林烟草CBL基因家族成员NsylCBL2的功能分析

发布时间：2018-07-11 12:54

本文选题：NsylCBL2 + 克隆　；参考：《中国农业科学院》2016年硕士论文

【摘要】：类钙调神经素B亚基蛋白(CBL)是植物中普遍存在的一种钙感受器家族。很多研究表明CBL在植物响应生物胁迫和非生物胁迫中起着至关重要的作用。因此,它被认为是基因抗逆育种工作中很重要的候选基因。本研究旨在从林烟草中分离CBL基因并对其进行功能验证。主要工作内容如下:1.利用同源克隆的策略,从林烟草的叶片中获得一个基因。进化树分析表明该基因与其它物种中的CBL2基因在同一个分支上。因此将其命名为林烟草CBL2基因。多序列比对分析表明林烟草CBL2蛋白与拟南芥CBL2蛋白的序列相似度高达89.38%,与杨树、水稻和玉米CBL2蛋白的序列相似度分别为86.22%、84.00%、48.21%。2.NsylCBL2的CDS序列全长为675 bp,编码一个由223个氨基酸组成的蛋白质,其基因组包括7个内含子和8个外显子。NsylCBL2的4个EF手型结构之间的距离分别为23、25和32个氨基酸。生物信息学分析表明NsylCBL2的结构和理化性质与其它物种中CBL2相似。3.通过浸花法将NsylCBL2转入拟南芥中,对T1代植株进行RCR阳性鉴定和RT-PCR表达量检测,对T2和T3代植株进行抗性筛选,T3代纯合株系用来进行NsylCBL2的功能验证,拟南芥cbl2突变体从TAIR上订购并进行了T-DNA插入验证。4.已有研究表明CBL2基因参与盐和镁的信号转导,因此本试验也进行了高盐和高镁处理,来检测NsylCBL2是否也具有这样的功能。但是萌发、表型观察和生理数据表明过表达NsylCBL2拟南芥植株、突变体与野生型在这些处理下没有明显差异。因此,NsylCBL2可能不参与盐与镁的信号转导。5.另一方面,pCHF3-NsylCBL2这个质粒通过叶盘转化法侵染到烟草里面。过表达转基因烟草就已经获得,并且表达量水平高于普通烟草。从转基因烟草收到种子后做特定的抗性筛选检测测试,获得符合3:1比例的T1代,然后种到温室获得T2代,供以后的实验做准备。6.获得NsylCBL2的启动子序列并对其进行了预测分析,发现其含有多种逆境和激素相关转录因子。构建了NsylCBL2启动子与GUS基因融合的表达载体pBI101-CBL2pro。本研究是对NsylCBL2基因的首次报道。因此,本研究期望能给NsylCBL2今后的研究提供一定的指导。
[Abstract]:Calmodulin B subunit protein (CBL) is a common calcium receptor family in plants. Many studies have shown that CBL plays an important role in plant response to biotic and abiotic stresses. Therefore, it is considered to be an important candidate gene in gene resistance breeding. The purpose of this study was to isolate CBL gene from forest tobacco and verify its function. The main work is as follows: 1. Using the strategy of homologous cloning, a gene was obtained from the leaves of forest tobacco. Phylogenetic tree analysis showed that the gene was in the same branch as CBL2 gene in other species. Therefore, it was named CBL2 gene of forest tobacco. The sequence similarity between CBL2 protein and CBL2 protein in Arabidopsis thaliana was 89.38 and 89.38 respectively. The sequence similarity of CBL2 protein in rice and maize is 86.22 and 84.00, respectively. The CDS sequence of NsylCBL2 is 675 BP, which encodes a 223 amino acid protein. The distance between the four EF structures of its genome including 7 introns and 8 exons. NsylCBL2 was 23 25 and 32 amino acids respectively. Bioinformatics analysis showed that the structure and physicochemical properties of NsylCBL2 were similar to those of other species. NsylCBL2 was transferred into Arabidopsis thaliana by flower soaking method. RCR positive identification and RT-PCR expression of T1 generation plants were detected, and resistance screening of T _ 2 and T _ 3 generation plants were carried out to verify the function of NsylCBL2 in Arabidopsis thaliana. Arabidopsis cbl2 mutants were ordered from TAIR and verified by T-DNA insertion. It has been shown that CBL2 gene is involved in the signal transduction of salt and magnesium. However, germinating, phenotypic observation and physiological data showed that NsylCBL2 was overexpressed in Arabidopsis thaliana plants, and there was no significant difference between mutant and wild type under these treatments. Therefore, NsylCBL2 may not be involved in the signal transduction of salt and magnesium. On the other hand, the plasmid pCHF 3-asylum CBL 2 infects tobacco by leaf disc transformation. Overexpression transgenic tobacco has been obtained, and the level of expression is higher than that of ordinary tobacco. After receiving the seeds from transgenic tobacco, a specific screening test of resistance was performed to obtain T1 generation with a ratio of 3:1, and then to plant in greenhouse to obtain T2 generation, which could be used to prepare for the later experiment. The promoter sequence of asylum CBL2 was obtained and predicted. It was found that it contained many stress and hormone related transcription factors. The expression vector pBI101-CBL2pro. was constructed. This study is the first report of NsylCBL 2 gene. Therefore, this study is expected to provide some guidance for the future study of asylum CBL 2.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S572

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