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），这是发现在血淋巴CP-1、CP-2、CP-3、CP-4和CP-4与鸡卵黄蛋白（CPegg）是由两种76 kD亚基CPalpha和CPbeta组成的同源和异源六聚体。（与CPegg相同）= α_6; CP-2 = α_4 β_2; CP-3 = α_2 β_4; CP-4 = β_6。这两个亚基的cDNA克隆和序列分析表明，CP亚基与其他昆虫储藏六聚体和节肢动物血蓝蛋白之间存在序列相似性。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含量及其合成活性随每次蜕皮而呈周期性波动。用碘标记的CP证明了CP（主要是CP-4）被晚期第5若虫的脂肪体优先隔离<125>。滞育成虫合成CP的速率较低。在非滞育的成年雌性中，CP-1在整个生殖阶段合成，而在雄性中没有发现CP合成。生殖雌虫血淋巴中积累的CP-1被发育中的卵巢隔离并以CPegg形式储存。卵巢对CP-1的选择性隔离由13 <125>I标记的CP证实。CP-4（beta_6）是滞育成虫的主要CP分子，但在保幼激素（JH）诱导的滞育后雌性中，CP-4（beta_6）合成被抑制，而CP-1（alpha_6）合成被激活。用北方印迹分析法测定的CP mRNA含量变化与合成活性变化一致。这表明CP的表达在转录水平上受到调节，并且JH诱导特定基因表达从CP β到CPalpha的转换。

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.