本文選題：SUMO化　切入點(diǎn)：SlSIZ1　出處：《山東農(nóng)業(yè)大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：植物在生長(zhǎng)發(fā)育過(guò)程中會(huì)遭受多種非生物脅迫,例如強(qiáng)光、干旱、極端溫度等,對(duì)植物的正常生理活動(dòng)和生長(zhǎng)發(fā)育產(chǎn)生顯著的影響。翻譯后修飾是一種重要調(diào)節(jié)機(jī)制,SUMO化修飾是近些年來(lái)發(fā)現(xiàn)的一種較新的翻譯后修飾。SUMO蛋白利用E1(激活酶)、E2(結(jié)合酶)、E3(連接酶)對(duì)底物蛋白進(jìn)行修飾。SUMO化修飾可以調(diào)節(jié)植物對(duì)高溫、低溫、干旱、鹽等非生物脅迫的響應(yīng),也可以調(diào)節(jié)植物的生長(zhǎng)發(fā)育、信號(hào)轉(zhuǎn)導(dǎo)及營(yíng)養(yǎng)代謝等。SIZ1是一種SUMO E3連接酶,主要作用是介導(dǎo)SUMO蛋白和底物蛋白的特異性結(jié)合,大多數(shù)的SUMO化都需要SUMO E3連接酶的介導(dǎo)。因此很多學(xué)者都通過(guò)對(duì)SIZ1的研究來(lái)探索植物體內(nèi)SUMO化的功能。SIZ1可以調(diào)節(jié)植物對(duì)高溫、低溫、干旱、過(guò)量銅離子等非生物脅迫的響應(yīng)。番茄是一種起源于熱帶的重要蔬菜作物,但是其高溫抗性并不強(qiáng),而且高溫常常伴隨著干旱,對(duì)番茄造成嚴(yán)重傷害,導(dǎo)致產(chǎn)量降低或者死亡。SIZ1可以通過(guò)介導(dǎo)SUMO化調(diào)節(jié)植物對(duì)高溫和干旱的響應(yīng),在番茄中尚無(wú)對(duì)SIZ1的研究。因此,探討SlSIZ1在調(diào)節(jié)番茄響應(yīng)高溫和干旱脅迫中的作用具有重要的理論和現(xiàn)實(shí)意義。我們從番茄中克隆得到SlSIZ1基因。以擬南芥、煙草和番茄為材料,研究SlSIZ1是否具有SUMO E3連接酶的特性,探討其在調(diào)節(jié)植物干旱和高溫抗性中的功能,主要結(jié)果如下:(1)對(duì)番茄進(jìn)行低溫(4°C)、高溫(42°C)、干旱(聚乙二醇,PEG)和氧化脅迫(MV,甲基紫精)處理,檢測(cè)SUMO化情況。發(fā)現(xiàn)經(jīng)過(guò)這幾種處理后番茄體內(nèi)的SUMO化程度都升高,在低溫、高溫和干旱脅迫處理下SUMO化程度增加較為明顯,而在氧化脅迫處理下,番茄體內(nèi)的SUMO化水平變化不明顯。(2)從番茄葉片中克隆得到SlSIZ1基因(GenBank注冊(cè)號(hào):KP323389),該基因開(kāi)放閱讀框長(zhǎng)2658bp,編碼886個(gè)氨基酸,預(yù)測(cè)蛋白分子量約為97.35kDa,等電點(diǎn)預(yù)測(cè)為4.92,生物信息學(xué)分析發(fā)現(xiàn)其位于番茄11號(hào)染色體上。(3)qRT-PCR分析發(fā)現(xiàn),SlSIZ1基因在番茄的根、莖、葉、花中均有表達(dá),在葉片中表達(dá)量最高。同時(shí)該基因受高溫、低溫、干旱、鹽等脅迫的誘導(dǎo)表達(dá),但變化不明顯。說(shuō)明SlSIZ1主要是通過(guò)介導(dǎo)SUMO化修飾,而不僅僅是通過(guò)其表達(dá)量的變化來(lái)調(diào)節(jié)植物對(duì)逆境的響應(yīng)。亞細(xì)胞定位分析顯示SlSIZ1定位于細(xì)胞核中。(4)對(duì)SlSIZ1進(jìn)行序列分析發(fā)現(xiàn)其具有SIZ1蛋白的五個(gè)保守結(jié)構(gòu)域,并且將SlSIZ1轉(zhuǎn)化擬南芥siz1-2突變體,可以部分恢復(fù)突變體植株矮小、葉片狹窄、對(duì)ABA敏感以及SUMO化積累降低等表型,可以調(diào)節(jié)和介導(dǎo)植物體內(nèi)的SUMO化,說(shuō)明SlSIZ1具有SUMO E3連接酶的活性。(5)干旱脅迫下,過(guò)表達(dá)SlSIZ1可以降低煙草中ROS的積累,增加滲透調(diào)節(jié)物質(zhì)(游離脯氨酸)的含量,減少生物膜損傷(MDA)、葉片壞死和失綠,上調(diào)逆境相關(guān)基因表達(dá),增加體內(nèi)SUMO化積累,維持較高的種子萌發(fā)率和生長(zhǎng)情況,提高轉(zhuǎn)基因煙草對(duì)干旱脅迫的抗性。(6)高溫脅迫下,過(guò)表達(dá)SlSIZ1可以減少番茄體內(nèi)ROS的積累,增加抗氧化酶活性,減少生物膜損傷(MDA)和細(xì)胞死亡,增加體內(nèi)SUMO化積累,過(guò)表達(dá)番茄中具有較高的葉綠素含量和小苗鮮重。同時(shí)SlSIZ1可以與SlHsfA1互作,介導(dǎo)SlHsfA1發(fā)生SUMO化修飾,促使SlHsfA1在高溫脅迫下具有更高的轉(zhuǎn)錄活性,上調(diào)下游高溫相關(guān)基因表達(dá)(SlHsfA2、SlHsp70、SlHsp70-3和SlHsp90)。高溫脅迫時(shí),過(guò)表達(dá)SlSIZ1番茄中Hsp70基因上調(diào)表達(dá),經(jīng)過(guò)放線菌酮處理后Hsp70蛋白降解程度較低,因此過(guò)表達(dá)番茄植株中含有更多的Hsp70,增加其高溫抗性。由于發(fā)現(xiàn)SlSIZ1和SlHsp70間可能存在較弱的蛋白互作,我們推測(cè)SlSIZ1介導(dǎo)的SUMO化可以修飾SlHsp70,從而維持其穩(wěn)定。因此,過(guò)表達(dá)SlSIZ1可以提高番茄的高溫抗性。
[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.

【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】：2017
【分類(lèi)號(hào)】：Q943.2;S641.2

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