玉米多梳蛋白MEZ1調(diào)控組蛋白修飾和DNA甲基化的研究
發(fā)布時(shí)間：2019-03-16 18:52
【摘要】：多梳家族蛋白(polycomb group proteins)是首先在果蠅中發(fā)現(xiàn)的一大類染色質(zhì)調(diào)節(jié)因子。PRC2(polycomb repressive complex 2)是其中最典型的一類復(fù)合體,在動(dòng)物和植物中是高度保守的。對(duì)PRC2的研究在動(dòng)物中最為廣泛,自2000年以來(lái)在擬南芥中也有很多報(bào)道,但是對(duì)其在玉米生長(zhǎng)發(fā)育中所發(fā)揮的作用卻是了解甚少。本研究利用反向遺傳學(xué),通過(guò)Crispr-Cas9敲除玉米PRC2核心組分ME探究MEZ1對(duì)玉米籽粒發(fā)育的影響。我們發(fā)現(xiàn),MEZ1是一個(gè)突變后有表型的母本表達(dá)的印記基因。攜帶母本allele的mez1/MEZ1在籽粒發(fā)育早期出現(xiàn)胚和胚乳的發(fā)育滯后,但是最終能夠恢復(fù)到野生型的水平,暗示MEZ1可能具有類似于擬南芥ME4的自我調(diào)控(autoregulation)的機(jī)制。純合的mez1/mez1的籽粒小、敗育。結(jié)合生物化學(xué)手段,我們首次證實(shí)了玉米MEZ1和FIE1處在一個(gè)約700 kDa的PRC2復(fù)合體中。在mez1/mmez1純合突變體中,胚乳中的H3K27me3水平幾乎完全喪失,證實(shí)了 MEZ1是一個(gè)有功能的H3K27me3甲基轉(zhuǎn)移酶。雖然籽粒中還同時(shí)表達(dá)MEZ1的兩個(gè)旁系同源物MEZ2和MEZ3,但是它們不能彌補(bǔ)MEZ1缺失帶來(lái)的影響,表明MEZ1在胚乳中發(fā)揮的作用是特異的。對(duì)轉(zhuǎn)錄組數(shù)據(jù)分析發(fā)現(xiàn)MEZ1調(diào)控了胚乳細(xì)胞化、脅迫響應(yīng)、激素響應(yīng)和模式形成(patterning)等過(guò)程;印記基因中的 PEG(paternally expressed imprinted genes)受 MEZ1 影響最大。在 BETL中特異表達(dá)的基因,受到MEZ1的間接調(diào)控。MEZ1可能是上游調(diào)控BETL特異表達(dá)基因的通用因子。H3K27me3的ChIP-seq結(jié)果表明,MEZ1-PRC2的靶基因表達(dá)量低,且具有組織表達(dá)特異性。靶基因富集在基因轉(zhuǎn)錄調(diào)控、細(xì)胞壁相關(guān)、脅迫響應(yīng)、脂肪酸代謝和氮代謝。同時(shí)一些家族的的轉(zhuǎn)錄因子基因也有富集。在胚乳發(fā)育過(guò)程中起著重要調(diào)控作用的MADS家族基因也是MEZ1的偏好靶點(diǎn)。全基因組亞硫酸氫鹽測(cè)序(BS-seq)發(fā)現(xiàn)與野生型相比,雖然整體上基因區(qū)的DNA甲基化水平升高幅度小,但是受MEZ1調(diào)控的轉(zhuǎn)錄因子和PEG的甲基化水平變化表現(xiàn)明顯。MEZ1可能是CHH island的上游調(diào)控因子。MEZ1主要影響的是TE上的DNA甲基化。mez1中TE上CG甲基化水平降低,而CHG和CHH甲基化水平升高。MEZ1可能起著抑制TE內(nèi)部CHH甲基化的作用。綜上,我們的研究揭示了 MEZ1在玉米籽粒發(fā)育中不可或缺的作用。
[Abstract]:(polycomb group proteins) is a major chromatin regulatory factor first found in Drosophila melanogaster. PRC2 (polycomb repressive complex 2 is one of the most typical complexes, which is highly conserved in animals and plants. The study of PRC2 is the most widespread in animals and has been reported in Arabidopsis since 2000. However, little is known about its role in the growth and development of maize. In this study, reverse genetics was used to explore the effect of MEZ1 on maize kernel development by Crispr-Cas9 knockout of ME, the core component of maize PRC2. We found that MEZ1 is a mutant imprinted gene expressed by the maternal parent. The development of embryo and endosperm of mez1/MEZ1 with maternal allele appeared lag in early grain development, but finally recovered to the level of wild type, suggesting that MEZ1 may have the mechanism of self-regulation (autoregulation) similar to Arabidopsis ME4. The seeds of homozygous mez1/mez1 were small and aborted. Combined with biochemical methods, we confirmed for the first time that maize MEZ1 and FIE1 are in a PRC2 complex of about 700 kDa. In the homozygous mutant of mez1/mmez1, the level of H3K27me3 in endosperm was almost completely lost, which confirmed that MEZ1 was a functional H3K27me3 methyltransferase. Although MEZ2 and MEZ3, two side-line homologues of MEZ1, were expressed simultaneously in grains, they could not make up for the effect of MEZ1 deletion, which indicated that MEZ1 played a special role in endosperm. Analysis of transcriptional data showed that MEZ1 regulated endosperm cellulation, stress response, hormone response and pattern formation of (patterning), and PEG (paternally expressed imprinted genes) in imprinted genes was most affected by MEZ1. The gene specifically expressed in BETL is indirectly regulated by MEZ1. MEZ1 may be a general factor for upstream regulation of BETL-specific expression gene. ChIP-seq results of H3K27me3 show that the target gene expression of MEZ1-PRC2 is low and has tissue-specific expression. Target genes are enriched in gene transcription regulation, cell wall correlation, stress response, fatty acid metabolism and nitrogen metabolism. At the same time, some family transcription factor genes are also enriched. MADS family genes, which play an important role in endosperm development, are also the preferred targets of MEZ1. Genome-wide bisulfite sequencing (BS-seq) found that although the overall DNA methylation level in the gene region was slightly higher than that in the wild type, MEZ1 may be the upstream regulatory factor of CHH island. The main effect of MEZ1 is DNA methylation on TE, and the decrease of CG methylation level on TE in MEZ1, but the change of transcription factor and methylation level of PEG regulated by MEZ1 is obvious. Mez-1 may play an important role in inhibiting CHH methylation in TE. CHG and CHH methylation levels are increased. In summary, our research reveals that MEZ1 plays an indispensable role in maize kernel development.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S513
本文編號(hào)：2441876
[Abstract]:(polycomb group proteins) is a major chromatin regulatory factor first found in Drosophila melanogaster. PRC2 (polycomb repressive complex 2 is one of the most typical complexes, which is highly conserved in animals and plants. The study of PRC2 is the most widespread in animals and has been reported in Arabidopsis since 2000. However, little is known about its role in the growth and development of maize. In this study, reverse genetics was used to explore the effect of MEZ1 on maize kernel development by Crispr-Cas9 knockout of ME, the core component of maize PRC2. We found that MEZ1 is a mutant imprinted gene expressed by the maternal parent. The development of embryo and endosperm of mez1/MEZ1 with maternal allele appeared lag in early grain development, but finally recovered to the level of wild type, suggesting that MEZ1 may have the mechanism of self-regulation (autoregulation) similar to Arabidopsis ME4. The seeds of homozygous mez1/mez1 were small and aborted. Combined with biochemical methods, we confirmed for the first time that maize MEZ1 and FIE1 are in a PRC2 complex of about 700 kDa. In the homozygous mutant of mez1/mmez1, the level of H3K27me3 in endosperm was almost completely lost, which confirmed that MEZ1 was a functional H3K27me3 methyltransferase. Although MEZ2 and MEZ3, two side-line homologues of MEZ1, were expressed simultaneously in grains, they could not make up for the effect of MEZ1 deletion, which indicated that MEZ1 played a special role in endosperm. Analysis of transcriptional data showed that MEZ1 regulated endosperm cellulation, stress response, hormone response and pattern formation of (patterning), and PEG (paternally expressed imprinted genes) in imprinted genes was most affected by MEZ1. The gene specifically expressed in BETL is indirectly regulated by MEZ1. MEZ1 may be a general factor for upstream regulation of BETL-specific expression gene. ChIP-seq results of H3K27me3 show that the target gene expression of MEZ1-PRC2 is low and has tissue-specific expression. Target genes are enriched in gene transcription regulation, cell wall correlation, stress response, fatty acid metabolism and nitrogen metabolism. At the same time, some family transcription factor genes are also enriched. MADS family genes, which play an important role in endosperm development, are also the preferred targets of MEZ1. Genome-wide bisulfite sequencing (BS-seq) found that although the overall DNA methylation level in the gene region was slightly higher than that in the wild type, MEZ1 may be the upstream regulatory factor of CHH island. The main effect of MEZ1 is DNA methylation on TE, and the decrease of CG methylation level on TE in MEZ1, but the change of transcription factor and methylation level of PEG regulated by MEZ1 is obvious. Mez-1 may play an important role in inhibiting CHH methylation in TE. CHG and CHH methylation levels are increased. In summary, our research reveals that MEZ1 plays an indispensable role in maize kernel development.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S513
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1 宋寧;玉米多梳蛋白MEZ1調(diào)控組蛋白修飾和DNA甲基化的研究[D];中國(guó)農(nóng)業(yè)大學(xué);2017年
，本文編號(hào)：2441876
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