Molecular Cloning And Characterization Of The Enzyme Genes Involved In The Final Steps Of Juvenile Hormone Biosynthesis In Apis Mellifera, And Their Expression During Caste Development

As a highly eusocial insect, the honey bee, Apis mellifera, has become a model organism in sociobiology. The reproductive division of labor in social insects, based on caste differentiation, is of great significance in evolution. People have been able to build a relatively complete regulatory network of honey bee caste differentiation with the exiting results. However, there are still many issues worthy of further exploration, such as the molecular mechanisms determining the expression of sub-phenotypes like body size, developmental duration, and ovary size, as well as the molecular pathways involved in the biosynthesis and actions of the important endocrine factors such as Juvenile hormone (JH). In this study, the next-generation sequencing (NGS) technology was employed to detect the gene expression profiles of queen and worker caste development. Three genes AmJHAMT, AmCYP15A1and AmFAMeT involved in the final steps of JH biosynthesis in honey bees were cloned and characterized, and their putative functions for honey bee caste development were investigated. The main findings were as follows:1. Next-generation sequencing-based digital gene expression profiles during honeybee caste differentiationIn this study, the digital gene expression profiling (DGE) was used to detect gene expression profiles responding to the queen and worker caste development. About7.6million and8.9million clean tags, with87,000and100,000distinct tags were obtained from the queen and worker DGE sequencing library, respectively.1,278and1,451genes were found to be significantly up-regulated and down-regulated in queen larvae compared with worker larvae. The majority of the top50significantly up-regulated genes in queen larvae were metabolic enzymes, and two juvenile hormone synthesis-related genes, JHAMT and CYP15A1were also included in this set. In contrast, only a few metabolic enzymes were detected in the50most significantly up-regulated gene list of worker larvae. There were62,417entities uniquely mapped to the honey bee genome, which failed to map to the reference tag database, and9,258candidate genes were obtained by gene prediction from these entities using Genscan program, of which3,566candidate genes were longer than300nt. GO analysis revealed that these candidate genes might be involved in the embryonic development, reproductive development and sexual reproduction.2. Molecular cloning and characterization of JHAMT in A. mellifera, and its functions during caste differentiationJuvenile hormone acid methyltransferase (JHAMT) is an enzyme involved in one of the final steps of juvenile hormone biosynthesis in insects, which transfers a methyl group from S-adenosyl-L-methionine (SAM) to the carboxyl group of either farnesoic acid (FA) or JH acid (JHA). RACE amplification method was used to clone JHAMT cDNA from the honey bee, Apis mellifera(AmJHAMT). The full length cDNA of AmJHAMT was1253bp long and encoded a278-aa protein that shared32%-36%identity with known JHAMTs. A SAM-binding motif, conserved in the SAM-dependent methyltransferase (SAM-MT) superfamily, was present in AmJHAMT. Its secondary structure also contained a typical SAM-MT fold. Most of the active sites bound with SAM and substrates (JHA or FA) were conserved in AmJHAMT as in other JHAMT orthologs. Purified recombinant AmJHAMT protein expressed in E. coli was used to produce polyclonal antibodies, and the identity of AmJHAMT was verified latter 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.3. Molecular cloning and characterization of CYP15A1in A. mellifera, and its expression during caste differentiationThe CYP15family members like CYP15A1of Cytochrome P450superfamily (CYP) catalyze the epoxidation reaction of the final steps of JH biosynthesis in insects, which transfers methyl farnesoate (MF) to JH. In this study, a putative CYP15A1gene, named AmCYP15A1, was cloned from the queen larvae of A. mellifera. It contained an1515bp ORF, which encoded an504aa protein. Multiple alignment analysis showed a relatively low identity between AmCYP15A1with most other insect CYP15s (<50%). However, AmCYP15A1and the other insect CYP15s all contained the motifs constituting the general CYP structure fold, and some other characteristic structures of CYPs, like N-terminal membrane anchor and hinges and so on. Phylogenetic analysis displayed that AmCYP15A1was successively clustered with the CYP15orthologs of insects from Hymenoptera, Diptera, Coleoptera, Orthoptera, Blattodea, and Hemiptera, which finally formed a major clade. However, the other major clade was constituted only by Lepidopteran CYP15s. The gene expression level of AmCYP15A1was determined by qPCR, and the result showed that the AmCYP15A1mRNA expression levels in queens were significantly higher than those in workers during most of developmental stages, especially for the physiologically critical temporal window of caste differentiation (also known as JH-sensitive period). On the other hand, the AmCYP15A1mRNA levels of both queens and workers peaked in the feeding stage of the5th instar, and then decreased rapidly to a low level until the white-eyed pupal stage, which was consist with JH biosynthesis activity.4. Molecular cloning and characterization of FAMeT in A. mellifera, and its expression during caste developmentFarnesoic acid methyl transferases (FAMeT) are thought to be involved in the terminal steps of JH biosynthesis, which catalyzes the methylation of FA to MF using the cofactor SAM. In this study, a putative FAMeT gene, namely AmFAMeT, was cloned from the queen larvae of western honey bees. It contained an891bp ORF which encoded an504aa protein. Multiple alignment analysis indicated that AmFAMeT had a higher identity with insects FAMeTs (54%-85%) when compared with crustacean ones (<50%). Comparison of AmFAMeT expression abundance between queen and workers showed that there were no significant differences for the most of caste developmental stages. Meanwhile, the trends of gene expression differed between the two castes, and were both not consist with the JH biosynthesis.In a word, our results suggest that:1) Honey bee caste differentiation is a process with significantly differential gene expression;2) AmJHAMT and AmCYP15A1are real JHAMT and CYP15enzyme genes in A. mellifera, which involve in the regulation of caste differentiation by control the terminal biosynthesis of JH;3) AmFAMeT is not related to the caste development, and may lose the function of catalyzing final reactions of JH biosynthesis.