番茄SUMO E3连接酶SlSIZ1的功能分析

发布时间：2018-03-17 08:41

本文选题：SUMO化　切入点：SlSIZ1　出处：《山东农业大学》2017年博士论文　论文类型：学位论文

【摘要】：植物在生长发育过程中会遭受多种非生物胁迫,例如强光、干旱、极端温度等,对植物的正常生理活动和生长发育产生显著的影响。翻译后修饰是一种重要调节机制,SUMO化修饰是近些年来发现的一种较新的翻译后修饰。SUMO蛋白利用E1(激活酶)、E2(结合酶)、E3(连接酶)对底物蛋白进行修饰。SUMO化修饰可以调节植物对高温、低温、干旱、盐等非生物胁迫的响应,也可以调节植物的生长发育、信号转导及营养代谢等。SIZ1是一种SUMO E3连接酶,主要作用是介导SUMO蛋白和底物蛋白的特异性结合,大多数的SUMO化都需要SUMO E3连接酶的介导。因此很多学者都通过对SIZ1的研究来探索植物体内SUMO化的功能。SIZ1可以调节植物对高温、低温、干旱、过量铜离子等非生物胁迫的响应。番茄是一种起源于热带的重要蔬菜作物,但是其高温抗性并不强,而且高温常常伴随着干旱,对番茄造成严重伤害,导致产量降低或者死亡。SIZ1可以通过介导SUMO化调节植物对高温和干旱的响应,在番茄中尚无对SIZ1的研究。因此,探讨SlSIZ1在调节番茄响应高温和干旱胁迫中的作用具有重要的理论和现实意义。我们从番茄中克隆得到SlSIZ1基因。以拟南芥、烟草和番茄为材料,研究SlSIZ1是否具有SUMO E3连接酶的特性,探讨其在调节植物干旱和高温抗性中的功能,主要结果如下:(1)对番茄进行低温(4°C)、高温(42°C)、干旱(聚乙二醇,PEG)和氧化胁迫(MV,甲基紫精)处理,检测SUMO化情况。发现经过这几种处理后番茄体内的SUMO化程度都升高,在低温、高温和干旱胁迫处理下SUMO化程度增加较为明显,而在氧化胁迫处理下,番茄体内的SUMO化水平变化不明显。(2)从番茄叶片中克隆得到SlSIZ1基因(GenBank注册号:KP323389),该基因开放阅读框长2658bp,编码886个氨基酸,预测蛋白分子量约为97.35kDa,等电点预测为4.92,生物信息学分析发现其位于番茄11号染色体上。(3)qRT-PCR分析发现,SlSIZ1基因在番茄的根、茎、叶、花中均有表达,在叶片中表达量最高。同时该基因受高温、低温、干旱、盐等胁迫的诱导表达,但变化不明显。说明SlSIZ1主要是通过介导SUMO化修饰,而不仅仅是通过其表达量的变化来调节植物对逆境的响应。亚细胞定位分析显示SlSIZ1定位于细胞核中。(4)对SlSIZ1进行序列分析发现其具有SIZ1蛋白的五个保守结构域,并且将SlSIZ1转化拟南芥siz1-2突变体,可以部分恢复突变体植株矮小、叶片狭窄、对ABA敏感以及SUMO化积累降低等表型,可以调节和介导植物体内的SUMO化,说明SlSIZ1具有SUMO E3连接酶的活性。(5)干旱胁迫下,过表达SlSIZ1可以降低烟草中ROS的积累,增加渗透调节物质(游离脯氨酸)的含量,减少生物膜损伤(MDA)、叶片坏死和失绿,上调逆境相关基因表达,增加体内SUMO化积累,维持较高的种子萌发率和生长情况,提高转基因烟草对干旱胁迫的抗性。(6)高温胁迫下,过表达SlSIZ1可以减少番茄体内ROS的积累,增加抗氧化酶活性,减少生物膜损伤(MDA)和细胞死亡,增加体内SUMO化积累,过表达番茄中具有较高的叶绿素含量和小苗鲜重。同时SlSIZ1可以与SlHsfA1互作,介导SlHsfA1发生SUMO化修饰,促使SlHsfA1在高温胁迫下具有更高的转录活性,上调下游高温相关基因表达(SlHsfA2、SlHsp70、SlHsp70-3和SlHsp90)。高温胁迫时,过表达SlSIZ1番茄中Hsp70基因上调表达,经过放线菌酮处理后Hsp70蛋白降解程度较低,因此过表达番茄植株中含有更多的Hsp70,增加其高温抗性。由于发现SlSIZ1和SlHsp70间可能存在较弱的蛋白互作,我们推测SlSIZ1介导的SUMO化可以修饰SlHsp70,从而维持其稳定。因此,过表达SlSIZ1可以提高番茄的高温抗性。
[Abstract]:Plants will suffer various abiotic stresses, such as high light, drought in the growth process, extreme temperatures, have a significant impact on the growth and physiological activity of plant development. Post translational modification is an important regulatory mechanism, modification of SUMO is discovered in recent years a new post-translational modification of.SUMO protein the use of E1, E2 (activating enzyme) (with E3 (enzyme), ligase) modified.SUMO modification can adjust plant of high temperature, low temperature, drought response to substrate protein, salt and other abiotic stresses, can also regulate plant growth and development, signal transduction and metabolism of.SIZ1 is a SUMO E3 ligase, the main role is to mediate the specific binding between SUMO protein and substrate protein, most of the SUMO need SUMO E3 ligase mediated. Therefore many scholars to explore the function of.SIZ1 in plant SUMO through the research of SIZ1 Can adjust the plants to high temperature, low temperature, drought, excessive copper ions in response to abiotic stress. Tomato is an important vegetable crop in a tropical in origin, but its high temperature resistance is not strong, often accompanied by high temperature and drought, causing serious damage to tomato, resulting in reduced death or.SIZ1 can regulate plant response the high temperature and drought mediated by SUMO in tomato is the research of SIZ1. Therefore, to investigate the regulation of SlSIZ1 in Tomato in response to high temperature and drought stress in effect has important theoretical and practical significance. We cloned the SlSIZ1 gene from tomato. In Arabidopsis, tobacco and tomato, whether the characteristics of SlSIZ1 with SUMO E3 ligase, on the regulation of plant drought and high temperature resistance function, the main results are as follows: (1) low temperature on Tomato (4 ~ C), high temperature (42 degrees C), drought (PEG, P EG (MV) and oxidative stress, methyl viologen), SUMO detection. After this treatment after SUMO found some degree of tomato were increased in low temperature, high temperature and drought stress degree of SUMO was increased markedly, and in oxidative stress, changes of SUMO level in the tomato obviously. (2) cloned from tomato SlSIZ1 gene (GenBank accession number: KP323389), the open reading frame of the gene 2658bp, encoding 886 amino acids. The predicted protein molecular weight of 97.35kDa and isoelectric point forecast of 4.92, bioinformatics analysis showed that it is located on chromosome 11 (tomato. 3) qRT-PCR analysis showed that SlSIZ1 gene in tomato roots, stems, leaves, flowers have expression, the highest expression level in leaves. At the same time the genes affected by high temperature, low temperature, drought, salt stress induced expression, but did not change significantly. The results showed that SlSIZ1 was mainly mediated through SU MO modification, and not just through its expression to modulate the response to abiotic stress. Subcellular localization showed that SlSIZ1 localized in the nucleus. (4) the sequence analysis showed that the SIZ1 protein has five conserved domains of SlSIZ1 and SlSIZ1 will be transformed into Arabidopsis siz1-2 mutant, it can restore the mutant plant is short, narrow leaves, sensitive to ABA and SUMO accumulation decreased phenotype can be regulated and mediated by plant SUMO, SlSIZ1 with SUMO E3 ligase activity. (5) under drought stress, overexpression of SlSIZ1 can reduce the accumulation of ROS in tobacco, increase osmolytes (proline) the content of reducing membrane damage (MDA), leaf necrosis and chlorosis, upregulation of the expression of stress related genes in vivo, increased SUMO accumulation, maintain the germination rate and growth of higher seed increased in transgenic tobacco Resistance to drought stress. (6) under high temperature stress, overexpression of SlSIZ1 can reduce the accumulation of ROS in tomato, increase the activity of antioxidant enzymes and reduce the membrane damage and cell death (MDA), an increase in SUMO accumulation, chlorophyll content and seedling with higher expression in tomato fresh weight. At the same time SlSIZ1 can cross as with SlHsfA1, SlHsfA1 mediated by SUMO modification occurred, prompting SlHsfA1 has higher transcriptional activity under high temperature stress, upregulation of the expression of downstream genes related to high temperature (SlHsfA2, SlHsp70, SlHsp70-3 and SlHsp90). The high temperature stress, over expression of Hsp70 SlSIZ1 in tomato gene expression after cycloheximide treatment after Hsp70 protein degradation degree is low, so the expression of tomato plants contained more Hsp70, increase its high temperature resistance. Due to the discovery of SlSIZ1 and SlHsp70 may have weak protein interaction, we detected SlSIZ1 mediated by SUMO SlHsp70 can be modified to maintain its stability. Therefore, overexpression of SlSIZ1 can improve the high temperature resistance of tomato.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2;S641.2

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