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Molecular cloning, prokaryotic expression, and purification of an alternatively spliced oligochitosan-induced Ser/Thr protein kinase in tobacco

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Oligochitosan elicitor has been reported to induce a variety of defense responses in plants. In our previous work, OIPK (oligochitosan induced protein kinase), which was a Ser/Thr protein kinase, was identified to be induced by oligochitosan through mRNA differential display. When OIPK was repressed in transgenic tobacco, decreased resistance to tobacco mosaic virus (TMV) was observed, which suggested that OIPK might play a role in resistance to TMV in tobacco. To investigate how OIPK functions in oligochitosan-induced tobacco resistance, here we cloned two spliced variants of OIPK gene: OIPKL and OIPKS. The OIPK and OIPKS sequences were almost identical with the OIPKL sequence, except that they lack some segments compared to OIPKL. The full-length OIPKL cDNA was inserted into pET-23b vector, prokaryotically expressed, and purified from inclusion bodies in E. coli strain BL21 (DE3). An anti-His antibody was able to recognize the His-tag in the OIPKL fusion protein, as revealed by immunoblotting. This prokaryotically expressed product could serve as a useful tool for further research on OIPK function in the signal transduction in tobacco resistance.

Affiliations: 1: Liaoning Provincial Key Laboratory of Carbohydrates, Biotechnology Department, Dalian Institute of Chemical Physics, Chinese Academy of Sciences ; 2: Graduate School of the Chinese Academy of Sciences


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