Cloning And Functional Analysis Of Soybean Gm1CE1Gene

Low temperature and chilling injury has been an important factor affecting plant growth and development and geographical distribution. Crop yield decreases caused by chilling and cold injury were great every year. Therefore it is significant to research and improve plant tolerance to chilling injury. ICE1gene in Arabidopsis thaliana was successfully cloned and applied to the area of plant cold resistance enhancement. Regulated and functional genes related with cold-induced have been found in many plants at present and transgenic cold-resistant plant may be gained by transgenic technology. Homologous gene GmICE1with ICE1gene in Arabidopsis thaliana was successfully cloned in soybean and plant expression vector pCAMBIA1304-35S-GwICE1-polyA was constructed, which was transferred into tobacco by method of Agrobacterium-mediated. Functions of improving cold resistance of GmICE1genes in soybean were determined by molecular biology and biological detection.The main research results of this experiment are as follows:(1) The total RNA was extracted using soybean variety He Feng35as experimental material, and the cDNA of soybean was gained by reverse transcription. The GmICE1gene was amplified by PCR, and the plant expression vector of soybean GmICE1gene controlled by CaMV35S constitutive promoter was constructed, which was named pCAMBIA1304-35S-GwICE1-polyA.(2) Plant expression vector pCAMBIA1304-35S-GmICE1-polyA was transferred into tobacco by method of Agrobacterium-mediated, and the selecting pressure of tobacco leaf discs hygromycin was5-8mg/L.52resistant transgenic tobacco plants were gained through the process of regeneration seedlings hygromycin resistance screening and transplanting, in which39%identified by PCR were positive.To generation of part PCR positive plants transferred35S the-GmICE1-polyA were also positive by Southern blot hybridization.(3) Low temperature stress experiment was carried out by using transgenic plants and non-GM plants of TO generation, and the cold tolerance of transgenic plants was significantly higher than CK. The experiment also determined several physiological indicators of transgenic plants and non-transgenic plants. The determination results of content of proline showed that there were not obvious differences between transgenic plants and the non-transgenic plants at room temperature. Content of proline of transgenic plants and non-transgenic plants both increased to varying degrees, however, the increasing degree of transgenic plants was significantly higher than that of non-transgenic plants after cold acclimation treatment. Content of MDA of non-transgenic plants increased dramatically under the cold stress conditions, which upward trend was almost linear; content of MDA of transgenic plants did not show a significant leaping trend under the same stress conditions, which indicated that to import GmICE1gene could not completely eliminate stress injury to plants, but it did effectively reduce the plant membrane lipid peroxidation and enhanced whole plant resistance. The level of antioxidant enzyme activities of transgenic plants and non-transgenic plants were almost the same before low temperature treatment. Tobacco antioxidant enzyme activities increased after low temperature stress. SOD, CAT and POD activities of transgenic plants in low-temperature treatment increased obviously, which indicated that antioxidant stress of GmICE1gene tobacco was higher than that of non-transgenic tobacco. The experimental results show that low temperature stress resistance of tobacco plants transferring GmICE1is stronger than that of non-transgenic plants. The soybean GmICE1gene has the same function as the AtICE1play an important role in soybean cold-induced gene expression.