Expression Analysis Of GAPDH In Wheat Changwu134and Promoter Activity Verification Of Its Gene Upstream Sequence

Enzyme sites are add to the GAPDH gene fragment was cloned into the pET-28aexpression vector to construct the prokaryotic expression vector and induced into expressionstrain BL21competent. After induction, the bacterial was sonicated in an ice bath to extracttotal protein. The supernatant was then purified by BIO-RED nickel column and gel cut forantibody preparation (rabbit serum) in Wuhan SanYing company.Total protein of wheat sprouts as materials was extracted for Western Blot test, takesamples when the plant is stressed with-1.2MPa PEG6000solution for0h,24h,48h,72h,rehydration24h, rehydration24h, rehydrated24h and then rehydrated48h. After SDS-PAGEelectrophoresis, transfer the protein by semi-dry method to a PCDF film. Block the film with5%skimmed milk powder dissolved with TBST, incubate with rabbit serum as first antibodyat1:500scale,37°C for1.5h then incubate with HRP-conjugated goat anti-rabbit secondaryantibody (Kang century), at1:10000scale,37°C for1h, and finally chromomeric with DAB.Based on the previous work of our laboratory, enzyme sites are add to1071bp GAPDHupstream sequence of wheat Changwu134through special digested primers. Then thefragment was cloned into the pBI121eukaryotic expression vector to construct the promoteractivity authentication vector and induced into into Top10competent cells for positiveselection and amplification. Extracted the plasmid for Arabidopsis protoplasts transduction.After incubation, take an improved GUS histochemical chromogenic method to check thepromoter activity.The Results are as follows:1. The expression conditions were optimized as: induction time7h, IPTG concentration:0.02mol/L, induction temperature:37°C. Soluble analysis shows that the target protein in thesupernatant. After centrifugation12000r/min,15min, the supernatant was obtained for nickelcolumn purification and further gel cut purification.2. Bands of Western Blot test proved the protein is indeed GAPDH. From differentstaining Results from the graph we can see different expression levels of GAPDHafter-1.2MPa PEG-6000stress at different times in wheat shoots. The trial found that droughtstress can increase the expression of GAPDH in wheat, revealing expression pattern of wheatGAPDH, the test proved GAPDH in Changwu134wheat contribute to drought resistance. 3. After incubation, the improved GUS histochemical chromogenic method to check thepromoter activity has a positive Result. Showing that our previous cloned GAPDH upstreamsequence having promoter activity.