本文選題：小麥(Triticum + aestivum��； 參考：《山東農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：小麥(Triticum aestivum L.)是我國(guó)重要的糧食作物。小麥生長(zhǎng)過(guò)程中常常遭受環(huán)境脅迫的危害,進(jìn)而導(dǎo)致生長(zhǎng)抑制和產(chǎn)量降低。通過(guò)基因工程技術(shù)提高小麥的逆境適應(yīng)性是小麥新品種培育的發(fā)展方向。研究逆境相關(guān)基因功能可以為小麥基因工程育種奠定理論基礎(chǔ),儲(chǔ)備基因資源。擴(kuò)展蛋白是一種調(diào)節(jié)細(xì)胞壁松弛和細(xì)胞延伸生長(zhǎng)的細(xì)胞壁蛋白,普遍存在于植物所有的細(xì)胞、組織和器官中。1989年美國(guó)賓夕法尼亞州立大學(xué)Cosgrove教授的實(shí)驗(yàn)室研究黃瓜下胚軸細(xì)胞延伸性生長(zhǎng),并首次發(fā)現(xiàn)了擴(kuò)展蛋白(Expansin)。近年來(lái),擴(kuò)展蛋白的研究成果不斷豐富�，F(xiàn)已證明擴(kuò)展蛋白參與多種植物生長(zhǎng)發(fā)育過(guò)程及逆境適應(yīng)性響應(yīng)。但是,由于小麥基因組較大、遺傳背景復(fù)雜以及轉(zhuǎn)化效率低等問(wèn)題,小麥擴(kuò)展蛋白基因功能的研究報(bào)道較少。擴(kuò)展蛋白由多基因家族編碼,家族成員之間在基因結(jié)構(gòu)、蛋白結(jié)構(gòu)、生化特性、進(jìn)化關(guān)系等方面存在多方面的異同。其中EXPA和EXPB是兩個(gè)最大、研究最清楚的基因家族。本實(shí)驗(yàn)室先前克隆了小麥EXPB家族基因Ta EXPB23,發(fā)現(xiàn)該基因的表達(dá)對(duì)多種非生物脅迫和激素信號(hào)產(chǎn)生響應(yīng)。組成型過(guò)表達(dá)、根特異性以及脅迫誘導(dǎo)表達(dá)該基因均增強(qiáng)了轉(zhuǎn)基因煙草的抗旱性。鑒于EXPA和EXPB家族成員存在多方面的異同,本研究中選擇小麥EXPA基因Ta EXPA2(Gen Bank accession number:AAT94292.1),利用過(guò)量表達(dá)技術(shù)研究該基因在植物生長(zhǎng)發(fā)育及逆境適應(yīng)中的作用,分析其生理機(jī)制,并與EXPB家族基因Ta EXPB23的功能相比較,為解釋不同家族基因功能上的異同點(diǎn)提供參考。首先克隆了Ta EXPA2的啟動(dòng)子區(qū)域,預(yù)測(cè)分析了該啟動(dòng)子的順式作用元件。分析了該基因的時(shí)空表達(dá)、激素響應(yīng)和逆境響應(yīng)表達(dá)模式;構(gòu)建Ta EXPA2基因表達(dá)載體,轉(zhuǎn)化煙草獲得了轉(zhuǎn)基因植株;利用過(guò)表達(dá)Ta EXPA2的轉(zhuǎn)基因煙草,研究了該基因在植物生長(zhǎng)發(fā)育調(diào)節(jié)、耐旱、耐鹽中的作用,并進(jìn)一步探討了其生理機(jī)制;最后,為研究深入探討過(guò)表達(dá)擴(kuò)展蛋白提高轉(zhuǎn)基因植株抗氧化能力的分子機(jī)制,利用擬南芥同源基因突變體atexpa2,并將Ta EXPA2在突變體atexpa2中進(jìn)行過(guò)表達(dá)回補(bǔ)實(shí)驗(yàn),初步分析了擴(kuò)展蛋白與細(xì)胞壁過(guò)氧化物酶之間的關(guān)系。研究主要結(jié)果與結(jié)論如下:(1)利用Tail-PCR克隆技術(shù)獲得了Ta EXPA2基因啟動(dòng)子區(qū)域1073bp的片段(Gen Bank ID:KP729264),用Plant CARE和PLACE功能預(yù)測(cè)軟件對(duì)其進(jìn)行了分析,結(jié)果表明該啟動(dòng)子區(qū)域含有多種光響應(yīng)元件、組織特異表達(dá)元件、多種激素響應(yīng)元件和逆境(干旱、ABA和高溫)響應(yīng)元件。(2)將Ta EXPA2::GFP融合蛋白在洋蔥表皮中瞬時(shí)表達(dá),證實(shí)Ta EXPA2蛋白定位于細(xì)胞壁。(3)q RT-PCR檢測(cè)了小麥中Ta EXPA2基因表達(dá)的組織特特異性以及對(duì)外源激素和非生物脅迫的響應(yīng)模式。結(jié)果表明,Ta EXPA2在小麥根、莖、葉中均有表達(dá),葉片中的表達(dá)量最高;Ta EXPA2基因表達(dá)對(duì)多種逆境脅迫和外源激素處理產(chǎn)生響應(yīng),干旱脅迫誘導(dǎo)基因表達(dá)水平上調(diào)。(4)構(gòu)建Ta EXPA2轉(zhuǎn)化載體,轉(zhuǎn)化煙草獲得了不同水平過(guò)表達(dá)Ta EXPA2的轉(zhuǎn)基因煙草株系。對(duì)轉(zhuǎn)基因煙草的生長(zhǎng)發(fā)育表型進(jìn)行觀察記錄,結(jié)果發(fā)現(xiàn)過(guò)表達(dá)Ta EXPA2提高了轉(zhuǎn)基因煙草的種子產(chǎn)量和果莢數(shù),但是沒(méi)有改變轉(zhuǎn)基因煙草營(yíng)養(yǎng)生長(zhǎng)表型及種子的大小。(5)對(duì)轉(zhuǎn)基因煙草進(jìn)行干旱脅迫處理,觀察了萌發(fā)率、幼苗生長(zhǎng)和成苗生長(zhǎng),結(jié)果發(fā)現(xiàn)過(guò)表達(dá)Ta EXPA2提高了轉(zhuǎn)基因煙草的抗旱性。從水分保持能力、光合作用特性、抗氧化特性等方面研究了轉(zhuǎn)基因煙草抗旱性提高的生理機(jī)制,發(fā)現(xiàn)轉(zhuǎn)基因煙草抗旱性的提高與根系發(fā)達(dá)、水分狀況維持能力強(qiáng)以及抗氧化能力較強(qiáng)有關(guān)�？寡趸芰Φ奶岣吲c相關(guān)基因表達(dá)上調(diào)有關(guān)。(6)對(duì)轉(zhuǎn)基因煙草進(jìn)行鹽脅迫處理,觀察了鹽脅迫種子萌發(fā)、幼苗生長(zhǎng)和成苗生長(zhǎng),結(jié)果發(fā)現(xiàn)過(guò)表達(dá)Ta EXPA2提高了轉(zhuǎn)基因煙草的抗鹽性。具體表現(xiàn)在鹽處理以后,轉(zhuǎn)基因煙草的萌發(fā)率更高,幼苗和成苗的生長(zhǎng)狀況較WT更好。從滲透脅迫耐性、離子脅迫耐性和抗氧化能力等方面探討了轉(zhuǎn)基因煙草耐鹽性提高的生理機(jī)制,結(jié)果發(fā)現(xiàn),煙草脅迫下轉(zhuǎn)基因煙草的水分維持能力強(qiáng),細(xì)胞內(nèi)Na+離子積累較WT少而K+積累較WT多,抗氧化能力較WT強(qiáng)。特別地,用轉(zhuǎn)基因煙草葉片細(xì)胞壁蛋白孵育的野生型葉圓片的抗鹽性增強(qiáng),且轉(zhuǎn)基因煙草葉圓片增強(qiáng)的抗鹽性可以被Ta EXPA2抗體抑制。結(jié)果表明,轉(zhuǎn)基因煙草抗鹽性的提高可能與鈉、鉀離子穩(wěn)態(tài)調(diào)節(jié)和抗氧化能力增強(qiáng)有關(guān)。(7)初步研究了ABA信號(hào)在Ta EXPA2介導(dǎo)的耐鹽性中的作用。用脫落酸(ABA)和Na Cl處理小麥幼苗,發(fā)現(xiàn)ABA和Na Cl均可以誘導(dǎo)小麥葉片中Ta EXPA2蛋白的積累,但是ABA的抑制劑氟啶酮(FLU)抑制小麥中Ta EXPA2蛋白的積累;同時(shí)使用Na Cl和FLU處理小麥時(shí),Ta EXPA2蛋白的積累受到抑制。說(shuō)明ABA信號(hào)與Na Cl誘導(dǎo)的Ta EXPA2蛋白積累有關(guān)。但是,轉(zhuǎn)基因煙草中,ABA的合成基因的表達(dá)水平無(wú)論在正常情況下還是鹽處理后都比野生型低。這些結(jié)果說(shuō)明,ABA信號(hào)參與了Ta EXPA2調(diào)控的轉(zhuǎn)基因煙草的抗鹽性。(8)利用擬南芥同源基因突變體atexpa2研究過(guò)表達(dá)Ta EXPA2提高轉(zhuǎn)基因煙草抗氧化能力的生理與分子機(jī)制。用H2O2處理小麥,檢測(cè)Ta EXPA2的表達(dá)水平,發(fā)現(xiàn)Ta EXPA2的表達(dá)受過(guò)氧化氫誘導(dǎo)上調(diào)。將Ta EXPA2基因在擬南芥中過(guò)表達(dá),發(fā)現(xiàn)過(guò)表達(dá)Ta EXPA2的轉(zhuǎn)基因擬南芥對(duì)過(guò)氧化氫引起的氧化脅迫的抗性明顯增強(qiáng)。研究轉(zhuǎn)基因擬南芥抗氧化能力提高的生理機(jī)制,發(fā)現(xiàn)轉(zhuǎn)基因擬南芥抗氧化能力的提高與植物細(xì)胞壁鍵連的過(guò)氧化物酶關(guān)系密切。用Ta EXPA2基因的擬南芥(Arabidopsis thaliana L.)同源基因突變體atexpa2進(jìn)一步驗(yàn)證發(fā)現(xiàn),與Col-0野生型相比,突變體atexpa2的細(xì)胞壁鍵連過(guò)氧化物酶活性明顯較低,抗氧化脅迫能力較Col-0顯著降低。將Ta EXPA2基因過(guò)表達(dá)回補(bǔ)到突變體atexpa2后,轉(zhuǎn)基因擬南芥的抗氧化能力增強(qiáng),并且擬南芥中三種過(guò)氧化物酶的活性增強(qiáng)。這些結(jié)果初步證明,Ta EXPA2與細(xì)胞壁POD之間關(guān)系密切。推測(cè)細(xì)胞壁擴(kuò)展蛋白Ta EXPA2可能通過(guò)與細(xì)胞壁鍵連的過(guò)氧化物酶互作,對(duì)轉(zhuǎn)基因植株的氧化脅迫能力產(chǎn)生影響。該結(jié)果對(duì)于解釋過(guò)表達(dá)擴(kuò)展蛋白提高植物的脅迫耐性具有重要意義。
[Abstract]:Wheat (Triticum aestivum L.) is an important grain crop in China. In the process of wheat growth, it is often damaged by environmental stress, which leads to growth inhibition and yield reduction. It is the direction of development of wheat new varieties to improve the adaptability of Wheat by genetic engineering technology. The function of adversity related genes can be a wheat gene. Engineering breeding lays a theoretical foundation and reserves genetic resources. Extended protein is a cell wall protein that regulates cell wall relaxation and cell elongation growth. It is ubiquitous in all plant cells, tissues and organs of Professor Cosgrove of Penn State University, Penn State University, to study the elongation of hypocotyls cells in cucumber hypocotyls. The extended protein (Expansin) has been discovered for the first time. In recent years, the research achievements of expanded protein have been enriched. It has been proved that the extended protein is involved in the growth and development process of various plants and the response to adversity adaptability. However, the function of wheat expanded protein gene function has been studied because of the large genome of wheat, complex genetic background and low conversion efficiency. There are few reports. The extended protein is encoded by the multi gene family. There are many similarities and differences between family members in gene structure, protein structure, biochemical characteristics and evolutionary relationships. Among them, EXPA and EXPB are the two largest and most clearly studied gene families. This laboratory previously cloned the wheat EXPB family gene Ta EXPB23, and found the gene. The expression has a response to a variety of abiotic stresses and hormone signals. Component overexpression, root specificity and stress induced expression of the gene enhanced the drought resistance of transgenic tobacco. In view of the diversity of EXPA and EXPB family members, the EXPA gene Ta EXPA2 (Gen Bank accession number:AAT94292.1) was selected in this study. The function of the gene in plant growth and adversity adaptation was studied by overexpression technology, and its physiological mechanism was analyzed and compared with the function of the EXPB family gene Ta EXPB23, to provide reference for explaining the similarities and differences of the function of different family genes. First, the promoter region of Ta EXPA2 was cloned, and the CIS of the promoter was predicted and analyzed. The spatial and temporal expression of the gene, the response to the hormone response and the expression pattern of adversity response were analyzed, and the Ta EXPA2 gene expression vector was constructed, and transgenic tobacco was obtained by transforming tobacco, and the transgenic tobacco expressing Ta EXPA2 was used to study the role of the gene in the regulation of plant growth and development, drought tolerance and salt tolerance, and further explored its growth. In the end, the molecular mechanism of the expression of extended protein to improve the antioxidant capacity of transgenic plants was discussed, and a homologous mutant atexpa2 of Arabidopsis thaliana was used, and Ta EXPA2 was used in the mutant atexpa2 for overexpression and supplementation. The relationship between expanded egg white and cell wall peroxidase was preliminarily analyzed. The results and conclusions are as follows: (1) the 1073bp fragment (Gen Bank ID:KP729264) of the promoter region of the Ta EXPA2 gene (Gen Bank ID:KP729264) was obtained by using the Tail-PCR cloning technique, and it was analyzed with the functional prediction software of Plant CARE and PLACE. The results showed that the promoter region contained a variety of light response components, tissue specific expression elements, and a variety of hormone response elements. And the response elements of the adversity (drought, ABA and high temperature). (2) the Ta EXPA2:: GFP fusion protein was instantaneously expressed in the onion epidermis, which confirmed that the Ta EXPA2 protein was located in the cell wall. (3) Q RT-PCR detected the specific specificity of the Ta EXPA2 gene expression in wheat and the response mode to the exogenous hormone and abiotic stress. The results showed that Ta EXPA2 was in wheat. The expression of the root, stem and leaf all was expressed, the expression of the leaf in the leaves was the highest; the expression of Ta EXPA2 gene expressed response to a variety of stress and exogenous hormone treatment, and the expression level of gene expression was up-regulated by drought stress. (4) the transformation vector of Ta EXPA2 was constructed, and transgenic tobacco plants with different levels of overexpressed Ta EXPA2 were obtained. The growth and development phenotype was observed. The results showed that overexpression of Ta EXPA2 increased the seed yield and pod number of transgenic tobacco, but did not change the growth phenotype and seed size of transgenic tobacco. (5) drought stress treatment of transgenic tobacco was carried out, germination rate, seedling growth and seedling growth were observed, and the results were found. The drought resistance of transgenic tobacco was increased by Ta EXPA2. The physiological mechanism of the increase of drought resistance in transgenic tobacco was studied from water retention ability, photosynthesis and antioxidant properties. It was found that the improvement of the drought resistance of transgenic tobacco was related to the development of root system, the strong ability of maintaining water condition and the strong antioxidant capacity. The improvement was related to the up-regulated expression of related genes. (6) salt stress treatment of transgenic tobacco was carried out to observe the seed germination, seedling growth and seedling growth of the transgenic tobacco. The results showed that the expression of Ta EXPA2 increased the salt resistance of transgenic tobacco. The physiological mechanism of salt tolerance in transgenic tobacco was discussed from the aspects of osmotic stress tolerance, ion stress tolerance and antioxidant capacity. The results showed that under tobacco stress, the water maintenance ability of transgenic tobacco was strong, the accumulation of Na+ ion in the cell was less than that of WT, and the accumulation of K + was more WT than that of WT, and the antioxidant capacity was stronger than that of WT. The salt resistance of the wild type leaf circular slices incubated with the cell wall protein of transgenic tobacco leaves was enhanced, and the salt resistance of the transgenic tobacco leaves could be inhibited by Ta EXPA2 antibody. The results showed that the salt resistance of the transgenic tobacco could be related to the stable regulation of sodium and potassium ions and the enhancement of the ability to resist oxygen. (7) the ABA signal was preliminarily studied. The effect of Ta EXPA2 on salt tolerance. The accumulation of Ta EXPA2 protein in wheat leaves could be induced by both ABA and Na Cl with abscisic acid (ABA) and Na Cl, but the accumulation of the protein in wheat was inhibited by the ABA inhibitor fluidizone (FLU). Inhibition. Indicating that ABA signals are associated with the accumulation of Ta EXPA2 protein induced by Na Cl. However, the expression level of ABA's synthetic genes in transgenic tobacco is lower than that of the wild type in both normal and salt treatments. These results suggest that ABA signals are involved in the salt resistance of genetically modified tobacco controlled by Ta EXPA2. (8) the use of Arabidopsis homologous genes. The mutant atexpa2 studied the physiological and molecular mechanisms of the expression of Ta EXPA2 to improve the antioxidant capacity of transgenic tobacco. The expression level of Ta EXPA2 was detected by H2O2 treatment and the expression of Ta EXPA2 was up regulated by hydrogen peroxide. Ta EXPA2 gene was overexpressed in Arabidopsis thaliana, and the transoxidation of transgenic Arabidopsis expressing Ta EXPA2 was found. The resistance to oxidative stress caused by hydrogen was obviously enhanced. The physiological mechanism of improving the antioxidant capacity of transgenic Arabidopsis was studied. It was found that the increase of antioxidant capacity of transgenic Arabidopsis was closely related to the peroxidase of plant cell wall bonds. The Ta EXPA2 gene Arabidopsis thaliana L. homologous gene mutant atexpa2 entered one. It was found that the cell wall bond peroxidase activity of the mutant atexpa2 was significantly lower than that of the Col-0 wild type, and the antioxidant capacity of the mutant atexpa2 was significantly lower than that of Col-0. After overexpression of the Ta EXPA2 gene, the antioxidant capacity of the transgenic Arabidopsis was enhanced and the activity of three peroxidase in Arabidopsis was increased. Strong. These results have preliminarily proved that Ta EXPA2 is closely related to the cell wall POD. It is presumed that the cell wall extension protein Ta EXPA2 may affect the oxidative stress of transgenic plants by interacting with the peroxidase linked to the cell wall bond. This result is important to explain the overexpression of extended protein to enhance the stress tolerance of plants. Righteousness.
【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】：2016
【分類號(hào)】：Q943.2;S512.1

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