Genetic Transformation And Drought Tolerance Analysis Of StRFP2 Gene In Potato

The ubiquitin-proteasome pathway(UPP)is a reversible major pathway for selective degradation of proteins in eukaryotic cells.The ubiquitin ligase(E3),which determines the specificity of the ubiquitination process,plays a vital role in regulating plant stress or stress response.In the previous RNA-seq study,our team found that the RING-type E3 StRFP2 gene was significantly up-regulated under drought stress in potato.In this study,the location of StRFP2 gene expression in cells was determined by subcellular localization;The tissue-specific expression of StRFP2 gene in potato and the response to drought stress were detected by qRT-PCR;Transgenic potatoes with over-expression and interference expression of StRFP2 gene were obtained by Agrobacterium-mediated methods,and further analysis of its drought resistance,thus providing a basis for further analysis of its drought resistance mechanism.The following main research results have been achieved:1.The transient expression vector of pCEGFP-StRFP2 fusion protein was constructed by homologous recombination and transformed into tobacco by Agrobacterium-mediated method.The StRFP2 protein was mainly located in cell membrane and cytoplasm by laser confocal scanning microscope.2.The tissue-specific expression and drought stress response of StRFP2 gene in potted potato seedlings "Atlantic" and "Qingshu 9" under greenhouse were analyzed by qRT-PCR.The results showed that the StRFP2 gene was differentially expressed in the roots,stems,and leaves of the two varieties,and the expression level was highest in the leaves.In the "Atlantic",the StRFP2 gene was more sensitive to drought.With the deepening of drought,the expression of StRFP2 gene showed a trend of increasing first and then decreasing,which indicated that StRFP2 gene responded to drought stress through positive regulation.3.Transgenic potato plants with pCPB-StRFP2 overexpression and transgenic plants with pCPB121-amiR-StRFP2 interference were obtained through Agrobacterium-mediated transformation.Phenotypic analysis of transgenic potatoes showed that the overexpression of StRFP2 gene positively regulated potato growth,while interfering with expression inhibited its growth.4.The relative expression of StRFP2 in transgenic plants under PEG osmotic stress was analyzed by qRT-PCR.The results showed that the expression of StRFP2 was up-regulated under PEG treatment.The expression level of StRFP2 gene in over-expressed plants was higher than that in non-transgenic plants(WT),while the expression level of StRFP2 gene in interference-expressed plants was lower than WT plants.5.Under PEG osmotic stress,the free proline(Pro)content,catalase(CAT)activity,malondialdehyde(MDA)content,and relative water content of the transgenic plants were determined.The results founded that the Pro content,CAT activity,and relative water content in over-expressed plants were higher than WT plants,and MDA was lower than WT plants,while the interference-expressed plants were the opposite.This indicates that the StRFP2 gene responds to drought stress by changing osmolytes.