Cloning And Functional Verification Of AlsCBF Gene Amygdalus Ledebouriana Schlecht In Xinjiang

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.