Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (12): 2599-2606.doi: 10.3864/j.issn.0578-1752.2013.12.022

• RESEARCH NOTES • Previous Articles     Next Articles

Localization of STK2 of Setosphaeria turcica in the Genome, Characterization of Its Protein Structure and Expression in Eukaryotic Cells

 GONG  Xiao-Dong, FAN  Yu, LI  Po, YANG  Yang, ZHANG  Chang-Zhi, TIAN  Lan, ZHANG  Xiao-Yu, FAN  Yong-Shan, HAN  Jian-Min, GU  Shou-Qin, DONG  Jin-Gao   

  1. 1.Mycotoxin and Molecular Plant Pathology Laboratory, Agricultural University of Hebei, Baoding 071001, Hebei
    2.Plant Protection Institute, Hebei Academy of Agriculture and Forestry Sciences, Baoding 071000, Hebei
    3.Department of Life Sciences, Tangshan Teachers’College, Tangshan 063000, Hebei
  • Received:2012-12-10 Online:2013-06-15 Published:2013-01-11

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Abstract: 【Objective】The objectives of this study is to determine the location in genome, and to investigate the protein structure of STK2 of Setosphaeria turcica. Meanwhile, a eukaryotic expression vector of STK2 was constructed and expressed in Pichia pastoris to obtain secreted protein in extracellular. 【Method】 The location of STK2 and protein structures were analyzed using bioinformatics method. Primers were designed according to multiple cloning sites in vector pPIC9K and the ORF sequence of STK2. A recombinant eukaryotic expression vector was constructed and transformed into host yeast strain GS115 by electroporation. The aiming protein expressed in yeast cell through induction and was identified by SDS-PAGE. 【Result】 The identity of STK2 was 91433, locating between 1561986 and 1563262 in the positive-strand of scaffold_3 in the genome of S. turcica. Stk2 shared characteristic conserved domains of MAPK-like protein kinase. α-helices and random coils were its main elements of the secondary structure of Stk2, β strands were lesser and mainly located in N ends in this structure. The 3D structure was composed of a smaller N-terminal lobe and a bigger C-terminal lobe. The eukaryotic expression vector of pPIC9K-STK2 was successfully constructed, and the transformants were screened according to their resistance. A protein, with its molecular weight of about 41 kD, was obtained after induced by methanol, while no corresponding proteins were found in control, suggesting STK2 had been secreted to extracellular of the yeast cells.【Conclusion】STK2 is located between 1561986 and 1563262 in the positive-strand of scaffold_3 in the genome of S. turcica. The characteristics of Stk2 were analyzed, and showed that Stk2 shared all of conserved domains of MAPK kinase. The results also revealed that STK2 was successfully expressed in yeast cell and soluble protein was got in the experiment. These results will lay a foundation for the preparation of Stk2 polyclonal antibody and for the study of the functions of STK2.

Key words: Setosphaeria turcica , bioinformatics method , eukaryotic expression , MAPK

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