小麦TaTLP8的抗体制备及其蛋白水平表达分析

doi:10.7668/hbnxb.20193736

摘要/Abstract

摘要：

为探究TaTLP8类甜蛋白在小麦抵御叶锈菌侵染过程中的功能,以小麦近等基因系TcLr26及其轮回亲本Thatcher(Tc)为材料,分别与叶锈菌生理小种260组成不亲和与亲和组合。经生物信息学分析、原核表达、亲和层析、动物免疫,制备了TaTLP8的兔源多克隆抗体,并使用制备的抗体,对小麦与叶锈菌互作的不亲和与亲和组合中TaTLP8的表达情况进行了分析。结果表明,小麦TaTLP8与大麦HvTLP8同源性较高且N-端含有信号肽。以无信号肽区段的TaTLP8基因(TaTLP8-nosp)构建重组质粒pET28a-TaTLP8-nosp,并以0.100 mmol/L的最适浓度IPTG对该蛋白在大肠杆菌BL21中进行诱导表达。以纯化后的TaTLP8-nosp重组蛋白免疫新西兰兔制备了抗体。Western Blotting检测发现,该抗体可与小麦中TaTLP8蛋白特异性结合。使用制备的抗体检测了小麦-叶锈菌不同亲和性组合中TaTLP8的表达水平,发现TaTLP8在不亲和组合中自接种叶锈菌8 h后启动表达,其表达量逐渐升高;在亲和组合中,直至叶锈菌接种后48 h才检测到TaTLP8的蛋白信号,并且其表达水平逐渐下降。总体而言,TaTLP8在不亲和组合中的表达水平高于亲和组合,说明TaTLP8在小麦抵御叶锈菌侵染的过程中可能发挥正调控作用。

关键词: 小麦, 叶锈菌, TaTLP8, 抗体制备, Western Blotting

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

刘超, 孙天杰, 刘娜, 陈琰, 王冬梅. 小麦TaTLP8的抗体制备及其蛋白水平表达分析[J]. 华北农学报, 2023, 38(3): 35-41. doi: 10.7668/hbnxb.20193736.

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.

http://www.hbnxb.net/CN/Y2023/V38/I3/35

图/表 5

图1 TaTLP8的生物信息学分析及原核表达载体的构建 A.TaTLP8基因编码区序列;B.TaTLP8蛋白氨基酸序列;C.TaTLP8信号肽预测;D.TaTLP8保守结构域; E.TaTLP8系统发育进化树分析;F.扩增TaTLP8-nosp片段;G.pET28a-TaTLP8-nosp重组质粒酶切验证。

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.

图2 TaTLP8-nosp蛋白的表达分析 A.不同浓度的IPTG诱导TaTLP8-nosp表达;B.不同温度诱导TaTLP8-nosp的表达;C.重组蛋白表达的Western Blotting检测。

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.

图3 TaTLP8-nosp蛋白的纯化与定量 A.TaTLP8-nosp蛋白纯化;B.TaTLP8-nosp蛋白定量分析。

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

图4 TaTLP8-nosp抗体的特异性检测 A.抗体对重组蛋白的特异性检测;B.抗体对小麦总蛋白中TaTLP8的特异性检测。

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.

图5 TaTLP8蛋白在叶锈菌侵染小麦过程中的表达变化

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

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