本文關(guān)鍵詞：NAC類轉(zhuǎn)錄因子調(diào)控香蕉果實(shí)誘導(dǎo)耐冷性及成熟的機(jī)制分析　出處：《華南農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 香蕉 耐冷性 果實(shí)成熟 調(diào)控網(wǎng)絡(luò) NAC類轉(zhuǎn)錄因子

【摘要】：香蕉是呼吸躍變型果實(shí),常溫下貯藏很快出現(xiàn)乙烯峰,導(dǎo)致果實(shí)成熟快,貨架期短。降低貯藏的溫度,可以減弱香蕉的呼吸強(qiáng)度,延長(zhǎng)貯藏期。然而,香蕉果實(shí)對(duì)低溫十分敏感,在低于13℃時(shí)即會(huì)出現(xiàn)冷害癥狀,降低商品價(jià)值。因此,深入研究香蕉果實(shí)的耐冷和成熟機(jī)制,可為提高香蕉果實(shí)品質(zhì)和延長(zhǎng)貨架期提供重要理論依據(jù)。NAC是一類植物特有的轉(zhuǎn)錄因子,在植物生長(zhǎng)發(fā)育和抗逆反應(yīng)中發(fā)揮重要作用。我們前期的研究發(fā)現(xiàn),外源丙烯(乙烯功能類似物)處理可以誘導(dǎo)香蕉果實(shí)耐冷性,乙烯和成熟誘導(dǎo)了香蕉MaNAC1和MaNAC2的表達(dá)。然而,MaNAC1/2是如何調(diào)控丙烯誘導(dǎo)的香蕉果實(shí)耐冷性以及成熟的,是否還有其它家族成員的NAC轉(zhuǎn)錄因子參與對(duì)果實(shí)成熟的調(diào)控,還不清楚。本論文中,我們深入分析了MaNAC1/2及其它家族成員的NAC轉(zhuǎn)錄因子在香蕉果實(shí)誘導(dǎo)耐冷性和成熟調(diào)控中的作用機(jī)制。主要結(jié)果如下:1.MaNAC1通過(guò)與ICE1-CBF冷信號(hào)通路互作來(lái)參與丙烯誘導(dǎo)的香蕉果實(shí)耐冷性。通過(guò)熒光定量分析,發(fā)現(xiàn)MaNAC1的表達(dá)受低溫誘導(dǎo),外源丙烯在減輕香蕉果實(shí)冷害的同時(shí)亦明顯促進(jìn)了MaNAC1在低溫下的表達(dá)。原生質(zhì)體瞬時(shí)表達(dá)分析顯示,低溫及外源乙烯均可以顯著誘導(dǎo)MaNAC1基因啟動(dòng)子的活性,這進(jìn)一步說(shuō)明MaNAC1能夠響應(yīng)低溫逆境,并與丙烯誘導(dǎo)的耐冷性有關(guān)。更為重要的是,通過(guò)酵母單雜交、凝膠阻滯分析和瞬時(shí)表達(dá)分析實(shí)驗(yàn),證實(shí)MaNAC1是MaICEl的直接靶基因,低溫增強(qiáng)了MaICE1結(jié)合MaNAC1啟動(dòng)子的能力,并且受到磷酸化修飾的影響。2.香蕉果實(shí)成熟過(guò)程中,MaNAC1/2與RING類E3泛素連接酶MaXB3相互拮抗地影響乙烯信號(hào)和合成。通過(guò)轉(zhuǎn)錄活性分析,發(fā)現(xiàn)MaNAC1和MaNAC2是轉(zhuǎn)錄抑制子。經(jīng)凝膠阻滯分析、染色質(zhì)免疫共沉淀和瞬時(shí)表達(dá)分析,表明MaNAC1和Ma NAC2可以直接結(jié)合乙烯信號(hào)負(fù)調(diào)控因子MaRTH1和MaERF11的啟動(dòng)子并抑制其表達(dá)。蛋白質(zhì)互作和泛素降解實(shí)驗(yàn)發(fā)現(xiàn),RING類E3泛素連接酶MaXB3與MaNAC2、MaACS1和MaACO1互作,并通過(guò)26S蛋白酶體途徑泛素化降解MaNAC2、MaACS1和MaACO1,進(jìn)而削弱MaNAC2的轉(zhuǎn)錄抑制能力和乙烯合成。更有意思的是,MaNAC1和MaNAC2還可以直接結(jié)合MaXB3的啟動(dòng)子并抑制其表達(dá),反饋抑制MaXB3介導(dǎo)的泛素化降解,維持MaNAC2、MaACS1和MaACO1的蛋白穩(wěn)定性。3.MaSPL1、MaMADS1和MaNAC10-12之間通過(guò)形成一個(gè)調(diào)控乙烯合成的級(jí)聯(lián)網(wǎng)絡(luò),來(lái)參與香蕉果實(shí)成熟過(guò)程。為了挖掘與MaNAC1/2作用機(jī)制不同的其它家族成員的NACs,從香蕉基因組中分離了11個(gè)NAC基因,命名為MaNAC7-17。通過(guò)表達(dá)分析和轉(zhuǎn)錄活性檢測(cè),從中挑選出了成熟過(guò)程中明顯上調(diào)的且為轉(zhuǎn)錄激活子的MaNAC10-11和明顯下調(diào)的且為轉(zhuǎn)錄抑制子的MaNAC12-14進(jìn)行后續(xù)分析。結(jié)果顯示,MaNAC10和MaNAC11協(xié)同性的直接激活MaACS1和MaACO1的表達(dá),MaNAC12則抑制MaACS1和MaACO1的表達(dá)。進(jìn)一步篩選MaNAC10-14的上游調(diào)控因子,發(fā)現(xiàn)MaSPL1可以結(jié)合MaNAC10/11的啟動(dòng)子并激活其表達(dá),而MaMADS1結(jié)合MaNAC12的啟動(dòng)子并抑制其表達(dá)。此外,MaXB3也與MaNAC10和MaNAC11互作,并泛素化降解MaNAC10和MaNAC11,進(jìn)而減弱MaNAC10/11激活MaACS1和MaACO1的能力。
[Abstract]:Banana is a respiratory climacteric fruit. The ethylene peak appears quickly in storage at normal temperature, which causes the fruit to mature quickly and the shelf life is short. Reducing the temperature of storage can reduce the respiratory intensity of bananas and prolong the storage period. However, banana fruit is very sensitive to low temperature. Cold injury symptoms will appear at less than 13 C and reduce the value of commodity. Therefore, the study of the mechanism of cold tolerance and maturation of banana fruit can provide an important theoretical basis for improving the quality of banana fruit and prolonging the shelf life. NAC is a kind of plant specific transcription factor, which plays an important role in plant growth and resistance. Our previous studies found that exogenous propylene (ethylene functional analogue) treatment could induce cold tolerance of banana fruit, and ethylene and maturation induced the expression of MaNAC1 and MaNAC2 in banana. However, how MaNAC1/2 regulates the cold tolerance and maturity of the banana fruit induced by propylene, and whether the NAC transcription factors of other family members are involved in the regulation of fruit ripening is not clear. In this paper, we analyzed the mechanism of NAC transcription factors of MaNAC1/2 and other family members in the regulation of cold tolerance and maturation of banana fruit. The main results are as follows: 1.MaNAC1 is involved in the interaction of the ICE1-CBF cold signal pathway to participate in the cold resistance of the banana fruit induced by propylene. By fluorescence quantitative analysis, it was found that the expression of MaNAC1 was induced by low temperature, while exogenous propylene reduced the chilling injury of banana fruit, and also promoted the expression of MaNAC1 at low temperature. Transient expression analysis of protoplasts showed that both low temperature and exogenous ethylene could significantly induce the activity of MaNAC1 gene promoter, which further indicated that MaNAC1 could respond to low temperature stress and is related to propylene induced chilling tolerance. More importantly, by yeast one hybrid, gel retardation analysis and transient expression analysis, it is confirmed that MaNAC1 is a direct target gene of MaICEl. Low temperature enhances the ability of MaICE1 to bind to MaNAC1 promoter and is affected by phosphorylation. 2. the interaction of MaNAC1/2 and RING E3 ubiquitin ligase MaXB3 affects ethylene signal and synthesis during the ripening of banana fruit. Through the analysis of transcriptional activity, MaNAC1 and MaNAC2 were found to be transcriptional suppressors. Gel retardation analysis, chromatin immunoprecipitation and transient expression analysis showed that MaNAC1 and Ma NAC2 could directly bind to ethylene signal negative regulator MaRTH1 and MaERF11 promoter and inhibit its expression. Protein interaction and ubiquitin degradation experiments showed that RING E3 ubiquitin ligase MaXB3 interacted with MaNAC2, MaACS1 and MaACO1, and ubiquitin degraded MaNAC2, MaACS1 and MaACO1 through 26S proteasome pathway, thereby weakening the transcriptional inhibition ability of ethylene and ethylene synthesis. More interesting is that MaNAC1 and MaNAC2 can also directly combine MaXB3 promoter and inhibit its expression, feedback inhibit MaXB3 mediated ubiquitination and maintain MaNAC2, MaACS1 and MaACO1 protein stability. 3.MaSPL1, MaMADS1 and MaNAC10-12 are involved in the ripening of banana fruit by forming a cascade network that regulates the synthesis of ethylene. In order to excavate NACs from other family members, different from the MaNAC1/2 mechanism, 11 NAC genes were isolated from the banana genome, named MaNAC7-17. Through expression analysis and transcriptional activity detection, we picked out MaNAC10-11 which was significantly up-regulated during maturation, and transcriptional activator, and downregulated transcriptional inhibitor MaNAC12-14. The results showed that the CO activation of MaNAC10 and MaNAC11 directly activates the expression of MaACS1 and MaACO1, and MaNAC12 inhibits the expression of MaACS1 and MaACO1. Further screening for upstream regulatory factors of MaNAC10-14 showed that MaSPL1 could bind to MaNAC10/11 promoter and activate its expression, while MaMADS1 combined with MaNAC12 promoter and inhibited its expression. In addition, MaXB3 also interacts with MaNAC10 and MaNAC11, and ubiquitin degradation of MaNAC10 and MaNAC11, thereby reducing the ability of MaNAC10/11 to activate MaACS1 and MaACO1.
【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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