發(fā)布時(shí)間：2018-08-01 11:11

【摘要】：香蕉是呼吸躍變型果實(shí),成熟啟動(dòng)后果實(shí)迅速軟化,給貯運(yùn)保鮮帶來很大困難。許多研究表明,ERF轉(zhuǎn)錄因子和組蛋白去乙�；�(HDAC)在植物的生長(zhǎng)發(fā)育過程中發(fā)揮著重要作用。我們前期的研究發(fā)現(xiàn),與成熟密切相關(guān)的MaERFs不僅可以反饋調(diào)控乙烯合成基因MaACS1和MaACO1,而且可以與MaACO1蛋白相互作用,從而參與香蕉果實(shí)成熟過程的轉(zhuǎn)錄調(diào)控。然而,HDAC是否參與以及怎樣參與ERF調(diào)控香蕉果實(shí)成熟機(jī)制目前并不清楚。本文在明確MaERF11參與香蕉果實(shí)成熟的基礎(chǔ)上,通過通過凝膠組織遷移實(shí)驗(yàn)(EMSA)、雙熒光素酶報(bào)告基因?qū)嶒?yàn)(DLR)、蛋白互作技術(shù)和染色質(zhì)免疫共沉淀(ChIP)等多種實(shí)驗(yàn)手段深入研究了HDAC參與ERF調(diào)控香蕉果實(shí)成熟機(jī)制,我們的研究結(jié)果拓寬了果實(shí)成熟的轉(zhuǎn)錄調(diào)控網(wǎng)絡(luò),尤其是在組蛋白去乙�；揎梾⑴c調(diào)控果實(shí)成熟機(jī)制方面加深了人們對(duì)果實(shí)成熟及其調(diào)控機(jī)制的認(rèn)識(shí)。本文獲得的主要研究結(jié)果和結(jié)論如下:1.MaERF11具轉(zhuǎn)錄抑制活性,它可以結(jié)合乙烯合成基因MaACO1和3個(gè)果實(shí)軟化相關(guān)基因MaEXP2/7/8的啟動(dòng)子,進(jìn)而參與調(diào)控香蕉果實(shí)的成熟過程。EMSA和ChIP實(shí)驗(yàn)表明,MaERF11可以結(jié)合乙烯合成基因MaACO1和3個(gè)果實(shí)軟化相關(guān)基因MaEXP2/7/8的啟動(dòng)子。另外,DLR檢測(cè)結(jié)果顯示,MaERF11具有轉(zhuǎn)錄抑制活性,能抑制下游基因MaACO1和MaEXP2/7/8的啟動(dòng)子活性。這些結(jié)果說明,MaERF11作為轉(zhuǎn)錄抑制子,可以通過對(duì)乙烯合成基因MaACO1和果實(shí)軟化相關(guān)基因MaEXP2/7/8的抑制作用來調(diào)控乙烯的合成和果實(shí)的軟化。2.成熟相關(guān)基因MaACO1和MaEXP2/7/8的表達(dá)及其組蛋白乙�；阶兓c果實(shí)成熟密切相關(guān)。熒光定量PCR(qRT-PCR)結(jié)果顯示,MaACO1和MaEXP2/7/8基因的表達(dá)在采后香蕉后熟進(jìn)程中均呈現(xiàn)不同程度的上升趨勢(shì)。ChIP結(jié)果顯示,成熟香蕉果實(shí)的MaACO1和MaEXP2/7/8基因染色質(zhì)上的組蛋白H3和H4的乙�；綍�(huì)高于未成熟的香蕉,這與基因的表達(dá)趨勢(shì)相吻合。這些結(jié)果說明,MaACO1和MaEXP2/7/8的表達(dá)及其染色質(zhì)上組蛋白的乙�；脚c香蕉果實(shí)成熟密切相關(guān)。3.MaERF11通過招募MaHDA1形成轉(zhuǎn)錄抑制復(fù)合體,共同調(diào)控MaACO1和MaEXP2/7/8的轉(zhuǎn)錄表達(dá)。酵母雙雜交(Y2H)、雙分子熒光互補(bǔ)(BiFC)和免疫共沉淀(Co-IP)的結(jié)果均說明MaERF11與MaHDA1之間存在相互作用關(guān)系,并且,MaHDA1具有HDAC保守域和組蛋白去乙�；富钚�,可以增強(qiáng)MaERF11對(duì)下游靶基因MaACO1和MaEXP2/7/8的抑制作用。這些結(jié)果共同說明,MaERF11與MaHDA1可以形成轉(zhuǎn)錄抑制復(fù)合體,共同調(diào)控后熟相關(guān)基因MaACO1和MaEXP2/7/8的表達(dá),從而參與調(diào)控香蕉果實(shí)的后熟衰老過程。4.從香蕉基因組序列中獲得了17個(gè)MaHDACs基因,可以分為RPD3/HDA1、HD2和SIR2三個(gè)不同亞族,并且它們?cè)谙憬豆麑?shí)后熟過程中呈現(xiàn)不同的表達(dá)模式。我們從香蕉基因組中鑒別出17個(gè)MaHDACs基因,通過進(jìn)化樹關(guān)系和氨基酸序列保守域分析發(fā)現(xiàn),其中的MaHDA1-12編碼的氨基酸序列都包含一個(gè)HDAC保守結(jié)構(gòu)域,屬于RPD3/HDA1亞家族;MaHDT1-3編碼的氨基酸序列在N端都有一個(gè)MEFWG保守域,屬于HD2亞家族;而MaSRT1/2編碼的氨基酸序列都有一個(gè)SIR2保守域,屬于SIR2亞家族。qRT-PCR分析結(jié)果顯示,17個(gè)MaHDACs在香蕉果實(shí)后熟進(jìn)程中呈現(xiàn)出不同的表達(dá)模式,其中,尤以MaHDA6的表達(dá)變化最明顯,在三個(gè)不同處理的香蕉果實(shí)中均表現(xiàn)出明顯的上調(diào)趨勢(shì)。5.探討了MaHDA6在乙烯信號(hào)轉(zhuǎn)導(dǎo)和果實(shí)成熟中的調(diào)控機(jī)制。研究表明,MaHDA6定位于細(xì)胞核內(nèi),具有組蛋白去乙�；富钚�,ChIP結(jié)果顯示,MaHDA6可以結(jié)合MaERF11/15的啟動(dòng)子,并且成熟香蕉果實(shí)的MaERF11/15基因染色質(zhì)上組蛋白乙�；乃降陀谖闯墒斓南憬�,這與它們的表達(dá)是一致的。這些結(jié)果說明,MaHDA6可能通過去乙�；饔脕碚{(diào)控下游基因MaERF11/15的表達(dá),從而參與調(diào)控乙烯信號(hào)轉(zhuǎn)導(dǎo)途徑和香蕉果實(shí)的后熟衰老。
[Abstract]:Bananas are respiratory climacteric fruits, and the fruits are rapidly softened after they are started. Many studies have shown that ERF transcription factors and histone deacetylase (HDAC) play an important role in the growth and development of plants. Our previous studies have shown that MaERFs, which is closely related to maturity, can not only be used for feedback. Regulate the ethylene synthesis gene MaACS1 and MaACO1, and interact with MaACO1 protein to participate in the transcriptional regulation of banana fruit ripening. However, it is not clear whether HDAC participates in and how to participate in ERF regulation of banana fruit ripening. EMSA, double luciferase reporter gene experiment (DLR), protein interaction technique and chromatin immunoprecipitation (ChIP) were used to study the mechanism of HDAC participation in the regulation of banana fruit ripening. Our results widened the transcriptional regulation network of fruit ripening, especially in histone deacetylation repair. The main research results and conclusions obtained in this paper are as follows: 1.MaERF11 has the transcriptional inhibitory activity, which can combine the MaACO1 of ethylene synthesis gene and the promoter of 3 fruit softening related genes MaEXP2/7/8, and then participate in the regulation of banana fruit. The actual maturation process.EMSA and ChIP experiments showed that MaERF11 could combine the ethylene synthesis gene MaACO1 and the promoter of 3 fruit softening related genes MaEXP2/7/8. In addition, DLR tests showed that MaERF11 had transcriptional inhibitory activity and could inhibit the activity of the promoter of the downstream gene MaACO1 and MaEXP2/7/8. These results suggest that MaERF11 is transcribed as a transcript. Suppressor, the expression of ethylene synthesis and fruit softening.2. maturity related genes MaACO1 and MaEXP2/7/8 can be regulated by the inhibition of ethylene synthesis gene MaACO1 and fruit softening related gene MaEXP2/7/8, and the changes in histone acetylation level are closely related to fruit maturity. Fluorescence quantitative PCR (qRT-PCR) results show that MaACO1 The expression of MaEXP2/7/8 gene in the post harvest banana ripening process showed an increasing trend in the process of post harvest banana ripening..ChIP results showed that the levels of histone H3 and H4 on the MaACO1 and MaEXP2/7/8 chromatin of mature banana fruit were higher than those of immature bananas, which coincided with the trend of gene expression. These results indicate that MaACO1 The expression of MaEXP2/7/8 and its chromatin histone acetylation level are closely related to the ripening of banana fruit..3.MaERF11 is used to form a transcriptional inhibition complex by recruiting MaHDA1 to regulate the transcriptional expression of MaACO1 and MaEXP2/7/8. Yeast two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and immunoprecipitation (Co-IP) result in MaERF There is a interaction between 11 and MaHDA1, and MaHDA1 has the HDAC conserved domain and histone deacetylase activity, which can enhance the inhibitory effect of MaERF11 on the downstream target gene MaACO1 and MaEXP2/7/8. These results show that MaERF11 and MaHDA1 can form a transcriptional inhibition complex and jointly regulate the back ripening related genes MaACO1 and MaEXP. The expression of 2/7/8, thus participating in the regulation of banana fruit ripening and senescence,.4. obtained 17 MaHDACs genes from the banana genome sequence, which can be divided into three different subgroups of RPD3/HDA1, HD2 and SIR2, and they present different expression patterns in the process of banana fruit ripening. We identified 17 MaHDACs genes from the banana genome. Through the evolutionary tree relationship and the conservative domain analysis of amino acid sequences, it is found that the MaHDA1-12 encoded amino acid sequences all contain a HDAC conservative domain, belonging to the RPD3/HDA1 subfamily, and the MaHDT1-3 encoded amino acid sequence has a MEFWG conservative domain at the N end, which belongs to the HD2 subfamily; and MaSRT1/2 encoded amino acid sequences have a SIR sequence. The 2 conservative domain, belonging to the.QRT-PCR analysis of the SIR2 subfamily, showed that 17 MaHDACs showed different expression patterns in the ripening process of banana fruit, in which the expression of MaHDA6 was most obvious, and all of the three different treated banana fruits showed obvious up-regulated.5., which discussed MaHDA6 in ethylene signal transduction and fruit formation. The study shows that MaHDA6 is located in the nucleus and has the histone deacetylase activity. The results of ChIP show that MaHDA6 can combine the promoter of MaERF11/15, and the level of the histone acetylation of the MaERF11/15 gene of the mature banana fruit is lower than that of the immature banana, which is in accordance with their expression. These results suggest that MaHDA6 may be used to regulate the expression of the downstream gene MaERF11/15 by deacetylation, thus participating in the regulation of ethylene signal transduction pathway and the ripening senescence of banana fruit.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S668.1

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