Identification of biliverdin binding protein receptor and its regulation by juvenile hormone in the bean bug, Riptortus clavatus

胆绿素结合蛋白、蓝蛋白 (CP)，在豆虫 Riptortus clavatus 的血淋巴（CP-1、2、3 和 4）和蛋黄 (CPegg) 中发现，是两种 76 kD 亚基 CPalpha 和 CPbeta 的同源和异源六聚体： CP-1（与 CPegg 相同）= alpha_6 ； CP-2 = alpha_4beta_2 ; CP-3 = alpha_2beta_4 ; CP-4 = beta_6。两个亚基的 cDNA 克隆和序列分析确定了 CP 亚基、其他昆虫储存六聚体和节肢动物血蓝蛋白之间的序列相似性。迄今为止测序的 CP 亚基和其他胆绿素结合蛋白之间没有发现相似之处。还进行了基因组 DNA 克隆，并证明 CPalpha 和 CPbeta 基因均由 11 个外显子构建。在若虫发育过程中，CP 含量及其合成活性根据每次幼虫蜕皮而周期性波动。通过使用^ 125 I标记的CP证明了第五若虫晚期的脂肪体对CP(主要是CP-4)的优先封存。滞育成虫合成CP的速度较低。在非绝经成年雌性中，CP-1在整个生殖阶段合成，而雄性中没有发现CP合成。 CP-1 积累在生殖雌性的血淋巴中，被发育中的卵巢隔离并以 CPegg 的形式储存。通过125 I-标记的CP证实了卵巢对CP-1的选择性隔离。 CP-4 (beta_6) 是滞育成年雌性中主要的 CP 分子，但在保幼激素 (JH) 处理诱导的滞育后雌性中，CP-4 (beta_6) 合成受到抑制，进而 CP-1 (alpha_6) 合成被激活。 Northern印迹分析测定的CP mRNA含量的变化与合成活性的变化非常一致。这表明 CP 的表达在转录水平受到调节，并且 JH 诱导特定基因表达从 CPbeta 转换为 CPalpha。

Biliverdin binding protein, cyanoprotein (CP), which were found in the hemolymph (CP-1, 2, 3 and 4) and the egg yolk (CPegg) of the bean bug, Riptortus clavatus, were homo- and heterohexamer of two sorts of 76 kD subunit, CPalpha and CPbeta : CP-1 (identical to CPegg) = alpha_6 ; CP-2 = alpha_4beta_2 ; CP-3 = alpha_2beta_4 ; CP-4 = beta_6. cDNA cloning and and sequence analyzes of the two subunit established the sequence similarities among the CP subunits, other insect storage hexamers and arthropod hemocyanins. No similarities were found between the CP subunits and other biliverdin-binding proteins so far sequenced. Genomic DNA cloning was also done and demonstrated that both CPalpha and CPbeta genes were constructed by 11 exons. During nymphal development CP content and its synthetic activity fluctuated cyclically in accordance with each larval molt. Preferential sequestration of CP(mainly CP-4) by the fat body of the late fifth nymph was demonstrated by using ^<125>I-labeled CPs. Diapause adults synthesized CP at a low rate. In non-deapause adult females, CP-1 was synthesized throughout the reproductive stages, while no CP synthesis was found in males. CP-1 accumulated in the hemolymph of the reproductive females was sequestered bythe developing ovary and stored as CPegg. Selective sequestration of CP-1 by the ovary was confirmed by ^<125>I-labeled CPs. CP-4 (beta_6) was the main CP molecule in the diapause adults females, but in the postdiapause females induced by juvenile hormone (JH) treatment CP-4 (beta_6) synthesis was suppressed and, in turn, CP-1 (alpha_6) synthesis activated. Changes of CP mRNA content determined by northern blot analyzes were well coincident with the changes of the synthetic activity. This suggested that the expression of CP is regulated at the level of transcription and that JH induces a switching of a specific gene expression from CPbeta to CPalpha.