小麦钙调磷酸酶B亚基类蛋白（CBL）的基因克隆及TaCBL1的功能研究
发布时间：2018-09-07 10:00
【摘要】：小麦是全球广泛种植的主要农作物之一,各种自然灾害,如旱、涝、盐碱等环境的急剧变化以及病虫害,导致小麦大量减产。因此,提高抗逆性对于保证小麦产量具有非常积极的意义。在植物中,很多外界环境的刺激都会引起细胞内钙离子浓度发生变化,从而传递外界信息进入细胞内进而引起各种细胞内和细胞间发生响应的生理生化反应,调整自身适应外界坏境以求在严苛的自然环境中生存下来。作为钙离子信号的感受器,钙离子结合蛋白家族在植物信号转导和抗逆过程中发挥重要作用。目前,植物中已发现的钙离子结合蛋白主要有三大类:钙调素(calmodulin,CaM)类蛋白、钙依赖蛋白激酶(calcium-dependent protein kinase,CDPK)以及钙调磷酸酶B类蛋白(calcineurin B-like protein,CBL)。本文旨在获得小麦CBL基因序列,检测其表达特性及亚细胞定位,以了解小麦CBL基因在抗逆中的作用,并对烟草进行遗传转化,进一步研究CBL的功能,为最终培育高抗性小麦奠定基础。本文主要获得的主要研究结果如下:(1)利用生物信息技术,将从数据库中获取的小麦CBL相关EST序列进行拼接,得到11条CBL基因序列(unigenes);利用分子生物学手段成功获得其中三个包含完整ORF(Open Reading Frame)的CBL基因cDNA,所获得的基因所编码的蛋白质分别与水稻的CBL1、CBL3和CBL6高度同源,因此对应的基因被命名为TaCBL1、TaCBL3和TaCBL6。对蛋白质进行结构分析也发现,小麦中的CBL蛋白与拟南芥、水稻、杨树、玉米等植物中相应的CBL蛋白结构极其相似。(2)通过实时荧光定量RT-PCR(Real-time Quantitative PCR,qRT-PCR)技术,分析小麦多个部位的多个发育阶段中TaCBL1、TaCBL3和TaCBL6的基因表达情况,并检测了小麦幼苗在模拟的低温、干旱、高盐胁迫及外源脱落酸(abscisic acid,ABA)刺激下的基因表达变化。实验结果显示,一个TaCBL基因可以响应多种逆境,而一种逆境信号可以引起多个TaCBL基因的响应。(3)分别构建了TaCBL1-GFP、TaCBL3-GFP和TaCBL6-GFP融合蛋白的表达载体并转化洋葱表皮细胞,在细胞膜上均观察到了TaCBL1、TaCBL3和TaCBL6的GFP融合蛋白。(4)构建了pBI-TaCBL1载体,利用农杆菌介导的遗传转化方法转化烟草(Nicotiana tabacum L.cv.Samsun)。通过对T2代转基因烟草进行抗逆分析发现,过表达TaCBL1可以提高烟草植株的抗旱性。转基因烟草植株的水含量提高,细胞损伤减轻,并且抗氧化能力增强。以上实验结果证明了小麦TaCBL1、TaCBL3、TaCBL6蛋白具有CBL蛋白家族的典型结构特征,参与了小麦对于逆境信号的响应;过表达TaCBL1基因可以通过增加植物抗氧化能力抵御细胞损伤,从而提高了转基因烟草对于干旱环境的适应能力。以上实验结果为研究和了解CBL基因及蛋白家族的特征、功能及其参与调控信号通路的机制奠定了基础,并为寻找培育高抗性作物的途径提供了新的思路。
[Abstract]:Wheat is one of the main crops widely planted in the world. All kinds of natural disasters, such as drought, waterlogging, salt alkali and other environmental changes, as well as pests and diseases, lead to a large number of wheat production. Therefore, improving the resistance to stress has a very positive significance to ensure wheat yield. In plants, the stimulation of many external environments will cause changes in intracellular calcium concentration, thus transmitting outside information into the cells and causing physiological and biochemical responses in various cells and between cells. Adapt yourself to the external environment in order to survive in the harsh natural environment. As a receptor of calcium signal, calcium binding protein family plays an important role in plant signal transduction and stress resistance. At present, there are three kinds of calcium binding proteins found in plants: calmodulin (calmodulin,CaM), calcium-dependent protein kinase (calcium-dependent protein kinase,CDPK) and calmodulin B protein (calcineurin B-like protein,CBL). The purpose of this study was to obtain the sequence of wheat CBL gene, to detect its expression characteristics and subcellular localization, to understand the role of wheat CBL gene in stress resistance, to conduct genetic transformation of tobacco, and to further study the function of CBL. It lays the foundation for the cultivation of high resistance wheat. The main results obtained in this paper are as follows: (1) the wheat CBL related EST sequences obtained from the database are spliced by bioinformatics. A total of 11 CBL gene sequences were obtained by using molecular biological methods. Three of the CBL gene cDNA, containing complete ORF (Open Reading Frame) were obtained, and the proteins encoded by CBL gene cDNA, were highly homologous to CBL1,CBL3 and CBL6 of rice, respectively. So the corresponding genes are named TaCBL1,TaCBL3 and TaCBL6.. It was also found that the structure of CBL protein in wheat was very similar to that in Arabidopsis thaliana, rice, poplar, corn and so on. (2) Real-time quantitative RT-PCR (Real-time Quantitative PCR,qRT-PCR) technique was used. The gene expression of TaCBL1,TaCBL3 and TaCBL6 in wheat seedlings at different developmental stages was analyzed, and the changes of gene expression under simulated low temperature, drought, high salt stress and exogenous abscisic acid (abscisic acid,ABA) stimulation were detected. The results showed that one TaCBL gene could respond to various stresses, and one stress signal could induce the response of multiple TaCBL genes. (3) expression vectors of TaCBL1-GFP,TaCBL3-GFP and TaCBL6-GFP fusion proteins were constructed and transformed into onion epidermal cells. The GFP fusion protein of TaCBL1,TaCBL3 and TaCBL6 was observed on the cell membrane. (4) the pBI-TaCBL1 vector was constructed and transformed into tobacco (Nicotiana tabacum L.cv.Samsun by Agrobacterium tumefaciens-mediated genetic transformation. Through the stress resistance analysis of T 2 transgenic tobacco, it was found that overexpression of TaCBL1 could improve the drought resistance of tobacco plants. The water content of transgenic tobacco plants was increased, cell damage was alleviated, and antioxidant capacity was enhanced. The results show that wheat TaCBL1,TaCBL3,TaCBL6 protein has the typical structural characteristics of CBL protein family and participates in the response of wheat to stress signal. Overexpression of TaCBL1 gene can resist cell damage by increasing the antioxidant ability of plants. Therefore, the adaptability of transgenic tobacco to drought environment was improved. These results provide a basis for the study and understanding of the characteristics, functions and mechanisms involved in the regulation of signaling pathways of CBL genes and proteins, and provide a new idea for the development of high resistant crops.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
本文编号：2227929
[Abstract]:Wheat is one of the main crops widely planted in the world. All kinds of natural disasters, such as drought, waterlogging, salt alkali and other environmental changes, as well as pests and diseases, lead to a large number of wheat production. Therefore, improving the resistance to stress has a very positive significance to ensure wheat yield. In plants, the stimulation of many external environments will cause changes in intracellular calcium concentration, thus transmitting outside information into the cells and causing physiological and biochemical responses in various cells and between cells. Adapt yourself to the external environment in order to survive in the harsh natural environment. As a receptor of calcium signal, calcium binding protein family plays an important role in plant signal transduction and stress resistance. At present, there are three kinds of calcium binding proteins found in plants: calmodulin (calmodulin,CaM), calcium-dependent protein kinase (calcium-dependent protein kinase,CDPK) and calmodulin B protein (calcineurin B-like protein,CBL). The purpose of this study was to obtain the sequence of wheat CBL gene, to detect its expression characteristics and subcellular localization, to understand the role of wheat CBL gene in stress resistance, to conduct genetic transformation of tobacco, and to further study the function of CBL. It lays the foundation for the cultivation of high resistance wheat. The main results obtained in this paper are as follows: (1) the wheat CBL related EST sequences obtained from the database are spliced by bioinformatics. A total of 11 CBL gene sequences were obtained by using molecular biological methods. Three of the CBL gene cDNA, containing complete ORF (Open Reading Frame) were obtained, and the proteins encoded by CBL gene cDNA, were highly homologous to CBL1,CBL3 and CBL6 of rice, respectively. So the corresponding genes are named TaCBL1,TaCBL3 and TaCBL6.. It was also found that the structure of CBL protein in wheat was very similar to that in Arabidopsis thaliana, rice, poplar, corn and so on. (2) Real-time quantitative RT-PCR (Real-time Quantitative PCR,qRT-PCR) technique was used. The gene expression of TaCBL1,TaCBL3 and TaCBL6 in wheat seedlings at different developmental stages was analyzed, and the changes of gene expression under simulated low temperature, drought, high salt stress and exogenous abscisic acid (abscisic acid,ABA) stimulation were detected. The results showed that one TaCBL gene could respond to various stresses, and one stress signal could induce the response of multiple TaCBL genes. (3) expression vectors of TaCBL1-GFP,TaCBL3-GFP and TaCBL6-GFP fusion proteins were constructed and transformed into onion epidermal cells. The GFP fusion protein of TaCBL1,TaCBL3 and TaCBL6 was observed on the cell membrane. (4) the pBI-TaCBL1 vector was constructed and transformed into tobacco (Nicotiana tabacum L.cv.Samsun by Agrobacterium tumefaciens-mediated genetic transformation. Through the stress resistance analysis of T 2 transgenic tobacco, it was found that overexpression of TaCBL1 could improve the drought resistance of tobacco plants. The water content of transgenic tobacco plants was increased, cell damage was alleviated, and antioxidant capacity was enhanced. The results show that wheat TaCBL1,TaCBL3,TaCBL6 protein has the typical structural characteristics of CBL protein family and participates in the response of wheat to stress signal. Overexpression of TaCBL1 gene can resist cell damage by increasing the antioxidant ability of plants. Therefore, the adaptability of transgenic tobacco to drought environment was improved. These results provide a basis for the study and understanding of the characteristics, functions and mechanisms involved in the regulation of signaling pathways of CBL genes and proteins, and provide a new idea for the development of high resistant crops.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
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1 周颐;小麦钙调磷酸酶B亚基类蛋白（CBL）的基因克隆及TaCBL1的功能研究[D];华中科技大学;2016年
，本文编号：2227929
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