Cloning Of Aldehyde Dehydrogenase Gene Of Leymus Chinensis And Researching On The Function Of Transcription Factor CBF2

Adversity stresses such as drought, high salt and freezing is a major factor that influences plant development. It has a serious impact on the output and quality of the crop, even on the environment. How to improve the ability to resist abiotic stress and to meet the increasingly demands of provision has become one of the key problems in the modern breeding works. It has became an important content in modern agriculture research to cultivating the new crop variety which can bear the stress and developing the tolerant resources and cloning the genes which are relevant to help plant to withstand the adversity. It is also an efficient strategy to keeping agricultural sustainable development. In this research, for further study the mechanism of stress tolerance in plant, Arabidopsis thaliana, tobacco and Leymus Chinensis were used as the main experimental materials to study the molecular mechanism of stress tolerance in plant and do some researches on the gene engineering. The main results are as follow:By measuring the relevant physiological index of grey green type Leymus Chinensis under the salt stress and comparing with yellow green type Leymus Chinensis, the variety trends of physiological indexes show that grey green type Leymus Chinensis is a high salt tolerant material and there are many genes related to salt tolerance in it. We get three read-through sequence LC-1,LC-2 and LC-3 with a conservation domain which belong to the aldehyde dehydrogenase family by RT-PCR method from Leymus Chinensis. Homologous comparison with their structure shows that LC-1 is a aldehyde dehydrogenase gene, LC-2 is a betaine aldehyde dehydrogenase gene and LC-3 is an unknown aldehyde dehydrogenase gene. These sequences are all enrolled in GeneBank and the number is DQ458294, DQ497618 and DQ458293, respectively.We clone the full-length aldehyde dehydrogenase gene which contain from Leymus Chinensis by RACE which use LC-1 as the target sequence. This gene contains 1503bp ORF including 66bp 5'-UTR and 144bp 3'-UTR, and codes 501 amino acid. There is a tailing signal AATAAA at the terminator downstairs 72bp and the registered number is EF492045 at GeneBank. LC-ALDH has an absolute conservative glutamic acid active site, cysteine residue and histidine residue of aldehyde dehydrogenase by bioinformatics analysis. There is 87% homology between the LC-ALDH and the OsALDHla gene in rice. Analysis of phylogenesis shows that LC-ALDH belongs to the aldehyde dehydrogenase gene in cytoplasm. The result of Real time RT-PCR shows that LC-ALDH is induced by drought, high salt and freezing and its expression increased prominently under these conditions. We confer that this gene plays an important role in restraining membrane lipid peroxidation and resisting the oxidation stress.After isolating the CBF2 gene and the rd29A promoter from Arabidopsis and constructing a plant expressive vector pBI-RD-CBF2 in which the CBF2 gene regulated by rd29A promoter, we transformed this vector to the tobacco Longjiang 851 by Agrobactorium. By the genetic analysis of the transgenic offspring, it shows that the exogenous gene inserts tobacco genome as one copy form and follows the Mendel 3:1 heredity separate law. The analysis of salt tolerance shows that transgenic tobacco can grow normally under the 150mM NaCI while the wild type tobacco represent harmfully under the 100ram NaCI. The result of the relative conductance shows that the damage extent of the cytoplasmic membrane in transgenic tobacco is lower than in the wild type tobacco. The experiment account for that the over expression of CBF2 gene is propitious to improve the plant salt tolerance. But the form of the TO generation of some transgenic tobaccos are obviously changed, including the plant stunting, leaf crimpling, color of the flower thinning, apical dominance weakening and node shortening, which was conferred that the changing is correlate to the AP2 conservative domain of CBF2 gene.