【摘要】：香蕉是呼吸躍變型果實,成熟啟動后果實迅速軟化,給貯運保鮮帶來很大困難。許多研究表明,ERF轉錄因子和組蛋白去乙�；�(HDAC)在植物的生長發(fā)育過程中發(fā)揮著重要作用。我們前期的研究發(fā)現(xiàn),與成熟密切相關的MaERFs不僅可以反饋調控乙烯合成基因MaACS1和MaACO1,而且可以與MaACO1蛋白相互作用,從而參與香蕉果實成熟過程的轉錄調控。然而,HDAC是否參與以及怎樣參與ERF調控香蕉果實成熟機制目前并不清楚。本文在明確MaERF11參與香蕉果實成熟的基礎上,通過通過凝膠組織遷移實驗(EMSA)、雙熒光素酶報告基因實驗(DLR)、蛋白互作技術和染色質免疫共沉淀(ChIP)等多種實驗手段深入研究了HDAC參與ERF調控香蕉果實成熟機制,我們的研究結果拓寬了果實成熟的轉錄調控網絡,尤其是在組蛋白去乙�；揎梾⑴c調控果實成熟機制方面加深了人們對果實成熟及其調控機制的認識。本文獲得的主要研究結果和結論如下:1.MaERF11具轉錄抑制活性,它可以結合乙烯合成基因MaACO1和3個果實軟化相關基因MaEXP2/7/8的啟動子,進而參與調控香蕉果實的成熟過程。EMSA和ChIP實驗表明,MaERF11可以結合乙烯合成基因MaACO1和3個果實軟化相關基因MaEXP2/7/8的啟動子。另外,DLR檢測結果顯示,MaERF11具有轉錄抑制活性,能抑制下游基因MaACO1和MaEXP2/7/8的啟動子活性。這些結果說明,MaERF11作為轉錄抑制子,可以通過對乙烯合成基因MaACO1和果實軟化相關基因MaEXP2/7/8的抑制作用來調控乙烯的合成和果實的軟化。2.成熟相關基因MaACO1和MaEXP2/7/8的表達及其組蛋白乙�；阶兓c果實成熟密切相關。熒光定量PCR(qRT-PCR)結果顯示,MaACO1和MaEXP2/7/8基因的表達在采后香蕉后熟進程中均呈現(xiàn)不同程度的上升趨勢。ChIP結果顯示,成熟香蕉果實的MaACO1和MaEXP2/7/8基因染色質上的組蛋白H3和H4的乙�；綍哂谖闯墒斓南憬�,這與基因的表達趨勢相吻合。這些結果說明,MaACO1和MaEXP2/7/8的表達及其染色質上組蛋白的乙�；脚c香蕉果實成熟密切相關。3.MaERF11通過招募MaHDA1形成轉錄抑制復合體,共同調控MaACO1和MaEXP2/7/8的轉錄表達。酵母雙雜交(Y2H)、雙分子熒光互補(BiFC)和免疫共沉淀(Co-IP)的結果均說明MaERF11與MaHDA1之間存在相互作用關系,并且,MaHDA1具有HDAC保守域和組蛋白去乙�；富钚�,可以增強MaERF11對下游靶基因MaACO1和MaEXP2/7/8的抑制作用。這些結果共同說明,MaERF11與MaHDA1可以形成轉錄抑制復合體,共同調控后熟相關基因MaACO1和MaEXP2/7/8的表達,從而參與調控香蕉果實的后熟衰老過程。4.從香蕉基因組序列中獲得了17個MaHDACs基因,可以分為RPD3/HDA1、HD2和SIR2三個不同亞族,并且它們在香蕉果實后熟過程中呈現(xiàn)不同的表達模式。我們從香蕉基因組中鑒別出17個MaHDACs基因,通過進化樹關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HDAC保守結構域,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SIR2保守域,屬于SIR2亞家族。qRT-PCR分析結果顯示,17個MaHDACs在香蕉果實后熟進程中呈現(xiàn)出不同的表達模式,其中,尤以MaHDA6的表達變化最明顯,在三個不同處理的香蕉果實中均表現(xiàn)出明顯的上調趨勢。5.探討了MaHDA6在乙烯信號轉導和果實成熟中的調控機制。研究表明,MaHDA6定位于細胞核內,具有組蛋白去乙�；富钚�,ChIP結果顯示,MaHDA6可以結合MaERF11/15的啟動子,并且成熟香蕉果實的MaERF11/15基因染色質上組蛋白乙�；乃降陀谖闯墒斓南憬�,這與它們的表達是一致的。這些結果說明,MaHDA6可能通過去乙�；饔脕碚{控下游基因MaERF11/15的表達,從而參與調控乙烯信號轉導途徑和香蕉果實的后熟衰老。
[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.
【學位授予單位】：華南農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S668.1

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