Research Of Cucumber 14-3-3 Proteins In Response To Powdery Mildew Infection

Cucumber powdery mildew belongs to air-borne diseases in the leaf, which is easy to result in serious economic loss because it is difficult to prevent and control when it happens. In our previous work, one 14-3-3 protein (gi|695767) which had a high-abundance expression was obtained for cucumber powdery mildew by two-dimensional electrophoresis and mass spectrometry. It was speculated that might be associated with resistance to powdery mildew in cucumber.14-3-3 protein family exists in eukaryotic and can be widely involved in cellular physiological activities. In this study, cucumber 14-3-3 family was analyzed by real-time quantitative and bioinformatics methods included phylogenetic tree, sequence alignment and predicted which one was the most likely in response to powdery mildew in cucumber. The coding region of Cs7 gene was cloned, and positive eukaryotic expression vector pBI121-Cs7 and negative eukaryotic expression vector pB7GWIWG2(I)-Cs7 were constructed by molecular biology techniques according to the bioinformatics analysis results. And both vectors were introduced into Agrobacterium (LBA4404) to provide experimental foundation for further study the resistance mechanism of Cs7 in response to powdery mildew infection. The main results were as follows:(1) Bioinformatics analysis showed that there were ten cucumber 14-3-3 genes which located in the cytoplasmic. The results of physical and chemical properties indicated that the relative molecular weight of cucumber 14-3-3 was between 28.2kD(Cs2) and 30.5kD(Cs9), and theoretical PI was between 4.66(Cs8) and 5.08(Cs2). The grand average of hydrophobicity of cucumber 14-3-3 proteins was between-0.649(Cs6) and-0.265(Cs1), suggested that cucumber 14-3-3 proteins were hydrophilic acidic proteins. The secondary structure of cucumber 14-3-3 proteins was analyzed that alpha helix was the main structure. The function of cucumber 14-3-3 protein family was forecasted primitively, and the results showed that Cs1, Cs3 and Cs8 might be the most beneficial in amino acid biosynthesis, but other proteins might play a greater role in translation and ten cucumber 14-3-3 proteins played least role in transport and binding. The phylogenetic tree of thirty-five 14-3-3 proteins from Arabidopsis, tomato and cucumber was constructed, indicated that cucumber 14-3-3 proteins were also divided into two groups, ε-group 14-3-3 proteins including Cs2, Cs6, Cs7, Cs9 and Cs1O, non ε-group containing Csl, Cs3, Cs4, Cs5 and Cs8. Phylogenetic tree of 14-3-3 protein obtained from early study in this laboratory, Arabidopsis GRF6 and cucumber 14-3-3 protein family were constructed by Mega5.0 software, and the results showed that cucumber 14-3-3 proteins Cs3, Cs4, Cs5 and Cs7 were most likely in response to powdery mildew infection.(2) Real-time PCR analysis showed that the appearance of the maximum amount of gene expression (except Cs4) in the resistant varieties(12 h and 24 h) was earlier than in the susceptible cultivar(48 h). Thus, cucumber Cs7, Cs3 and Cs5 might play a role in response to powdery mildew fungus infection. Combined with previous laboratory results, Cs7 gene was selected to do follow-up experiments.(3) Over-expression vector of pBI121-Cs7 and RNAi vector of pB7GWIWG2(I)-Cs7 were constructed. The target fragment was cloned by PCR, and the vectors were identified by colony PCR and restriction enzyme digestion. And two vectors have been successfully introduced into Agrobacterium (LBA4404), which would provide experimental foundation for further study the mechanism of cucumber Cs7 in response to powdery mildew infection.