Analysis Of Differentially Expressed Proteins And Cloning Of The Candidate Gene Under Water Stress In Wheat Seedling Leaves

Drought is one of the serious ecological problems. Agricultural economic losses causedby the drought equivalent to those caused by other natural disasters. It have importantsignificance on drought tolerance research of crops to relieve water resource shortage, enlargewheat planting area, increase the yield of wheat, and alleviate commissariat problem.The class of proteins expression that called water stress response protein will be reducedor increased when plant is under water stress.The proteins plays a protective role on plantstressing adaptation, and it can significantly improve the plant resistance to stress. Waterstress D-response protein is closely related to drought. It has been reported that the responsiveprotein were significantly correlated with ecological types of cultivars or advanced lines, andit can be regarded as an indicator of wheat drought resistance identification.Wheat droughtresistance is a complex quantitative trait controlled by multiple genes, and the functions ofeach gene are interlaced to form complex network system.In order to make clear the waterstress D-response protein’s types, properties of response to water stress, molecularmechanisms, this study applied the SDS-PAGE, two-dimensional gel electrophoresis andcombined with mass spectrometry (MALDI-TOF/MS, LC-MS/MS) to investigate water stressresponse protein, and at a global level, under-0.5MPa PEG-6000solution stress, differentiallyexpressed proteins were identified, the main conclusions are as follows:1、It was analyzed that wheat seedling leaf protein differential expression profile underthe normal water supply and48h-0.5MPaPEG-6000stress in.In a repeatable detected850±45protein spots,31differentially expressed protein spots were detected, among them14spotsis down regulated expression，while17spots up regulation. These spots are mainly involvedin energy metabolism, amino acid metabolism, photosynthesis, stress defense and signaltransduction processes. The study can help us understand plant the response to molecularmechanism under drought condition, at the same time for for drought resistance molecularmarkers provide reference development.2、SDS-PAGE results showed that the protein was found after treatment of6h, compared to normal water supply, and had the highest expression quantity under-0.5MPa PEG-6000permeation stress in the48h of the treatment. Along with the extension of stress time, theexpression of protein gradually declined; When stressed for48h, the seeding was treatedwith normal water supply. The bind of D-response protein was disappeared when rehydratedfor72h. The target protein bands under normal water supply and water stress were excisedfrom the gel and identified by mass spectrometry after the protein bands were digested bytrypsin. After mass spectrometry identification we have obtained two positive results, and theresults from two different treatment methods was identified. The results showed that the targetprotein bands were composed of Ribulose-1,5-bisphosphate carboxylase (Rubisco) largesubunit and Phosphoglycerate mutase by mass spectrum analysis(P <0.05). The Mass scoreswere1632,88and coverage30%,21%, respectively. Among the matches peptides, there were65peptides that belong to ribulose-1,5-bishosphate carboxylase (Rubisco) big subunit,among them35peptides’s credibility scores were more than threshold credibility scores;There were five peptides which belong to Phosphoglycerate Mutase, and four of themcredibility peptides were more than threshold credibility scores.The reasonable implicationsof the drought responsive protein were that it was Rubisco large subunit3、Rubisco small subunit of different drought resistance cultivars was cloned. Thesequence analysis shows that the genes in the different drought resistance of two varieties isnot difference, and rubisco small subunit may be not the cause of water stress D-responseprotein different expression.It was the first time for the domestic large area to popularize Dryland Wheat VarietyJinmai47were under drought stress differential proteomics analysis, clearly involved indrought stress response protein species, and for the first time water stress D-response proteinwere identification by mass spectrometry analysis, which helps us to explore well droughtresistance mechanism in wheat, at the same time as the breeding for drought resistancemolecular markers’development provide reference.