摘要：

旨在克隆小麦抗叶锈病相关基因 TaRLKS.2,并检测其表达蛋白的激酶活性,为进一步探讨TaRLKS.2在小麦抵抗叶锈菌侵染中的作用机制奠定基础,从而为小麦抗叶锈病育种提供新基因资源。以抗叶锈病近等基因系 TcLr26 的cDNA为模板,克隆 TaRLKS.2 编码区全长;利用GSDS 2.0软件分析TaRLKS.2蛋白的结构域;构建pET28a-TaRLKS.2原核表达载体,利用 IPTG诱导TaRLKS.2-His 融合蛋白的表达,并采用Ni-NTA亲和层析的方法纯化表达蛋白;利用磷酸化底物 MBP 和Western Blotting检测表达蛋白TaRLKS.2的激酶活性。结果表明,TaRLKS.2 编码区全长2 244 bp,其编码蛋白包含2个激酶结构域,含保守的激酶活性中心和ATP结合位点,具有典型的RLCK家族特征。在28 ℃、0.25 mmol/L IPTG条件下,TaRLKS.2-His重组蛋白被诱导形成可溶性蛋白,在纯化后的重组蛋白中加入MBP底物、ATP、磷酸缓冲液以及蛋白酶抑制剂后,30 ℃反应30 min,利用抗-磷酸化-Ser/Thr-Pro抗体检测,证明 TaRLKS.2具备蛋白激酶活性,推测其可能通过磷酸化下游信号组分调控小麦免疫通路,增强小麦对叶锈菌的抗性。

关键词: 小麦, 叶锈病, 原核表达, 激酶活性

Abstract:

This study aimed to clone the wheat leaf rust resistance-related gene TaRLKS.2 and detect the kinase activity of its expressed protein,in order to lay a foundation for further exploring the mechanism of TaRLKS.2 in wheat resistance against Puccinia triticina infection and to provide new genetic resources for wheat leaf rust resistance breeding.Using cDNA from the leaf rust-resistant near-isogenic line TcLr26 as a template,the full-length coding region of TaRLKS.2 was cloned.The domain structure of the TaRLKS.2 protein was analyzed using GSDS 2.0 software.A pET28a-TaRLKS.2 prokaryotic expression vector was constructed,and the TaRLKS.2-His fusion protein was induced with IPTG,followed by purification using Ni-NTA affinity chromatography.The kinase activity of the expressed TaRLKS.2 protein was detected using the phosphorylation substrate MBP and Western Blotting.The results showed that the coding region of TaRLKS.2 was 2 244 bp in length,encoding a protein containing two kinase domains with conserved kinase active centers and ATP-binding sites,exhibiting typical characteristics of the RLCK family.Under conditions of 28 ℃ and 0.25 mmol/L IPTG,the TaRLKS.2-His recombinant protein was induced to form soluble protein.After adding the substrate MBP,ATP,phosphate buffer,and protease inhibitors to the purified recombinant protein,the reaction was carried out at 30 ℃ for 30 min.Detection with an anti-phospho-Ser/Thr-Pro antibody confirmed that TaRLKS.2 possesses protein kinase activity.It is speculated that TaRLKS.2 may regulate wheat immune pathways by phosphorylating downstream signaling components,thereby enhancing wheat resistance to leaf rust.

Key words: Wheat, Leaf rust, Prokaryotic expression, Kinase activity

中图分类号: 

• S512.1小麦

• S435.121.43