碱基，开放阅读框架编码377个氨基酸。研究发现，该DNA序列与已报道的六色曼陀罗复眼感光色素之一ManipI有很高的同源性。我们将这种家蚕脑中的视蛋白样蛋白命名为Boceropis(Bombyx Brain Opsin)。合成了一个与博尔罗布酶的羧基末端25个氨基酸对应的多肽。免疫细胞化学显示，家蚕脑中的某些细胞对该多肽的兔抗血清表现出较强的免疫反应。这些免疫反应细胞被认为是参与家蚕光周期反应的化蛹感光细胞。

PCR was carried out using cDNA prepared from brain of the silkworm, Bombyx mori with primers by which opsin encoding genes of the compound eyes can be amplified. We got an amplification of a band corresponding about 200 bases, and then subcloned a fragment of 210 bases DNA (70 amino acids) encoding a opsin-like gene. On the base of this DNA sequence, we conducted 3'-5'-Race method to determine all length of ORF region. The cloned DNA sequence had about 1600 bases and the ORF coded 377 amino acids. It was found that the DNA sequence has a high homology with ManipI reported as one of the photoreceptive pigments in the Manduca sexta compound eyes. We named this opsin like protein in the silkworm brain Boceropisn (bombyx cerebral opsin). One peptide corresponding to the carboxyterminal 25 amino acids of Boceropsin was synthesized. And immunocytochemistry revealed that some pairs of cells of the silkworm brain showed potent immunoreactive to the rabbit antisera of the peptide. These immunoreactive cells is suggested to be a pupative photoreceptor involved in the photoperiodic response in the silkworm.