| It is well known that the genome contains information in two forms,genetic and epigenetic.The genetic information provides the blueprint for the manufacture of all the proteins necessary to create a living thing while the epigenetic information provides instructions on how,where,and when the genetic information should be used.The most important and widespread epigenetic mechanism in diverse taxa is the methylation of DNA.In eukaryotes,DNA methylation is commonly occurred to the 5 position of cytosine catalyzed by the conserved enzymes,which were named as DNA cytosine methyltransferases(Dnmts).The growing body of studies have already demonstrated that DNA methylation mediated by Dnmts plays a decisive role in normal cell differentiation,individual growth,phenotypic changes and other important metabolic processes of eukaryotes.Therefore,giving a deep insight of DNA methylation toolkit can further clarify the important role of DNA methylation in the developmental and metabolic regulation.The two-spotted spider mite(Tetranychus urticae Koch)is a worldwide agricultural mites that can damage approximately 1,100 species of plants.Now,with its unique advantages such as rapid development and easy to maintain,the two-spotted spider mite is widely used as a genetic model in the laboratory.Although the development of the two-spotted spider mite is rapid,it undergoes a more complex developmental plasticity.In addition,the two-spotted spider mite has a haplo-diploid sex determination mechanism,and several differences between adult females and male are deemed as a typical sexual dimorphism.More intriguing,the two-spotted spider mite often consists of two color forms:one consists of the greenish females,while the other consists of the reddish females,also known as carmine spider mite(T.cinnabarinus(Boisduval)).Based on the numerous evidence supporting the similar genetic background between these two color forms,it is most likely that one genome outputs two phenotypes by DNA methylation.DNA methylation has been demonstrated that it is closely related to the phenotypic changes.Thus,it is worth to explore whether DNA methylation can function in the ontogeny and coloration variation of T.urticae.In order to tackle the questions mentioned above,we first cloned the de novo methyltransferase 3 gene(Tudnmt3)in the two-spotted spider mite by RACE technique and performed a series of computational analyses on the primary,secondary and tertiary structure of this gene.The results of primary analyses clearly showed that this enzyme possesses the necessary conserved motifs for the catalytic activity of de novo methylation of DNA.As well,bioinformatics results of secondary and tertiary structure revealed that the structure of TuDNMT3 is highly conserved,with several vital amino acid residues for the activation and stabilization of its confirmation.Together,these results reflect that Tudnmt3 gene in T.urticae does belong to the de novo methyltransferase family and is especially possibly more similar to human DNMT3 A in terms of function.Since Tudnmt3 possessed the active motif for the capability of de novo methylation,it is expected to that Tudnmt3 might have a crucial role in the metabolism and development of the two-spotted spider mites.In this study,we investigated the potential role of DNA methyltransferase 3 in the development of Tetrany,chus urticae Koch.Real-time PCR revealed that Tudnmt3 was expressed ubiquitously and throughout the life cycle of two-spotted spider mite.However,the pattern of Tudnmt3 expression was sex-dependent during the adult stage.Whole in situ hybridization provided supportive evidence that Tudnmt3 is linked to differentiation of gonads in adult females and males.MSAP(methylation-sensitive amplification polymorphism)analyses of 119 loci showed that the status of DNA methylation is partially different between adult females and males the DNA methylation frequency was higher in adult males than in adult females,raising the possibility that sex-dependent DNA methylation pattern is mediated by different methylation activity of Tudnmt3.To better elucidate the functional role of Tudnmt3,its protein expression and localization were subjected to more detailed analyses.Western blot analyses revealed that this protein is expressed in both genders,with higher expression in adult females,which is inconsistent with the gene expression,suggesting translational regulation of Tudnmt3.Subsequent immunodetection provided supportive evidence for higher expression of the TuDNMT3 protein in adult females and indicated that this protein was generally localized in the cytoplasm and that its expression was predominantly confined to the genital region of spider mites,further supporting the hypothesis that de novo methylation mediated by Tudnmt3 in gonad development or gametogenesis may have a different mechanism from maintenance methyltransferase.Previous results demonstrated that de novo methyltransferase can play a critical role in the development of T.urticae.Based on the evidence that DNA methylation is closely related to caste variation in social insects,it is required for determining whether this gene functions in the color variation of T.urticae two forms.The present study investigated the mRNA expression throughout the whole life cycle of T.urticae red form(TuR)and green form(TuG).qRT-PCR results showed that Tudnmt3 mRNA was present in all examined developmental stages,and exhibited a similar expression pattern in both two forms accompanying with a progressively dropping trend.The variation of expression level among the four development stages in one form was statistically significant(P<0.05).In detail,the highest expression of Tudnmt3 was in the embryo stage with a significantly higher than that in the adult stage(P<0.05).Compared with the same developmental stage between two forms,the Tudnmt3 transcript level corresponding to each stage of TuR was lower than that in TuG,and with only one statistically significant in adult stage(P<0.05).The different expression pattern in adult stage of T.urticae red form and green form suggested a potential role of de novo methylation in body coloration variation.De novo methylation probably affects the body coloration variation of the two-spotted spider mite,but it is unknown that which pigmentation genes can be targeted by methylation and whether the differential expression of these genes can be regulated by methylation.Thus,for addressing this remaining question,we explored the differentially expressed genes related to pigment synthesis in T.urticae two forms in aid of transcriptome sequencing technology.Transcriptome data indicated that a total of 83 pigment gene shared in two color forms,of which 11 are significantly differential expression(FDR<0.05).Real-time PCR was carried out for examining the expression pattern of 10 pigmentation genes across the coloration stages of the two color forms,and results showed that the majority of genes exhibits a higher expression in red form when compared to the same stage in green form.Meanwhile,bisulfite sequencing method discovered that two genes,i.e.a kynurenine-3-hydroxylase gene and a serine protease inhibitor gene 27A,were higher methylated in red form than that in green form,but with no significant difference(P>0.05).Finally,the methylation inhibitor—5-azacytidine treatments of red forms revealed a most significant down-regulation of kynurenine 3-hydroxylase gene and the serine protease inhibitor gene 27A(P<0.001),directly proving that gene-body methylation has a direct impact on the expression of pigment genes.Unexpectedly,this genomic hypomethylation failed to influence the body color or other physiological index of T.urticae red form. |
PDF Full Text Request