本文選題：玉米　切入點：非生物脅迫　出處：《甘肅農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：非生物脅迫是致使世界范圍內(nèi)產(chǎn)生糧食危機(jī)的主要因素之一,全球每年因非生物脅迫至少能導(dǎo)致農(nóng)作物減產(chǎn)50%。玉米是重要的糧食、飼料及工業(yè)原料,其生育期極易受干旱、高鹽、低溫等多種脅迫因子的干擾,是逆境敏感型植物。在面臨著全球總?cè)藬?shù)不斷上升而可種植面積不斷減少的窘境下,探索植物對非生物脅迫的應(yīng)答原理以及挖掘能夠參與調(diào)節(jié)植物生長抗逆過程的基因是非常必要的。MYB是植物中最為普遍存在的轉(zhuǎn)錄因子家族,幾乎參與調(diào)控了植物的全部生命活動,在植物抵抗非生物脅迫的過程當(dāng)中也發(fā)揮出著重要的調(diào)控作用。本研究從玉米自交系F83中克隆得到一個MYB轉(zhuǎn)錄基因,并通過對該基因進(jìn)行生物學(xué)信息分析、實時熒光定量PCR以及轉(zhuǎn)基因植株的抗性分析,初步研究了Zm MYBR58基因的基本性質(zhì)及其生物學(xué)功能。研究結(jié)果如下:1利用RT-PCR法克隆,從玉米自交系F83中得到Zm MYBR58基因,該基因編碼的蛋白共包括433個氨基酸,包含了一個全長1302 bp的開放閱讀框;預(yù)測蛋白理論分子量為46.0966 KD,理論等電點為9.52,理論半衰期為30 h,屬于不穩(wěn)定蛋白,親水性氨基酸多于疏水性氨基酸,是一種親水性蛋白;同源性比較結(jié)果顯示該基因分別與高粱、谷子、小麥、水稻等作物的MYB蛋白具有一定的相似性。2蛋白分析顯示Zm MYBR58基因編碼的蛋白序列僅具有一個保守的SANT結(jié)構(gòu),能形成3個α螺旋結(jié)構(gòu),幫助折疊成正確的蛋白結(jié)構(gòu),屬于MYB-related家族。3根據(jù)Zm MYBR58基因上游序列搜索其順式作用元件發(fā)現(xiàn)存在光誘導(dǎo)、激素誘導(dǎo)、干旱誘導(dǎo)、低溫誘導(dǎo)、組織特異表達(dá)等一系列相關(guān)元件,預(yù)測Zm MYBR58基因可能能夠被干旱、低溫、激素等脅迫因子誘導(dǎo),從而參與到植物對逆境的響應(yīng)過程中。4表達(dá)分析結(jié)果顯示Zm MYBR58基因在玉米各檢測組織中均有表達(dá),其中在玉米胚芽中的表達(dá)量顯著高于其他組織,具有組織特異性;Zm MYBR58基因的表達(dá)模式受干旱、高鹽、ABA、低溫以及高溫的誘導(dǎo),且其表達(dá)模式受不同誘導(dǎo)的響應(yīng)程度不同,說明該基因可能參與調(diào)控著植物抗逆機(jī)制中的激素/逆境信號傳遞通路。5成功構(gòu)建了CPB-Zm MYBR58重組載體,利用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化擬南芥,經(jīng)過除草劑篩選和PCR鑒定得到Zm MYBR58轉(zhuǎn)基因擬南芥的抗性植株,干旱處理下轉(zhuǎn)基因抗性植株表現(xiàn)出較野生型擬南芥更高的耐旱性。以上研究結(jié)果均表明Zm MYBR58基因能夠參與調(diào)節(jié)植物的非生物脅迫耐受性,本研究初步分析了Zm MYBR58基因的結(jié)構(gòu)和功能,為進(jìn)一步分析該基因參與的植物脅迫響應(yīng)調(diào)節(jié)機(jī)制奠定了理論依據(jù),也為今后利用基因工程相關(guān)技術(shù)培育玉米抗逆品種提供了一個優(yōu)良的候選基因。
[Abstract]:Abiotic stress is one of the main factors leading to the food crisis in the world.Maize is an important food, feed and industrial raw material. It is susceptible to drought, high salt, low temperature and other stress factors in its growth period.Faced with a growing global population and declining plantable areas,It is necessary to explore the principles of plant response to abiotic stress and to dig up genes that can regulate plant growth and stress resistance. MYB is the most common transcription factor family in plants.It plays an important role in the process of plant resistance to abiotic stress.In this study, a MYB transcription gene was cloned from maize inbred line F83 and analyzed by biological information, real-time quantitative PCR and resistance analysis of transgenic plants.The basic properties and biological functions of Zm MYBR58 gene were preliminarily studied.The results were as follows: 1. The Zm MYBR58 gene was cloned by RT-PCR from maize inbred line F83. The protein encoded by the gene contained 433 amino acids and contained an open reading frame with a length of 1302 BP.The theoretical molecular weight of the predicted protein is 46.0966 KD, the theoretical isoelectric point is 9.52, the theoretical half-life is 30 h, the predicted protein belongs to unstable protein, hydrophilic amino acid is more than hydrophobic amino acid, and it is a kind of hydrophilic protein.The MYB protein of millet, wheat, rice and other crops showed that the MYB protein sequence encoded by Zm MYBR58 gene had only one conserved SANT structure, and could form three 偽 helical structures, helping to fold into the correct protein structure.According to the upstream sequence of Zm MYBR58 gene, a series of related elements, such as light induction, hormone induction, drought induction, hypothermia induction and tissue specific expression, were found in MYB-related family. It was predicted that Zm MYBR58 gene might be drought induced.The results showed that Zm MYBR58 gene was expressed in all the tested tissues of maize, and the expression level of Zm MYBR58 in maize germ was significantly higher than that in other tissues.The expression pattern of Zm MYBR58 gene with tissue specificity was induced by drought, high salinity, low temperature and high temperature, and its expression pattern was different in response to different induction.These results suggest that the gene may be involved in regulating the hormone / stress signaling pathway in plant stress resistance mechanism. 5. The recombinant vector of CPB-Zm MYBR58 was successfully constructed and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens.The resistant plants of transgenic Arabidopsis thaliana (Arabidopsis thaliana) with Zm MYBR58 gene were obtained by herbicide screening and PCR identification. The transgenic plants showed higher drought resistance than wild type Arabidopsis thaliana under drought treatment.All the results indicated that Zm MYBR58 gene was involved in the regulation of abiotic stress tolerance in plants. The structure and function of Zm MYBR58 gene were preliminarily analyzed in this study.It provides a theoretical basis for further analysis of the regulation mechanism of plant stress response in which the gene is involved, and also provides a good candidate gene for the cultivation of stress resistant maize varieties by genetic engineering techniques in the future.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S513

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