Antibody Preparation of TaMAPK5 Protein and Its Expression Characteristics in the Interaction Between Wheat and Puccinia triticina
In order to further explore the function of TaMAPK5 in the interaction between wheat and Puccinia triticina,the CDS region of TaMAPK5 gene was cloned,and the prokaryotic expression vector pET28a-TaMAPK5 was constructed and transformed into E.coli BL21(DE3).The optimal concentration,induction time and induction temperature of isopropyl β-D-thiogalactoside(IPTG)induced expression of the target protein were explored,and the target protein was purified by Ni-NTA affinity chromatography.The purified recombinant protein was used to immu-nize New Zealand rabbits to prepare TaMAPK5 polyclonal antibody.The results showed that the full length of the CDS region of TaMAPK5 was 1 110 bp,and the TaMAPK5 recombinant protein was induced with IPTG at a final concentration of 0.050 mmol/L and incubated at 16 ℃ for 48 h.The recombinant protein was used as an antigen to immunize New Zealand rabbits,and a polyclonal antibody capable of specifically recognizing TaMAPK5 was suc-cessfully prepared.The antibody titer was 1:51 200.The results of Western Blot showed that TaMAPK5 was induced by P.triticina infection in wheat and P.triticina incompatible combinations.The recombinant protein TaMAPK5 was successfully expressed and purified,and its polyclonal antibody was prepared.It was revealed that TaMAPK5 protein may positively regulate wheat resistance to leaf rust infection.
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