本文選題：蘋果 + YTP��； 參考：《西北農(nóng)林科技大學(xué)》2017年博士論文

【摘要】：RNA結(jié)合蛋白參與植物逆境響應(yīng)和生長(zhǎng)發(fā)育過程。包含YT521-B homology(YTH)結(jié)構(gòu)域的RNA結(jié)合蛋白(YTH domain containing RNA binding protein;YTP)最初在小鼠中發(fā)現(xiàn),與缺氧脅迫相關(guān)。相比于酵母和動(dòng)物,植物中關(guān)于YTP的研究很少。第一個(gè)在植物上發(fā)現(xiàn)的YTP是擬南芥AtCPSF30(cleavage and polyadenylation specificity factor 30;剪切/多聚腺苷酸化特異性因子30),為植物多聚腺苷酸化復(fù)合體的一部分,參與氧化脅迫等逆境響應(yīng);初步研究也表明擬南芥YTP家族基因能夠響應(yīng)多種外界環(huán)境刺激。蘋果(Malus domestica Borkh.)是世界上廣泛種植和消費(fèi)的水果之一,但其生長(zhǎng)發(fā)育受到外界脅迫和自身生長(zhǎng)狀態(tài)的影響。研究蘋果YTP對(duì)各種逆境的響應(yīng)和與蘋果生長(zhǎng)發(fā)育的關(guān)系,能夠?yàn)樘O果抗性改良和生長(zhǎng)發(fā)育調(diào)控等提供理論依據(jù)。本論文首先從蘋果基因組中鑒定出YTP家族成員,并探究了各個(gè)成員對(duì)葉片衰老和各種逆境的響應(yīng)。綜合分析蘋果YTP基因在不同逆境下的表達(dá)模式,挑選出MdYTP1和MdYTP2兩個(gè)同源基因進(jìn)行后續(xù)研究。從蘋果屬植物平邑甜茶(Malus hupehensis(Pamp.)Rehd.)中克隆獲得基因全長(zhǎng),分別命名為MhYTP1和MhYTP2,并鑒定了MhYTP1和MhYTP2在非生物脅迫、生物脅迫、葉片衰老和果實(shí)發(fā)育中的功能。獲得的主要結(jié)果如下:1.蘋果YTP基因家族鑒定及平邑甜茶MhYTP1和MhYTP2基因克隆和功能分析。蘋果(M.domestica)基因組中共包含15個(gè)能夠在不同組織中表達(dá)的YTP基因。15個(gè)基因家族成員在葉片衰老過程中被誘導(dǎo)表達(dá),也能夠響應(yīng)各種環(huán)境脅迫。挑選出YTP1和YTP2作為后續(xù)研究對(duì)象,從平邑甜茶(M.hupehensis)中克隆獲得YTP1和YTP2基因全長(zhǎng)和啟動(dòng)子序列。MhYTP1和MhYTP2在整個(gè)細(xì)胞中均有表達(dá),具有RNA結(jié)合活性。MhYTP1和MhYTP2的啟動(dòng)子序列均包含多種與逆境響應(yīng)和生長(zhǎng)發(fā)育相關(guān)的順式作用元件,說明MhYTP1和MhYTP2可能在脅迫響應(yīng)和生長(zhǎng)發(fā)育過程中發(fā)揮著重要作用。組織定位結(jié)果表明,MhYTP1主要在莖中表達(dá),而MhYTP2主要在根中表達(dá)。MhYTP1和MhYTP2雖為同源基因,但它們啟動(dòng)子序列所含順式作用元件及組織定位的不同,預(yù)示著MhYPT1和MhYTP2在逆境響應(yīng)和生長(zhǎng)發(fā)育中功能的不同。2.MhYTP1和MhYTP2在擬南芥及蘋果中過表達(dá)能夠影響植株對(duì)外源激素刺激和脅迫的響應(yīng)。MhYTP1和MhYTP2響應(yīng)外源脫落酸(abscisic acid;ABA)誘導(dǎo),相比于野生型植株,基因過表達(dá)擬南芥和蘋果GL-3植株均表現(xiàn)出對(duì)ABA的不敏感性。ABA信號(hào)途徑在干旱脅迫響應(yīng)中發(fā)揮重要作用。MhYTP1和MhYTP2也能被干旱環(huán)境誘導(dǎo)表達(dá)。MhYTP1或MhYTP2過表達(dá)使蘋果葉片氣孔密度和開張度減小,從而提高植株抗旱性。進(jìn)一步深入研究了MhYTP1和MhYTP2參與干旱脅迫響應(yīng)的分子機(jī)制。通過酵母單雜交和酵母雙雜交等試驗(yàn),篩選互作蛋白,MhRD22能夠結(jié)合MhYTP1的啟動(dòng)子來調(diào)控其基因的表達(dá);MhYTP2和MhSCE1相互作用共同參與ABA信號(hào)途徑;結(jié)合第二章組織定位結(jié)果,MhYTP1主要在莖中表達(dá),MhYTP2主要在根中表達(dá),說明MhYTP1和MhYTP2通過不同的方式,在植物不同的組織部位發(fā)揮主要功能,從而參與干旱脅迫響應(yīng)。除干旱脅迫外,MhYTP1和MhYTP2還參與水澇、高低溫、高鹽和低營養(yǎng)等非生物脅迫應(yīng)答,也參與響應(yīng)蘋果褐斑病病菌侵染。MhYTP1和MhYTP2過表達(dá)能夠提高植物對(duì)水澇(氧化脅迫)、低溫和低營養(yǎng)脅迫的抗性,也能通過影響JA和SA信號(hào)途徑降低蘋果對(duì)褐斑病的抗性;但轉(zhuǎn)基因擬南芥和蘋果在高鹽和高溫脅迫下的表型存在差異,因此,MhYTP1和MhYTP2在植物抗高鹽和高溫方面的功能尚不能明確。3.MhYTP1和MhYTP2在葉片衰老和果實(shí)成熟過程中的作用。大部分蘋果YTP基因家族成員能夠響應(yīng)自然衰老,MhYTP1和MhYTP2的表達(dá)量隨著自然衰老逐漸升高,衰老后期,表達(dá)量隨著自然衰老逐漸降低。MhYTP1或MhYTP2過表達(dá)能夠促進(jìn)擬南芥離體葉片衰老;在蘋果GL-3中過表達(dá),也能促進(jìn)蘋果葉片離體衰老和自然衰老。果實(shí)發(fā)育過程也是果實(shí)不斷衰老的過程,MhYTP1或MhYTP2過表達(dá)不僅能夠促進(jìn)葉片衰老,還能夠促進(jìn)果實(shí)成熟。MhYTP1或MhYTP2轉(zhuǎn)基因番茄果實(shí)成熟所需時(shí)間少于野生型,同一生長(zhǎng)時(shí)期過表達(dá)植株果實(shí)果皮類胡蘿卜素含量、果實(shí)中類胡蘿卜素合成基因、乙烯合成基因和乙烯響應(yīng)基因表達(dá)量均高于野生型番茄。進(jìn)一步探究了MhYTP1和MhYTP2參與葉片衰老和果實(shí)成熟過程的分子機(jī)制。通過酵母雙雜交試驗(yàn),發(fā)現(xiàn)MhYTP2能夠與MhARD4互作。ARD4參與甲硫氨酸循環(huán),甲硫氨酸循環(huán)與乙烯合成相關(guān)。因此,MhYTP1和MhYTP2可能影響乙烯生物合成,從而促進(jìn)葉片衰老,縮短果實(shí)成熟所需時(shí)間。
[Abstract]:RNA binding proteins participate in plant stress response and growth and development. The RNA binding protein containing the YT521-B homology (YTH) domain (YTH domain containing RNA binding protein; YTP) was first found in mice and related to hypoxia stress. Compared to yeast and animals, there are few studies in plants. TP is AtCPSF30 (cleavage and polyadenylation specificity factor 30; shear / polyadenosine acidification specific factor 30), a part of plant polyadenylation complex, involved in oxidative stress and other adverse responses. Preliminary studies also indicate that the Arabidopsis YTP family base can respond to a variety of external environmental stimuli. Apple (Malus DOM) Estica Borkh.) is one of the most widely cultivated and consumed fruits in the world, but its growth is influenced by external stress and its own growth state. The study of the response of apple YTP to various adversity and the relationship with the growth and development of apple can provide theoretical basis for the improvement of Apple Resistance and control of growth and development. The YTP family members were identified in the genome, and the responses of each member to leaf senescence and various adversities were explored. The expression patterns of apple YTP genes under different stresses were analyzed, and two homologous genes of MdYTP1 and MdYTP2 were selected for subsequent studies. The clone obtained from the Malus hupehensis (Pamp.) Rehd. of the apple plant (Malus hupehensis (Pamp.) Rehd.) The whole length of the gene was named MhYTP1 and MhYTP2, and the functions of MhYTP1 and MhYTP2 in abiotic stress, biological stress, leaf senescence and fruit development were identified. The main results were as follows: 1. apple YTP gene family identification and the cloning and functional analysis of MhYTP1 and MhYTP2 genes in Pingyi sweet tea. The genome of apple (M.domestica) contains 1 5.15 gene family members of the YTP gene that can be expressed in different tissues can be induced and expressed in the process of leaf senescence, and can also respond to various environmental stresses. YTP1 and YTP2 are selected as a follow-up object, and the whole length of YTP1 and YTP2 primes and promoter sequences.MhYTP1 and MhYTP2 are obtained from Pingyi sweet tea (M.hupehensis). All the cells were expressed in the cells. The promoter sequences with RNA binding activity.MhYTP1 and MhYTP2 contain a variety of cis acting elements related to stress response and growth and development. It shows that MhYTP1 and MhYTP2 may play an important role in the process of stress response and growth and development. The results of tissue localization show that MhYTP1 is mainly expressed in the stem, while MhYTP is mainly expressed in the stem. 2 mainly expressed.MhYTP1 and MhYTP2 as homologous genes in the root, but the difference between the cis acting elements and the tissue localization of the promoter sequences indicates that the different.2.MhYTP1 and MhYTP2 functions of MhYPT1 and MhYTP2 in the Arabidopsis and the apple in adversity response and growth can affect the stimulation and threat of the plant to the exogenous hormone. Forced response to the response of.MhYTP1 and MhYTP2 to exogenous abscisic acid (abscisic acid; ABA) induction, compared to wild type plants, genes overexpressed Arabidopsis and apple GL-3 plants all showed an insensitivity to ABA and played an important role in the drought stress response..MhYTP1 and MhYTP2 could also be induced to be induced by drought environment to express.MhYTP1 or MhYTP2. The overexpression reduced the stomatal density and opening degree of apple leaves, thus improving the drought resistance of the plants. Further in-depth study of the molecular mechanism of MhYTP1 and MhYTP2 in response to drought stress was further studied. The interaction proteins were screened by yeast single hybridization and yeast two hybrid experiments. MhRD22 could regulate the expression of its genes by combining the promoter of MhYTP1; MhYTP The interaction of 2 and MhSCE1 participates in the ABA signal pathway together; in combination with the results of the second chapter, MhYTP1 is mainly expressed in the stem, and MhYTP2 is mainly expressed in the root. It shows that MhYTP1 and MhYTP2 play the main functions in different tissue parts of the plant, and thus participate in the response to drought stress. In addition to drought stress, MhYTP1 and MhYTP2 are in addition to drought stress. It also participates in the abiotic stress response such as waterlogging, high and low temperature, high salt and low nutrition. It also participates in the response to the overexpression of.MhYTP1 and MhYTP2 in the infection of the pathogen of Apple brown spot, which can improve the resistance of plants to waterlogging (oxidative stress), low temperature and low nutrition stress, and can also reduce the resistance of apple to brown spot by affecting the JA and SA signaling pathway. The phenotypes of South mustard and apple are different under high salt and high temperature stress. Therefore, the functions of MhYTP1 and MhYTP2 in plant resistance to high salt and high temperature are not yet clear about the role of.3.MhYTP1 and MhYTP2 in leaf senescence and fruit ripening. Most of the members of the apple YTP gene family can respond to natural aging, MhYTP1 and MhYTP2 expression. With the gradual increase of natural aging, the expression of.MhYTP1 or MhYTP2 over expression can promote the senescence of leaves of Arabidopsis in the later period of aging. Over expression in apple GL-3 can also promote the senescence and natural senescence of apple leaves in vitro. The process of fruit development is also the process of continuous senescence of fruit, MhYTP1 or MhYTP2 over table. It can not only promote leaf senescence, but also promote the fruit maturity of.MhYTP1 or MhYTP2 transgenic tomato less than the wild type, the content of carotenoid content in the fruit peel, the carotenoid synthesis gene, the ethylene synthase gene and the ethylene response gene in the fruit are higher than that in the wild. The molecular mechanism of MhYTP1 and MhYTP2 involved in the process of leaf senescence and fruit ripening was further explored. Through the yeast two hybrid test, it was found that MhYTP2 could interact with MhARD4 to participate in methionine cycle, and the methionine cycle was related to ethylene synthesis. Therefore, MhYTP1 and MhYTP2 may affect ethylene biosynthesis and thus promote leaves. It will take time to reduce the maturity of the fruit.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S661.1

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4 李慧峰;泰沂山區(qū)蘋果屬植物系統(tǒng)學(xué)研究[D];沈陽農(nóng)業(yè)大學(xué);2012年

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8 高建杰;蘋果兩個(gè)MYB轉(zhuǎn)錄因子基因的克隆與功能分析[D];南京農(nóng)業(yè)大學(xué);2011年

9 馬小衛(wèi);蘋果屬植物種間水分利用效率的差異及其機(jī)理研究[D];西北農(nóng)林科技大學(xué);2009年

10 趙玲玲;蘋果MdSAMDC2和MdICE1基因的功能鑒定以及抗逆性研究[D];山東農(nóng)業(yè)大學(xué);2007年

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