Cloning And Expression Functional Analysis Of Fe Superoxide Dismutase (FeSOD) Genes From Triticum Aestivum

Plants will produce many harmful free radicals in the process of plant growth and development when a variety of environmental stress subject to plant. At present, cultivating new crop varieties resistant to stress by isolating plant defense genes through biotechnology methods and then lerting it express in transgenic plants, has become an effective way to increase crop yield. Superoxide dismutase (SOD) is an important antioxidant enzyme which can clear reactive oxygen species in plant. There are some studies finding that SOD activity level in plants was closely related to plant stress tolerance, and over expression of SOD gene can significantlyimprove plant resilience to salt stress. In this paper, the wheat yangnong 19 was selected as the experimental material. Wheat FeSOD gene was cloned using RACE technology and its sequence was analysis; Then Constructed of prokaryotic expression vector pETDuetl/FeSOD and made it express in E. coli, and furthermore optimized inducing expression conditions; At last analyzed of the gene expression differences under different environmental stress using quantitative PCR. The results were as follows:1. Wheat FeSOD gene cloned with degenerate primers and RACE technology. The total length of wheat FeSOD (GenBank registration number:JX398977) was 1397bp. The open reading frame length was 594bp, encoding 197 amino acids presumably. Its protein molecular weight was about 22.8KDa, isoelectric point (PI) was 5.382. Its amino acid sequence had a high degree of homology with corn, rice and other species.2. Designed a pair of primers based on the obtained gene sequence and introduced NotI and BamHI restriction sites in the primers to clone the FeSOD coding region. Constructed Prokaryotic expression vector pETDuetl/FeSOD, and then transformed it into E. coli Rosetta (DE3) to be induced express. The expressed protein bands detected by SDS-PAGE in line with expectation size. Western Blot successfully detected recombinant protein. The Objective protein was successfully expressed. The best inducing expression conditions for recombinant bacteria were 0.5mmol/L of IPTG, induction of 5 hours,37℃, which were determined by optimizing IPTG concentration, induction time and induction temperature.3. Using fluorescence quantitative PCR to analyse the gene expression differences under different environmental stress. The results showed that:Under stress conditions, the expression of FeSOD generally increased firstly and then decreased. The expression level of FeSOD gene which were under 37℃,4℃ low temperature, different salt concentrations, 300mmol salt,30% PEG-6000 and 100μmol ABA stress conditions, were the highest when at 3h, 1h,200mmol,6h,48h and 24h, respectively. And they were 34 times,4.5 times,4.3 times,5.8 times,13.5 times and 3.3 times of control group, respectively, which all reached the significant level.