烟草类胡萝卜素降解关键基因NCED3的克隆及功能研究

发布时间：2018-05-19 19:49

本文选题：烟草 + 类胡萝卜素　；参考：《河南农业大学》2016年硕士论文

【摘要】：烟叶中的类胡萝卜素以及它的降解生成物是引起烟叶挥发香味物质的主要前体物质,其中降解产物的类型以及浓度值都能够作为烟草质量的评价指标,因此,研究烟叶内部的类胡萝卜素降解有关的基因情况,确定这些基因的重要功能,对于提升烟叶质量具有重大的科研价值。类胡萝卜素裂解双加氧酶是植物类胡萝卜素降解代谢途径中的关键基因,CCDs家族有9个成员,其中包括NCED2,NCED3,NCED5,NCED6,NCED9等,本研究通过同源克隆方法获得烟草品种K326类胡萝卜素降解关键基因NCED3两个c DNA全长序列,并对其进行生物详细的生物信息学分析,采用干旱胁迫研究基因的表达情况,同时采用in-fusion和gateway方法分别构建了过表达和抑制表达载体,农杆菌介导法转化烟草植株,研究结果为进一步分析NCED3在烟草抗旱性的功能研究方面提供一定的基础。主要研究结果如下:1克隆得到烤烟品种K326类胡萝卜素降解关键基因NCED3 2个拷贝NCED3-1、NCED3-2,并提交NCBI Genbank,登录号KM605434和KM605435。序列分析表明:K326NCED3-1和NCED3-2基因分别包含一个1842bp和1830bp的开放读码框(ORF),各编码613个和609个氨基酸。跨膜区预测、亲水性和信号肽分析表明很可能是定位于线粒体膜上的亲水性跨膜蛋白。二级结构预测模型显示此蛋白结构以β折叠和无规则卷曲为主。蛋白三级结构预测分析表明NCED3-1与NCED3-2空间结构极为相似。2 PEG6000干旱胁迫分析表明,干旱胁迫可以诱导NCED3基因表达以及内源ABA的积累。且在处理12h之前,NCED3表达与ABA累积速度均呈现升高趋势,之后逐渐降低。3构建了K326 NCED3基因的过表达和RNAi表达载体,并采用农杆菌介导法转化烟草植株。获得了NCED3-1过表达和抑制表达阳性植株。
[Abstract]:Carotenoids in tobacco leaves and their degradation products are the main precursors causing volatile aroma substances in tobacco leaves. The types and concentrations of degradation products can be used as indicators of tobacco quality evaluation. It is of great scientific value to study the genes related to carotenoid degradation in tobacco leaves and to determine the important functions of these genes in order to improve the quality of tobacco leaves. Carotenoid cleavage dioxygenase is a key gene in the pathway of plant carotenoid degradation and metabolism. There are nine members of the CCDs family, including NCED2, NCED3, NCED5, NCED6, NCED9, and so on. In this study, the full-length sequence of NCED3 two c DNA, a key gene of carotenoid degradation in tobacco variety K326, was obtained by homologous cloning method, and its bioinformatics was analyzed in detail, and the expression of the gene was studied by drought stress. At the same time, the overexpression and inhibition expression vectors were constructed by in-fusion and gateway methods respectively. Agrobacterium tumefaciens mediated transformation of tobacco plants. The results provided a basis for further analysis of the function of NCED3 in the study of tobacco drought resistance. The main results were as follows: 1. The two copies of the key gene NCED3 of carotenoid degradation in flue-cured tobacco variety K326 were cloned as follows: NCED3-1, NCED3-2, and submitted to NCBI Genbank, accession numbers KM605434 and KM605435. Sequence analysis showed that the gene K326NCED3-1 and NCED3-2 contained an open reading frame of 1842bp and 1830bp, encoding 613 and 609 amino acids respectively. The hydrophilicity and signal peptide analysis indicated that the transmembrane proteins were probably located on the mitochondrial membrane. The prediction model of secondary structure showed that the protein structure was mainly 尾-folding and irregular crimp. Protein tertiary structure prediction analysis showed that the spatial structure of NCED3-1 and NCED3-2 was very similar to that of NCED3-2. 2 PEG6000 drought stress analysis showed that drought stress could induce the expression of NCED3 gene and the accumulation of endogenous ABA. The expression of NCED3 and the accumulation rate of ABA increased before treatment for 12 h, then the overexpression of K326 NCED3 gene and the expression of RNAi were gradually decreased. The vector was transformed into tobacco plants by Agrobacterium tumefaciens (Agrobacterium tumefaciens). NCED3-1 overexpression and inhibition expression positive plants were obtained.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S572

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