Vermunt A M, Koopmanschap A B, Vlak J M, de Kort C A
Department of Entomology, Wageningen Agricultural University, The Netherlands.
Insect Biochem Mol Biol. 1997 Nov;27(11):919-28. doi: 10.1016/s0965-1748(97)00073-8.
In the Colorado potato beetle, Leptinotarsa decemlineata, reproduction and diapause are mediated by the juvenile hormone (JH) titer in the hemolymph. This titer is controlled by JH synthesis in the corpora allata and by JH degradation. The main pathway of JH degradation is by JH esterase in the hemolymph. The native JH esterase appeared to be a dimer consisting of two 57 kDa subunits (Vermunt et al., 1997). The 57 kDa subunit of JH esterase was digested with endoproteinase Lys-C and the digestion products were separated by reversed phase HPLC. Three different peptides were collected and sequenced. The amino acid sequence of one peptide showed high similarity to fragments of other insect esterases. Based on the amino acid sequence of these peptides, degenerate primers were constructed for RT-PCR. A PCR product of 1.3 kb was obtained and sequenced. This product was used to screen a cDNA library for a complete cDNA copy and to analyze the messenger RNA from larvae and adult beetles. The size of the messenger RNA was 1.7 kb. The complete amino acid sequence of the protein was deduced from the nucleotide sequence of overlapping clones from a cDNA library and a 5'RACE product. An open reading frame (ORF) of 1545 base pairs encoded a 57 kDa protein with a predicted pI of 5.5. The ORF contained the sequence of the three peptides. It showed no significant homology to other proteins present in databases, but it did contain several functional esterase motifs.
在科罗拉多马铃薯甲虫（Leptinotarsa decemlineata）中，繁殖和滞育由血淋巴中的保幼激素（JH）滴度介导。该滴度由咽侧体中的JH合成以及JH降解控制。JH降解的主要途径是通过血淋巴中的JH酯酶。天然的JH酯酶似乎是由两个57 kDa亚基组成的二聚体（Vermunt等人，1997年）。用内肽酶Lys-C消化JH酯酶的57 kDa亚基，消化产物通过反相高效液相色谱分离。收集并测序了三种不同的肽。一种肽的氨基酸序列与其他昆虫酯酶的片段具有高度相似性。基于这些肽的氨基酸序列，构建了简并引物用于RT-PCR。获得了一个1.3 kb的PCR产物并进行了测序。该产物用于筛选cDNA文库以获得完整的cDNA拷贝，并分析幼虫和成虫甲虫的信使RNA。信使RNA的大小为1.7 kb。从cDNA文库的重叠克隆和5'RACE产物的核苷酸序列推导出该蛋白质的完整氨基酸序列。一个1545个碱基对的开放阅读框（ORF）编码一个57 kDa的蛋白质，预测的pI为5.5。该ORF包含这三种肽的序列。它与数据库中存在的其他蛋白质没有显著同源性，但确实包含几个功能性酯酶基序。
