本文選題：擬南芥　切入點：花藥不開裂　出處：《華中農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：雄性不育在高等植物中廣泛存在,它是作物雜種優(yōu)勢利用最重要的途徑之一。雄性不育指雄蕊發(fā)育不正常,不能完成受精作用,其中包括花藥不開裂。擬南芥擁有大量雄性不育突變體,這些突變體為我們研究花的發(fā)育、小孢子的產(chǎn)生、花粉的分化、花藥的開裂等現(xiàn)象以及其相關基因功能研究提供了理想的實驗材料。甘藍型油菜和擬南芥同屬蕓薹科植物,擬南芥雄性不育機理的研究能為油菜雄性不育和雜種優(yōu)勢利用相關研究提供參考。本研究以擬南芥轉(zhuǎn)基因花藥不開裂突變體M6和M24為材料,進行了全基因組DNA甲基化測序和small RNA的測序,對這些數(shù)據(jù)進行了生物信息學分析,闡釋了突變體花藥不開裂與甲基化、miRNA的關系。主要研究結(jié)果如下:1.全基因組甲基化水平分析結(jié)果表明,野生型、突變體M6和M24全基因組甲基化水平分別為:11.5%、12.7%和12.4%,突變體全基因組甲基化水平相比于野生型呈現(xiàn)上升趨勢。進一步分析基因編碼區(qū)和啟動子區(qū)的甲基化水平,我們發(fā)現(xiàn)突變體中CHH序列環(huán)境下差異位點遠多于其他兩種序列,這說明M6,M24位點的突變可能是與RdDM途徑相關。這一現(xiàn)象印證了:RdDM路徑上的突變體rdr2-2,dcl3,drm1/2,nrpd1a-3,nrpd1b-11作為父本與M6和M24分別進行雜交實驗,F1代的表型恢復的結(jié)果。2.本研究比較了M6和M24的差異甲基化基因,發(fā)現(xiàn)有269個hyper-CHHme的差異甲基化基因。其中有11個基因與花發(fā)育相關,例如SAUR-like,F-box家族基因。通過RT-PCR對部分候選基因進行表達量驗證,發(fā)現(xiàn)基因區(qū)CHH甲基化水平升高的基因表達量下降,與甲基化分析結(jié)論相吻合。3.我們對突變體M6和M24進行small RNA測序,結(jié)合DNA甲基化數(shù)據(jù)進行綜合分析,結(jié)果表明突變體M6與野生型間差異表達的miRNA有9個,突變體M24與野生型間差異表達的miRNA有11個,其中ath-miR398b-3p、ath-miR398c-3p、ath-miR8175、ath-miR408-3p、ath-miR869.2是在兩個突變體中都差異表達的。部分與花發(fā)育的相關的基因的CHH甲基化水平升高,并伴隨著24nt-sRNA表達量升高,例如F-box/RNI-like超家族蛋白SADHU3-1。這一結(jié)論進一步說明突變體M6和M24是小RNA介導的DNA甲基化增強,導致了部分花發(fā)育相關基因表達下降,從而產(chǎn)生花藥不開裂。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2

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本文編號：1561691