Prokaryotic Expression Of Wheat TaWRKY9 Gene And Its Polyclonal Antibody Preparation

Wheat is one of the most essential crops in the world. However, abiotic stresses like drought, low temperature and high salinity, biotic stresses like microbial pathogens and harmful insects affect the growth and yield of wheat seriously. Therefore, it is important to improve wheat biotic stress and abiotic stress resistance. WRKY transcription factors play a key role in the physiological and biochemical responses of plants under the harsh external environment. WRKY transcription factors involve in regulating plant growth, metabolism,response to the biotic and abiotic stresses. In addition, it can regulate disease-related,damage-inducible and senescence-related genes expression to enhance stress resistance ability of plants. Therefore, the study of WRKY gene might provide a theoretical basis for wheat resistance.In previous study, TaWRKY9 transgenic tobacco plants have been identified in DNA and RNA levels, but not in the protein level. In the present research, Chinese Spring wheat seedlings were cultivated, the cDNA was obtained after the total RNA was extracted from wheat leaves. Appropriate primers were designed for TaWRKY9 gene amplification by RT-PCR. Then TaWRKY9 gene was cloned into expression vector pET32 a to construct the prokaryotic expression vector. After that, the constructed plasmid was transformed into E.coli BL21(DE3) strain to induce the expression of target protein. Through exploring of several conditions such as OD600, IPTG final concentration and temperature, a molecular weight of 67.93 kDa recombinant protein was successfully expressed by induction. It was found that the recombinant protein was the inclusion body protein through soluble detection test. After that, the recombinant protein was extracted by denaturing solution A which contains urea, and was purified by SDS-PAGE gel extraction experiment. Then, the recombinant protein was concentrated to a certain degree by ultrafiltration, and was used as antigen to immune the New Zealand rabbit in Wuhan Institute of Virology to produce the polyclonal antibody. The polyclonal antibody can be used to identify TaWRKY9 transgenic tobacco at the protein level by Western-blot, thereby laying the foundation of TaWRKY9 biological function for further study, also provide a basis for research on transgenic wheat stress resistance.