蜡梅CpNAC8转录因子基因的克隆及功能初步鉴定

发布时间：2018-02-26 18:48

本文关键词： NAC转录因子表达特性分析转录激活转基因过表达非生物胁迫　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：蜡梅[Chimonanthus praecox(L.)Link]为蜡梅科蜡梅属的落叶乔灌木植物,是我国特有的传统名贵观赏花木,作为冬季重要的园林花卉和鲜切花,具有较高的经济价值和观赏价值,对于培育抗逆的蜡梅品种是一项非常重要的任务。NAC转录因子是植物体内特有的一类能够调节植物生长发育,应答生物和非生物胁迫的重要转录因子,它能与植物体内的顺式作用元件发生特异结合,从而激活或抑制下游目标基因的表达。目前对于NAC基因的研究主要集中在模式植物中,而在非模式植物中非常有限,本研究将对蜡梅中NAC转录因子Cp NAC8的功能进行初步探索。主要实验结果如下1.Cp NAC8的克隆及分子特性从实验室的蜡梅转录组数据库获得NAC转录因子家族成员的部分基因序列,设计特异引物,通过RACE技术获得其中一个NAC基因的c DNA全长,命名为Cp NAC8。得到的基因全长1583bp,含有1479bp的最大开放阅读框(ORF),编码492个氨基酸残基。构建Cp NAC8系统进化树,并将蜡梅Cp NAC8编码的蛋白与其它NAC蛋白进行同源比对,表明Cp NAC8属于II/ONAC4亚家族,且与马铃薯的亲缘关系较近。2.Cp NAC8基因在蜡梅中的表达特征分析采取实时荧光定量PCR技术,对Cp NAC8基因在蜡梅不同组织,不同时期,不同胁迫处理(干旱、高盐、高温、低温、ABA、GA)的表达特性进行分析。表明Cp NAC8基因在蜡梅的各个组织中均有表达,但在成熟叶和雌蕊中表达量最高,推测该基因可能参与植物叶的形态建成以及雌蕊的发育。Cp NAC8基因在萌动期表达量最高,盛开期几乎不表达。Cp NAC8基因在干旱、高盐、高温、ABA、GA胁迫条件下都上调表达,但不同胁迫条件下,呈现最高表达量的时间点不同。结果推测该基因可能响应干旱、高盐、高温胁迫,可能参与ABA、GA的信号转导途径。3.CpNAC8转录激活特性验证构建酵母表达载体p GBKT7-Cp NAC8-ORF、p GBKT7-Cp NAC8-VP16,转化酵母菌株AH109,表明CpNAC8基因具有转录激活性,且是转录激活子。4.Cp NAC8基因转拟南芥的表型研究构建植物超表达载体pCAMBIA1300-CpNAC8,利用花序侵染法转化拟南芥,通过筛选得到纯合的T3代转基因拟南芥,选择表达量高,中,稍弱的三个株系进行表现观察和胁迫处理。结果表明,将蜡梅Cp NAC8基因导入野生型拟南芥,不会对其生长造成不良影响;转基因株系拟南芥种子比野生型拟南芥种子对Na Cl的敏感性更强,且土壤盐胁迫中Cp NAC8基因对拟南芥的耐盐性起着逆调控的作用;土壤干旱胁迫中Cp NAC8基因能够增强转基因拟南芥的抗旱性。初步判定CpNAC8基因与耐旱性和耐盐性相关。
[Abstract]:Chimonanthus praecox(L.)Link, a deciduous tree and shrub plant of the genus Calymaceae, is a traditional and precious ornamental flower in China. As an important garden flower and fresh cut flower in winter, Chimonanthus praecox(L.)Link has high economic value and ornamental value. NAC transcription factor is a kind of important transcription factors that can regulate plant growth and development and respond to biological and abiotic stress. It can specifically bind to cis-acting elements in plants, thus activating or inhibiting the expression of downstream target genes. At present, the study of NAC genes is mainly concentrated in model plants, but very limited in non-model plants. The main results of this study are as follows: 1. Cloning and molecular characteristics of CP NAC8 obtained some gene sequences of NAC transcription factor family from the transcriptional database of Waxberry in laboratory. A specific primer was designed to obtain the full length of c DNA of one of the NAC genes by RACE technique, named CpNAC8. the total length of the gene was 1583 BP, containing 1479bp maximum open reading frame (ORF), encoding 492 amino acid residues. The phylogenetic tree of CP NAC8 was constructed. The results showed that CP NAC8 belonged to the II/ONAC4 subfamily and had close relationship with potato. 2.The expression characteristics of CP NAC8 gene in Wintersweet were analyzed by real-time fluorescence quantitative PCR technique. The expression characteristics of CP NAC8 gene in different tissues, different periods and different stress treatments (drought, high salt, high temperature, low temperature) were analyzed. However, the highest expression level was found in mature leaves and pistil, suggesting that the gene might be involved in the morphogenesis of plant leaves and the expression of .Cp NAC8 gene in pistil development. However, in flowering stage, the expression of .Cp NAC8 gene was almost non-expressed in drought and high salt. The expression of ABA was up-regulated under GA stress, but the time point of the highest expression was different under different stress conditions. The results suggested that the gene might respond to drought, high salt and high temperature stress. 3. The transcriptional activation characteristics of CpNAC8 may be involved in the signal transduction pathway of ABAA-GA. 3. The construction of yeast expression vector p GBKT7-Cp NAC8-ORFN p GBKT7-Cp NAC8-VP16 and the transformation of yeast strain AH109 indicate that the CpNAC8 gene has transcriptional activation. The phenotype of transgenic Arabidopsis thaliana with the transcriptional activator .4.The plant superexpression vector pCAMBIA1300-CpNAC8 was constructed and transformed into Arabidopsis thaliana by inflorescence inflorescence. The homozygous T3 generation transgenic Arabidopsis thaliana was obtained by screening. The results showed that the introduction of CP NAC8 gene into wild-type Arabidopsis thaliana would not cause adverse effects on the growth of wild Arabidopsis thaliana. Transgenic Arabidopsis seeds are more sensitive to NaCl than wild type Arabidopsis seeds, and CP NAC8 gene plays an inverse role in salt tolerance of Arabidopsis thaliana. CpNAC8 gene could enhance drought resistance of transgenic Arabidopsis thaliana during soil drought stress. It was preliminarily determined that CpNAC8 gene was related to drought tolerance and salt tolerance.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S685.99;Q943.2

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