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

CLC Number: 

• S512.1小麦

• S435.121.43