Vermunt A M, Koopmanschap A B, Vlak J M, de Kort C A
Department of Entomology, Wageningen Agricultural University, The Netherlands.
Insect Mol Biol. 1998 Nov;7(4):327-36. doi: 10.1046/j.1365-2583.1998.740327.x.
Juvenile hormone esterase (JHE) activity in the haemolymph of the Colorado potato beetle is necessary to initiate pupation in larvae as well as diapause in adults. The enzyme appears in the haemolymph as a dimer consisting of two 57 kDa subunits. The sequence of an encoding cDNA, JHE.A, is distinct from lepidopteran JHEs. In this study, RT-PCR using primers designed on the basis of the 5'- and 3'-ends of the coding region revealed the existence of a related gene, JHE.B. The presence of two JHE-related genes was also shown by PCR amplification on genomic DNA from different individual beetles followed by restriction enzyme analysis. Both forms, probably paralogues, were transcribed since they could be amplified on messenger RNA from fat bodies. The size of the PCR products generated with mRNA and genomic DNA were both 1.6 kb, suggesting the absence of introns in the genomic JHE coding sequence. The sequence of a genomic clone, which encoded JHE.B, was 77% identical and 82% similar in amino acids compared to JHE.A. No introns were found in the coding sequence of these coleopteran JHE-related genes, in contrast to lepidopteran JHE genes. Southern blot analysis of digested genomic DNA confirmed the presence of two JHE-related genes.
科罗拉多马铃薯甲虫血淋巴中的保幼激素酯酶（JHE）活性对于幼虫开始化蛹以及成虫进入滞育是必需的。该酶以由两个57 kDa亚基组成的二聚体形式出现在血淋巴中。编码cDNA JHE.A的序列与鳞翅目JHE不同。在本研究中，使用基于编码区5'端和3'端设计的引物进行RT-PCR，揭示了一个相关基因JHE.B的存在。通过对来自不同个体甲虫的基因组DNA进行PCR扩增，随后进行限制性酶切分析，也表明存在两个与JHE相关的基因。两种形式可能是旁系同源物，因为它们可以从脂肪体的信使RNA上扩增出来。用mRNA和基因组DNA产生的PCR产物大小均为1.6 kb，表明基因组JHE编码序列中不存在内含子。与JHE.A相比，编码JHE.B的基因组克隆序列在氨基酸上有77%的同一性和82%的相似性。与鳞翅目JHE基因不同，在这些鞘翅目JHE相关基因的编码序列中未发现内含子。对消化后的基因组DNA进行Southern印迹分析，证实了存在两个与JHE相关的基因。
