干旱诱导基因TaDLea3的功能及在小麦不同生长期的时空表达规律研究

发布时间：2018-04-04 22:13

本文选题：小麦　切入点：LEA蛋白　出处：《西北农林科技大学》2016年博士论文

【摘要】：干旱,高盐和低温等非生物胁迫是影响作物生长和产量的重要因素。当作物受到非生物胁迫时,为了减轻其对作物的伤害会诱导一些蛋白表达。植物胚胎发育晚期丰富蛋白(Late embryogenesis abundant protein,LEA)是一类与逆境胁迫相关的蛋白。LEA蛋白可分为7族,其中第3族LEA(LEA3)蛋白含有11个氨基酸串联重复序列。LEA3蛋白可以形成α-螺旋结构,在非生物胁迫中保护生物大分子,减轻对植物造成的伤害。然而LEA3蛋白的保护功能及其作用机制仍不清晰。本研究从小麦中克隆了1条新的LEA3族基因,通过亚细胞定位,体外保护乳酸脱氢酶活性、转基因在拟南芥中过表达验证以及对非生物胁迫的响应等评价该基因的耐旱功能;同时对2种不同耐旱小麦品种的4个生长时期(苗期、分蘖期、拔节期和开花期)进行不同程度的干旱胁迫和复水处理,从转录水平和翻译水平上研究该基因在小麦全生育期的变化规律以及对干旱的响应,主要研究结果如下:1.通过电子克隆和RT-PCR方法,从小麦陕合6号中克隆1条新的LEA3家族基因(命名为TaDlea3基因)和4条新的LEA3基因同源性较高TaWSI18基因。生物信息学分析表明,TaDlea3蛋白和4个TaWSI18蛋白均具有亲水性,定位于细胞质中,无跨膜结构域。小麦TaDlea3和TaWSI18蛋白与其他物种的LEA3蛋白有高度同源性。通过原核表达TaDlea3蛋白,纯化该蛋白并制备了特异性较高的多克隆抗体。2.通过农杆菌注射法将亚细胞定位载体注射到烟草叶片,并制备原生质体,结果表明,TaDlea3蛋白定位于细胞质中。在高温和低温胁迫下,TaDlea3蛋白对LDH均有一定的保护作用。不同逆境胁迫处理小麦陕合6号和郑引1号,结果表明2种小麦中TaDlea3基因对不同的逆境胁迫均有响应。陕合6号TaDlea3基因对干旱胁迫的响应较显著,而郑引1号TaDlea3基因对ABA胁迫的响应较显著。3.通过农杆菌介导法,获得转基因拟南芥植株。正常生长条件下,转基因和野生型的拟南芥在表型和生理指标上无差异,干旱胁迫后,转TaDlea3基因拟南芥的叶片保持正常,而野生型的拟南芥叶片有明显的萎焉现象。多种生理指标的变化结果表明,在干旱胁迫下,转基因拟南芥中丙二醛含量和相对电导率均低于野生型,而转基因拟南芥中的脯氨酸和三个氧化酶的含量均显著高于野生型,说明TaDlea3基因的过表达提高了转基因的拟南芥抗旱能力。4.对2种耐旱型不同品种的陕合6号(干旱耐受型)和郑引1号(干旱敏感型)小麦,分别在4个生长时期进行干旱胁迫和复水处理,实时定量RT-PCR和Western blot分析TaDlea3基因在转录和翻译水平上的表达。结果表明,4个不同的生长时期的TaDlea3基因在转录和翻译水平上的变化趋势一致,干旱程度越严重,该基因和蛋白的表达量越高,复水后该基因和蛋白的表达量下降。开花期的TaDlea3基因和蛋白的表达量均低于其它3个生长时期。干旱条件下,2个不同品种小麦的4个生长时期的生理指标丙二醛、相对电导率、脯氨酸和叶绿素含量的变化趋势基本一致。对产量的分析表明苗期和分蘖期干旱对小麦的产量影响较轻,而拔节期和开花期干旱则严重影响小麦产量。结果表明TaDlea3基因和蛋白表达和干旱胁迫有一定的相关性。
[Abstract]:Drought, high salt and low temperature abiotic stress is an important factor affecting the growth and yield of crops. When crops under abiotic stress, in order to reduce the crop damage can induce the expression of some proteins. Late embryogenesis abundant protein (Late embryogenesis abundant protein, LEA) is a kind of stress-related protein.LEA proteins can be divided into 7 groups, including third LEA (LEA3) protein containing 11 amino acid tandem repeat.LEA3 protein can form alpha helix structure, protect biological macromolecules in abiotic stress, reduce the cause plant damage. However the protection function and mechanism of LEA3 protein is still not clear. Study on the cloning of 1 new LEA3 genes from wheat, the subcellular localization of in vitro conservation of lactic dehydrogenase activity in Arabidopsis, transgenic expression validation and response to abiotic stresses. To evaluate the drought resistant gene function; while the 4 growth period of 2 different drought resistant wheat varieties (seedling, tillering, jointing and anthesis) of different degrees of drought stress and rewatering, study the gene variation in the whole growth period of wheat and response to drought from transcription and translation level, the main results are as follows: 1. by electronic clone and RT-PCR method from wheat Shanhe No. 6 cloning 1 new LEA3 gene (named TaDlea3 gene) and 4 new LEA3 gene homology of TaWSI18 gene. Bioinformatics analysis showed that TaDlea3 protein and TaWSI18 protein 4 are hydrophilic, located in cytoplasm and no transmembrane domain. TaWSI18 protein and TaDlea3 in wheat and other species of LEA3 protein with high homology. The expression of TaDlea3 protein by prokaryotic high specificity, preparation and purification of this protein and preparation The polyclonal antibody.2. subcellularlocalization vector injected into tobacco leaves via Agrobacterium injection method and protoplast preparation, the results show that the TaDlea3 protein located in the cytoplasm. In high temperature and low temperature stress, the protective effect of TaDlea3 protein on LDH were different. Some adversity stress wheat Shanhe No. 6 and Zheng 1, the results show that 2 kinds of wheat TaDlea3 gene in different stress response. Both Shanhe No. 6, TaDlea3 gene significantly in response to drought stress, and Zhengyin 1 TaDlea3 gene in response to ABA stress significantly.3. by Agrobacterium mediated method. Transgenic Arabidopsis plants normal growth conditions. Next, transgenic and wild type Arabidopsis differences in phenotypic and physiological indexes, after drought stress, the leaves of TaDlea3 transgenic Arabidopsis remains normal and wild type Arabidopsis leaves have obvious wilting phenomenon. The changes of physiological indices showed that under drought stress, MDA content and relative conductivity of transgenic Arabidopsis were lower than the wild type, while the content of proline in transgenic Arabidopsis and three enzymes were significantly higher than that of the wild type, that over expression of TaDlea3 gene increased the transgenic Arabidopsis thaliana.4. on 2 different drought resistant type variety Shanhe No. 6 (drought resistant) and Zhengyin No. 1 (drought sensitive) wheat, respectively under drought stress and Rewatering in 4 growth periods, the expression of Western blot and real-time quantitative RT-PCR analysis of TaDlea3 gene in transcription and translation level. The results show that the consistent variation trend of TaDlea3 gene 4 different growth periods at the levels of transcription and translation of the drought is more serious, the expression level of gene and protein is higher, the expression level of gene and protein decreased after rewatering. The expression of TaDlea3 mRNA and protein in the flowering stage were lower than that of the other 3 growth period. Under drought conditions, the relative conductivity of physiological indexes of MDA, 4 growth period of wheat in 2 different varieties, the change trend of proline and chlorophyll content are basically the same. The analysis shows that the effects on Yield Yield drought seedling and tillering stage of wheat the lighter, and the drought at jointing stage and anthesis stage is seriously affected wheat yield. The results showed that the TaDlea3 gene and protein expression and drought stress had certain relevance.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S512.1;Q943.2

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