| The ERFs (Ethylene-Responsive Factors), are unique transcript factor of plants, play an important role in plant growth and stress response. It can improve plant resistance to diseases through interaction with GCC box which present in promoters of many pathogenesis-related (PR) genes. In addition, ERFs can response to abiotic stress, such as cold, drought, ABA signaling pathway through interaction with non GCC box containing genes.StERF3 is an EAR-motif containing suppress transcription regulation gene associated with late blight resistance which was cloned by our lab previously. It could be induced by SA, JA and ETH, and was conferred that it may act as an important element among the network of signal pathways to modulate PR genes expression which lead to the defense response to Phytophthora infestans. In this study we further investigated its functions against late blight resistance and abiotic stress through the overexpression and RNA interference methods, promoter cis elements and downstream regulated genes analysis. The main findings are as follows:1. Overexpression vector of StERF3 was constructed and transformed into two potato varieties E3 and Jx. Thirty four E3 and 18 Jx overexpression transgenic lines was obtained separately. At the same time, RNA specific interference and RNA family interference vectors was also constructed. We got 26 RNA specific interference transgenic lines and 21 family interference transgenic lines of E3, 5 specific interference and 4 family interference transgenic lines of Jx. Southern blot analysis indicated that most transgenic lines are positive and gene copy number ranged frome 1 to 4.2. RT-PCR analysis of transgenic plant showed that the expression of StERF3 in over-expression transgenic lines are higher than that in E3 and Jx control, but with lower level in RNA specific interference and RNA family interference transgenic lines compared with control. Expression of PR1,PR1b,PR4b,CHIB, the downstream gene of StERF3 with GCC-box containing in their promoters, were increased in RNA specific interference transgenic lines. In RNA family interference transgenic lines, besides StERF3 interference, the AP2/EREBP family homologous gene StEREBP1, CIP353 and STWAAEIRD shown silenced in different degree.3. Detached leaves inoculation of P. infestans showed that the lesion growth rate of 9 overexpression transgenic lines are significantly higher than that of control which hold 36% of the total inoculated overexpression transgenic lines. The lesion growth rate of 12 RNA specific interference transgenic lines are lower than that of control which stand 46.2% of the total inoculated RNA specific interference transgenic lines.4. Under 150 mM NaCl salt stress condition, the growth of overexpression and RNA specific interference transgenic lines were both inhibited, but the former is more serious than that of latter. After 4℃cold treatment, the top of E3 overexpression transgenic lines are necrosis and wilting, while specific interference transgenic lines grows well as normal. This indicated that resistance of specific interference transgenic lines increased in a certain degree.5. Using hiTAIL-PCR, 4 StERF3 promoter sequences were cloned, and one of 1214 bp in length sequence was analyzed in detail. In addition to a number of basal promoter structural elements, there are a wide range of biotic and abiotic stress-related cis elements in it, such as pyrimidine-box response to ABA and drought stress, LTRE-1 response to cold, GT-1,ABRE,MYBCORE,MYC,W-box,GCC-box response to disease and salt stress.To sum up, the late blight resistance level of StERF3 specific interference transgenic lines are increased; at the same time salt stress and low-temperature tolerance are raised at a certain extent. Thus, it can be inferred that StERF3, as a transcription inhibitor, regulated biotic resistant and abiotic stress response in potato mainly by negativly regulate disease-resistant and stress-related genes.
|
PDF Full Text Request