| Plants always suffer from various stresses,mainly including drought,high salinity,high temperature,the chilling injury,diseases and insect pests,during the whole life span which affect their normal growth.Adversity can harm the cells in numerous ways,even could lead to the death of the plant directly.The injuries of adversity to the plant are as follows: cause cell dehydration,destroy membrane system,affect enzyme activity and lead to intracellular metabolism disorders.Plants have also developed a variety of defense mechanisms to counter the influences caused by the adverse factors in their long-term evolution processes.Late embryogenesis abundant(LEA)protein,as a kind of stress-related protein,plays an important role in plant stress physiology regulation.To explore the functions of LEA protein in plant under adversity conditions,the group 2 LEA protein WZY1-2 and the group 3 LEA protein WRAB18 were considered as the research objects to detect their functions in plants under adversity stresses in this paper.WZY1-2 protein,as one of the group 2 LEA protein(also known as dehydrin)members,it has the typical conservative sequence of this protein family: K-segments and S-segment,and belongs to the SK3 type dehydrins,it can be induced by variety of adversity signal molecules.In this study,the subcellular localizations and existing forms of WZY1-2 protein and its truncated derivatives without K-segments or S-segment in plant cell were detected respectively for the first time,results also showed the influence of lack of the K-segments or S-segment on WZY1-2 protein's subcellular localization and existing form.Through overexpressed WZY1-2 protein in Escherichia coli and Nicotiania benthamiana,we explored its functions on both prokaryotic and eukaryotic organisms under drought,high salt,high temperature and low temperature conditions.The co-immunoprecipitation assay was carried out to explore the interactions of WZY1-2 protein within the cells.The results of the study are as follows:1.In this research,the ORF sequence of wzy1-2 was isolated from the Zhengyin 1 cultivar of Triticum aestivum and WZY1-2 protein was expressed in Escherichia coli.The molecular weight of WZY1-2 in SDS-PAGE showed a double size of its theoretical value,Bimolecular Fluorescence Complementation(BiFC)approach demonstrated that WZY1-2 was existed in the form of homologous dimers in cells.Considered the ORF sequence of wzy1-2 gene as the template,using overlap extension PCR technology to delete the segment(s)of K,S,respectively,obtained its truncated derivatives without K-segments wzy1-2?K or S-segment wzy1-2?S,according to the results of BiFC,truncated derivative protein missing K-segments or S-segment still existed in the form of dimers in the leave cells of tobacco,this indicated that neigher K-segments nor S-segment do work on the dimer formation of WZY1-2 protein.2.GFP fusion protein expression vectors were built for protein WZY1-2,WZY1-2?K and WZY1-2?S,respectively,and expressed transiently in tobacco leave to investigate their subcellular localizations,results showed that WZY1-2 and WZY1-2?K were localized in the nucleus.Parts of WZY1-2?S still had the localization in nucleus,while there were amount of WZY1-2?S proteins localized in the inner membrance and cytoplasm of cells.It followed that the K-segments made no efforts on the nuclear localization of WZY1-2,yet the S-segment involved in WZY1-2's proper nuclear positioning,nonetheless,it was not the determinant.3.Overexpressed WZY1-2 protein in strain BL21(DE3)of Escherichia coli and treated the strains which harbored WZY1-2 protein and empty vectors with four abiotic stresses(drought,high salt,high temperature and low temperature).By measuring the OD value under 600 nm,results indicated that overexpression of WZY1-2 protein could enhance the viability of E.coli during stress conditions.Built a transgenic vector of wzy1-2 gene,agrobacterium-mediated semal impregnation method was used to obtain the transgenic tobacco.Wild type tobacco was considered as control,by analyzing the growth phenotype,root length,germination rate,survival rate and stress-related enzymatic activities of tobaccos under different adversity stresses,data showed that overexpression of WZY1-2 increased the stress tolerance of transgenic plants.4.Using the Ni2 + resin column to purify the WZY1-2 protein expressed from BL21(DE3)of Escherichia coli,and prepared the WZY1-2 protein antibody to implement the co-immunoprecipitation assay,after incubated WZY1-2 protein with genomic DNA of wheat leave which have been treated under low temperature for 36 h,results showed that WZY1-2 could bind to DNA.WRAB18 protein,has the conservative amino acid sequence(TAQAAKEKAGE)of group 3 LEA proteins,it belongs to the group 3 LEA protein family.In this study,we explored the subcellular localization of WRAB18 protein in plant cells and the protective effect towards lactate dehydrogenase(LDH)in vitro against adverse stresses including drought,high salinity,and high and low temperatures.Then WRAB18 was overexpressed in both Escherichia coli and tobacco to explore its function under multiple stresses.In addition,the genomic sequence of WRAB18 gene and its promoter sequence were isolated and the sequence analysis was carried out.Results of the study are as follows:1.A C-terminal GFP recombinant vector was generated to examine subcellular localization of the WRAB18 protein in vivo.The GFP::WRAB18 fusion protein was injected into N.benthamiana leaves and transiently expressed.The fluorescence signals under the 488 nm GFP channel and m Cherry channel(pt-rk CD3-999)were observed in protoplast cells under a confocal microscope and indicated WRAB18 localization in the plastids of protoplast cells.2.Incubated the purified WRAB18 protein with LDH solution under the drought,high salt,high temperature and low temperature stresess,compared with controls,WRAB18 showed greater protection of LDH activity during adversity conditions.3.Overexpressed WRAB18 protein in both Escherichia coli BL21(DE3)and tobacco plants,treated the overexpression strains and lanes under drought,high salt,high temperature and low temperature stresses respectively.Results suggested that WRAB18 protein has protective effects in both prokaryotic and eukaryotic cells under various abiotic stresses compared with controls.4.Based on the full-length cDNA sequence of WRAB18,the 610 bp genomic sequence of WRAB18 gene containing one intron(100 bp)and two exons which length was 60 bp and 450 bp respectively was isolated.In addition,using wheat genomic DNA as template,2000 bp sequence upstream WRAB18 gene was obtained,PlantCARE Database analysis showed that the sequence contained the promoter conservative elements,such as TATA-box and CAAT-box.It also contained cis-acting elements: ABREs,GARE,LTRE,MBS,etc.The quantitative PCR analysis demonstrated that the transcript accumulation of WRAB18 gene occurred in response to drought,high salt,high temperature and low temperature treatments. |
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