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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Juvenile hormone acid methyltransferase: A key regulatory enzyme for insect metamorphosis.
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Juvenile hormone acid methyltransferase: A key regulatory enzyme for insect metamorphosis.

机译：少年激素甲基转移酶：昆虫变态的关键调控酶。

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摘要

Juvenile hormone (JH) acid methyltransferase (JHAMT) is an enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis pathway in insects. By fluorescent mRNA differential display, we have cloned a cDNA encoding JHAMT from the corpora allata (CA) of the silkworm, Bombyx mori (BmJHAMT). The BmJHAMT cDNA encodes an ORF of 278 aa with a calculated molecular mass of 32,544 Da. The predicted amino acid sequence contains a conserved S-adenosyl-l-methionine (SAM) binding motif found in the family of SAM-dependent methyltransferases. Purified N-terminal 6xHis-tagged recombinant BmJHAMT protein expressed in Escherichia coli catalyzed conversion of farnesoic acid and JH acids I, II, and III to their cognate methyl esters in the presence of SAM, confirming that this cDNA encodes a functional JHAMT. Putative orthologs, DmJHAMT and AgJHAMT, were identified from the genome sequence of the fruit fly Drosophila melanogaster, and a malaria vector, Anopheles gambiae, respectively. Northern blot and quantitative RT-PCR analyses revealed that the BmJHAMT gene was expressed specifically in the CA throughout the third and fourth instar. At the beginning of the last (fifth) instar, the expression level of BmJHAMT declined rapidly and became undetectable by day 4 and remained so until pupation. Correlation of the BmJHAMT gene expression and the JH biosynthetic activity in the CA suggests that the transcriptional suppression of the BmJHAMT gene is crucial for the termination of JH biosynthesis in the CA, which is a prerequisite for the initiation of metamorphosis.
机译：少年激素（JH）酸性甲基转移酶（JHAMT）是一种在昆虫的JH生物合成途径的最后一步将JH酸或JHs的非活性前体转化为活性JHs的酶。通过荧光mRNA差异显示，我们从家蚕Bombyx mori（BmJHAMT）的全集（CA）中克隆了编码JHAMT的cDNA。 BmJHAMT cDNA编码一个278aa的ORF，计算的分子量为32544 Da。预测的氨基酸序列包含在SAM依赖性甲基转移酶家族中发现的保守S-腺苷-1-蛋氨酸（SAM）结合基序。在SAM的存在下，在大肠杆菌中表达的纯化的N末端6xHis标记的重组BmJHAMT蛋白催化了法呢酸和JH酸I，II和III向其同源甲基酯的转化，从而证实该cDNA编码了功能性JHAMT。从果蝇果蝇（Drosophila melanogaster）和疟疾媒介冈比亚按蚊（Anopheles gambiae）的基因组序列中分别鉴定出假定的直系同源物DmJHAMT和AgJHAMT。 Northern印迹和定量RT-PCR分析显示，BmJHAMT基因在第三和第四龄期的CA中特异性表达。在最后一个（第5龄）开始时，BmJHAMT的表达水平迅速下降并在第4天变得不可检测，并一直保持直至化脓。 BmJHAMT基因表达与CA中JH生物合成活性的相关性表明，BmJHAMT基因的转录抑制对于CA中JH生物合成的终止至关重要，这是启动变态的先决条件。

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