苹果YTH结构域RNA结合蛋白基因MhYTP1和MhYTP2的功能研究

发布时间：2018-04-26 04:03

  本文选题：苹果 + YTP　； 参考：《西北农林科技大学》2017年博士论文

【摘要】：RNA结合蛋白参与植物逆境响应和生长发育过程。包含YT521-B homology(YTH)结构域的RNA结合蛋白(YTH domain containing RNA binding protein;YTP)最初在小鼠中发现,与缺氧胁迫相关。相比于酵母和动物,植物中关于YTP的研究很少。第一个在植物上发现的YTP是拟南芥AtCPSF30(cleavage and polyadenylation specificity factor 30;剪切/多聚腺苷酸化特异性因子30),为植物多聚腺苷酸化复合体的一部分,参与氧化胁迫等逆境响应;初步研究也表明拟南芥YTP家族基因能够响应多种外界环境刺激。苹果(Malus domestica Borkh.)是世界上广泛种植和消费的水果之一,但其生长发育受到外界胁迫和自身生长状态的影响。研究苹果YTP对各种逆境的响应和与苹果生长发育的关系,能够为苹果抗性改良和生长发育调控等提供理论依据。本论文首先从苹果基因组中鉴定出YTP家族成员,并探究了各个成员对叶片衰老和各种逆境的响应。综合分析苹果YTP基因在不同逆境下的表达模式,挑选出MdYTP1和MdYTP2两个同源基因进行后续研究。从苹果属植物平邑甜茶(Malus hupehensis(Pamp.)Rehd.)中克隆获得基因全长,分别命名为MhYTP1和MhYTP2,并鉴定了MhYTP1和MhYTP2在非生物胁迫、生物胁迫、叶片衰老和果实发育中的功能。获得的主要结果如下:1.苹果YTP基因家族鉴定及平邑甜茶MhYTP1和MhYTP2基因克隆和功能分析。苹果(M.domestica)基因组中共包含15个能够在不同组织中表达的YTP基因。15个基因家族成员在叶片衰老过程中被诱导表达,也能够响应各种环境胁迫。挑选出YTP1和YTP2作为后续研究对象,从平邑甜茶(M.hupehensis)中克隆获得YTP1和YTP2基因全长和启动子序列。MhYTP1和MhYTP2在整个细胞中均有表达,具有RNA结合活性。MhYTP1和MhYTP2的启动子序列均包含多种与逆境响应和生长发育相关的顺式作用元件,说明MhYTP1和MhYTP2可能在胁迫响应和生长发育过程中发挥着重要作用。组织定位结果表明,MhYTP1主要在茎中表达,而MhYTP2主要在根中表达。MhYTP1和MhYTP2虽为同源基因,但它们启动子序列所含顺式作用元件及组织定位的不同,预示着MhYPT1和MhYTP2在逆境响应和生长发育中功能的不同。2.MhYTP1和MhYTP2在拟南芥及苹果中过表达能够影响植株对外源激素刺激和胁迫的响应。MhYTP1和MhYTP2响应外源脱落酸(abscisic acid;ABA)诱导,相比于野生型植株,基因过表达拟南芥和苹果GL-3植株均表现出对ABA的不敏感性。ABA信号途径在干旱胁迫响应中发挥重要作用。MhYTP1和MhYTP2也能被干旱环境诱导表达。MhYTP1或MhYTP2过表达使苹果叶片气孔密度和开张度减小,从而提高植株抗旱性。进一步深入研究了MhYTP1和MhYTP2参与干旱胁迫响应的分子机制。通过酵母单杂交和酵母双杂交等试验,筛选互作蛋白,MhRD22能够结合MhYTP1的启动子来调控其基因的表达;MhYTP2和MhSCE1相互作用共同参与ABA信号途径;结合第二章组织定位结果,MhYTP1主要在茎中表达,MhYTP2主要在根中表达,说明MhYTP1和MhYTP2通过不同的方式,在植物不同的组织部位发挥主要功能,从而参与干旱胁迫响应。除干旱胁迫外,MhYTP1和MhYTP2还参与水涝、高低温、高盐和低营养等非生物胁迫应答,也参与响应苹果褐斑病病菌侵染。MhYTP1和MhYTP2过表达能够提高植物对水涝(氧化胁迫)、低温和低营养胁迫的抗性,也能通过影响JA和SA信号途径降低苹果对褐斑病的抗性;但转基因拟南芥和苹果在高盐和高温胁迫下的表型存在差异,因此,MhYTP1和MhYTP2在植物抗高盐和高温方面的功能尚不能明确。3.MhYTP1和MhYTP2在叶片衰老和果实成熟过程中的作用。大部分苹果YTP基因家族成员能够响应自然衰老,MhYTP1和MhYTP2的表达量随着自然衰老逐渐升高,衰老后期,表达量随着自然衰老逐渐降低。MhYTP1或MhYTP2过表达能够促进拟南芥离体叶片衰老;在苹果GL-3中过表达,也能促进苹果叶片离体衰老和自然衰老。果实发育过程也是果实不断衰老的过程,MhYTP1或MhYTP2过表达不仅能够促进叶片衰老,还能够促进果实成熟。MhYTP1或MhYTP2转基因番茄果实成熟所需时间少于野生型,同一生长时期过表达植株果实果皮类胡萝卜素含量、果实中类胡萝卜素合成基因、乙烯合成基因和乙烯响应基因表达量均高于野生型番茄。进一步探究了MhYTP1和MhYTP2参与叶片衰老和果实成熟过程的分子机制。通过酵母双杂交试验,发现MhYTP2能够与MhARD4互作。ARD4参与甲硫氨酸循环,甲硫氨酸循环与乙烯合成相关。因此,MhYTP1和MhYTP2可能影响乙烯生物合成,从而促进叶片衰老,缩短果实成熟所需时间。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S661.1

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