Construction Of Overexpressing Vector Of HOG1 Gene From Yeast And Transformation In Arabidopsis Thaliana

Soil salinization is one of the worest factors that limit the yield and quality of crops. Analysizing the response to salt stress and exploring the salt-related gene to uncover the salt-tolerant mechanism in plants are not only very important in theory, but also in cultivating salt-tolerant crops in practice. Saccharomyces cerevisiae is a model organism and has higher tolerance to salt stress. The study on the regulation mechanism of gene expression and signal transduction and the mechanism of ion transportion of yeast is helpful to clarify the mechanism of salt tolerance of higher eukaryotes. HOG1 gene is an important regulatory factor involving in resistance to high osmotic stress in yeast. Most genes which response to salt stress are highly or completely dependent on HOG1, but HOG1 heterologous over-expression in higher plants and their impact has not been reported. In this study, the HOG1 gene was cloned successfully from S. cerevisiae by PCR technique and then transferred to the model plant Arabidopsis to indentify its function. Those results showed that signfiance through genetic engineering to improve plant resistance, and had found the basis to cultivate salt-tolerant plants. The main results of this study are as follows:1. Cloning the HOG1 gene and construction the plant expression vector. Genomic DNA of S. cerevisiae as template, the HOG1 gene was cloned by PCR method with one pair of primers designed according to the HOG1 sequence on the NCBI （L06279） . Digested by the BamHⅠand the SacⅠ, the HOG1 gene was ligased to the expression vector pBI121 digested by the corresponding enzemies. After checked by PCR and restriction digest, the plant expression vector pBI121-HOG1 was constructed successfully. The recombinant plasmid pBI121-HOG1was then successfully transformed into Agrobacterium tumefaciens GV3101 through the electropotation.2. Transformation of Arabidopsis thaliana and molecular biology detection: The expression vector pBI121-HOG1 was transformed into Arabidopsis through Floral Dip method by screening with Kan resistance. 22 plants were positive to Kan and the transformation efficiency was 2.2%. The genomic DNA was isolated from T₀ generation resistant seedlings to conduct PCR selection and a total of 19 plants could be detected. Further, the target bands can be amplified from the T₃ homozygous transformed Arabidopsis by RT-PCR detection.3. Analysis of the salt tolerance of transgenic Arabidopsis: To discover the relationship between the HOG1 gene and salt tolerance of higher plants, the salt tolerance of T₃ generation was studied on the MS plates with different concentrations of NaCl respectively（0, 50,100,150,200 mM）. The results domenstrated that with the increase of NaCl concentration, the germination rate of transgenic plants was significantly higher than that of wild-type Arabidopsis thaliana, and more time for stress, the growth status of transgenic plants was significantly better than that of wild-type. Analysis the salt-tolerance of T₃ generation transgenic plants in the soil indicated that transgenic plants grow faster than the wild type, which means poisonation by the salt to a lesser extent, which indicated that transformation into Arabidopsis with HOG1 gene could improve the salt tolerance of the plants.