Juvenile hormone receptor and molecular mechanism of gene expresion

为了研究昆虫保幼激素(JH)调控基因表达的分子机制，我们利用mRNA差异显示技术分析了菜豆幼虫保幼激素(JH)应答基因。用JH类似物甲基丁二烯处理滞育雌成虫，从脂肪体中提取RNA。在mRNA差异显示中，我们发现了JHA和溶剂丙酮处理的动物的许多差异条带。我们提取了经JH处理后被诱导或抑制的基因。利用这些基因对JH或丙酮处理的脂肪体RNA进行了Northern Blot分析，发现了两个JH诱导基因(JI-1和JI-2)和三个JH抑制基因(JR-1、JR-2和JR-3)。部分序列分析和数据库同源性搜索表明，JI-1和JI-2分别与氰蛋白-a和卵黄蛋白原有很高的同源性，我们已经发现了血淋巴储存六聚体和蛋黄蛋白前体。JR-1在脊椎动物和昆虫中与转铁蛋白有很高的同源性。另一方面，我们没有找到与JR-2和JR-3有同源性的基因，认为这些基因是新的。我们克隆了JI-2和JR-1的全长cDNA，并进行了测序，并用Northern Blot分析了JH处理后的mRNA水平的变化。我们还克隆了JI-1(CP-a)、JI-2(VG)和JR-1(Transferrin)的基因组DNA，并对其上游调控基因结构进行了分析。目前已有3个JH可诱导基因和3个可抑制基因可用于比较JH在相同激素条件下对同一昆虫的表达调控。

To study the molecular mechanism of gene expression regulation by insect juvenile hormone (JH), we have analyzed JH responsive genes by mRNA Differential Display in the bean bug, Riptortus clavatus. JH analog, methoprene, was applied on the diapause female adults and extracted RNA from the fat body. We found many cDNA bands which showed differnence between JHA and solvent acetone treated animals in mRNA Differential Display. We picked up the cDNA which were induced or repressed cDNA by JH treatment. Using these cDNA we performed Northern Blot analyzes for JH or acetone treated fat body RNA,and identified two JH inducible genes (JI-1and JI-2) and three JH repressible genes (JR-! , JR-2 and JR-3). The analysis of the partial sequeces of these cDNA and data base homology search indicated that JI-1 and JI-2 had high homology to cyanoprotein-a and vitellogenin, respectively, which we have showed hemolymph storage hexamer and egg yolk protein precursor. JR-1 was showed high homology to transferrin in vertebrate and insects. On the other hand we could not find any genes which have homology to JR-2 and JR-3 and concluded these gene are novel. We cloned and sequenced full length of cDNA of JI-2 and JR-1 and analyzed the changes of mRNA level after JH tretment by NOrthern Blot analyzes. We also cloned genomic DNA and analyzed the upstream regulatory gene structure of JI-1 (CP-a), JI-2 (Vg) and JR-1 (Transferrin). Now three JH inducible and three repressible genes are available to compare the regulation of expression by JH in the same insect under the same hormonal condition.