A Preliminary Study On The Molecular Mechanism Of MAPK1 In Response To Drought Stress And The Drought Resistance Of Its Interaction Protein BnABA1

Oilseed rape（Brassica napus）is an important oil crop and one of the most important sources of vegetable oil.However,with the global climate change,rapeseed suffers from various stresses during its growth and development,which severely affects the development and yield.Among them,drought stress is one of the most serious ones,which can cause oxidative stress,disrupt the homeostasis of water and ions in plants,and influence the function of related proteins,thereby seriously affecting the growth and yield of rape.Therefore,to study the drought resistance mechanism and to explore the regulatory network responsible for drought stress are essential for breeding drought-resistant and high-yield rape varieties.The MAPK cascade pathway is a very conservative signaling pathway for eukaryotes.This pathway is involved in biological processes such as gene expression,cell growth and development,and signal transduction.It is the hub of various regulatory networks.The former study showed that BnMAPK1 can promote the drought resistance in rapeseed,confirming the interaction between BnABA1 and BnMAPK1,and the ABA1 gene encoding Zeaxanthin cyclooxygenase ZEP plays an important role in plant growth and development and abiotic stress response.Based on this,this study used quantitative proteomics and phosphorylated proteomics techniques to preliminary analyze the molecular mechanism of MAPK1 in response to drought stress;screened the interaction region between BnABA1 and BnMAPK1,and predicted the phosphorylation site;and obtain genetic-modified oilseed rape with overexpression and inhibited expression of BnABA1,In addition,BnABA1overexpression transgenic Arabidopsis thaliana were obtained,and the effect of BnABA1 on drought tolerance of A.thaliana was evaluated.The main results are as follows:1.MAPK1 expression influenced quantitative proteomics and phosphorylated proteomicsin A.thaliana exposed to drought stressUsing Atmapk1 mutant and wild-type A.thaliana as materials,the molecular mechanism of MAPK1 in response to drought stress simulated by 40%PEG6000 was studied.It was found that the inhibition expression of MAPK1 would increase the wilting symptoms of the plants under drought stress.Quantitative proteomics and phosphorylated proteomics based on TMT-labeled were performed on A.thaliana exposed to drought stress.Compared with the wild type,proteomics identified 6,814quantifiable proteins,including 87 differentially expressed proteins（P-value<0.05,FOLD CHANGE 1.2）,42 proteins were up-regulated and 45 proteins were down-regulated.Functional analysis of differentially expressed proteins indicated that down-regulated proteins were significantly enriched in the photosynthetic pathway,while up-regulated proteins were mainly enriched in galactose metabolism,α-linolenic acid metabolism,catabolism of glucosamine-containing compounds,cell wall decomposition,and plant senescence.It shows that MAPK1 slows the growth of plants and the expression of senescence-related proteins to make them less prone to senescence,alleviates the degradation of chlorophyll,promotes the biosynthesis of chlorophyll,electron transfer,and the expression and combination of light-harvesting proteins to make the photosynthesis process more stable and inhibit the redox process,polysaccharide catabolism process,cell wall decomposition and other biological processes related protein expression,thereby enhancing plant drought resistance.Phosphoproteomics analysis screened out in a total of 94 differentially phosphorylated proteins（112 phosphorylation sites）,of which 56 proteins（64 sites）were significantly up-regulated and 38 proteins（48 sites）were phosphorylated.Function analysis of differentially phosphorylated proteins showed that the up-regulated differentially phosphorylated proteins were significantly enriched in the photosynthesis pathway,and the down-regulated differentially phosphorylated proteins were mainly enriched in macromolecular glycosylation,glycoprotein biosynthesis and metabolism,RNA transport,Translation initiation,calcium-mediated signaling and other pathways.In addition,the phosphorylation level of aquaporin is also significantly increased.The results showed that the phosphorylation of MAPK1 may be mainly related to the synthesis of proteins related to biosynthesis of macromolecules such as photosynthetic pathway-related proteins,aquaporins,proteins involved in the transcription and translation of proteins,glycoproteins,and calcium-mediated signaling pathway Protein phosphorylation levels are used to mediate plant tolerance to drought stress.2.Screening of the interaction segment between BnABA1 and BnMAPK1Specific primers were synthesized to clone the BnABA1 gene in B.napus,and two copies were obtained:BnABA1A07 and BnABA1C07.The protein domains of BnABA1A07 and BnABA1C07 were predicted to contain phosphopeptide recognition domain FHA.On this basis,we constructed pGADT7-Prey recombinant expression vectors with different lengths of BnABA1,using yeast two-hybrid technology,and finally screened out the 575-630 aa segment of BnABA1A07 and the 613-668 aa segment of BnABA1C07,which interacts with BnMAPK1,respectively.Predicting the phosphorylation sites of the two interacting segments,the results showed that both contain 6 identical serine/threonine residues that are easily phosphorylated,indicating that BnMAPK1 may phosphorylates BnABA1 at these serine/threonine residues site to interact.3.Overexpression of BnABA1 in napus and the acquisition of RNAi plantsUsing Gateway technology,pEarleyGate101-BnABA1A07 and pEarleyGate101-BnABA1C07 over-expression vectors were constructed;RNAi technology was used to construct BnABA1A07 and BnABA1C07 co-inhibition expression vector p FGC5941-BnABA1.With transformation of Zhongshuang11（B.napus）by agrobacterium-mediated transformation,and identification of T₀ transgenic plants by PCR and qRT-PCR,respectively,we obtained 3 positive OE-BnABA1A07plants,9 positive OE-BnABA1C07 plants,and 6 positive RNAi plants-BnABA1plants.This will provide materials for our subsequent research on the function of BnABA1,and lay a foundation for the in-depth exploration of the signal network mediated by BnMAPK1 and BnABA1.4.Acquisition of Arabidopsis BnABA1-overexpression plants and analysis of their tolerance to drought stressThe overexpression-BnABA1A07 and overexpression-BnABA1C07 recombina nt vectors were transformed into A.thaliana（Col-0）plants by dipping flower,a nd the T₁ generation plants were screened using Basta screening,PCR and qRT-PCR identification.A total of 35 positive AtOE-BnABA1A07 Arabidopsis plant s were obtained,and the BnABA1A07 gene expression was about 150-4600 time s that of WT.A total of 7 positive AtOE-BnABA1C07 Arabidopsis plants were obtained,and the BnABA1C07 gene expression was about 900-7800 times that of WT.The T₄ generation homozygous AtOE-BnABA1A07 and AtOE-BnABA1C07 Arabidopsis lines（A07-19/-21/-51 and C07-10/-11/-12）and WT pl ants were treated with mannitol and natural drought And dehydration treatment.The results showed that under the treatment of mannitol,the germination rate a nd relative root length of transgenic lines were higher than WT;under natural d rought,the survival rate of transgenic lines were significantly higher than WT,and the water retention capacity of transgenic lines were all greater than WT In addition,under the treatment of dehydration,the transgenic plant’s detached lea ves’water loss rate was significantly lower than that of WT.The results showe d that overexpression of BnABA1 in A.thaliana can enhance the drought tolera nce of plants.exposed to drought stress.