本文選題：蘋果 + MdEIL1�。� 參考：《山東農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：在擬南芥中,EIN3/EILs轉(zhuǎn)錄因子是一類典型的轉(zhuǎn)錄因子,通過結(jié)合到乙烯信號(hào)傳導(dǎo)途徑的下游基因的啟動(dòng)子上,調(diào)控下游基因的轉(zhuǎn)錄,完成乙烯信號(hào)響應(yīng)。同時(shí),它也可以在轉(zhuǎn)錄水平調(diào)控其他途徑基因的啟動(dòng)子,從而調(diào)控植物生長(zhǎng)和發(fā)育。本研究中,以蘋果‘嘎拉’(Malus×domestic‘Royal Gala’)為試材,分離了乙烯信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的MdEIL1基因(基因序列號(hào):MDP0000423881)。序列分析表明,該基因包含1 980 bp完整的開放閱讀框,編碼658個(gè)氨基酸的蛋白。進(jìn)化樹分析表明MdEIL1屬于EIN3/EIL轉(zhuǎn)錄因子家族蛋白,與白梨(XP_009341360.1)、枇杷(ACM89299.1)、碧桃(ABK35086.1)、梅(XP_008231880.1)、川桑(XP_010106128.1)、葡萄(XP_002276380.1)的同源性較高。接著,我們利用生物信息學(xué)分析發(fā)現(xiàn)在蘋果中,共包括12個(gè)MdEILs,根據(jù)它們處于染色體上的位置和基因特點(diǎn),分別命名為MdEIL1、MdEIL02、MdEIL03、MdEIL04、MdEIL05、MdEIL06、MdEIL07、MdEIL08、MdEIL09、MdEIL10、Md EIL11、MdEIL12。基因結(jié)構(gòu)分析發(fā)現(xiàn)基因家族成員之間外顯子的數(shù)量不同,MdEIL1、MdEIL02、MdEIL03、MdEIL06、MdEIL09、MdEIL11、MdEIL12只含有1個(gè)外顯子,MdEIL104、MdEIL05、MdEIL07、MdEIL08、MdEIL10含有2個(gè)外顯子。MdEILs的同源性分析表明,EILs廣泛存在高等植物中,單子葉植物、雙子葉植物、苔蘚植物和蕨類植物中都有EILs基因的存在,但是綠藻中不存在該類基因。qRT-PCR分析發(fā)現(xiàn),MdEILs在蘋果的根、莖、葉、花和果實(shí)中有不同程度的表達(dá),在葉片中的表達(dá)量一般高于其他組織。說明MdEILs的表達(dá)模式呈組成性表達(dá),且具有一定的組織差異性。qRT-PCR分析還發(fā)現(xiàn),在葉片不同的發(fā)育時(shí)期,MdEILs的表達(dá)量呈規(guī)律性變化。暗示MdEILs可能參與葉片的衰老過程。MdEIL1過表達(dá)的轉(zhuǎn)基因愈傷中,衰老相關(guān)基因(SAGs)的表達(dá)量升高。擬南芥中異位表達(dá)MdEIL1,葉片表現(xiàn)為早衰的表型。進(jìn)一步研究發(fā)現(xiàn),MdEIL1能夠直接結(jié)合到miR164、ORE1、NYC1、NYE1和PAO基因啟動(dòng)子,促進(jìn)擬南芥葉片提早衰老。EIN3/EILs的作為植物中一類轉(zhuǎn)錄因子,不僅可以綁定到不同的下游基因啟動(dòng)子上調(diào)控基因的表達(dá),而且可以通過與不同植物蛋白互作,調(diào)控自身或其他蛋白的功能,進(jìn)而調(diào)控植物的生長(zhǎng)發(fā)育和逆境響應(yīng)。實(shí)驗(yàn)室以MdBT2為誘餌進(jìn)行酵母雙雜篩庫發(fā)現(xiàn)MdEIL1是MdBT2的一個(gè)互作蛋白。我們進(jìn)一步通過酵母雙雜交、pull-down和Co-IP的實(shí)驗(yàn)證明了MdEIL1和MdBT1、MdBT2的蛋白互作,并且MdBT1/2的TAZ結(jié)構(gòu)域和MdEIL1的C末端區(qū)域是兩者互作所必需的。利用體外降解實(shí)驗(yàn),驗(yàn)證了MdBT2負(fù)調(diào)控MdEIL1蛋白積累水平,加入26S蛋白酶體抑制劑MG132后,MdBT2負(fù)調(diào)控MdEIL1蛋白積累的能力減弱。利用瞬時(shí)表達(dá)系統(tǒng),驗(yàn)證了Md EIL1蛋白能夠發(fā)生泛素化,并且這個(gè)過程是受MdBT2所調(diào)控的。另外,病毒載體處理35S::MdEIL1-HA愈傷,愈傷中衰老相關(guān)基因的表達(dá)量下降,暗示MdBT2可以通過MdEIL1抑制葉片衰老。
[Abstract]:In Arabidopsis thaliana (Arabidopsis thaliana), EIN3 / Eils transcription factor is a typical transcriptional factor. By binding to the promoter of downstream gene in ethylene signaling pathway, the transcription of downstream gene is regulated to complete the ethylene signal response. At the same time, it can regulate the promoter of other pathway genes at the transcriptional level, thus regulating plant growth and development. In this study, the MdEIL1 gene (sequence number: MDP00423881) related to ethylene signal transduction was isolated from apple 'Malus 脳 domestic'Royal Gala'. Sequence analysis showed that the gene contained a complete open reading frame of 1 980 BP, encoding 658 amino acids. Phylogenetic tree analysis showed that MdEIL1 belonged to the EIN3/EIL transcription factor family protein, and had a higher homology with XP201, ACM89299.1, ABK35086.1, XP008231880.1, XP0106128.1, XP002276380.1) of the white pear, ACM89299.1, ABK35086.1, XP0106128.1, XP002276380.1 of the white pear, ACM89299.1, ABK35086.1, XP00823880.1, XP002276380.1, XP0106128.1, XP002276380.1, respectively. Then, using bioinformatics analysis, we found that there are 12 MdEILs in apple. According to their position on chromosomes and genetic characteristics, we named MdEIL1 MdEIL02MdEIL03 MdEIL04 MdEIL05MdEIL07MdEIL08MdEIL09 MdEIL10MIDEIL12. Gene structure analysis showed that the number of exons of MdEIL1 and MdEIL02MdEIL03MdEIL03MdEIL06MdEIL09 MdEIL12 contained only one exon MdEIL104MdEIL05MdEIL08MdEIL10 containing 2 exons. MdEILs were widely distributed in higher plants, monocotyledonous plants, dicotyledonous plants, Monocotyledonous plants, dicotyledonous plants, EILs gene was found in bryophytes and pteridophytes, but there was no such gene in green algae. QRT-PCR analysis showed that MdEILs were expressed in different degrees in apple roots, stems, leaves, flowers and fruits. The expression level in leaves was generally higher than that in other tissues. The results showed that the expression pattern of MdEILs was constitutively expressed, and the expression level of MdEILs was regularly changed at different developmental stages of leaves. It was suggested that MdEILs might be involved in the senescence process of leaves. The expression of senescence related gene (sags) was increased in transgenic calli with overexpression of MdEIL1. MdEIL 1 was expressed ectopic in Arabidopsis thaliana. It was further found that MdEIL1 could directly bind to the promoter of the NYE1 and PAO genes of miR164ORE1 / NYC1and promote the early senescence of Arabidopsis thaliana leaves. EIN3 / Eils was a kind of transcription factor in plants, which could not only bind to the regulation of gene expression on different downstream gene promoters, but also promote the early senescence of Arabidopsis leaves. Moreover, the function of self or other proteins can be regulated by interaction with different plant proteins, and then the growth and development of plants and the response to adversity can be regulated. The yeast double sieve library using MdBT2 as bait found that MdEIL1 is an interaction protein of MdBT2. We further demonstrated the interaction between MdEIL1 and MdBT1MdBT2 protein by yeast two-hybrid pull-down and Co-IP experiments, and the TAZ domain of MdBT1/2 and the C-terminal region of MdEIL1 were necessary to interact with each other. In vitro degradation experiments demonstrated that MdBT2 negatively regulated the accumulation of MdEIL1 protein, and the ability of MdBT2 to negatively regulate the accumulation of MdEIL1 protein was decreased after adding 26s proteasome inhibitor MG132. The transient expression system was used to verify that Md EIL1 protein was ubiquitin, and the process was regulated by MdBT2. In addition, when 35S::MdEIL1-HA callus was treated with virus vector, the expression of senescence related genes in callus decreased, suggesting that MdBT2 could inhibit leaf senescence through MdEIL1.
【學(xué)位授予單位】：山東農(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)】：S661.1

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