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

菜豆Riptortus clavatus血淋巴(CP-1、2、3、4)和卵黄(CPegg)中发现的胆绿素结合蛋白(CP)是CPalpha和CPbeta两种76kD亚基的同源和异源六聚体：CP-1(与CPega相同)=α_6；CP-2=α_4beta_2；CP-3=α_2beta_4；CP-4=β_6。克隆和序列分析证实了CP亚基与其他昆虫储藏六聚体和节肢动物血蓝蛋白的序列相似性。到目前为止，CP亚基与其他胆绿素结合蛋白之间没有发现相似之处。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幼虫的每一次蜕皮而周期性波动。用~&lt；125&gt；I标记的CPS证实了CP(主要是Cp-4)被后5龄若虫的脂肪体优先吸收。滞育成虫合成CP的速率较低。在非滞育的雌性成虫中，CP-1在整个生殖阶段都有合成，而雄性则没有。CP-1积聚在生殖雌体的血淋巴中，被发育中的卵巢隔离并储存为CP卵。~(125)I标记的CPS证实了CP-1在卵巢的选择性滞留。在滞育成虫中，CP-4(β_6)是主要的CP分子，但在保幼激素(JH)诱导的滞育后，CP-4(β_6)的合成受到抑制，而CP-1(α_6)的合成又被激活。Northern印迹分析表明，蛋白质含量的变化与合成活性的变化具有很好的一致性。这表明，CP的表达是在转录水平上调节的，JH诱导特定基因的表达从CPbeta切换到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.