Antibody Preparation and Protein Expression Analysis of TaTLP8 in Wheat

doi:10.7668/hbnxb.20193736

Abstract

Abstract:

In order to investigate the function of thaumatin-like protein 8(TaTLP8)in wheat resistance to Puccinia triticina infection,the incompatible and compatible combinations of the wheat near-isogenic line TcLr26 and its recurrent parent Thatcher(Tc)with P.triticina physiological race 260 were studied.Following bioinformatics analysis,prokaryotic expression,affinity purification,and immunization,a rabbit-derived polyclonal antibody to TaTLP8 was prepared,and the expression of TaTLP8 in the incompatible and compatible combinations of wheat and P.triticina was detected.The results showed that wheat TaTLP8 was highly homologous to the HvTLP8 in barley and contained a signal peptide at its N-terminus.The recombinant plasmid pET28a-TaTLP8-nosp was generated with the TaTLP8 gene excluding its signal peptide region(TaTLP8-nosp),and the protein was expressed at an optimal concentration of 0.100 mmol/L IPTG in E.coli BL21.The purified TaTLP8-nosp recombinant protein was used to immunize New Zealand rabbits to prepare an antibody,which was found to bind specifically to TaTLP8 protein in wheat by Western Blotting.The prepared antibody was used to detect the expression of TaTLP8 in wheat-P.triticina incompatible and compatible interactions.TaTLP8 started to express 8 h after inoculation in the incompatible combination,and its expression level gradually increased.In the compatible combination,the expression of TaTLP8 was not detected until 48 h after P.triticina inoculation,and its expression level gradually decreased.In addition,the expression of TaTLP8 in the incompatible combination was higher than that in the compatible combination,indicating that TaTLP8 may play a positive regulatory role in the resistance of wheat to P.triticina infection.

Key words: Wheat, Puccinia triticina, TaTLP8, Antibody preparation, Western Blotting

LIU Chao, SUN Tianjie, LIU Na, CHEN Yan, WANG Dongmei. Antibody Preparation and Protein Expression Analysis of TaTLP8 in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(3): 35-41. doi: 10.7668/hbnxb.20193736.

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Figures/Tables 5

Fig.1 Bioinformatics analysis and construction prokaryotic expression vector of TaTLP8 A.TaTLP8 gene coding sequence;B.TaTLP8 protein amino acid sequence;C.TaTLP8 signal peptide prediction;D.TaTLP8 conservative domain; E.TaTLP8 phylogenetic tree analysis;F.Amplification TaTLP8-nosp fragment;G.pET28a-TaTLP8-nosp recombinant plasmid enzyme-cut.

Fig.2 Expression analysis of TaTLP8-nosp protein A.Different concentrations of IPTG induced of TaTLP8-nosp;B.The expression of TaTLP8-nosp was induced by different temperature;C.Western Blotting detection of recombinant protein expression.

Fig.3 Purification and quantification of TaTLP8-nosp protein A.Purification of TaTLP8-nosp protein;B.Quantitative analysis of TaTLP8-nosp protein.

Fig.4 Specific detection of TaTLP8-nosp antibody A.Specific detection of antibody against recombinant protein;B.Specific detection of antibodies against TaTLP8 in wheat total proteins.

Fig.5 Expression changes of TaTLP8 protein during Puccinia triticina infect wheat

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