Isolation And Functional Study Of Stress-Related StMAPKs Gene In Potato

Mitogen activated protein kinase(MAPK)cascade is a highly conserved signaling transduction model in eukaryotes.This cascade consists of MAPKKK,MAPKK and MAPK modules.This cascade amplifies the sensed exogenous signaling and successively transduces the stimuli through these three kinases to mediate cell proliferation,differentiation and death.Increasing drought and salinization have caused a decrease in potato(Solanum tuberosum L.)production.At present,the research on the gene function of plant MAPK under drought,salt and osmotic stress is very limited.So far,it has not been systematically studied whether StMAPKs responses to abiotic stress,and the expression patterns of StMAPKs has not been investigated in hormone-treated plants either.Moreover,the expression pattern of StMAPKs in different tissues and organs of potato is not clear.This study mainly analyzes whether StMAPKs mediate a variety of stress-related signal transduction pathways,whether StMAPKs work under drought,salt and osmotic stress,and the underlying mechanisms.This study analyzed the expression patterns of StMAPKs in potato plants treated with different abiotic stresses and phytohormone.Meanwhile,we also analyzed the expression patterns of StMAPKs in different tissues and organs.Salinity-and osmosis-related gene StMAPK3 and drought-related gene StMAPK11 were identified in potato cultivar‘Atlantic'.Next,we detected the subcellular localization of StMAPK3 and St MAPK11 protein.To further study the function of StMAPK3 and St MAPK11,we constructed the yeast two-hybrid system for detecting the interacted proteins,followed by confirmation using bimolecular fluorescence complementation.Then gain-of-function or loss-of-function transfection in potato plants were established for investigating gene function in biological activities,photosynthesis,and phosphorylation-mediated signaling transduction.The obtained results were presented as follows.1.The expression patterns of 15 StMAPKs in potato under different abiotic stress and phytohormone treatments,as well as in different tissues and organs were analyzed by qRT-PCR technology.The results showed that the expression of 15 StMAPKs in potato not only differed significantly under abiotic stress and phytohormone treatment,but also had significant differences in specific expression in different tissues and organs.2.Two mitogen-activated protein kinase genes StMAPK3 and St MAPK11 were cloned from potato.Among them,StMAPK3 is related to salt and osmotic stress,and StMAPK11 is related to drought stress,The pCAM35s-GFP-StMAPK3 and pCAM35s-GFP-St MAPK11 Subcellular localization vectors were constructed and transiently expressed in tobacco epidermal cells.The results showed that both StMAPK3 and StMAPK11 are located in the cell membrane and cell nucleus.3.The prokaryotic recombinant expression vector pET28b-StMAPK11 was constructed and then transduced into Escherichia coli BL21(DE3).IPTG was used to induce StMAPK11 expression,followed by protein purification for preparing rabbit anti-StMAPK11 polyclonal antibodies.Western blot results showed that the prepared antibodies can specifically recognize StMAPK11 protein.4.Yeast two-hybrid and bimolecular fluorescence complementary vectors were constructed respectively,and verified by yeast two-hybrid and bimolecular fluorescence complementation experiments.The results showed that StMAPK3 can interact with StDof1,StSR2,StACS5,St MEK2 and StMAPKK1,among which StMAPK3 and StSR2 interacts in the nucleus.StMAPK11 can interact with StbZIP2,StPTP1 and StVQP9,among which StMAPK11 and StbZIP2 interacts in the nucleus.5.Functional analysis of StMAPK3 related to salt and osmotic stress was carried out.Under salt and osmotic stress conditions,compared with non-transgenic StMAPK3 plants,the enzymatic activity of catalase(CAT),peroxidase(POD),superoxide dismutase(SOD)and proline content increased in plants overexpressing StMAPK3,of which results were contrary in RNA interference-mediated StMAPK3knockdown plants.However,in StMAPK3 overexpressing plants,the content of H₂O₂and malondialdehyde(MDA)is reduced,and the content of H₂O₂ and MDA in StMAPK3 knockdown plants is increased.At the same time,overexpression of StMAPK3 increases the transpiration rate,net photosynthetic rate,stomatal conductance and water use efficiency of plants under salt and osmotic stress.On the contrary,in RNA interference expressing plants,these photosynthetic indicators are reduced to varying degrees compared with non-transgenic plants.It was also found that the transduction of StMAPK3 gene under salt and osmotic stress has different effects on the stomatal closure,plant height,stem thickness,fresh weight,root length and lateral root number of potato plants.It showed that StMAPK3 can significantly improve the resistance of potato plants to salt and osmotic stress.6.Functional analysis of StMAPK11 related to drought stress was carried out.Under drought stress conditions,compared with non-transgenic StMAPK11 plants(potato,tobacco and Arabidopsis),the enzyme activity of CAT,POD,SOD and proline content is increased in plants overexpressing StMAPK11.In RNA interference expressing potato plants,the results are opposite.H₂O₂ and MDA content in StMAPK11-overexpressing plants(potato,tobacco and Arabidopsis)are reduced,but H₂O₂ and MDA content in RNA interference expression potato plants is increased.At the same time,overexpression of StMAPK11 increases the transpiration rate,net photosynthetic rate,stomatal conductance and water use efficiency of potato plants under drought stress.On the contrary,in RNA interference expressing plants,these photosynthetic indicators are reduced to varying degrees compared with non-transgenic plants.It was also found that StMAPK11 has different effects on the fresh weight,dry weight,root fresh weight and root dry weight of potato plants under drought stress.It showed that StMAPK11 can significantly improve the resistance of plants(potato,tobacco and Arabidopsis)to drought stress.7.Phosphoproteomic analysis of StMAPK3 overexpression and RNA interference expression potato plants identified 2180 phosphorylated proteins.Through the quantitative analysis of modification sites,2922 modification sites were jointly identified in the two groups.Among them,a total number of 78 motifs were differentially modified;thereinto,49 phosphorylated proteins were up-regulated and29 were down-regulated.Through phosphorylated protein difference analysis,2023proteins were identified in the two groups,of which 15 were up-regulated and 14 were down-regulated.Using GO,KEGG,and COG databases to annotate the identified phosphorylated proteins,the results showed that these proteins mainly play functions in plant growth,development,signal transduction and metabolic pathways.