Molecular Mechanism of Juvenile Hormone Action

菜豆根结线虫(Riptortus Clavatus)、CP-1、CP-2、CP-3和CP-4的氰基蛋白是由CPA和CPB两个亚基组成的六聚体蛋白。从滞育和滞育雌性成虫的脂肪体中分离到Poly(A)+RNA，构建了cDNA文库。分别用抗CPA和CPB抗血清筛选编码CPA和CPB的cDNA克隆。CPA和CPB插入片段(a：2217bp，b：2163bp)的核苷酸序列预测CPA和CPB分别含有693和686个残基。两个CP亚基的氨基酸序列同源性为68.0%。同源性搜索表明，CPA和CPB与昆虫条纹六角体和节肢动物血蓝蛋白关系密切。本研究从基因水平研究了棒状圆锥虫血蓝蛋白(CP)的表达。从5龄若虫脂肪体中提取总RNA，并用保幼激素(JH)处理滞育雌雄。以CPA和CPB亚基的cDNA为探针，进行Northern印迹分析。对RND进行体外翻译分析。若虫体内两种mRNAs在第2天开始出现，然后开始增加。CPA信使核糖核酸下降到蜕皮，而CPB信使核糖核酸仍大量保留到发育末期。在生殖雌虫中，CPA在羽化后第3天开始表达，然后逐渐增加，而在蜕皮中未检测到CPA基因的表达。至于CPB，在生殖虫中只观察到微量的表达。在对滞育雌虫的处理中，JH处理导致了CP表达从CPB到CPA的筛选，而JH处理完全抑制了滞育雄虫CPA和CPB的表达。

Cyanoproteins of the bean bug, Riptortus clavatus, CP-1,2,3, and 4 are hexameric proteins composed of two different subunits, CPa and CPb. The cDNA libraries were constructed from poly (A) +RNA isolated from the fat body fromdiapause and diapause female adults. The cDNA clones encoding CPa and CPb were screened by using antisera against CPa and CPb, respectively. The nucleotide sequences of CPa and CPb cDNA inserts (a : 2217bp, b : 2163bp) predicted that CPa and CPb contain 693 and 686 residues, respectively. The identities at the level of amino acid sequence between the two CP subunits were calculated to be 68.0%. Homology search revealed that CPa and CPb were closely related to insect strage hexamers and arthropod hemocyanins. The author concluded that the hemocyanins of R.clavatus fall into the so-celled 'hexamerin' superfamily.The expression of Cyanoprotein (CP) was studied at the level of mRNA in the bean bug, Riptortus clavatus. Total RNA was extracted from the fat body of 5th nymphal stages, reproductive females and males, and juvenile hormone (JH) treated diapause females and males. The RNA was analyzed by northern blot analysis using cDNAs of CPa and CPb subunits as probes. The RND was also analyzed by in vitro translation. In the nymphs, both mRNAs appeared first on day-2 and then increased. CPa mRNA decreased to the molt while large amounts of CPb mRNA still remained at the end of the stage. In the reproductive females CPa expression was first detected on day-3 after emergence, then increased onward while no CPa mRNA was detected in the molts. As for CPb, a only trace expression was observed in the reproductive bugs. In the JH treatment to the diapause females resulted in the sift of CP expression from CPb to CPa while the treatment supressed completely the expression of both CPa and CPb in the diapause males.