本文選題：小麥　切入點(diǎn)：LEA蛋白　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：干旱,高鹽和低溫等非生物脅迫是影響作物生長(zhǎng)和產(chǎn)量的重要因素。當(dāng)作物受到非生物脅迫時(shí),為了減輕其對(duì)作物的傷害會(huì)誘導(dǎo)一些蛋白表達(dá)。植物胚胎發(fā)育晚期豐富蛋白(Late embryogenesis abundant protein,LEA)是一類與逆境脅迫相關(guān)的蛋白。LEA蛋白可分為7族,其中第3族LEA(LEA3)蛋白含有11個(gè)氨基酸串聯(lián)重復(fù)序列。LEA3蛋白可以形成α-螺旋結(jié)構(gòu),在非生物脅迫中保護(hù)生物大分子,減輕對(duì)植物造成的傷害。然而LEA3蛋白的保護(hù)功能及其作用機(jī)制仍不清晰。本研究從小麥中克隆了1條新的LEA3族基因,通過(guò)亞細(xì)胞定位,體外保護(hù)乳酸脫氫酶活性、轉(zhuǎn)基因在擬南芥中過(guò)表達(dá)驗(yàn)證以及對(duì)非生物脅迫的響應(yīng)等評(píng)價(jià)該基因的耐旱功能;同時(shí)對(duì)2種不同耐旱小麥品種的4個(gè)生長(zhǎng)時(shí)期(苗期、分蘗期、拔節(jié)期和開花期)進(jìn)行不同程度的干旱脅迫和復(fù)水處理,從轉(zhuǎn)錄水平和翻譯水平上研究該基因在小麥全生育期的變化規(guī)律以及對(duì)干旱的響應(yīng),主要研究結(jié)果如下:1.通過(guò)電子克隆和RT-PCR方法,從小麥陜合6號(hào)中克隆1條新的LEA3家族基因(命名為TaDlea3基因)和4條新的LEA3基因同源性較高TaWSI18基因。生物信息學(xué)分析表明,TaDlea3蛋白和4個(gè)TaWSI18蛋白均具有親水性,定位于細(xì)胞質(zhì)中,無(wú)跨膜結(jié)構(gòu)域。小麥TaDlea3和TaWSI18蛋白與其他物種的LEA3蛋白有高度同源性。通過(guò)原核表達(dá)TaDlea3蛋白,純化該蛋白并制備了特異性較高的多克隆抗體。2.通過(guò)農(nóng)桿菌注射法將亞細(xì)胞定位載體注射到煙草葉片,并制備原生質(zhì)體,結(jié)果表明,TaDlea3蛋白定位于細(xì)胞質(zhì)中。在高溫和低溫脅迫下,TaDlea3蛋白對(duì)LDH均有一定的保護(hù)作用。不同逆境脅迫處理小麥陜合6號(hào)和鄭引1號(hào),結(jié)果表明2種小麥中TaDlea3基因?qū)Σ煌哪婢趁{迫均有響應(yīng)。陜合6號(hào)TaDlea3基因?qū)Ω珊得{迫的響應(yīng)較顯著,而鄭引1號(hào)TaDlea3基因?qū)BA脅迫的響應(yīng)較顯著。3.通過(guò)農(nóng)桿菌介導(dǎo)法,獲得轉(zhuǎn)基因擬南芥植株。正常生長(zhǎng)條件下,轉(zhuǎn)基因和野生型的擬南芥在表型和生理指標(biāo)上無(wú)差異,干旱脅迫后,轉(zhuǎn)TaDlea3基因擬南芥的葉片保持正常,而野生型的擬南芥葉片有明顯的萎焉現(xiàn)象。多種生理指標(biāo)的變化結(jié)果表明,在干旱脅迫下,轉(zhuǎn)基因擬南芥中丙二醛含量和相對(duì)電導(dǎo)率均低于野生型,而轉(zhuǎn)基因擬南芥中的脯氨酸和三個(gè)氧化酶的含量均顯著高于野生型,說(shuō)明TaDlea3基因的過(guò)表達(dá)提高了轉(zhuǎn)基因的擬南芥抗旱能力。4.對(duì)2種耐旱型不同品種的陜合6號(hào)(干旱耐受型)和鄭引1號(hào)(干旱敏感型)小麥,分別在4個(gè)生長(zhǎng)時(shí)期進(jìn)行干旱脅迫和復(fù)水處理,實(shí)時(shí)定量RT-PCR和Western blot分析TaDlea3基因在轉(zhuǎn)錄和翻譯水平上的表達(dá)。結(jié)果表明,4個(gè)不同的生長(zhǎng)時(shí)期的TaDlea3基因在轉(zhuǎn)錄和翻譯水平上的變化趨勢(shì)一致,干旱程度越嚴(yán)重,該基因和蛋白的表達(dá)量越高,復(fù)水后該基因和蛋白的表達(dá)量下降。開花期的TaDlea3基因和蛋白的表達(dá)量均低于其它3個(gè)生長(zhǎng)時(shí)期。干旱條件下,2個(gè)不同品種小麥的4個(gè)生長(zhǎng)時(shí)期的生理指標(biāo)丙二醛、相對(duì)電導(dǎo)率、脯氨酸和葉綠素含量的變化趨勢(shì)基本一致。對(duì)產(chǎn)量的分析表明苗期和分蘗期干旱對(duì)小麥的產(chǎn)量影響較輕,而拔節(jié)期和開花期干旱則嚴(yán)重影響小麥產(chǎn)量。結(jié)果表明TaDlea3基因和蛋白表達(dá)和干旱脅迫有一定的相關(guān)性。
[Abstract]:Drought, high salt and low temperature abiotic stress is an important factor affecting the growth and yield of crops. When crops under abiotic stress, in order to reduce the crop damage can induce the expression of some proteins. Late embryogenesis abundant protein (Late embryogenesis abundant protein, LEA) is a kind of stress-related protein.LEA proteins can be divided into 7 groups, including third LEA (LEA3) protein containing 11 amino acid tandem repeat.LEA3 protein can form alpha helix structure, protect biological macromolecules in abiotic stress, reduce the cause plant damage. However the protection function and mechanism of LEA3 protein is still not clear. Study on the cloning of 1 new LEA3 genes from wheat, the subcellular localization of in vitro conservation of lactic dehydrogenase activity in Arabidopsis, transgenic expression validation and response to abiotic stresses. To evaluate the drought resistant gene function; while the 4 growth period of 2 different drought resistant wheat varieties (seedling, tillering, jointing and anthesis) of different degrees of drought stress and rewatering, study the gene variation in the whole growth period of wheat and response to drought from transcription and translation level, the main results are as follows: 1. by electronic clone and RT-PCR method from wheat Shanhe No. 6 cloning 1 new LEA3 gene (named TaDlea3 gene) and 4 new LEA3 gene homology of TaWSI18 gene. Bioinformatics analysis showed that TaDlea3 protein and TaWSI18 protein 4 are hydrophilic, located in cytoplasm and no transmembrane domain. TaWSI18 protein and TaDlea3 in wheat and other species of LEA3 protein with high homology. The expression of TaDlea3 protein by prokaryotic high specificity, preparation and purification of this protein and preparation The polyclonal antibody.2. subcellularlocalization vector injected into tobacco leaves via Agrobacterium injection method and protoplast preparation, the results show that the TaDlea3 protein located in the cytoplasm. In high temperature and low temperature stress, the protective effect of TaDlea3 protein on LDH were different. Some adversity stress wheat Shanhe No. 6 and Zheng 1, the results show that 2 kinds of wheat TaDlea3 gene in different stress response. Both Shanhe No. 6, TaDlea3 gene significantly in response to drought stress, and Zhengyin 1 TaDlea3 gene in response to ABA stress significantly.3. by Agrobacterium mediated method. Transgenic Arabidopsis plants normal growth conditions. Next, transgenic and wild type Arabidopsis differences in phenotypic and physiological indexes, after drought stress, the leaves of TaDlea3 transgenic Arabidopsis remains normal and wild type Arabidopsis leaves have obvious wilting phenomenon. The changes of physiological indices showed that under drought stress, MDA content and relative conductivity of transgenic Arabidopsis were lower than the wild type, while the content of proline in transgenic Arabidopsis and three enzymes were significantly higher than that of the wild type, that over expression of TaDlea3 gene increased the transgenic Arabidopsis thaliana.4. on 2 different drought resistant type variety Shanhe No. 6 (drought resistant) and Zhengyin No. 1 (drought sensitive) wheat, respectively under drought stress and Rewatering in 4 growth periods, the expression of Western blot and real-time quantitative RT-PCR analysis of TaDlea3 gene in transcription and translation level. The results show that the consistent variation trend of TaDlea3 gene 4 different growth periods at the levels of transcription and translation of the drought is more serious, the expression level of gene and protein is higher, the expression level of gene and protein decreased after rewatering. The expression of TaDlea3 mRNA and protein in the flowering stage were lower than that of the other 3 growth period. Under drought conditions, the relative conductivity of physiological indexes of MDA, 4 growth period of wheat in 2 different varieties, the change trend of proline and chlorophyll content are basically the same. The analysis shows that the effects on Yield Yield drought seedling and tillering stage of wheat the lighter, and the drought at jointing stage and anthesis stage is seriously affected wheat yield. The results showed that the TaDlea3 gene and protein expression and drought stress had certain relevance.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S512.1;Q943.2

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1 陳娟;干旱誘導(dǎo)基因TaDLea3的功能及在小麥不同生長(zhǎng)期的時(shí)空表達(dá)規(guī)律研究[D];西北農(nóng)林科技大學(xué);2016年

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