【作者】 李宁；

【导师】 罗淑萍；

【作者基本信息】 新疆农业大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 低温冷害使许多植物生存受到严重危害,全球每年因低温冷害造成的农作物损失高达数千亿元,提高植物抗寒能力的研究具有重要的理论意义和实践意义。由于我国野扁桃相关研究工作起步较晚,其功能基因的发掘和分子育种有待进一步深入研究。为了揭示野扁桃抗逆分子机制,获得优良抗逆基因,本文克隆了野扁桃抗寒相关基因-AlsCBF基因,并对其进行特异性表达分析和功能验证。主要研究内容如下：1.采用RT-PCR结合RACE方法,获得了全长为1171bp的新疆野扁桃(Amygdalus ledebouriana Schlecht)抗寒基因,命名为AlsCBF (GenBank登录号为HQ908653),该基因ORF长713bp,编码一个由237个氨基酸组成的蛋白。生物信息学分析结果表明,该蛋白分子量26.5581kD,理论等电点6.76,为亲水性蛋白,位于细胞膜外,α-螺旋和无规则卷曲是AlsCBF的大量结构元件,而β-转角和延伸链则散布于整个蛋白质中。根据氨基酸序列构建的系统进化树显示,发现AlsCBF与中国梅的CBF蛋白亲缘关系比较近。2.利用实时荧光定量PCR方法分析了野扁桃AlsCBF基因在低温胁迫处理下的表达差异,结果显示AlsCBF基因的相对表达量在4℃处理24h时达到峰值,基因的表达量随胁迫时间的延长呈现先升后降再升的趋势。3.以野扁桃cDNA为模板,以BamHⅠ和XbaⅠ酶切位点的特异性引物进行PCR获得该基因的编码框,并将其构建到植物表达载体pCAMBIA1301中,构建了重组表达载体pCA1301-AlsCBF,冻融法将其导入根癌农杆菌LBA4404,并通过叶盘法转化烟草,获得转基因烟草T1代种子。4.通过DNA-PCR和RT-PCR对T2代转基因烟草进行分子检测,结果表明野扁桃AlsCBF基因已整合到烟草基因组中,并在转录水平上表达。通过对转基因和非转基因植株在低温胁迫处理下生理指标的测定,结果显示通过表型低温试验、电导率、丙二醛和保护酶活性的变化来看转基因植株的抗寒能力较对照植株有明显的提高。由此可以认为植物抗寒力的提高是低温诱导所引起的转录调控因子CBF表达水平以及各种生理生化代谢适应性变化的综合结果。更多还原

【Abstract】 Chilling damage is seriously harmful at the survival of many plants, and the global crop losser caused by chilling damage every year is up to several hundred billion Yuan. Therefore, the research that improving the ability of plant fighting cold has important theoretical and realistic significances. Due to the related research work on Chinese Amygdalus ledebouriana Schlecht starts later, its excavation of function gene and molecular breeding need further research. In order to reveal the resistance molecular mechanism of Amygdalus ledebouriana Schlecht, and to gain good resistance genes, this paper clones AlsCBF genes of Xinjiang Amygdalus ledebouriana Schlecht, and does analysis and functional verification to their specificity. The main research contents are as follows:Firstly, this paper adopts the RT-PCR combined with RACE methods to get Xinjiang Amygdalus ledebouriana Schlecht resistance cold gene with1171bP long, named for AlsCBF (the registration number of GenBank is HQ908653), and the ORF of this gene is with713bP long, which encodes a protein composed of237amino acids. The analysis results of Bioinformatics show that the protein molecular weights26.5581kD, and the isoelectric point of which is6.76, which results to be hydrophilic protein. It is located in the cell membrane, and large quantities of structural components of AlsCBF are alpha helix and inconstant crispation, while beta corner and extension are scattered in the whole protein. According to the system evolutionary tree of amino acid sequence construction, the genetic relationship between AlsCBF and CBF protein of Chinese mei is nearer.Next, using the method of real-time fluorescence quantitative polymerase chain reaction (PCR) analysis, this paper analyzes the expression differences of Amygdalus ledebouriana Schlecht AlsCBF gene in the cryogenic stress, and the result shows that the relative expression amount of AlsCBF gene peaks processed24hours in4℃. With the stress of the extension of time, the trend of gene presents expression first increases and drops then rises again.Then, taking the wild prunus communis cDNA as template, this paper carries on PCR for the specificity primer of BamH Ⅰ and Xba Ⅰ enzyme cut, and gains the gene encoding frame, which will be built into the plant expression vector pCAMBIA1301, and constructs the restructuring pCA1301-AlsCBF expression vectors. This paper will import the root cancer agrobacterium-mediated LBA4404through freeze-thaw law, and transforms tobacco through the dordrecht method to get the seeds of transgenic tobacco T1.At last, this paper makes molecular testing on T2generation of genetically modified tobacco through the DNA-PCR and RT-PCR, and the result indicates that the wild AlsCBF gene of Amygdalus ledebouriana Schlecht has integrated into tobacco genomes and expresses in transcription. Through the physiological indexes determination of the genetically modified and the transgenic plants in the low temperature stress, the result shows that the ability of transgenic plants fighting cold is more improved than control plants through the low temperature test, phenotype conductivity and the enzyme activity changes of protection mda. Therefore, it can be thought that the enhancement of plants KangHanLi is a comprehensive result of the transcription regulation factor CBF expression level caused by cold induced and various physiological and biochemical metabolic adaptability changes.更多还原