小麦LEAⅢ族TaDRLea3-2基因在大肠杆菌和拟南芥中的功能研究
发布时间：2018-11-16 12:18
【摘要】：小麦是人类重要的粮食资源,干旱是导致小麦产量降低的重要原因之一。LEA蛋白作为一种重要的抗逆蛋白,在植物遭受外界不良条件时大量表达,可以减轻植物细胞受害,发挥对植物的保护功能。LEA3蛋白是LEA蛋白家族的重要成员,具有高度亲水性,已有实验证明该族LEA蛋白与植物抗旱能力相关。本文以实验室已经克隆得到的“郑引1号”小麦中的lea3基因TaDRLea3-2为基础,通过转化原核生物大肠杆菌和拟南芥,研究小麦TaDRLea3-2蛋白在非生物胁迫下对大肠杆菌和拟南芥抗逆能力的影响。并通过体外酶活实验研究在高温和冷冻处理下TaDRLea3-2蛋白对LDH酶活的保护作用。为进一步了解LEA蛋白在抵抗非生物胁迫中的功能和作用机制奠定了基础。通过研究获得以下结果:1.在已经克隆得到小麦TaDRLea3-2基因的基础上,成功构建了原核表达载体pET28a-TaDRLea3-2,热激法转化大肠杆菌BL21(DE3)。SDS-PAGE检测大肠杆菌表达的重组蛋白的分子量在23 kD附近。用组氨酸抗体对纯化后的蛋白进Weatern Blot鉴定,表明得到的蛋白为TaDRLea3-2蛋白。2.体外酶活保护实验表明TaDRLea3-2蛋白可以保护LDH酶在液氮冻融处理和45℃高温处理下酶活性。3.通过对含有空载的对照菌BL21-DE3(pET28a)和含有目的基因的重组菌BL21-DE3(pET28a-TaDRLea3-2)进行高温,低温,高盐和高渗处理,表达TaDRLea3-2蛋白的大肠杆菌对这些非生物胁迫的耐受性较对照菌增加。4.通过农杆菌介导的遗传转化,将构建成功的植物表达载体pBI121-TaDRLea3-2转化野生型拟南芥,对转基因拟南芥筛选和鉴定,成功得到T3代转基因拟南芥的种子。以野生型和T3转基因拟南芥的种子为材料进行抗旱性研究。在幼苗期,在浓度为150 mM、200 mM、250 mM甘露醇处理下,转基因拟南芥的根长大于野生型拟南芥。在成苗期,通过自然干旱处理,转基因株系表现出更高的叶片含水量和叶绿素含量,低的丙二醛含量和高的脯氨酸的积累量。表明小麦TaDRLea3-2基因在拟南芥中过表达提高了转基因拟南芥的抗旱性。
[Abstract]:Wheat is an important food resource for human being. Drought is one of the important reasons leading to the decrease of wheat yield. As an important stress resistance protein, LEA protein can be expressed in large quantities when plants suffer from external adverse conditions, which can alleviate the damage of plant cells. LEA3 protein is an important member of LEA protein family and has high hydrophilicity. It has been proved that the LEA protein of this family is related to plant drought resistance. Based on the lea3 gene TaDRLea3-2 of "Zhengyin 1" wheat which has been cloned in the laboratory, the transformation of Escherichia coli and Arabidopsis thaliana was carried out by the transformation of E. coli and Arabidopsis thaliana. To study the effect of wheat TaDRLea3-2 protein on stress resistance of Escherichia coli and Arabidopsis thaliana under abiotic stress. The protective effect of TaDRLea3-2 protein on LDH activity was studied by enzyme activity in vitro. It lays a foundation for further understanding the function and mechanism of LEA protein in resisting abiotic stress. The results are as follows: 1. Based on the cloning of wheat TaDRLea3-2 gene, the prokaryotic expression vector pET28a-TaDRLea3-2, heat shock method was successfully constructed to transform Escherichia coli BL21 (DE3). The molecular weight of the recombinant protein detected by SDS-PAGE was around 23 kD. The purified protein was identified by Weatern Blot with histidine antibody, and the protein was identified as TaDRLea3-2 protein. 2. 2. In vitro enzyme protection experiments showed that TaDRLea3-2 protein could protect the enzyme activity of LDH under liquid nitrogen freeze-thaw treatment and 45 鈩，
本文编号：2335503
[Abstract]:Wheat is an important food resource for human being. Drought is one of the important reasons leading to the decrease of wheat yield. As an important stress resistance protein, LEA protein can be expressed in large quantities when plants suffer from external adverse conditions, which can alleviate the damage of plant cells. LEA3 protein is an important member of LEA protein family and has high hydrophilicity. It has been proved that the LEA protein of this family is related to plant drought resistance. Based on the lea3 gene TaDRLea3-2 of "Zhengyin 1" wheat which has been cloned in the laboratory, the transformation of Escherichia coli and Arabidopsis thaliana was carried out by the transformation of E. coli and Arabidopsis thaliana. To study the effect of wheat TaDRLea3-2 protein on stress resistance of Escherichia coli and Arabidopsis thaliana under abiotic stress. The protective effect of TaDRLea3-2 protein on LDH activity was studied by enzyme activity in vitro. It lays a foundation for further understanding the function and mechanism of LEA protein in resisting abiotic stress. The results are as follows: 1. Based on the cloning of wheat TaDRLea3-2 gene, the prokaryotic expression vector pET28a-TaDRLea3-2, heat shock method was successfully constructed to transform Escherichia coli BL21 (DE3). The molecular weight of the recombinant protein detected by SDS-PAGE was around 23 kD. The purified protein was identified by Weatern Blot with histidine antibody, and the protein was identified as TaDRLea3-2 protein. 2. 2. In vitro enzyme protection experiments showed that TaDRLea3-2 protein could protect the enzyme activity of LDH under liquid nitrogen freeze-thaw treatment and 45 鈩，
本文编号：2335503
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2335503.html
最近更新
教材专著
