本文選題：小麥 + SUMO蛋白酶��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：小麥?zhǔn)且环N重要的糧食作物。鹽旱等逆境嚴(yán)重影響小麥產(chǎn)量,因而鑒定耐逆相關(guān)基因,并應(yīng)用于耐逆小麥分子育種非常必要。本實驗室利用不對稱體細(xì)胞雜交技術(shù),以普通小麥濟(jì)南177(JN177)為親本,培育了小麥漸滲系耐鹽抗旱新品種山融3號(SR3)。通過轉(zhuǎn)錄組分析,鑒定了一個SR3中鹽脅迫響應(yīng)更顯著SUMO蛋白酶基因TaDSU。SUMO化是重要的可逆性蛋白質(zhì)修飾化,在生長發(fā)育和逆境響應(yīng)中發(fā)揮重要作用。SUMO化的蛋白質(zhì)可通過SUMO蛋白酶去除。但是,SUMO蛋白酶在非生物逆境響應(yīng)的作用機(jī)制還知之甚少。為了認(rèn)識SUMO蛋白酶在非生物脅迫應(yīng)答中的功能,實驗室前期構(gòu)建了 TaDSU異源表達(dá)擬南芥轉(zhuǎn)基因株系,并初步分析了 TaDSU在非生物脅迫應(yīng)答中的功能。本論文在前期研究的基礎(chǔ)上,利用TaDSU異源表達(dá)擬南芥轉(zhuǎn)基因株系,開展了一下研究:1、進(jìn)一步分析了TaDSU的耐逆功能及其生理基礎(chǔ)與野生型相比,TaDSU過表達(dá)擬南芥具有以下特點:在萌發(fā)和幼苗生長階段的耐鹽能力均明顯增強(qiáng),鹽脅迫下鈉離子含量更低、鉀離子含量和鉀鈉比更低;抗旱性提高,失水率降低;對甘露醇模擬的滲透脅迫耐受性增強(qiáng),可溶性糖和脯氨酸等滲透調(diào)節(jié)物含量增加;對外源H2O2的抗性及過氧化氫酶活性明顯提高,脅迫下葉片中H202水平降低;對ABA的敏感性降低。結(jié)果顯示,TaDSU過表達(dá)通過對離子平衡維持、滲透平衡調(diào)節(jié)、抗氧化等抗逆生理基礎(chǔ)的綜合影響,提高了廣譜性的耐逆能力。為了驗證TaDSU在小麥?zhǔn)欠裉岣邚V譜性的耐逆能力,構(gòu)建了 TaDSU轉(zhuǎn)基因小麥,共獲得了 19株T2代陽性轉(zhuǎn)基因株系。2、鑒定了TaDSU互作蛋白結(jié)合體外酶活實驗和western bloting分析,初步證實了 TaDSU具有體外和體內(nèi)酶活性。結(jié)合體外pull-down實驗、質(zhì)譜鑒定和生物信息學(xué)分析,篩選了 6個候選的TaDSU互作蛋白DIP1-DIP6,并通過酵母雙雜交初步驗證了 3個互作蛋白。TaDSU定位于細(xì)胞核,并且利用雙分子熒光互補(bǔ)實驗發(fā)現(xiàn),TaDSU和DIP2在細(xì)胞核中相互作用。鹽脅迫下,TaDSU過表達(dá)增強(qiáng)了 DIP2的富集及DIP2下游靶基因的表達(dá)。結(jié)果暗示,TaDSU通過和DIP2互作,影響DIP2的SUMO化和富集水平,調(diào)控DIP2下游基因的表達(dá)。3、初步鑒定了調(diào)控TaDSU表達(dá)的調(diào)控因子克隆了 1.1 kb的TaDSU啟動子。構(gòu)建了 TaDSU啟動子-GUS轉(zhuǎn)基因擬南芥,GUS染色分析顯示,TaDSU在不同時期的多個組織中表達(dá)。TaDSU啟動子含有DIP2結(jié)合元件,凝膠遷移實驗顯示,DIP2可以結(jié)合TaDSU啟動子的DIP2結(jié)合元件區(qū)。綜合以上結(jié)果推測,非生物脅迫誘導(dǎo)DIP2大量合成,后者促進(jìn)TaDSU表達(dá),TaDSU和DIP2結(jié)合,調(diào)控DIP2的SUMO化水平,提高DIP2下游基因表達(dá),增強(qiáng)耐逆能力。
[Abstract]:Wheat is an important food crop. Salt drought and other stresses seriously affect the yield of wheat, so it is necessary to identify the stress tolerance related genes and to apply them to the molecular breeding of stress tolerant wheat. Using asymmetrical somatic cell hybridization technique and common wheat Jinan 177 (JN177) as parents, a new salt-tolerant and drought-resistant wheat variety, Shanrong No. 3, was developed in our laboratory. By transcriptome analysis, it was identified that the SUMO protease gene TaDSU.SUMO was an important reversible protein modification in a SR3. The proteins that play an important role in growth, development and stress response can be removed by SUMO protease. However, little is known about the mechanism of Sumo protease in response to abiotic stress. In order to understand the function of SUMO protease in abiotic stress response, TaDSU heterologous expression Arabidopsis transgenic lines were constructed in early laboratory, and the function of TaDSU in abiotic stress response was preliminarily analyzed. On the basis of previous studies, TaDSU was used to express Arabidopsis transgenic lines. The stress tolerance function and physiological basis of TaDSU were further analyzed. Compared with wild type, the overexpressed Arabidopsis thaliana with TaDSU had the following characteristics: salt tolerance of Arabidopsis thaliana during germination and seedling growth was significantly enhanced. Under salt stress, the content of sodium ion was lower, the content of potassium ion and the ratio of potassium and sodium were lower, the drought resistance was increased, the rate of water loss was decreased, the tolerance to osmotic stress simulated by mannitol was enhanced, and the content of osmotic regulators such as soluble sugar and proline increased. The resistance to exogenous H2O2 and the activity of catalase increased significantly, and the level of H202 in leaves decreased and the sensitivity to ABA decreased under stress. The results showed that the over-expression of TaDSU enhanced the broad-spectrum stress tolerance through the comprehensive effects on the physiological basis of stress resistance, such as ion balance maintenance, osmotic balance regulation and oxidation resistance. In order to verify the ability of TaDSU to improve broad-spectrum stress tolerance in wheat, TaDSU transgenic wheat was constructed, 19 T2 generation positive transgenic lines were obtained, and TaDSU interaction protein binding enzyme activity and western bloting analysis in vitro were identified. It was preliminarily confirmed that TaDSU had enzyme activity in vitro and in vivo. In combination with in vitro pull-down assay, mass spectrometry and bioinformatics analysis, six candidate TaDSU interaction proteins DIP1-DIP6 were screened, and three interacting proteins. TaDSU were preliminarily confirmed to be located in the nucleus by yeast two-hybrid. Furthermore, the interaction between TaDSU and DIP2 in the nucleus was found by bimolecular fluorescence complementary experiment. Overexpression of TaDSU enhanced the enrichment of DIP2 and the expression of target genes downstream of DIP2 under salt stress. The results suggested that TaDSU affected the level of SUMO and enrichment of DIP2 by interacting with DIP2, and regulated the expression of DIP2 downstream gene. The TaDSU promoter of 1.1 kb was identified as the regulator of TaDSU expression. Gus staining analysis of TaDSU promoter Gus showed that TaDSU expressed DIP2 binding elements in several tissues at different stages. Gel migration experiments showed that DIP2 could bind to the DIP2 binding element region of TaDSU promoter. These results suggest that abiotic stress induces a large amount of DIP2 synthesis, which promotes the binding of TaDSU to DIP2, regulates the SUMO level of DIP2, enhances the downstream gene expression of DIP2 and enhances the ability of stress tolerance.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;S512.1

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