小麥脫水素基因WZY2-1的克隆及功能分析
發(fā)布時間：2019-01-16 05:25
【摘要】：干旱、低溫、鹽等逆境脅迫都影響植物生長和發(fā)育,甚至降低作物產(chǎn)量。這些脅迫因子都與脫水脅迫相關(guān),水分脅迫會導(dǎo)致植物細胞產(chǎn)生一系列的生理生化應(yīng)答反應(yīng)。植物應(yīng)答脫水反應(yīng)會積累許多滲透調(diào)節(jié)復(fù)合物,包括許多親水蛋白,如脫水素。研究表明,脫水素的高效表達能夠提高植物的抗逆能力。小麥作為世界三大糧食作物,但小麥的產(chǎn)量長期受各種逆境脅迫的影響。因此,研究小麥抗逆分子機制對于提高小麥產(chǎn)量具有重大意義。本研究以鄭引1#小麥為材料,根據(jù)研究目的,從小麥中成功克隆了WZY2-1基因,通過熒光定量PCR技術(shù),分析了各種脅迫處理下WZY2-1基因在小麥葉片中的表達模式,并以GFP為報告基因,對WZY2-1基因進行亞細胞定位;將WZY2-1基因轉(zhuǎn)化大腸桿菌,研究WZY2-1蛋白對大腸桿菌的保護作用,同時純化獲得WZY2-1蛋白,發(fā)現(xiàn)WZY2-1蛋白具有保護乳酸脫氫酶的功能。我們還將目的基因在擬南芥中過表達,以進一步揭示脫水蛋白在逆境脅迫下的調(diào)控機制。通過研究,獲得了如下結(jié)果:1.從鄭引1#小麥中成功克隆了脫水素基因WZY2-1,基因編碼區(qū)序列長1740bp,編碼579個氨基酸,并且含有9個保守的K片段,屬于Kn型脫水素。2.通過生物信息學(xué)分析,發(fā)現(xiàn)WZY2-1基因與大麥DHN5具有較高同源性,過對WZY2-1疏水性和無序化程度預(yù)測,發(fā)現(xiàn)WZY2-1是具有極強的親水性的高度無序蛋白。3.實時定量PCR分析了WZY2-1基因的表達模式,發(fā)現(xiàn)該基因響應(yīng)干旱、低溫和鹽脅迫,但不能被ABA誘導(dǎo)。4.將WZY2-1基因在大腸桿菌中原核表達,分析了WZY2-1蛋白對大腸桿菌的保護作用,發(fā)現(xiàn)該蛋白能夠提高大腸桿菌非生物脅迫的耐受性。5.構(gòu)建了pBI121-WZY2-1-GFP亞細胞定位載體,并轉(zhuǎn)化洋蔥表皮細胞,觀察表明,WZY2-1蛋白定位于細胞核和細胞膜。6.我們純化了WZY2-1蛋白,將該蛋白與乳酸脫氫酶混合,結(jié)果表明,WZY2-1蛋白能夠保護溫度脅迫下乳酸脫氫酶活性。7.實驗構(gòu)建了pBI121-WZY2-1植物表達載體,采用農(nóng)桿菌浸染法轉(zhuǎn)化擬南芥,通過抗生素、基因組DNA PCR和RT-PCR進行篩選,成功獲得了轉(zhuǎn)WZY2-1基因的擬南芥植株,傳代培養(yǎng)獲得T4代植株,通過比較野生型擬南芥,發(fā)現(xiàn)WZY2-1基因能夠提高擬南芥的抗旱能力。
[Abstract]:Drought, low temperature, salt and other stresses all affect plant growth and development, and even reduce crop yield. These stress factors are related to dehydration stress, and water stress can lead to a series of physiological and biochemical responses of plant cells. Plant response to dehydration accumulates many osmotic regulatory complexes, including many hydrophilic proteins, such as dehydrins. The study showed that the high expression of dehydrin could improve the stress resistance of plants. Wheat is one of the three major food crops in the world, but its yield is affected by various stresses for a long time. Therefore, it is of great significance to study the molecular mechanism of wheat stress resistance for increasing wheat yield. In this study, the WZY2-1 gene was cloned successfully from wheat, and the expression pattern of WZY2-1 gene in wheat leaves under various stresses was analyzed by fluorescence quantitative PCR. Using GFP as reporter gene, subcellular localization of WZY2-1 gene was carried out. The WZY2-1 gene was transformed into Escherichia coli to study the protective effect of WZY2-1 protein on Escherichia coli. At the same time, WZY2-1 protein was purified and obtained. It was found that WZY2-1 protein had the function of protecting lactate dehydrogenase. We also overexpressed the target gene in Arabidopsis thaliana to further reveal the regulatory mechanism of dehydrated protein under stress. The results are as follows: 1. The nucleotide sequence of WZY2-1, gene encoding 579 amino acids was cloned successfully from Zhengyin1# wheat. The sequence was composed of 9 conserved K fragments and belonged to Kn type dehydrin. 2. By bioinformatics analysis, we found that WZY2-1 gene has high homology with barley DHN5, and predicted the degree of hydrophobicity and disorder of WZY2-1, and found that WZY2-1 is a highly disordered protein with strong hydrophilicity. The expression pattern of WZY2-1 gene was analyzed by real-time quantitative PCR. It was found that the gene was responsive to drought, low temperature and salt stress, but could not be induced by ABA. The prokaryotic expression of WZY2-1 gene in Escherichia coli was carried out, and the protective effect of WZY2-1 protein on Escherichia coli was analyzed. It was found that the protein could improve the tolerance of E. coli to abiotic stress. PBI121-WZY2-1-GFP subcellular localization vector was constructed and transformed into onion epidermal cells. The results showed that WZY2-1 protein was located in nucleus and cell membrane. WZY2-1 protein was purified and mixed with lactate dehydrogenase. The results showed that WZY2-1 protein could protect lactate dehydrogenase activity under temperature stress. The plant expression vector of pBI121-WZY2-1 was constructed and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens. The transgenic Arabidopsis plants were obtained by antibiotic, genomic DNA PCR and RT-PCR screening. Through the comparison of wild type Arabidopsis thaliana, it was found that WZY2-1 gene could improve the drought resistance of Arabidopsis thaliana.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;S512.1
，
本文編號：2409510
[Abstract]:Drought, low temperature, salt and other stresses all affect plant growth and development, and even reduce crop yield. These stress factors are related to dehydration stress, and water stress can lead to a series of physiological and biochemical responses of plant cells. Plant response to dehydration accumulates many osmotic regulatory complexes, including many hydrophilic proteins, such as dehydrins. The study showed that the high expression of dehydrin could improve the stress resistance of plants. Wheat is one of the three major food crops in the world, but its yield is affected by various stresses for a long time. Therefore, it is of great significance to study the molecular mechanism of wheat stress resistance for increasing wheat yield. In this study, the WZY2-1 gene was cloned successfully from wheat, and the expression pattern of WZY2-1 gene in wheat leaves under various stresses was analyzed by fluorescence quantitative PCR. Using GFP as reporter gene, subcellular localization of WZY2-1 gene was carried out. The WZY2-1 gene was transformed into Escherichia coli to study the protective effect of WZY2-1 protein on Escherichia coli. At the same time, WZY2-1 protein was purified and obtained. It was found that WZY2-1 protein had the function of protecting lactate dehydrogenase. We also overexpressed the target gene in Arabidopsis thaliana to further reveal the regulatory mechanism of dehydrated protein under stress. The results are as follows: 1. The nucleotide sequence of WZY2-1, gene encoding 579 amino acids was cloned successfully from Zhengyin1# wheat. The sequence was composed of 9 conserved K fragments and belonged to Kn type dehydrin. 2. By bioinformatics analysis, we found that WZY2-1 gene has high homology with barley DHN5, and predicted the degree of hydrophobicity and disorder of WZY2-1, and found that WZY2-1 is a highly disordered protein with strong hydrophilicity. The expression pattern of WZY2-1 gene was analyzed by real-time quantitative PCR. It was found that the gene was responsive to drought, low temperature and salt stress, but could not be induced by ABA. The prokaryotic expression of WZY2-1 gene in Escherichia coli was carried out, and the protective effect of WZY2-1 protein on Escherichia coli was analyzed. It was found that the protein could improve the tolerance of E. coli to abiotic stress. PBI121-WZY2-1-GFP subcellular localization vector was constructed and transformed into onion epidermal cells. The results showed that WZY2-1 protein was located in nucleus and cell membrane. WZY2-1 protein was purified and mixed with lactate dehydrogenase. The results showed that WZY2-1 protein could protect lactate dehydrogenase activity under temperature stress. The plant expression vector of pBI121-WZY2-1 was constructed and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens. The transgenic Arabidopsis plants were obtained by antibiotic, genomic DNA PCR and RT-PCR screening. Through the comparison of wild type Arabidopsis thaliana, it was found that WZY2-1 gene could improve the drought resistance of Arabidopsis thaliana.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;S512.1
，
本文編號：2409510
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2409510.html
最近更新
教材專著
