| Transposable elements occupy a large fraction of plant genomes, and play vital roles in studying plant genome composition, evolution and regulation of gene expression. The studies on transposon discovering, application and activity regulating in recent years have become hot spots of plant breeding and molecular biology in plants. The studies have shown that epigenetic change under stress may be an important mechanism in regulating the variation of transposon activity in plants. Based on the researches of model plants such as Arabidopsis and rice, a preliminary understanding about epigenetic phenomenon of regulating plant transposon activity under stress has been obtained, but the study on the mechanism of transposon activity regulation is still very limited. In this study, low-energy ion beam as an abiotic stress was used to induce the maize single-copy Mu transposon system for reactivation, and then its characteristics and variation of transposable activity was explored to further reveal the relationship between Mu transposon activity and epigenetic changes. Furthermore, the identification and expression analysis of relative gene families associated about RNA-mediated DNA methylation pathway in maize were performed by applying some bioinformatic methods. The results were shown as following:1. By low energy N+ion implantation, pollen grains of maize from a special line with a single-copy Mu transposon system were implanted with30keV nitrogen ion beams at doses ranging from2.4×1015ions/cm2to3.6xβγ1015ions/cm2to study the effect of Mu transposon activity variation. The results revealed that N+ion implantation significantly reactivated the silence Mutator transposable systems. By genetic hybridization and screening mutant progeny, two main kernels with different phenotypic characteristics of purple spots, as indicator traits, were found. One was designated as partially activated mutant (PA) when the silenced Mu transposon was reactivated at the dose2.4x1015ions/cm2N+ion implantation, which resulted in12moderately spotted kernels, and the other was fully activated (FA) mutant when at the dose3.6x1015ions/cm2N+ion implantation, which resulted in10heavily spotted kernels.2. Southern hybridization, semi-quantitative RT-PCR and real-time fluorescence quantitative PCR method were performed to detect the transposable characteristics of the nonautonomous Mul elements and differences in expression levels of the autonomous MuDR elements among the control and mutants. The results showed that the polymorphism of Mul elements from two mutants with the emergence of two characteristic sizes of restriction fragments (1.7kb and1.3kb), was significantly higher than from control plants indicating that Mu transposon systems from two mutants were reactivated, which resulted in the transposition of the nonautonomous Mu1elements. Since the activity of the Mu transposable system is usually controlled by two genes from the autonomous MuDR element, mudrA (encoding the transposase protein MURA) and mudrB (encoding the helper protein MURB), the analysis of expression level of these two genes directly related to MuDR activity at different periods of growth and development and from different tissues revealed that the mudrA and mudrB from mature somatic tissues from two mutants did not exhibit the significantly increased level of gene expression. However, the mudrA and mudrB from germinal tissues showed significantly increased level of gene expression. Therefore, the reactivation of MuDR transposon mainly occurred in the formation of gametes. Through Real-time PCR, in mature pollen tissues, the expression levels of mudrA and mudrB genes significantly increased1.8to4.3times and0.8to1.6times compared with the controls, respectively, indicating that the activity of the MuDR transposons from male gametes were reactivated significantly.3. By using restriction enzyme and bisulfite sequencing, the status of DNA methylation sites of TIRA and TIRB terminal regions of the maize MuDR transposon was detected qualitatively and quantitatively. The results showed that three different types of cytosine sites (CpQ CpHpG and CpHpH) of TIRA and TIRB at MuDR transposon terminals from the mutants had demethylation variation at different degrees. Among them, the cytosine methylation at the symmetrical CpG and CpHpG sites occur with a more significant variation, lower than the control plants35.3%～55.9%and38.9%～60.0%, respectively; the cytosine methylation of non-symmetrical CpHpH sites occur with a smaller variation, lower than control plants3.0%～6.1%. In addition, the analysis from single sites of cytosine methylation variation revealed that some important regions such as the MURA-binding site (MBS) composed of a highly conserved32-bp motif of the MuDR element TIR, a characteristic SacI restriction sites and the transcription start sites of mudrA and mudrB genes etc., have been demonstrated to influence MuDR activity, and the demethylation of cytosine loci within these regions occurred with a significant variation, which might directly affect the activity of MuDR transposon in this study. In addition, the extent of DNA methylation variation between two mutants had an obvious difference. The mutant with a higher transposition activity had a larger variation of DNA demethylation.4. By using bioinformatics methods, we identified several key gene families related with the RNA-mediated DNA methylation pathway from the maize B73genome, and by semi-quantitative RT-PCR, the expression levels of these genes were also detected. Based on sequence comparison and evolutionary analyses, we found that the predicted Zmdcl(5), Zmago(18), Zmrdr(5) and Zmet(8) genes encoding the respective DCL, AGO, RDR and DMT proteins from the RNA-mediated DNA methylation pathway and most of these genes had highly conserved sequences with highly conserved motifs and important functional domains, compared with the homologous genes in Arabidopsis and rice. This result revealed that these genes might share similar functions with their counterparts in Arabidopsis and rice. Phylogenetic analyses clustered each of these genes families in maize, rice and Arabidopsis into four different subfamilies, but during the course of evolution, the homologous genes in maize and rice had more closed phylogenetic relationship. Furthermore, the transcripts of these genes were detected in the leaves by two different abiotic stress treatments using semi-quantitative RT-PCR, to predict their functions of different members of these gene families in the RNA-mediated regulation of DNA methylation pathway, and to provide a theoretical basis for cloning and verifying these genes for the next step. |
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