【摘要】：小麥?zhǔn)侨驈V泛種植的主要農(nóng)作物之一,各種自然災(zāi)害,如旱、澇、鹽堿等環(huán)境的急劇變化以及病蟲害,導(dǎo)致小麥大量減產(chǎn)。因此,提高抗逆性對(duì)于保證小麥產(chǎn)量具有非常積極的意義。在植物中,很多外界環(huán)境的刺激都會(huì)引起細(xì)胞內(nèi)鈣離子濃度發(fā)生變化,從而傳遞外界信息進(jìn)入細(xì)胞內(nèi)進(jìn)而引起各種細(xì)胞內(nèi)和細(xì)胞間發(fā)生響應(yīng)的生理生化反應(yīng),調(diào)整自身適應(yīng)外界壞境以求在嚴(yán)苛的自然環(huán)境中生存下來。作為鈣離子信號(hào)的感受器,鈣離子結(jié)合蛋白家族在植物信號(hào)轉(zhuǎn)導(dǎo)和抗逆過程中發(fā)揮重要作用。目前,植物中已發(fā)現(xiàn)的鈣離子結(jié)合蛋白主要有三大類:鈣調(diào)素(calmodulin,CaM)類蛋白、鈣依賴蛋白激酶(calcium-dependent protein kinase,CDPK)以及鈣調(diào)磷酸酶B類蛋白(calcineurin B-like protein,CBL)。本文旨在獲得小麥CBL基因序列,檢測(cè)其表達(dá)特性及亞細(xì)胞定位,以了解小麥CBL基因在抗逆中的作用,并對(duì)煙草進(jìn)行遺傳轉(zhuǎn)化,進(jìn)一步研究CBL的功能,為最終培育高抗性小麥奠定基礎(chǔ)。本文主要獲得的主要研究結(jié)果如下:(1)利用生物信息技術(shù),將從數(shù)據(jù)庫(kù)中獲取的小麥CBL相關(guān)EST序列進(jìn)行拼接,得到11條CBL基因序列(unigenes);利用分子生物學(xué)手段成功獲得其中三個(gè)包含完整ORF(Open Reading Frame)的CBL基因cDNA,所獲得的基因所編碼的蛋白質(zhì)分別與水稻的CBL1、CBL3和CBL6高度同源,因此對(duì)應(yīng)的基因被命名為TaCBL1、TaCBL3和TaCBL6。對(duì)蛋白質(zhì)進(jìn)行結(jié)構(gòu)分析也發(fā)現(xiàn),小麥中的CBL蛋白與擬南芥、水稻、楊樹、玉米等植物中相應(yīng)的CBL蛋白結(jié)構(gòu)極其相似。(2)通過實(shí)時(shí)熒光定量RT-PCR(Real-time Quantitative PCR,qRT-PCR)技術(shù),分析小麥多個(gè)部位的多個(gè)發(fā)育階段中TaCBL1、TaCBL3和TaCBL6的基因表達(dá)情況,并檢測(cè)了小麥幼苗在模擬的低溫、干旱、高鹽脅迫及外源脫落酸(abscisic acid,ABA)刺激下的基因表達(dá)變化。實(shí)驗(yàn)結(jié)果顯示,一個(gè)TaCBL基因可以響應(yīng)多種逆境,而一種逆境信號(hào)可以引起多個(gè)TaCBL基因的響應(yīng)。(3)分別構(gòu)建了TaCBL1-GFP、TaCBL3-GFP和TaCBL6-GFP融合蛋白的表達(dá)載體并轉(zhuǎn)化洋蔥表皮細(xì)胞,在細(xì)胞膜上均觀察到了TaCBL1、TaCBL3和TaCBL6的GFP融合蛋白。(4)構(gòu)建了pBI-TaCBL1載體,利用農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化方法轉(zhuǎn)化煙草(Nicotiana tabacum L.cv.Samsun)。通過對(duì)T2代轉(zhuǎn)基因煙草進(jìn)行抗逆分析發(fā)現(xiàn),過表達(dá)TaCBL1可以提高煙草植株的抗旱性。轉(zhuǎn)基因煙草植株的水含量提高,細(xì)胞損傷減輕,并且抗氧化能力增強(qiáng)。以上實(shí)驗(yàn)結(jié)果證明了小麥TaCBL1、TaCBL3、TaCBL6蛋白具有CBL蛋白家族的典型結(jié)構(gòu)特征,參與了小麥對(duì)于逆境信號(hào)的響應(yīng);過表達(dá)TaCBL1基因可以通過增加植物抗氧化能力抵御細(xì)胞損傷,從而提高了轉(zhuǎn)基因煙草對(duì)于干旱環(huán)境的適應(yīng)能力。以上實(shí)驗(yàn)結(jié)果為研究和了解CBL基因及蛋白家族的特征、功能及其參與調(diào)控信號(hào)通路的機(jī)制奠定了基礎(chǔ),并為尋找培育高抗性作物的途徑提供了新的思路。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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1 周頤;小麥鈣調(diào)磷酸酶B亞基類蛋白（CBL）的基因克隆及TaCBL1的功能研究[D];華中科技大學(xué);2016年

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本文編號(hào)：2227929