苹果MdBTs与MdEIL1互作调控叶片衰老的研究

发布时间：2018-05-30 03:45

本文选题：苹果 + MdEIL1　；参考：《山东农业大学》2016年博士论文

【摘要】：在拟南芥中,EIN3/EILs转录因子是一类典型的转录因子,通过结合到乙烯信号传导途径的下游基因的启动子上,调控下游基因的转录,完成乙烯信号响应。同时,它也可以在转录水平调控其他途径基因的启动子,从而调控植物生长和发育。本研究中,以苹果‘嘎拉’(Malus×domestic‘Royal Gala’)为试材,分离了乙烯信号转导相关的MdEIL1基因(基因序列号:MDP0000423881)。序列分析表明,该基因包含1 980 bp完整的开放阅读框,编码658个氨基酸的蛋白。进化树分析表明MdEIL1属于EIN3/EIL转录因子家族蛋白,与白梨(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)的同源性较高。接着,我们利用生物信息学分析发现在苹果中,共包括12个MdEILs,根据它们处于染色体上的位置和基因特点,分别命名为MdEIL1、MdEIL02、MdEIL03、MdEIL04、MdEIL05、MdEIL06、MdEIL07、MdEIL08、MdEIL09、MdEIL10、Md EIL11、MdEIL12。基因结构分析发现基因家族成员之间外显子的数量不同,MdEIL1、MdEIL02、MdEIL03、MdEIL06、MdEIL09、MdEIL11、MdEIL12只含有1个外显子,MdEIL104、MdEIL05、MdEIL07、MdEIL08、MdEIL10含有2个外显子。MdEILs的同源性分析表明,EILs广泛存在高等植物中,单子叶植物、双子叶植物、苔藓植物和蕨类植物中都有EILs基因的存在,但是绿藻中不存在该类基因。qRT-PCR分析发现,MdEILs在苹果的根、茎、叶、花和果实中有不同程度的表达,在叶片中的表达量一般高于其他组织。说明MdEILs的表达模式呈组成性表达,且具有一定的组织差异性。qRT-PCR分析还发现,在叶片不同的发育时期,MdEILs的表达量呈规律性变化。暗示MdEILs可能参与叶片的衰老过程。MdEIL1过表达的转基因愈伤中,衰老相关基因(SAGs)的表达量升高。拟南芥中异位表达MdEIL1,叶片表现为早衰的表型。进一步研究发现,MdEIL1能够直接结合到miR164、ORE1、NYC1、NYE1和PAO基因启动子,促进拟南芥叶片提早衰老。EIN3/EILs的作为植物中一类转录因子,不仅可以绑定到不同的下游基因启动子上调控基因的表达,而且可以通过与不同植物蛋白互作,调控自身或其他蛋白的功能,进而调控植物的生长发育和逆境响应。实验室以MdBT2为诱饵进行酵母双杂筛库发现MdEIL1是MdBT2的一个互作蛋白。我们进一步通过酵母双杂交、pull-down和Co-IP的实验证明了MdEIL1和MdBT1、MdBT2的蛋白互作,并且MdBT1/2的TAZ结构域和MdEIL1的C末端区域是两者互作所必需的。利用体外降解实验,验证了MdBT2负调控MdEIL1蛋白积累水平,加入26S蛋白酶体抑制剂MG132后,MdBT2负调控MdEIL1蛋白积累的能力减弱。利用瞬时表达系统,验证了Md EIL1蛋白能够发生泛素化,并且这个过程是受MdBT2所调控的。另外,病毒载体处理35S::MdEIL1-HA愈伤,愈伤中衰老相关基因的表达量下降,暗示MdBT2可以通过MdEIL1抑制叶片衰老。
[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.
【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S661.1

【参考文献】