Cloning And Functional Analysis Of A Catalase Gene CsCAT3 From Cucumber

As a key scavenger of hydrogen peroxide,catalase(CAT)plays an important role in tolerance to abiotic stress in many different organisms.Catalase was encoded by CAT gene and has been cloned in a series of species.Cucumber is an important vegetable crop of Cucurbitaceae,and the function of CAT enzyme is little known.In order to study the function of CAT enzyme in cucumber,the CAT family genes from North China cucumber 9930 were analyzed by bioinformatic approach.And their expression pattern profiles of different tissues and under various stresses were analyzed by RT-PCR and q RT-PCR,respectively.In addition,the prokaryotic expression vector of Cs CAT3 was constructed and transformed into the E.coli BL21 strain.The resistance of the Cs CAT3 gene was analyzed under various stress conditions.The main results were as follows: 1.A total of 4 non-redundant CAT genes were identified.According to their ID numbers,they are named as Cs CAT1-Cs CAT4,respectively.Phylogenetic analysis results showed that Cs CAT1 and Cs CAT2 genes belong to III and I subclasses,respectively,while the other 2 genes(Cs CAT3 and Cs CAT4)belong to II subclass.The cucumber CAT genes are closer to CAT genes of Arabidopsis than those of rice and poplar.2.The cucumber CAT genes displayed specific expression levels in different tissues.The RT-PCR results showed that Cs CAT1-Cs CAT3 were expressed in roots,stems,leaves,flowers and fruits,while Cs CAT4 is not expressed in these tissues.The expression pattern of Cs CAT1 is similar to Cs CAT2,and they had high transcriptional signal in roots,leaves and fruits.Cs CAT3 was abundantly expressed in stems,leaves,flowers and fruits,and weakly expressed in roots.3.Fluorescence Quantitative real-time PCR analysis showed that: The results showed that the expression of Cs CAT3 was induced under different stress treatments(cold,PEG,Na Cl,H2O2 and ABA).4.The Cs CAT3 gene was cloned by RT-PCR,and the prokaryotic expression vector was successfully constructed and highly expressed in BL21.The molecular weight of Cs CAT3 was about 77 KD.Prokaryotic expression analysis showed that the best induction time of p ET-Cs CAT3 was 4 h,IPTG concentration was 1 m M,the best concentration of bacterial solution was OD600=0.6.Cs CAT3 was a soluble protein.5.The results of Escherichia coli resistance showed that: Cs CAT3 recombinant protein can enhance the growth ability of E.coli under different stress conditions,Cs CAT3 has important tolerance to abiotic stress.