Study On Transformation Of HAL1 Gene Related To Salt-Tolerance In Crops

The HAL1 gene is an important salt-tolerance factor in yeast. To acquire seed resources of crops with salt tolerance, the gene of HAL1 was transferred into tobacco leaf disks , wheat ovary and wheat immature embryo callus, respectively with the help of Ti-plasmid of A. tumefaction, pollen tube pathway and particle projectile. Mean the while, some factors affecting transformation efficiency of the means was studied. The result is following.Factors including different mediums, growth state of A. tumefaction, component and concentration of transformation solution, the amount of DNA, the time frozen in liquid N2 ,and so on affecting preparation of competent A. tumefaction cells and transformation frequency by the freeze-thaw method were evaluated. The results suggested that the best competent cells can be obtained when A. tumefaction grew at OD600=0.5-0.6 with YEB medium, prepared with 50mmol/L-70mmol/LCaCl₂. LBA4404 competent cells mixed with 10ng plasmid DNA at 0℃for 30min, then frozen by liquid N₂ for 5min, followed heat shock at 37℃for 5min,the transformation frequency reached 1.4×10⁵ transformants/ugDNA. But C₅₈C₁ competent cells mixed with 20ng plasmid for 10min, frozened for 1min, heat shock at 37℃for 1min, the transformation frequency reached 1.33×10⁵ transformants/ugDNA.Tobacco leaf disks were rooted in MS medium containing kanamycin and Carbenicillin after pre-culture, agroinfection and co-culture in 2-3 months. In this experiment, it was found there were many effect factors. The effect of agro-infection on plant in different grow period was different and time of transgene were affected by different lines. The culture and dilution of A. tumefaction influence transgenic efficiency. The co-culture of plant and A. tumefaction also influence transgenic efficiency. It was important for root of seedling to be screened that could select transgene tobacco.The strain of A. tumefaction with pROKâ…¡carring NPTâ…¡gene and HAL1 gene was used to transfer tobacco cv. Qinxuan no.1 and Qinyan95. The resistant buds were selected by kanamycin 20mg/L for Qinxuan no.1 and 30mg/L for Qinyan95. 23 resistant plants were obtained selected with kanamycin 30mg/L in rooted medium, and 6 plants were confirmed to be positive by PCR amplification with the primer of HAL1 gene. The salt tolerance of...