小麦扩展蛋白基因TaEXPA2的功能分析

发布时间：2018-05-16 21:24

本文选题：小麦(Triticum + aestivum　；参考：《山东农业大学》2016年博士论文

【摘要】：小麦(Triticum aestivum L.)是我国重要的粮食作物。小麦生长过程中常常遭受环境胁迫的危害,进而导致生长抑制和产量降低。通过基因工程技术提高小麦的逆境适应性是小麦新品种培育的发展方向。研究逆境相关基因功能可以为小麦基因工程育种奠定理论基础,储备基因资源。扩展蛋白是一种调节细胞壁松弛和细胞延伸生长的细胞壁蛋白,普遍存在于植物所有的细胞、组织和器官中。1989年美国宾夕法尼亚州立大学Cosgrove教授的实验室研究黄瓜下胚轴细胞延伸性生长,并首次发现了扩展蛋白(Expansin)。近年来,扩展蛋白的研究成果不断丰富。现已证明扩展蛋白参与多种植物生长发育过程及逆境适应性响应。但是,由于小麦基因组较大、遗传背景复杂以及转化效率低等问题,小麦扩展蛋白基因功能的研究报道较少。扩展蛋白由多基因家族编码,家族成员之间在基因结构、蛋白结构、生化特性、进化关系等方面存在多方面的异同。其中EXPA和EXPB是两个最大、研究最清楚的基因家族。本实验室先前克隆了小麦EXPB家族基因Ta EXPB23,发现该基因的表达对多种非生物胁迫和激素信号产生响应。组成型过表达、根特异性以及胁迫诱导表达该基因均增强了转基因烟草的抗旱性。鉴于EXPA和EXPB家族成员存在多方面的异同,本研究中选择小麦EXPA基因Ta EXPA2(Gen Bank accession number:AAT94292.1),利用过量表达技术研究该基因在植物生长发育及逆境适应中的作用,分析其生理机制,并与EXPB家族基因Ta EXPB23的功能相比较,为解释不同家族基因功能上的异同点提供参考。首先克隆了Ta EXPA2的启动子区域,预测分析了该启动子的顺式作用元件。分析了该基因的时空表达、激素响应和逆境响应表达模式;构建Ta EXPA2基因表达载体,转化烟草获得了转基因植株;利用过表达Ta EXPA2的转基因烟草,研究了该基因在植物生长发育调节、耐旱、耐盐中的作用,并进一步探讨了其生理机制;最后,为研究深入探讨过表达扩展蛋白提高转基因植株抗氧化能力的分子机制,利用拟南芥同源基因突变体atexpa2,并将Ta EXPA2在突变体atexpa2中进行过表达回补实验,初步分析了扩展蛋白与细胞壁过氧化物酶之间的关系。研究主要结果与结论如下:(1)利用Tail-PCR克隆技术获得了Ta EXPA2基因启动子区域1073bp的片段(Gen Bank ID:KP729264),用Plant CARE和PLACE功能预测软件对其进行了分析,结果表明该启动子区域含有多种光响应元件、组织特异表达元件、多种激素响应元件和逆境(干旱、ABA和高温)响应元件。(2)将Ta EXPA2::GFP融合蛋白在洋葱表皮中瞬时表达,证实Ta EXPA2蛋白定位于细胞壁。(3)q RT-PCR检测了小麦中Ta EXPA2基因表达的组织特特异性以及对外源激素和非生物胁迫的响应模式。结果表明,Ta EXPA2在小麦根、茎、叶中均有表达,叶片中的表达量最高;Ta EXPA2基因表达对多种逆境胁迫和外源激素处理产生响应,干旱胁迫诱导基因表达水平上调。(4)构建Ta EXPA2转化载体,转化烟草获得了不同水平过表达Ta EXPA2的转基因烟草株系。对转基因烟草的生长发育表型进行观察记录,结果发现过表达Ta EXPA2提高了转基因烟草的种子产量和果荚数,但是没有改变转基因烟草营养生长表型及种子的大小。(5)对转基因烟草进行干旱胁迫处理,观察了萌发率、幼苗生长和成苗生长,结果发现过表达Ta EXPA2提高了转基因烟草的抗旱性。从水分保持能力、光合作用特性、抗氧化特性等方面研究了转基因烟草抗旱性提高的生理机制,发现转基因烟草抗旱性的提高与根系发达、水分状况维持能力强以及抗氧化能力较强有关。抗氧化能力的提高与相关基因表达上调有关。(6)对转基因烟草进行盐胁迫处理,观察了盐胁迫种子萌发、幼苗生长和成苗生长,结果发现过表达Ta EXPA2提高了转基因烟草的抗盐性。具体表现在盐处理以后,转基因烟草的萌发率更高,幼苗和成苗的生长状况较WT更好。从渗透胁迫耐性、离子胁迫耐性和抗氧化能力等方面探讨了转基因烟草耐盐性提高的生理机制,结果发现,烟草胁迫下转基因烟草的水分维持能力强,细胞内Na+离子积累较WT少而K+积累较WT多,抗氧化能力较WT强。特别地,用转基因烟草叶片细胞壁蛋白孵育的野生型叶圆片的抗盐性增强,且转基因烟草叶圆片增强的抗盐性可以被Ta EXPA2抗体抑制。结果表明,转基因烟草抗盐性的提高可能与钠、钾离子稳态调节和抗氧化能力增强有关。(7)初步研究了ABA信号在Ta EXPA2介导的耐盐性中的作用。用脱落酸(ABA)和Na Cl处理小麦幼苗,发现ABA和Na Cl均可以诱导小麦叶片中Ta EXPA2蛋白的积累,但是ABA的抑制剂氟啶酮(FLU)抑制小麦中Ta EXPA2蛋白的积累;同时使用Na Cl和FLU处理小麦时,Ta EXPA2蛋白的积累受到抑制。说明ABA信号与Na Cl诱导的Ta EXPA2蛋白积累有关。但是,转基因烟草中,ABA的合成基因的表达水平无论在正常情况下还是盐处理后都比野生型低。这些结果说明,ABA信号参与了Ta EXPA2调控的转基因烟草的抗盐性。(8)利用拟南芥同源基因突变体atexpa2研究过表达Ta EXPA2提高转基因烟草抗氧化能力的生理与分子机制。用H2O2处理小麦,检测Ta EXPA2的表达水平,发现Ta EXPA2的表达受过氧化氢诱导上调。将Ta EXPA2基因在拟南芥中过表达,发现过表达Ta EXPA2的转基因拟南芥对过氧化氢引起的氧化胁迫的抗性明显增强。研究转基因拟南芥抗氧化能力提高的生理机制,发现转基因拟南芥抗氧化能力的提高与植物细胞壁键连的过氧化物酶关系密切。用Ta EXPA2基因的拟南芥(Arabidopsis thaliana L.)同源基因突变体atexpa2进一步验证发现,与Col-0野生型相比,突变体atexpa2的细胞壁键连过氧化物酶活性明显较低,抗氧化胁迫能力较Col-0显著降低。将Ta EXPA2基因过表达回补到突变体atexpa2后,转基因拟南芥的抗氧化能力增强,并且拟南芥中三种过氧化物酶的活性增强。这些结果初步证明,Ta EXPA2与细胞壁POD之间关系密切。推测细胞壁扩展蛋白Ta EXPA2可能通过与细胞壁键连的过氧化物酶互作,对转基因植株的氧化胁迫能力产生影响。该结果对于解释过表达扩展蛋白提高植物的胁迫耐性具有重要意义。
[Abstract]:Wheat (Triticum aestivum L.) is an important grain crop in China. In the process of wheat growth, it is often damaged by environmental stress, which leads to growth inhibition and yield reduction. It is the direction of development of wheat new varieties to improve the adaptability of Wheat by genetic engineering technology. The function of adversity related genes can be a wheat gene. Engineering breeding lays a theoretical foundation and reserves genetic resources. Extended protein is a cell wall protein that regulates cell wall relaxation and cell elongation growth. It is ubiquitous in all plant cells, tissues and organs of Professor Cosgrove of Penn State University, Penn State University, to study the elongation of hypocotyls cells in cucumber hypocotyls. The extended protein (Expansin) has been discovered for the first time. In recent years, the research achievements of expanded protein have been enriched. It has been proved that the extended protein is involved in the growth and development process of various plants and the response to adversity adaptability. However, the function of wheat expanded protein gene function has been studied because of the large genome of wheat, complex genetic background and low conversion efficiency. There are few reports. The extended protein is encoded by the multi gene family. There are many similarities and differences between family members in gene structure, protein structure, biochemical characteristics and evolutionary relationships. Among them, EXPA and EXPB are the two largest and most clearly studied gene families. This laboratory previously cloned the wheat EXPB family gene Ta EXPB23, and found the gene. The expression has a response to a variety of abiotic stresses and hormone signals. Component overexpression, root specificity and stress induced expression of the gene enhanced the drought resistance of transgenic tobacco. In view of the diversity of EXPA and EXPB family members, the EXPA gene Ta EXPA2 (Gen Bank accession number:AAT94292.1) was selected in this study. The function of the gene in plant growth and adversity adaptation was studied by overexpression technology, and its physiological mechanism was analyzed and compared with the function of the EXPB family gene Ta EXPB23, to provide reference for explaining the similarities and differences of the function of different family genes. First, the promoter region of Ta EXPA2 was cloned, and the CIS of the promoter was predicted and analyzed. The spatial and temporal expression of the gene, the response to the hormone response and the expression pattern of adversity response were analyzed, and the Ta EXPA2 gene expression vector was constructed, and transgenic tobacco was obtained by transforming tobacco, and the transgenic tobacco expressing Ta EXPA2 was used to study the role of the gene in the regulation of plant growth and development, drought tolerance and salt tolerance, and further explored its growth. In the end, the molecular mechanism of the expression of extended protein to improve the antioxidant capacity of transgenic plants was discussed, and a homologous mutant atexpa2 of Arabidopsis thaliana was used, and Ta EXPA2 was used in the mutant atexpa2 for overexpression and supplementation. The relationship between expanded egg white and cell wall peroxidase was preliminarily analyzed. The results and conclusions are as follows: (1) the 1073bp fragment (Gen Bank ID:KP729264) of the promoter region of the Ta EXPA2 gene (Gen Bank ID:KP729264) was obtained by using the Tail-PCR cloning technique, and it was analyzed with the functional prediction software of Plant CARE and PLACE. The results showed that the promoter region contained a variety of light response components, tissue specific expression elements, and a variety of hormone response elements. And the response elements of the adversity (drought, ABA and high temperature). (2) the Ta EXPA2:: GFP fusion protein was instantaneously expressed in the onion epidermis, which confirmed that the Ta EXPA2 protein was located in the cell wall. (3) Q RT-PCR detected the specific specificity of the Ta EXPA2 gene expression in wheat and the response mode to the exogenous hormone and abiotic stress. The results showed that Ta EXPA2 was in wheat. The expression of the root, stem and leaf all was expressed, the expression of the leaf in the leaves was the highest; the expression of Ta EXPA2 gene expressed response to a variety of stress and exogenous hormone treatment, and the expression level of gene expression was up-regulated by drought stress. (4) the transformation vector of Ta EXPA2 was constructed, and transgenic tobacco plants with different levels of overexpressed Ta EXPA2 were obtained. The growth and development phenotype was observed. The results showed that overexpression of Ta EXPA2 increased the seed yield and pod number of transgenic tobacco, but did not change the growth phenotype and seed size of transgenic tobacco. (5) drought stress treatment of transgenic tobacco was carried out, germination rate, seedling growth and seedling growth were observed, and the results were found. The drought resistance of transgenic tobacco was increased by Ta EXPA2. The physiological mechanism of the increase of drought resistance in transgenic tobacco was studied from water retention ability, photosynthesis and antioxidant properties. It was found that the improvement of the drought resistance of transgenic tobacco was related to the development of root system, the strong ability of maintaining water condition and the strong antioxidant capacity. The improvement was related to the up-regulated expression of related genes. (6) salt stress treatment of transgenic tobacco was carried out to observe the seed germination, seedling growth and seedling growth of the transgenic tobacco. The results showed that the expression of Ta EXPA2 increased the salt resistance of transgenic tobacco. The physiological mechanism of salt tolerance in transgenic tobacco was discussed from the aspects of osmotic stress tolerance, ion stress tolerance and antioxidant capacity. The results showed that under tobacco stress, the water maintenance ability of transgenic tobacco was strong, the accumulation of Na+ ion in the cell was less than that of WT, and the accumulation of K + was more WT than that of WT, and the antioxidant capacity was stronger than that of WT. The salt resistance of the wild type leaf circular slices incubated with the cell wall protein of transgenic tobacco leaves was enhanced, and the salt resistance of the transgenic tobacco leaves could be inhibited by Ta EXPA2 antibody. The results showed that the salt resistance of the transgenic tobacco could be related to the stable regulation of sodium and potassium ions and the enhancement of the ability to resist oxygen. (7) the ABA signal was preliminarily studied. The effect of Ta EXPA2 on salt tolerance. The accumulation of Ta EXPA2 protein in wheat leaves could be induced by both ABA and Na Cl with abscisic acid (ABA) and Na Cl, but the accumulation of the protein in wheat was inhibited by the ABA inhibitor fluidizone (FLU). Inhibition. Indicating that ABA signals are associated with the accumulation of Ta EXPA2 protein induced by Na Cl. However, the expression level of ABA's synthetic genes in transgenic tobacco is lower than that of the wild type in both normal and salt treatments. These results suggest that ABA signals are involved in the salt resistance of genetically modified tobacco controlled by Ta EXPA2. (8) the use of Arabidopsis homologous genes. The mutant atexpa2 studied the physiological and molecular mechanisms of the expression of Ta EXPA2 to improve the antioxidant capacity of transgenic tobacco. The expression level of Ta EXPA2 was detected by H2O2 treatment and the expression of Ta EXPA2 was up regulated by hydrogen peroxide. Ta EXPA2 gene was overexpressed in Arabidopsis thaliana, and the transoxidation of transgenic Arabidopsis expressing Ta EXPA2 was found. The resistance to oxidative stress caused by hydrogen was obviously enhanced. The physiological mechanism of improving the antioxidant capacity of transgenic Arabidopsis was studied. It was found that the increase of antioxidant capacity of transgenic Arabidopsis was closely related to the peroxidase of plant cell wall bonds. The Ta EXPA2 gene Arabidopsis thaliana L. homologous gene mutant atexpa2 entered one. It was found that the cell wall bond peroxidase activity of the mutant atexpa2 was significantly lower than that of the Col-0 wild type, and the antioxidant capacity of the mutant atexpa2 was significantly lower than that of Col-0. After overexpression of the Ta EXPA2 gene, the antioxidant capacity of the transgenic Arabidopsis was enhanced and the activity of three peroxidase in Arabidopsis was increased. Strong. These results have preliminarily proved that Ta EXPA2 is closely related to the cell wall POD. It is presumed that the cell wall extension protein Ta EXPA2 may affect the oxidative stress of transgenic plants by interacting with the peroxidase linked to the cell wall bond. This result is important to explain the overexpression of extended protein to enhance the stress tolerance of plants. Righteousness.
【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2;S512.1

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