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

在豆虫的血淋巴（CP-1、2、3和4）和蛋黄（CPegg）中发现的胆绿素结合蛋白、氰蛋白（CP）是两种76 kD亚基（CPalpha和CPbeta）的同异六聚体：CP-1（与CPegg相同）= alpha_6；CP-2 = alpha_4beta_2；CP-3 = alpha_2beta_4；CP-4 = beta_6。对这两个亚基的cDNA克隆和序列分析确定了CP亚基与其他昆虫贮藏六聚体和节肢动物血青素的序列相似性。到目前为止，CP亚基与其他胆汁素结合蛋白之间没有相似之处。基因组DNA克隆结果表明，CPalpha和CPbeta基因均由11个外显子组成。在若虫发育过程中，CP含量及其合成活性随每次蜕皮呈周期性波动。通过使用^<125> i标记的CP，证实了晚期五若虫脂肪体对CP（主要是CP-4）的优先吸收。滞育成虫合成CP的速率较低。在非滞育的成年雌性中，CP-1在整个生殖阶段都有合成，而在雄性中没有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cpeg的形式储存。通过^<125> i标记的CPs证实了卵巢对CP-1的选择性吸收。在滞育雌成虫体内，CP-4 （beta_6）是主要的CP分子，但在滞育后雌虫体内，幼虫激素（JH）诱导的CP-4 （beta_6）合成受到抑制，而CP-1 （alpha_6）合成被激活。northern blot检测的CP mRNA含量变化与合成活性变化吻合较好。这表明CP的表达在转录水平上受到调控，JH诱导了特定基因表达从CPbeta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.