小麦脱水素基因WZY2-1的克隆及功能分析
发布时间：2019-01-16 05:25
【摘要】：干旱、低温、盐等逆境胁迫都影响植物生长和发育,甚至降低作物产量。这些胁迫因子都与脱水胁迫相关,水分胁迫会导致植物细胞产生一系列的生理生化应答反应。植物应答脱水反应会积累许多渗透调节复合物,包括许多亲水蛋白,如脱水素。研究表明,脱水素的高效表达能够提高植物的抗逆能力。小麦作为世界三大粮食作物,但小麦的产量长期受各种逆境胁迫的影响。因此,研究小麦抗逆分子机制对于提高小麦产量具有重大意义。本研究以郑引1#小麦为材料,根据研究目的,从小麦中成功克隆了WZY2-1基因,通过荧光定量PCR技术,分析了各种胁迫处理下WZY2-1基因在小麦叶片中的表达模式,并以GFP为报告基因,对WZY2-1基因进行亚细胞定位;将WZY2-1基因转化大肠杆菌,研究WZY2-1蛋白对大肠杆菌的保护作用,同时纯化获得WZY2-1蛋白,发现WZY2-1蛋白具有保护乳酸脱氢酶的功能。我们还将目的基因在拟南芥中过表达,以进一步揭示脱水蛋白在逆境胁迫下的调控机制。通过研究,获得了如下结果:1.从郑引1#小麦中成功克隆了脱水素基因WZY2-1,基因编码区序列长1740bp,编码579个氨基酸,并且含有9个保守的K片段,属于Kn型脱水素。2.通过生物信息学分析,发现WZY2-1基因与大麦DHN5具有较高同源性,过对WZY2-1疏水性和无序化程度预测,发现WZY2-1是具有极强的亲水性的高度无序蛋白。3.实时定量PCR分析了WZY2-1基因的表达模式,发现该基因响应干旱、低温和盐胁迫,但不能被ABA诱导。4.将WZY2-1基因在大肠杆菌中原核表达,分析了WZY2-1蛋白对大肠杆菌的保护作用,发现该蛋白能够提高大肠杆菌非生物胁迫的耐受性。5.构建了pBI121-WZY2-1-GFP亚细胞定位载体,并转化洋葱表皮细胞,观察表明,WZY2-1蛋白定位于细胞核和细胞膜。6.我们纯化了WZY2-1蛋白,将该蛋白与乳酸脱氢酶混合,结果表明,WZY2-1蛋白能够保护温度胁迫下乳酸脱氢酶活性。7.实验构建了pBI121-WZY2-1植物表达载体,采用农杆菌浸染法转化拟南芥,通过抗生素、基因组DNA PCR和RT-PCR进行筛选,成功获得了转WZY2-1基因的拟南芥植株,传代培养获得T4代植株,通过比较野生型拟南芥,发现WZY2-1基因能够提高拟南芥的抗旱能力。
[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.
【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】：2017
【分类号】：Q943.2;S512.1
，
本文编号：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.
【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】：2017
【分类号】：Q943.2;S512.1
，
本文编号：2409510
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