發(fā)布時間：2018-03-30 09:34

  本文選題：蘋果　切入點：MdCBL3　出處：《山東農(nóng)業(yè)大學》2017年碩士論文

【摘要】：植物在生長過程中經(jīng)常會遇到高鹽、干旱、水澇和低溫等非生物逆境脅迫。植物感知到非生物脅迫后會產(chǎn)生一些防御機制從而避免傷害發(fā)生。植物CBL基因在植物響應逆境脅迫過程中發(fā)揮十分重要的作用。擬南芥中對CBL研究較多,CBL能夠響應多種脅迫,尤其是鹽脅迫,但在果樹等木本植物中研究較少。本研究從‘嘎拉’蘋果(Malus×domestica Borkh.‘Gala’)中克隆了一個CBL基因MdCBL3(序列號:MDP0000155124),并對其進行生物信息學分析,利用定量PCR技術檢測其在不同組織和逆境條件下的表達水平。通過農(nóng)桿菌介導法獲得轉(zhuǎn)基因蘋果愈傷組織、擬南芥,并對轉(zhuǎn)基因愈傷、擬南芥進行功能鑒定。旨在為進一步深入研究蘋果CBL的功能奠定基礎。具體研究工作和結(jié)果如下:1.克隆了蘋果MdCBL3基因,其由852 bp堿基組成,編碼284個氨基酸殘基的多肽鏈。基因組結(jié)構分析表明MdCBL3基因位于1號染色體上,含有8個外顯子和7個內(nèi)含子。2.熒光定量PCR分析表明MdCBL3基因在蘋果‘嘎拉’中的表達情況,發(fā)現(xiàn)MdCBL3基因在所有組織中都有表達,在根中表達量最高。3.蛋白同源比對分析顯示,MdCBL3含有3個保守的EF手形結(jié)構域EF-hand calcium binding motif,與擬南芥AtCBL3蛋白序列類似,表明蘋果CBL3與擬南芥CBL3具有較高的同源性。4.利用Plant Care數(shù)據(jù)庫對MdCBL3進行啟動子順式作用元件的預測分析,MdCBL3啟動子序列中存在低溫、干旱、光、生長素、赤霉素、水楊酸、防御等響應元件。表明MdCBL3可能受低溫、干旱、光、激素等多種環(huán)境的調(diào)控,通過參與復雜的的生物學過程來調(diào)控其特定的生長發(fā)育過程。5.熒光定量PCR分析MdCBL3基因在不同脅迫的響應,發(fā)現(xiàn)MdCBL3基因?qū)}、干旱、低溫有一定的響應。6.構建MdCBL3表達載體并利用農(nóng)桿菌介導法侵染蘋果愈傷組織、野生型擬南芥。半定量和定量RT-PCR證明MdCBL3在蘋果愈傷組織中過量表達,在擬南芥中得到異源表達。表型分析表明與野生型愈傷組織和擬南芥對照相比,在鹽脅迫下轉(zhuǎn)基因蘋果愈傷和擬南芥生長更好,抗性更強。表明在蘋果愈傷中過量表達MdCBL3,在擬南芥中異源表達MdCBL3能明顯提高對鹽脅迫的抗性。
[Abstract]:Plants often experience high salt and drought in the course of their growth. Abiotic stresses, such as waterlogging and low temperature, produce some defense mechanisms to avoid injury. Plant CBL gene plays a very important role in plant response to stress stress. In Arabidopsis thaliana, more studies on CBL can respond to multiple stresses. In this study, a CBL gene MdCBL3 (sequence number: MDP00155124) was cloned from Malus 脳 domestica Borkh.Gala, and analyzed by bioinformatics. The expression level of transgenic apple callus, Arabidopsis thaliana (Arabidopsis thaliana) was obtained by means of Agrobacterium tumefaciens mediated by quantitative PCR. Function identification of Arabidopsis thaliana was carried out in order to lay a foundation for further study on the function of apple CBL. The specific research work and results are as follows: 1.The MdCBL3 gene of apple was cloned and composed of 852bp base. The genomic structure analysis showed that the MdCBL3 gene was located on chromosome 1, containing 8 exons and 7 introns. Fluorescence quantitative PCR analysis showed the expression of MdCBL3 gene in apple 'Gala'. It was found that MdCBL3 gene was expressed in all tissues, and the highest expression was found in root. The homology analysis of MdCBL3 showed that MdCBL3 contained three conserved EF chiral domain EF-hand calcium binding motif, which was similar to Arabidopsis AtCBL3 protein sequence. The results showed that apple CBL3 had high homology with Arabidopsis thaliana CBL3. 4.Using Plant Care database to predict the promoter cis-acting element of MdCBL3, there were low temperature, drought, light, auxin, gibberellin and salicylic acid in the promoter sequence of MdCBL3. Defense and other response elements. Suggests that MdCBL3 may be regulated by low temperature, drought, light, hormones, etc. By participating in complex biological processes to regulate their specific growth and development process. 5. Fluorescence quantitative PCR analysis of the response of MdCBL3 gene to different stresses, found that MdCBL3 gene to salt, drought, The expression vector of MdCBL3 was constructed and infected with Agrobacterium tumefaciens to infect apple callus and wild type Arabidopsis thaliana. Semi-quantitative and quantitative RT-PCR showed that MdCBL3 was overexpressed in apple callus. Phenotypic analysis showed that transgenic apple callus and Arabidopsis grew better under salt stress than wild type callus and Arabidopsis control. The results showed that overexpression of MdCBL3 in apple callus and heterologous expression of MdCBL3 in Arabidopsis could significantly increase the resistance to salt stress.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S661.1

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