Antibody Preparation of TaMAPK5 Protein and Its Expression Characteristics in the Interaction Between Wheat and Puccinia triticina

doi:10.7668/hbnxb.20194872

Abstract

Abstract:

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 immunize 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 successfully 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.

Key words: Wheat, TaMAPK5, Prokaryotic expression, Protein purification, Polyclonal antibody

SHAO Wenxian, WANG Dongmei. Antibody Preparation of TaMAPK5 Protein and Its Expression Characteristics in the Interaction Between Wheat and Puccinia triticina[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5): 186-192. doi: 10.7668/hbnxb.20194872.

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Figures/Tables 7

Fig.1 TaMAPK5 prokaryotic expression vector construction A.Amplification of CDS region of TaMAPK5 gene:M.DL2000 DNA Marker,1.TaMAPK5 prokaryotic expression fragment;B.The detection of digesting positive plasmid:M.DL15000 DNA Marker,1.pET28a-TaMAPK5 vector plasmid,2.pET28a-TaMAPK5 plasmid digest.The red arrow.The target fragment.

Fig.2 Screening of conditions for soluble expression of TaMAPK5 recombinant protein A.Screening of IPTG induction concentration:M.Protein Marker,1.Total protein of TaMAPK5 without IPTG induction,2-10.Total protein of TaMAPK5 induced by 0.001,0.005,0.010,0.025,0.050,0.075,0.100,0.125,0.150 mmol/L IPTG;B.Screening of IPTG induction temperature:M.Protein Marker,1-4.Supernatant of uninduced,16,28,37 ℃-induced,5-8.Precipitate of uninduced,16,28,37 ℃-induced.The red arrow.The target protein.

Fig.3 Protein electrophoresis analysis and Western Blot test of purified TaMAPK5 A.Screening of imidazole elution concentration:M.Protein Marker,1.Clear supernatant,2.Effluent,3-8.0,20,50,100,250,500 mmol/L imidazole elution;B.Western Blot test:M.Protein Marker,1.BSA,2.TaMAPK5;The red arrow.TaMAPK5 recombinant protein.

Fig.4 SDS-PAGE for TaMAPK5 antibody purity M. Protein Marker;1. Anti-TaMAPK5.

Fig.5 ELISA for TaMAPK5 polyclonal antibody potency

Fig.6 Specificity detection of TaMAPK5 polyclonal antibody A.Western Blot detection of TaMAPK5 recombinant protein:M.Protein Marker,1.TaMAPK5,2.BSA;B.Western Blot detection in total protein of TcLr26 at 48 h after inoculation:M.Protein Marker;1.TcLr26,2.BSA.

Fig.7 Western Blot was used to detect the expression of TaMAPK5 protein at different time points after inoculation

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