Cloning and expression of a novel juvenile hormone-metabolizing epoxide hydrolase during larval-pupal metamorphosis of the cabbage looper, Trichoplusia ni
Cloning and expression of a novel juvenile hormone-metabolizing epoxide hydrolase during larval-pupal metamorphosis of the cabbage looper, Trichoplusia ni
复制标题
DOI：
10.1046/j.1365-2583.1999.810085.x
复制
发表时间：
1999-02-01
影响因子：
2.6
通讯作者：
Roe, RM
中科院分区：
文献类型：
--
作者：
Harris, SV;Thompson, DM;Roe, RM
在JH环氧化物水解酶活性最大的发育时间，从最后一只甘蓝粉纹夜蛾（Trichoplusia ni）脂肪体构建的cDNA文库中分离到编码微粒体保幼激素（JH）代谢环氧化物水解酶（TmEH-1）的全长cDNA，TmEH-1长1887个碱基对，开放阅读框为1389个碱基对，编码463个氨基酸。氨基酸序列分析表明，TmEH-1是最相似的，并包含确切的催化三联体（Asp-226，Glu-403和His-430）中发现的微粒体环氧化物水解酶。TmEH-1特异性信息沿着JH环氧化物水解酶活性存在于摄食（第1和2天）和游走（第3天）幼虫的脂肪体中，信息水平的峰值在JH环氧化物水解酶活性的峰值之前1天。当TmEH-1在杆状病毒感染的草地贪夜蛾细胞中表达时，在SDS-PAGE上出现了46，000分子量的蛋白质，其对应于由TmEH-1信息编码的预测大小，并且与JH III环氧化物水解酶活性高于野生型对照的活性的增加正相关。在亚细胞分布研究中，保幼激素III环氧化物水解酶活性的58%是在不溶性馏分。杆状病毒表达的TmEH-1表现出比一般EH底物顺式和反式二苯乙烯氧化物更高的JH III特异性活性。Southern杂交分析表明，T.倪
A full-length cDNA encoding for a microsomal juvenile hormone (JH)-metabolizing epoxide hydrolase (TmEH-1) was isolated from a cDNA library constructed from fat body of last stadium (wandering) cabbage loopers, Trichoplusia ni, at the exact developmental time of maximum JH epoxide hydrolase activity, TmEH-1 was 1887 base pairs in length with a 1389 base pair open reading frame encoding 463 amino acids. Amino acid sequence analysis showed that TmEH-1 was most similar to and contained the exact catalytic triad (Asp-226, Glu-403 and His-430) found in microsomal epoxide hydrolases. TmEH-1-specific message was present along with JH ill epoxide hydrolase activity in fat body in feeding (days 1 and 2) and wandering (day 3) larvae with the peak in message level preceding the peak in JH epoxide hydrolase activity by 1 day. When TmEH-1 was expressed in baculovirus-infected Spodoptera frugiperda cells, a 46,000 molecular weight protein appeared on SDS-PAGE which corresponded to the predicted size coded by the TmEH-1 message and which was positively correlated with increases in JH ill epoxide hydrolase activity above that of wild-type controls. In subcellular distribution studies, 58% of the juvenile hormone III epoxide hydrolase activity was in the insoluble fractions. Baculovirus expressed TmEH-1 demonstrated a higher specific activity for JH ill as compared to the general EH substrates, cis- and trans-stilbene oxide. Southern blot analyses suggested that multiple epoxide hydrolase genes are present in T. ni.