拟南芥转基因雄性不育突变体DNA甲基化研究

发布时间：2018-03-03 15:53

本文选题：拟南芥　切入点：花药不开裂　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：雄性不育在高等植物中广泛存在,它是作物杂种优势利用最重要的途径之一。雄性不育指雄蕊发育不正常,不能完成受精作用,其中包括花药不开裂。拟南芥拥有大量雄性不育突变体,这些突变体为我们研究花的发育、小孢子的产生、花粉的分化、花药的开裂等现象以及其相关基因功能研究提供了理想的实验材料。甘蓝型油菜和拟南芥同属芸薹科植物,拟南芥雄性不育机理的研究能为油菜雄性不育和杂种优势利用相关研究提供参考。本研究以拟南芥转基因花药不开裂突变体M6和M24为材料,进行了全基因组DNA甲基化测序和small RNA的测序,对这些数据进行了生物信息学分析,阐释了突变体花药不开裂与甲基化、miRNA的关系。主要研究结果如下:1.全基因组甲基化水平分析结果表明,野生型、突变体M6和M24全基因组甲基化水平分别为:11.5%、12.7%和12.4%,突变体全基因组甲基化水平相比于野生型呈现上升趋势。进一步分析基因编码区和启动子区的甲基化水平,我们发现突变体中CHH序列环境下差异位点远多于其他两种序列,这说明M6,M24位点的突变可能是与RdDM途径相关。这一现象印证了:RdDM路径上的突变体rdr2-2,dcl3,drm1/2,nrpd1a-3,nrpd1b-11作为父本与M6和M24分别进行杂交实验,F1代的表型恢复的结果。2.本研究比较了M6和M24的差异甲基化基因,发现有269个hyper-CHHme的差异甲基化基因。其中有11个基因与花发育相关,例如SAUR-like,F-box家族基因。通过RT-PCR对部分候选基因进行表达量验证,发现基因区CHH甲基化水平升高的基因表达量下降,与甲基化分析结论相吻合。3.我们对突变体M6和M24进行small RNA测序,结合DNA甲基化数据进行综合分析,结果表明突变体M6与野生型间差异表达的miRNA有9个,突变体M24与野生型间差异表达的miRNA有11个,其中ath-miR398b-3p、ath-miR398c-3p、ath-miR8175、ath-miR408-3p、ath-miR869.2是在两个突变体中都差异表达的。部分与花发育的相关的基因的CHH甲基化水平升高,并伴随着24nt-sRNA表达量升高,例如F-box/RNI-like超家族蛋白SADHU3-1。这一结论进一步说明突变体M6和M24是小RNA介导的DNA甲基化增强,导致了部分花发育相关基因表达下降,从而产生花药不开裂。
[Abstract]:Male sterility is widespread in higher plants and is one of the most important ways to utilize crop heterosis. These include anthers that are not dehiscent. Arabidopsis has a large number of male sterile mutants that are used to study flower development, microspore production, pollen differentiation, Studies on anther dehiscence and its related gene function provide an ideal experimental material. Brassica napus and Arabidopsis thaliana belong to Brassicaceae. The study on the mechanism of male sterility in Arabidopsis thaliana can provide a reference for the study on the utilization of male sterility and heterosis in rape. In this study, the transgenic anthers M6 and M24 of Arabidopsis thaliana were used as materials. Genomic DNA methylation sequencing and small RNA sequencing were performed, and bioinformatics analysis was performed on these data. The relationship between anther nondehiscence and methylated miRNA was explained. The main results were as follows: 1. The whole genome methylation level analysis showed that wild type, The total genomic methylation levels of mutant M6 and M24 were 12. 7% and 12. 4%, respectively. Compared with wild type, the methylation level of the whole genome of mutant M6 and M24 was higher than that of wild type, and the methylation level of gene coding region and promoter region was further analyzed. We found that the difference sites in the CHH sequence environment were much more than those in the other two sequences. This indicates that the mutation at M6 / M24 may be related to the RdDM pathway. This phenomenon confirms the results of phenotypic recovery of F1 generation by cross experiment with M6 and M24 respectively as male parent, rdr2-2dcl3dcl3drm1 / 2nrpd1a-3nrpd1b-11. The results of this study were compared with that of M6 and M24. The differential methylation gene of M24, A total of 269 differentially methylated genes of hyper-CHHme were found, 11 of which were related to flower development, such as SAUR-like / F-box family genes. The expression of some candidate genes was verified by RT-PCR, and it was found that the expression of genes with higher CHH methylation level in the gene region decreased. We sequenced the small RNA of the mutants M6 and M24, combined with the DNA methylation data. The results showed that there were 9 miRNA differentially expressed between the mutant M6 and the wild type. There were 11 miRNA differentially expressed between mutant M24 and wild type, among which ath-miR398b-3pnath-miR398c-3path-miR8175nath-miR408-3pnath-miR869.2 was differentially expressed in both mutants. The CHH methylation level of some floral related genes was increased, accompanied by an increase in 24nt-sRNA expression. For example, F-box / RNI-like superfamily protein SADHU3-1.This conclusion further indicates that M6 and M24 are small RNA mediated DNA methylation enhancement, resulting in a decrease in the expression of some floral development-related genes, resulting in anther non-cracking.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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