Analysis Of Function And Regulation Characteristics Of Potato StERF3 Gene

Potato late blight, caused by Phytophthora infestans, is one of the most devastating diseases of potato, which causes huge economic losses every year. To investigate the mechanisms of potato late blight resistance, a potato gene StERF3 was cloned in our laboratory previously. StERF3 was induced by SA, MJ and ETH significantly. Sequence analysis showed that the C-terminus of StERF3 contains-L/FDLNL/F(x) p motif (EAR motif) which is a motif of active repressor. Preliminary studies showed that the StERF3 gene may be negatively regulated potato late blight resistance. To further look into its function, late blight resistance of transgenic lines overexpressed and interfered StERF3 were tested and confirmed in the present study. The regulation property of StERF3 promoter was determined by means of promoter and GUS fusion method. At the same time, yeast two-hybrid protocol was used to screen proteins potentially interact with StERF3. The reason of some transgenic lines mutations which showed dwarf phenotype were investigated as well. The main results are as follows:1. Expression level of StERF3 in overexpressed and interfered transgenic lines was tested by RT-PCR. The results showed that expression level is differing from deferent lines and the really overexpressed and silenced transgenic lines were selected for late blight resistance test. The inoculation results demonstrated that disease lesion area in overexpressed lines of E3 and J was bigger than that of control. While the lesion area on the leaves of specific and non-specific interference lines of E3 and J was smaller than control. These results implied that StERF3 gene may play a role of negative regulation in potato late blight resistance.2. Promoter prediction showed that StERF3 promoter contain several cis-acting elements responsible for pathogen, SA, MJ, ETH, low temperature and salt stress induction. Fusion vector of the promoter and GUS reporter gene was constructed and transformed into Nicotiana tobacco and Nicotiana benthamiana mediated by Agrobacterium. After PCR identification,16 N.benthamiana and 3 N. tobacco transgenic plants were obtained. GUS staining showed that leaves of three N. benthamiana transgenic plants presented different levels of blue color after 48 h of P. infestans inoculation. Blue color also appeared on the leaves of N. tobacco transgenic plants which treated by SA, ETH and salt stress for 8 h,24 h and 4 h respectively. However, the leaves of control did not show blue color after GUS staining. Above results confirmed that StERF3 promoter is an inducible promoter which could response to P. infestans, SA, MJ, ETH and salt stress.3. For the purpose of screening of interact protein with StERF3, yeast bait vector pGBKT7-StERF3 was constructed. It has non-toxic or self-activated.69 dark blue clones (Ade+, His+) appeared 8 h after selection on SD/-Ade/-His/-Leu/-Trp/-X-α-gal plates. At the end,6 candidate clones was obtained after second round selection on SD/-Ade/-His/-Leu/-Trp/X-β-gal plates for 8 h. Nucleotide and protein sequence alignment analysis showed that one full length cDNA shares high similarity with tomato CONSTANS interacting protein 3 which may associated with flower development. Another truncated sequence shares similarity with a castor cyanate salt synthasee protein which mainly involved in defense-related gene expression.4. Phenotype observation, StERF3 expression level test, histological and auxin experiment were performed on some dwarf transgenic plants which transformed by overexpression vecter. The results showed that gene expression level in dwarf plants was lower than that of control. Most of the dwarf lines contain 3-4 copies. Multi-copy insertion of overexpression vector may result transgene silencing in transgenic plants.However, StERF3 gene expression decreased significantly in interference lines compared to control and no dwarfing phenomenon appeared. The results implied that dwarf phenomenon may have no relationship with StERF3 gene expression. Medium supplemented with exogenous GA3 could improve the growth of dwarf plants in vitro to a certain extent. In conclusion, the dwarf might caused by multiple copies insertion on the genomic DNA which interrupted the genes associated with GA3 pathway.