Gene Expression And Cloning Of GAPDH In Changwu 134 Wheat With Drought Resistance

Two winter wheat cultivars differing in drought resistance (Changwu 134 was stronger than Shan 253 in drought resistance ) were used to study the ratio of root and bud,relative water content (RWC) and the rate of free water and bound water under–1.2 MPa PEG6000 solution. The Changwu134 wheat seedling was treated with–1.2 MPa PEG6000 solution, 0.9%NaCl single salt, 300μmol/L ABA, 4℃low temperature. The changes of GAPDH protein content in wheat seedling were studied under the stress for 0h, 24h, 48h, 72h, rewatering (rewarming) for 48h, then treating for 48h again. The RNA of the wheat seedling with–1.2 MPa PEG6000 solutionwas extracted. Through the RT - PCR, TA cloning, the full length sequence of GAPDH protein gene was obtained in the wheat seedling. The aim was to the deeper understanding the relationship of GAPDH protein and drought resistance in the wheat seedling under dry conditions. The physiology-biochemistry mechanisms of drought resistance of wheat were analysed. Provide a theoretical basis for wheat drought breeding and agricultural water-saving irrigation.The results showed:1. The wheat with different drought tolerances was treated with PEG. The wheat increased value of root and bud ratio, reduced RWC and the value of free water and bound water rate adaptation to water stress. The trend in Changwu 134 was more obvious than Shan 253. The Changwu 134 wheat seedling was stronger in water-holding capacity.2. SDS-PAGE electrophoresis results showed that the effect of the wheat protein expression was different with different treatments. But in various simulation of adversity, they all can induce the expression of 39.5kDa GAPDH protein to change.3. Three kinds of adversity (expect low temperature), GAPDH protein begained to increase at 24h under treating. The increase of GAPDH protein continued at 48h under treating. The protein content reached a maximum velue at 72h under treating. The protein gradually was degradated at rewatering 48h, and the expression reduced. The expression increased at treating 48h again. Low temperature inhibited GAPDH protein expression. 4. Total RNA of the ChangWu 134 wheat seedling treated by - 1.2 MPaPEG6000 was extracted using Trizol method. Total RNA integrity was good. After using it to make template do RT-PCR, the complete of GAPDH gene with 1014bp was gained. The sequence was sequenced. The obtained sequencing was compared using Blast in NCBI. The results showed: the similarity between the sequencing and the known sequence(scode: EF592180.1)was 94%. The result showed that the sequence was exactly GAPDH gene.5. The gel recovery product was sequenced after the TA cloning , and get the full length sequences of the ChangWu 134 wheat varieties of the GAPDH. The obtained sequencing was compared using Blast in NCBI. The results showed: the similarity between the sequencing and the known sequences(code: EF592180.1)was 99.7%. The result showed that the sequence was exactly GAPDH gene.