小麦抗逆相关基因TaFKBP62c-2B的克隆及功能验证

发布时间：2018-05-19 09:19

本文选题：小麦 + FKBP基因　；参考：《西北农林科技大学》2016年硕士论文

【摘要】：小麦是世界上最重要的粮食作物之一,其种植面积和总产量均居粮食作物前列。但随着全球气候变暖,高温、干旱频繁发生,特别是灌浆前期的热旱交迫严重影响小麦生产。培育耐热抗旱小麦新品种是御灾保产最为经济有效的途径。然而,小麦的耐热、耐旱性属于数量性状,机理复杂,加上小麦基因组庞大,导致传统育种方法很难在小麦抗逆育种上取得突破性进展。现代基因工程和分子生物学则给作物抗逆育种带来了新的契机。近年来一些研究表明,FK506结合蛋白(FK506 bindingprotein,FKBP)家族成员参与植物的生长发育及抗逆性调控。目前关于植物FKBP的研究主要集中在拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa L.)、玉米(Zea mays)等少数植物,而小麦(Triticum aestivum L.)FKBP基因的克隆与功能分析则鲜有报道。本研究克隆了一个小麦FKBP基因,命名为TaFKBP62c-2B,分析了其在高温和干旱胁迫条件下的表达模式,并通过转化拟南芥进行了功能分析。获得的主要研究结果如下:1.从普通小麦cDNA中克隆得到了TaFKBP62c-2B基因,全长1926bp,编码的氨基酸长度为642aa。生物信息学分析,TaFKBP62c-2B包含有三个FKBP_C保守域(FK506 binding protein_C conserved domain)和三个TPR结构域(tetratricopeptide repeat,三十四肽重复序列),属于多域FKBP蛋白,TaFKBP62c-2B在C端存在一个跨膜区域。进化分析表明,普通小麦与水稻、玉米、高粱等9个物种的FKBP62c氨基酸序列同源性在86%-96%之间,其中与乌拉尔图小麦的同源性最高。2.利用荧光定量PCR(qRT-PCR)技术分析了TaFKBP62c-2B基因在高温和干旱胁迫条件下的表达模式,发现TaFKBP62c-2B基因同时受高温、干旱胁迫的诱导。受高温胁迫时,TaFKBP65-2B的表达量先迅速上升,在2h时达到最高值,然后逐渐下降;受干旱胁迫诱导时,TaFKBP62c-2B的表达量迅速上升并在1 h时达到峰值,然后逐渐下降,4 h达到最低,然后再次逐渐上升。3.利用荧光定量PCR(qRT-PCR)技术分析了TaFKBP62c-2B基因在抽穗两周左右小麦的根、茎、叶、整穗、小穗、外稃皮和内稃皮组织的表达模式,发现TaFKBP62c-2B在以上被检测组织中均有表达,其表达丰度在茎中最高,整穗、小穗、外稃皮和内稃皮中呈中等丰度表达,根和叶中最低。4.构建了亚细胞定位载体TaFKBP62c-2B::16318GFP,以16318GFP空载体作为对照,利用PEG诱导法将其转入小麦叶肉原生质体,利用激光共聚焦显微镜观察目标蛋白和16318GFP空载体的亚细胞定位情况。根据本研究结果,参照在线工具WOLF PSORT的预测和水稻OsFKBP62c的定位信息,判定TaFKBP62c-2B定位于内质网。5.构建了植物表达载体pCAMBIA1302::TaFKBP62c-2B,并利用土壤农杆菌(Agrobacterium)介导的拟南芥转化技术获得转TaFKBP62c-2B基因的拟南芥植株。对转TaFKBP62c-2B基因的拟南芥T3进行了抗旱性鉴定,结果发现,在8%PEG和15%PEG胁迫条件下,转基因株系的萌发率高于野生型,说明TaFKBP62c-2B基因的表达增强了拟南芥的苗期抗旱能力。但对其耐热功能还需进一步研究。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S512.1;Q943.2

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