苹果类钙调磷酸酶B亚基蛋白MdCBL3基因的克隆与功能鉴定

发布时间：2018-03-30 09:34

本文选题：苹果　切入点：MdCBL3　出处：《山东农业大学》2017年硕士论文

【摘要】：植物在生长过程中经常会遇到高盐、干旱、水涝和低温等非生物逆境胁迫。植物感知到非生物胁迫后会产生一些防御机制从而避免伤害发生。植物CBL基因在植物响应逆境胁迫过程中发挥十分重要的作用。拟南芥中对CBL研究较多,CBL能够响应多种胁迫,尤其是盐胁迫,但在果树等木本植物中研究较少。本研究从‘嘎拉’苹果(Malus×domestica Borkh.‘Gala’)中克隆了一个CBL基因MdCBL3(序列号:MDP0000155124),并对其进行生物信息学分析,利用定量PCR技术检测其在不同组织和逆境条件下的表达水平。通过农杆菌介导法获得转基因苹果愈伤组织、拟南芥,并对转基因愈伤、拟南芥进行功能鉴定。旨在为进一步深入研究苹果CBL的功能奠定基础。具体研究工作和结果如下:1.克隆了苹果MdCBL3基因,其由852 bp碱基组成,编码284个氨基酸残基的多肽链。基因组结构分析表明MdCBL3基因位于1号染色体上,含有8个外显子和7个内含子。2.荧光定量PCR分析表明MdCBL3基因在苹果‘嘎拉’中的表达情况,发现MdCBL3基因在所有组织中都有表达,在根中表达量最高。3.蛋白同源比对分析显示,MdCBL3含有3个保守的EF手形结构域EF-hand calcium binding motif,与拟南芥AtCBL3蛋白序列类似,表明苹果CBL3与拟南芥CBL3具有较高的同源性。4.利用Plant Care数据库对MdCBL3进行启动子顺式作用元件的预测分析,MdCBL3启动子序列中存在低温、干旱、光、生长素、赤霉素、水杨酸、防御等响应元件。表明MdCBL3可能受低温、干旱、光、激素等多种环境的调控,通过参与复杂的的生物学过程来调控其特定的生长发育过程。5.荧光定量PCR分析MdCBL3基因在不同胁迫的响应,发现MdCBL3基因对盐、干旱、低温有一定的响应。6.构建MdCBL3表达载体并利用农杆菌介导法侵染苹果愈伤组织、野生型拟南芥。半定量和定量RT-PCR证明MdCBL3在苹果愈伤组织中过量表达,在拟南芥中得到异源表达。表型分析表明与野生型愈伤组织和拟南芥对照相比,在盐胁迫下转基因苹果愈伤和拟南芥生长更好,抗性更强。表明在苹果愈伤中过量表达MdCBL3,在拟南芥中异源表达MdCBL3能明显提高对盐胁迫的抗性。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S661.1

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