Cloning of a Dehydrin Gene and Preparation of Polyclonal Antibody of WZY1-1 in Wheat

Abstract: LEA proteins and Lea genes were one of the hot fields in plant embryology and stress physiology.Late embryogenesis abundant(lea)genes are a large and diverse group of genes.T- he lea proteins are plant proteins that synthesized at the onset of desiccation in maturing seeds and some of them can be induced by drought,high salt,low temperature and ABA application.Six major groups have been established based on regions of significant similarity between LEA proteins of different species. Dehydrins,which is known as groupⅡof plant late embryogenesis abundant(LEA)proteins,usually accumulate in plant during late embryogenesis or under stresses which cause dehydration.They are hydrophilic proteins with high thermostability,characterized by three highly conserved domains,the K,Y,and S segments. Dehydrin is a kind of protective agent of dehydration,which can protect biological macromol- ecules and maintain the cell membrane stability from drought and other water stresses of environment, in order to against dehydration-induced injure.At present there are many researches about dehydrin proteins in Arabidopsis thaliana,so- ybean,cotton,maize,barley,rice or wheat and barley was studied as a model plants.The target of the experiment are cloning of dehydrin of wheat and construction of the expression vector of dehydrin in prokaryotic organisms,and using the polyclonal antibody induced by the purified fusion protein aimed at studying the expression of dehydrin gene during different growth and immnocytochemical localization which can explain the functional characteristics of dehydrins through the researches of their distribution in the tissue and cell.In this experiment recombination expression plasmid PET-32a(+)-wzy1-1 was construct- ed according to recombination of cloned vector PUCM-T-WZY1-1 of wheat dehydrin gene WZY1-1,then transformed into the host strain E.coli BL21(DE3) which was induced by IPTG and target protein was got.The purified fusion protein was used to induce the production of polyclonal antibody in rabbits,at the same time study the situation of expression of dehydrin gene during the course of seedling dry reirrigation.The following results were obtained:1. A cloning of dehydrin gene wzy1-1from wheat was got.Construction of the expression vector of dehydrin wzy1-1 in prokaryotic,and a functiomal fusion protein through IPTG’s induction were obtained.It’s very easy to purify the protein because of its water solubility expression.2. The fusion protein was expressed at high level in BL21(DE3).After purification by Ni-NTA resin affinity chromatography and electroelution in bagfilter, the fusion protein was used to induce the production of polyclonal antibody in rabbits and ELISA detection showed the antigenicity of the fusion protein was satisfactory. Western blot showed the antiserum raised against the recombination dehydrin protein in rabbits could react to the protein expressed specifically,and antiserum could react to the dehydrin protein expressed in wheat leaf specifically under drought stress,these demonstrated its good antigenicity.3.The protein of zheng yin 1# wheat under drought stress was extracted, through the SDS-PAGE, Western blot assays revealed that there is a protein band with a relative molecular weight 28kd,during the extension process of water deficit by PEG6000,the expression of the gene which encoded the protein with Mr 28000 was intense and electrophoresis protein bands became deep,the signal of being hybrided was stronger than ever,which indicate that the content of soluble proteins were increased.After reirrigation,the protein with Mr 28000 in the leaves was measuted still but gradually reduced.4. Under drought stress,design the premier and the cloned gene through RT-PCR was got which both on the basis of the one of ORF template of L29152 mRNA,and the target gene was sequenced which is 843bp,then constructed the expression vector of target gene,and functiomal fusion protein through IPTG’s induction were obtained, which indicated that this can expressed under drought environment as well the cold stress…
Key words: dehydrin protein; prokaryotic expression; preparation of polyclonal antibody

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