發(fā)布時間：2018-05-11 09:49

  本文選題：蘋果 + sRNA��； 參考：《中國農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：蘋果是我國和世界重要果樹之一,為多年生木本植物。在其花誘導(dǎo)期內(nèi),生長點(diǎn)內(nèi)由營養(yǎng)生長到生殖生長的成花轉(zhuǎn)變是影響花芽數(shù)量和質(zhì)量、決定下一年產(chǎn)量的關(guān)鍵環(huán)節(jié),解析成花轉(zhuǎn)變的分子機(jī)制對研究蘋果生理與發(fā)育具有重要理論意義。sRNA是一類非編碼的sRNA,可通過調(diào)控靶位點(diǎn)進(jìn)而調(diào)控相關(guān)分子路徑和基因網(wǎng)絡(luò),在植物生長發(fā)育、逆境響應(yīng)、激素水平等發(fā)面發(fā)揮重要作用。目前果樹中對sRNA調(diào)控成花的研究主要集中在miR156和miR172的調(diào)控機(jī)制,而對其他miRNA以及siRNA在其中的作用了解尚少;同時,在木本植物中,對sRNA合成加工路徑中關(guān)鍵因子的功能也知之甚少。本研究以'金冠'蘋果為試材,對其葉芽和花芽進(jìn)行了 sRNA文庫構(gòu)建、測序和生物信息學(xué)分析;并克隆了sRNA合成加工路徑的關(guān)鍵因子MdAGO1和MdAGO10,將其轉(zhuǎn)化擬南芥相應(yīng)突變體進(jìn)行功能互補(bǔ)分析。主要結(jié)果如下:1通過對'金冠'蘋果葉芽和花芽進(jìn)行sRNA測序,發(fā)現(xiàn)除miR156/miR172外,還有33個差異表達(dá)的已知miRNA、6個新miRNA也參與了成花轉(zhuǎn)變,其中大部分miRNA在成花轉(zhuǎn)變和前人研究的蘋果階段轉(zhuǎn)換中存在不同的表達(dá)模式。進(jìn)一步生物信息學(xué)分析發(fā)現(xiàn),差異表達(dá)miRNA主要與SPL基因、逆境響應(yīng)、生長素和GA途徑相關(guān)。根據(jù)靶基因分析,蘋果的成花轉(zhuǎn)變一方面可能在植物體內(nèi)產(chǎn)生了相應(yīng)的生理變化,誘導(dǎo)了相關(guān)逆境響應(yīng)miRNA的差異表達(dá),并進(jìn)一步誘導(dǎo)了生長素相關(guān)miRNA的差異表達(dá);另一方面,成花轉(zhuǎn)變還誘導(dǎo)了大量siRNA在花芽中高表達(dá),因此表明花芽中相應(yīng)位點(diǎn)的DNA甲基化水平升高。綜上所述,這些差異表達(dá)的miRNA可能通過調(diào)控SPL基因和GA路徑調(diào)控營養(yǎng)生長,通過逆境響應(yīng)路徑和生長素路徑調(diào)控生殖生長,siRNA則可能主要通過提高花芽內(nèi)DNA甲基化水平促進(jìn)成花轉(zhuǎn)變。2本研究從蘋果中克隆了AGO 和AGO10基因,其均具有AGO蛋白保守的PAZ和PIWI結(jié)構(gòu)域,AGO10在各植物中高度保守,而AGO1則在多年生木本果樹中存在特異的多次序列插入。MdAGO1和MdAGO10均主要定位于細(xì)胞核和質(zhì)膜。之后我們分別構(gòu)建了擬南芥AGO1和AGO10啟動子驅(qū)動的MdAGO1和MdAGO10表達(dá)載體。功能互補(bǔ)分析發(fā)現(xiàn),MdAGO1和MdAGO10可分別互補(bǔ)擬南芥突變體ago1-27和pnh-2的缺失表型,并使突變體中miRNA表達(dá)恢復(fù)至野生型水平。然而,與擬南芥AGO1不同,MdAGO1也可互補(bǔ)pnh-2的缺失表型,但未能恢復(fù)其miRNA水平至野生型。通過表型分離統(tǒng)計(jì),我們再次確認(rèn)了轉(zhuǎn)基因植株的pnh-2背景并確定該恢復(fù)表型來自于MdAGO1的過表達(dá)。進(jìn)一步分析認(rèn)為,MdAGO1功能上不同于擬南芥AGO1的原因可能來自于其蛋白N端插入的多年生木本果樹特異序列。
[Abstract]:Apple is one of the most important fruit trees in China and the world, and it is a perennial woody plant. During the flower induction period, the floral transformation from vegetative growth to reproductive growth in the growing point is the key link that affects the number and quality of flower buds and determines the yield of the next year. The molecular mechanism of floral transformation is of great theoretical significance for the study of apple physiology and development. SRNA is a kind of non-coding sRNAs, which can regulate the molecular pathways and gene networks by regulating the target sites, and respond to plant growth and stress. Hormone levels and other hair surface play an important role. At present, the studies on the regulation of flower formation by sRNA in fruit trees are mainly focused on the regulatory mechanisms of miR156 and miR172, but little is known about the role of other miRNA and siRNA in them, meanwhile, in woody plants, Little is known about the function of key factors in the sRNA synthesis path. In this study, the leaf buds and flower buds of 'Golden Crown' apple were constructed by sRNA library, sequenced and analyzed by bioinformatics. MdAGO1 and MdAGO10, the key factors of sRNA synthesis pathway, were cloned and transformed into Arabidopsis thaliana corresponding mutants for functional complementation analysis. The main results were as follows: 1 by sequencing the sRNA of the leaf buds and flower buds of 'Golden Crown' apple, we found that in addition to miR156/miR172, there were 33 known miRNAs differentially expressed, and six new miRNA were also involved in the flower formation transition. Most of the miRNA have different expression patterns in flowering transition and apple stage transition. Further bioinformatics analysis showed that differential expression of miRNA was mainly related to SPL gene, stress response, auxin and GA pathway. According to the target gene analysis, on the one hand, the flowering transformation of apple may produce corresponding physiological changes in the plant, induce the differential expression of miRNA in response to related stresses, and further induce the differential expression of auxin related miRNA in the plant, on the other hand, Floral transformation also induced a large number of siRNA overexpression in flower bud, which indicated that the DNA methylation level of the corresponding site in flower bud was increased. In conclusion, these differentially expressed miRNA may regulate vegetative growth by regulating SPL gene and GA pathway. Regulation of reproductive growth by stress response pathway and auxin pathway may promote floral transformation by increasing the level of DNA methylation in flower buds. 2. In this study, AGO and AGO10 genes were cloned from apple. The conserved PAZ and PIWI domain AGO10 of AGO protein were highly conserved in all plants, while AGO1 had specific multiple sequence insertions in perennial woody fruit trees. MdAGO1 and MdAGO10 were mainly located in nucleus and plasma membrane. Then we constructed Arabidopsis thaliana AGO1 and AGO10 promoter driven MdAGO1 and MdAGO10 expression vectors. Functional complementation analysis showed that MdAGO1 and MdAGO10 could complement the deletion phenotypes of ago1-27 and pnh-2 in Arabidopsis thaliana mutant, and restore miRNA expression to wild-type level. However, unlike Arabidopsis thaliana AGO1, MdAGO1 could complement the deletion phenotype of pnh-2, but could not recover its miRNA level to wild-type. By phenotypic isolation statistics, we reconfirmed the pnh-2 background of transgenic plants and confirmed that the recovery phenotype was derived from overexpression of MdAGO1. It is suggested that the reason why MdAGO1 is functionally different from Arabidopsis AGO1 may be due to the specific sequence of perennial woody fruit trees with N-terminal insertion of MdAGO1 from Arabidopsis thaliana.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S661.1

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