本文選題：小麥 + FKBP基因�。� 參考：《西北農(nóng)林科技大學(xué)》2016年碩士論文

【摘要】：小麥?zhǔn)鞘澜缟献钪匾募Z食作物之一,其種植面積和總產(chǎn)量均居糧食作物前列。但隨著全球氣候變暖,高溫、干旱頻繁發(fā)生,特別是灌漿前期的熱旱交迫嚴(yán)重影響小麥生產(chǎn)。培育耐熱抗旱小麥新品種是御災(zāi)保產(chǎn)最為經(jīng)濟(jì)有效的途徑。然而,小麥的耐熱、耐旱性屬于數(shù)量性狀,機(jī)理復(fù)雜,加上小麥基因組龐大,導(dǎo)致傳統(tǒng)育種方法很難在小麥抗逆育種上取得突破性進(jìn)展。現(xiàn)代基因工程和分子生物學(xué)則給作物抗逆育種帶來了新的契機(jī)。近年來一些研究表明,FK506結(jié)合蛋白(FK506 bindingprotein,FKBP)家族成員參與植物的生長發(fā)育及抗逆性調(diào)控。目前關(guān)于植物FKBP的研究主要集中在擬南芥(Arabidopsis thaliana)、水稻(Oryza sativa L.)、玉米(Zea mays)等少數(shù)植物,而小麥(Triticum aestivum L.)FKBP基因的克隆與功能分析則鮮有報(bào)道。本研究克隆了一個(gè)小麥FKBP基因,命名為TaFKBP62c-2B,分析了其在高溫和干旱脅迫條件下的表達(dá)模式,并通過轉(zhuǎn)化擬南芥進(jìn)行了功能分析。獲得的主要研究結(jié)果如下:1.從普通小麥cDNA中克隆得到了TaFKBP62c-2B基因,全長1926bp,編碼的氨基酸長度為642aa。生物信息學(xué)分析,TaFKBP62c-2B包含有三個(gè)FKBP_C保守域(FK506 binding protein_C conserved domain)和三個(gè)TPR結(jié)構(gòu)域(tetratricopeptide repeat,三十四肽重復(fù)序列),屬于多域FKBP蛋白,TaFKBP62c-2B在C端存在一個(gè)跨膜區(qū)域。進(jìn)化分析表明,普通小麥與水稻、玉米、高粱等9個(gè)物種的FKBP62c氨基酸序列同源性在86%-96%之間,其中與烏拉爾圖小麥的同源性最高。2.利用熒光定量PCR(qRT-PCR)技術(shù)分析了TaFKBP62c-2B基因在高溫和干旱脅迫條件下的表達(dá)模式,發(fā)現(xiàn)TaFKBP62c-2B基因同時(shí)受高溫、干旱脅迫的誘導(dǎo)。受高溫脅迫時(shí),TaFKBP65-2B的表達(dá)量先迅速上升,在2h時(shí)達(dá)到最高值,然后逐漸下降;受干旱脅迫誘導(dǎo)時(shí),TaFKBP62c-2B的表達(dá)量迅速上升并在1 h時(shí)達(dá)到峰值,然后逐漸下降,4 h達(dá)到最低,然后再次逐漸上升。3.利用熒光定量PCR(qRT-PCR)技術(shù)分析了TaFKBP62c-2B基因在抽穗兩周左右小麥的根、莖、葉、整穗、小穗、外稃皮和內(nèi)稃皮組織的表達(dá)模式,發(fā)現(xiàn)TaFKBP62c-2B在以上被檢測組織中均有表達(dá),其表達(dá)豐度在莖中最高,整穗、小穗、外稃皮和內(nèi)稃皮中呈中等豐度表達(dá),根和葉中最低。4.構(gòu)建了亞細(xì)胞定位載體TaFKBP62c-2B::16318GFP,以16318GFP空載體作為對(duì)照,利用PEG誘導(dǎo)法將其轉(zhuǎn)入小麥葉肉原生質(zhì)體,利用激光共聚焦顯微鏡觀察目標(biāo)蛋白和16318GFP空載體的亞細(xì)胞定位情況。根據(jù)本研究結(jié)果,參照在線工具WOLF PSORT的預(yù)測和水稻OsFKBP62c的定位信息,判定TaFKBP62c-2B定位于內(nèi)質(zhì)網(wǎng)。5.構(gòu)建了植物表達(dá)載體pCAMBIA1302::TaFKBP62c-2B,并利用土壤農(nóng)桿菌(Agrobacterium)介導(dǎo)的擬南芥轉(zhuǎn)化技術(shù)獲得轉(zhuǎn)TaFKBP62c-2B基因的擬南芥植株。對(duì)轉(zhuǎn)TaFKBP62c-2B基因的擬南芥T3進(jìn)行了抗旱性鑒定,結(jié)果發(fā)現(xiàn),在8%PEG和15%PEG脅迫條件下,轉(zhuǎn)基因株系的萌發(fā)率高于野生型,說明TaFKBP62c-2B基因的表達(dá)增強(qiáng)了擬南芥的苗期抗旱能力。但對(duì)其耐熱功能還需進(jìn)一步研究。
[Abstract]:Wheat is one of the most important grain crops in the world. Its planting area and total yield are in the forefront of grain crops. However, with the global warming, high temperature and drought frequently occur, especially the hot and drought stress at the early stage of grain filling, which seriously affect the production of wheat. The heat resistance and drought resistance of wheat belong to quantitative characters, the mechanism is complex and the genome of wheat is huge, which makes it difficult to make breakthrough in wheat breeding by traditional breeding methods. Modern genetic engineering and molecular biology have brought new opportunities for crop resistance breeding. In recent years, some studies have shown that FK506 binding protein (FK506 bind) Ingprotein, FKBP) family members participate in the growth and control of plant resistance. At present, the research on plant FKBP is mainly concentrated in the Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa L.), corn (Zea mays) and other plants, and the cloning and functional analysis of the wheat (Triticum aestivum) gene is rarely reported. A wheat FKBP gene, named TaFKBP62c-2B, was cloned, and its expression patterns under high temperature and drought stress were analyzed and functional analysis of Arabidopsis thaliana was carried out. The main results obtained were as follows: 1. the TaFKBP62c-2B gene was cloned from the common wheat cDNA, with a full-length 1926bp and the encoded amino acid length of 642aa.. Bioinformatics analysis, TaFKBP62c-2B contains three FKBP_C conservative domains (FK506 binding protein_C conserved domain) and three TPR domains (tetratricopeptide repeat, thirty-four peptide repeat sequences), which belong to the multidomain FKBP protein. There is a trans membrane region in the TaFKBP62c-2B. The homology of FKBP62c amino acid sequences of 9 species, such as sorghum, is between 86%-96%, among which the highest homology with Ural map wheat.2. uses fluorescence quantitative PCR (qRT-PCR) technique to analyze the expression pattern of TaFKBP62c-2B gene under high temperature and drought stress, and it is found that the TaFKBP62c-2B gene is induced by high temperature and drought stress. When stress, the expression of TaFKBP65-2B first increased rapidly, reached the highest value at 2h, and then declined gradually. When induced by drought stress, the expression of TaFKBP62c-2B increased rapidly and reached its peak at 1 h, then gradually decreased, the 4 h reached the lowest, and then gradually increased.3. by using fluorescence quantitative PCR (qRT-PCR) technology to analyze TaFKBP62c-2B. The expression pattern of the gene in the root, stem, leaf, panicle, spikelet, lemma skin and palea of the wheat at about two weeks of heading, found that TaFKBP62c-2B was expressed in the above detected tissues, and the expression abundance was highest in the stem, the whole ear, the spikelet, the palea skin and the palea, and the lowest.4. in the root and leaf. Carrier TaFKBP62c-2B:: 16318GFP, taking 16318GFP empty carrier as control, using PEG induction method to transfer it to wheat mesophyll protoplast, using laser confocal microscope to observe the subcellular localization of target protein and 16318GFP empty carrier. According to the results of this study, the prediction of WOLF PSORT and the positioning letter of rice OsFKBP62c according to the results of this study. It was determined that TaFKBP62c-2B was located in the endoplasmic reticulum.5. to construct plant expression vector pCAMBIA1302:: TaFKBP62c-2B, and to obtain Arabidopsis plants with TaFKBP62c-2B gene by using Arabidopsis transformation technology mediated by soil Agrobacterium tumefaciens (Agrobacterium). The drought resistance of Arabidopsis T3 with TaFKBP62c-2B gene was identified. The results were found to be in 8%PEG and 1. Under the stress of 5%PEG, the germination rate of the transgenic lines was higher than that of the wild type, indicating that the expression of TaFKBP62c-2B gene enhanced the drought resistance ability of Arabidopsis thaliana at seedling stage, but the heat tolerance needed to be further studied.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S512.1;Q943.2

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1 吳迪;小麥抗逆相關(guān)基因TaFKBP62c-2B的克隆及功能驗(yàn)證[D];西北農(nóng)林科技大學(xué);2016年

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本文編號(hào)：1909547