Molecular cloning and characterization of juvenile hormone acid methyltransferase in the honey bee, Apis mellifera, and its differential expression during caste differentiation.
Molecular cloning and characterization of juvenile hormone acid methyltransferase in the honey bee, Apis mellifera, and its differential expression during caste differentiation.
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DOI：
10.1371/journal.pone.0068544
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Su S
中科院分区：
文献类型：
--
作者：
Li W;Huang ZY;Liu F;Li Z;Yan L;Zhang S;Chen S;Zhong B;Su S
保幼激素酸性甲基转移酶（JHAMT）是一种参与昆虫保幼激素生物合成最后步骤之一的酶。它将S-腺苷-L-甲硫氨酸（SAM）的甲基转移到法呢酸（FA）或JH酸（JHA）的羧基上。JHAMT基因在昆虫变态和生殖过程中起着重要作用。然而，JHAMT在水翅目昆虫中的意义尚不清楚。本研究采用RACE扩增技术从意大利蜜蜂（Apis mellifera，AmJHAMT）中克隆了JHAMT cDNA。我们克隆的AmJHAMTs全长cDNA为1253 bp，编码278个氨基酸，与已知的JHAMTs同源性为32-36%。SAM依赖性甲基转移酶（SAM-MT）超家族中保守的SAM结合基序存在于AmJHAMT中。其二级结构也含有典型的SAM-MT折叠。与SAM和底物（JHA或FA）结合的大多数活性位点在AmJHAMT中与在其他JHAMT直系同源物中一样是保守的。系统发育分析将AmJHAMT与其他来自Hyplutera的直系同源物聚在一起，形成系统发育树中的一个主要分支。纯化的重组AmJHAMT蛋白在E.大肠杆菌中制备多克隆抗体，并通过免疫印迹和质谱鉴定AmJHAMT的身份。定量RT-PCR和免疫印迹分析表明，皇后幼虫含有显着较高水平的AmJHAMT mRNA和蛋白质比工人幼虫在种姓的发展时期。AmJHAMTmRNA和蛋白质在蜂王和工蜂体内的表达与JH的合成具有相似的规律。这些结果表明，我们克隆的基因编码一个功能性的JHAMT，催化蜜蜂JH生物合成的最终反应。此外，AmJHAMT可能在蜜蜂的种姓分化中起重要作用。
Juvenile hormone acid methyltransferase (JHAMT) is an enzyme involved in one of the final steps of juvenile hormone biosynthesis in insects. It transfers a methyl group from S-adenosyl-L-methionine (SAM) to the carboxyl group of either farnesoic acid (FA) or JH acid (JHA). Several genes coding for JHAMT have been cloned and characterized from insects from different orders, and they have been shown to play critical roles in metamorphosis and reproduction. However, the significance of JHAMT in Hymenopteran insects is unknown. We used RACE amplification method to clone JHAMT cDNA from the honey bee, Apis mellifera (AmJHAMT). The full length cDNA of AmJHAMT that we cloned is 1253bp long and encodes a 278-aa protein that shares 32-36% identity with known JHAMTs. A SAM-binding motif, conserved in the SAM-dependent methyltransferase (SAM-MT) superfamily, is present in AmJHAMT. Its secondary structure also contains a typical SAM-MT fold. Most of the active sites bound with SAM and substrates (JHA or FA) are conserved in AmJHAMT as in other JHAMT orthologs. Phylogenetic analysis clustered AmJHAMT with the other orthologs from Hymenoptera to form a major clade in the phylogenetic tree. Purified recombinant AmJHAMT protein expressed in E. coli was used to produce polyclonal antibodies and to verify the identity of AmJHAMT by immunoblotting and mass spectrometry. Quantitative RT-PCR and immunoblotting analyses revealed that queen larvae contained significantly higher levels of AmJHAMT mRNA and protein than worker larvae during the periods of caste development. The temporal profiles of both AmJHAMT mRNA and protein in queens and workers showed a similar pattern as the JH biosynthesis. These results suggest that the gene that we cloned codes for a functional JHAMT that catalyzes the final reactions of JH biosynthesis in honey bees. In addition, AmJHAMT may play an important role in honey bee caste differentiation.
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通讯作者：
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