Identification And Annotation Of GMC Oxidoreductase Family And Functional Analysis Of Genes Involved In The Ecdysone C3-epimerization Pathway In The Silkworm, Bomby Mori

GMC (Glucose-Methanol-Choline) oxidoreductase family is one member of the numerous oxidoreductases in organisms. The enzymes widely exist in a varity of organisms, and some of these enzymes play roles in developmental or physiological process, such as glucose and choline metabolism. GMC oxidoreductases constitute a large gene family in insects, and have several conserved characters in different species. Nevertheless, little is known about the function of the insect GMC family. Using the silkworm as the model, this study analyzed the evolutionary process and expression feature of the silkworm GMC genes. In addition, we also used the RNA interference to survey the function of the GMC genes for the silkworm to defense against the pathogens. Ecdysone oxidase (EO) is also the member of the GMC family, and is the rate-limiting enzyme of the insect ecdysone C3-epimerization pathway. This pathway is one of major inactivation pathways of the ecdysone in insects. The regulation of the degradated pathway of the ecdysteroid is essential for maintain the precise fluctuation of the ecdysteroid. Thus, we cloned two key genes(EO and3DE-3a-reducase) involved in the ecdysone C3-epimerization pathway from the silkworm. The heterologously expressed proteins of the two genes in yeast showed related enzyme activities. Furthermore, we also examined the function of these two genes in the feeding stage and metamorphosis of the silkworm. The main results are as follows:1. Identification and annotation of GMC oxidoreductase family in the silkworm genomeWe identified43GMC genes in the silkworm genome, the largest number of GMC genes among all the insect genomes sequenced to date. Similar to the other insects, there is a highly conserved GMC cluster within the second intron of the silkworm flotillin-2(flo-2) gene. However, the silkworm GMC genes outside of the conserved GMC cluster have experienced a large expansion. Phylogenetic analysis suggested that the silkworm GMCB subfamily contained22copies. Eighteen of the22members of the silkworm GMCB subfamily are located outside of the conserved GMC cluster, and are known as silkworm expansion genes (SEs). The large gene number of GMCB subfamily made a major contribution to expansion of the silkworm GMC genes. SEs were randomLy located in the differnet genome regions. Relative rate tests showed that SEs evolved significantly faster than the GMCB genes inside the conserved GMC cluster. The elevated evolutionary rate of the silkworm GMCB genes outside of the conserved GMC cluster may reflect the evolution of function diversity.2. The expression pattern of the silkworm GMC genes and the function of these genes in the silkworm immunityRT-PCR was performed to determine the temporal and spatial expression profiles of the silkworm GMC genes, especially for the GMCB genes. The results suggested that each silkworm GMC gene had a distinct expression pattern. Even the silkworm GMCB expansion genes (SEs) that were closely related to one another on the phylogenetic tree had different expression patterns. We retrieved the microarray data from microbial induced silkworm for the silkworm GMC genes. After infection by four microorganisms,16silkworm GMC genes were upregulated by the different pathogens. Interestingly, fourteen of these16GMC genes belonged to the GMCB subfamily. Moreover, silkworms whose three representative GMCB genes (BmGMC1, BmGMCI2and BmGMCβ3) were knocked down exhibited higher susceptibilities to pathogens. This indicated that the expansion of the silkworm GMCB subfamily may be involved in resistance to pathogenic microorganisms.3. Characterization of the BmEO and Bm3DE-3a-reducase from the silkwormBased on the previously characterized cDNAs in Spodoptera littoralis, we identified EO and3DE-3a-reducta.se candidate genes in the silkworm. Then we obtained the full-length of these two genes by RACE. The complete length of putative BmEO gene was2353bp, and the coding region was2007bp. For putative Bm3DE-3a-reducta.se gene, the complete length of this gene was851bp, and the coding region was753bp. The heterologously expressed proteins of the two genes in yeast showed the ecdysone oxidase and3DE-3a-reductase activities, respectively. Expression of BmEO was only detected in the midgut at transcriptional and translational levels. We also localized EO within the midgut goblet cell cavities. For Bm3DE-3a-reductase gene, RT-PCR and Western blot showed that it was expressed in the midgut and the Malpighian tubules. Moreover, we localized3DE-3a-reductase within the midgut goblet cell cavities and the cytosol of principal cells of the Malpighian tubules. These two genes have similar expression profiles during different developmental stages. Both genes were highly expressed at the beginning of the5th instar, and remained a relative low level during the feeding stage, and then were highly expressed at the wandering stage. All these results showed that the profiles of the two genes were well correlated with the ecdysteroid titer.4. Functional analysis of the BmEO and Bm3DE-3a-reducase in the silkwormBmEO and Bm3DE-3a-reducase gene have expression during the feeding and wandering stage. As the key genes of the ecdysone C3-epimerization pathway, this result revealed that this pathway may be very important in the two stages. We used several technologies to detect the function of this pathway in these two stages. In the feeding stage, we found that the ecdysone C3-epimerization pathway may take part in the degradation of phytoecdysteroids from mulberry. When this pathway is abnormal, the silkworm may induce or promote the Cypl8al activity which is involved in the ecdysone C26hydroxylation pathway to degrade the phytoecdysteroids to rescue the deficient BmEO function. Taken together, silkworm has functionally redundant in degrading the phytoecdysteroids from mulberry. In the wandring stage, we knocked down the expression of BmEO and Bm3DE-3a-reducase gene, respectively. The results showed that the ecdysteroid titer from the RNAi group was significantly higher than that from control, and the expression pattern of several hormone receptors was also changed. The mortality of the silkworm at wandering and pupal stages was much higher than that from the control which indicated that the ecdysone C3-epimerization pathway is essential for the silkworm metamorphosis.From all the results shown above, we found that GMC oxidoreductase family gene exhibited the large functional diversification. These enzymes are not only take part in the physiological and developmental processes, but also play important role in plant-insect interaction. GMC oxidoreductase family is a large family in insect, and it experienced a burst expansion in the silkworm. According to our results, the expansion of the silkworm GMC genes is associated with the silkworm immunity. Taken together, we suggested that the lineage-specific expansion of the insect GMC oxidoreductase family may be benefit for insects to adaptive different enviroment.