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Cloning, prokaryotic expression, and subcellular localisation of the TaMAPK10-like gene in common wheat

Author:
Nie, Yingbin, Ma, Songmei, Ji, Wanquan
Source:
Canadian journal of plant science 2019 v.99 no.4 pp. 460-466
ISSN:
1918-1833
Subject:
Aegilops tauschii, Setaria italica, Sorghum bicolor, Triticum, amino acids, bacteria, cell membranes, genes, genetic vectors, germplasm, microtubules, mitogen-activated protein kinase, molecular cloning, open reading frames, phylogeny, powdery mildew, protein content, wheat
Abstract:
Mitogen-activated protein kinase (MAPK/MPK) is a group of serine-threonine protein kinases that are activated by different extracellular stimuli. To explore the function of MAPK in wheat infected with powdery mildew, a new wheat germplasm N9134 was used to obtain the full-length MAPK gene and the MAPK sequence was used to identify its prokaryotic expression and subcellular localisation. Wheat MAPK was obtained by homologous gene cloning and designated as TaMAPK10-like. The open reading frame of TaMAPK10-like was 1638 bp, which coded a deduced protein of 545 amino acids. Phylogenetic analysis revealed that TaMAPK10-like was most closely related to MAPK10-like of Aegilops tauschii at the protein level. It had high nucleotide similarity with the reported MAPK gene in A. tauschii, Sorghum bicolor, and Setaria italica, and had features typical of MAPK family genes. Subcellular localisation showed that TaMAPK10-like was mainly located in the cytoplasm along the microtubules, and a small number were located in the cell membrane and the nucleus. A pMD-MAPK10-like fusion expression vector was constructed and the TaMAPK10-like fusion protein was 70 kDa. The expressions of protein in bacteria were best obtained using 0.5 mmol L⁻¹ isopropyl β-d-1-thiogalactopyranoside at 37 °C for 12 h. These results provide the basic data for further understanding the biological function of the TaMAPK10-like gene.
Agid:
6559855