关键词: 诱导, hrpNEcc, 蛋白合成, 生物农药

Abstract:

【Objective】 HrpNEcc protein is a kind of cellular signal transduction elicitor, which can induce plants acquiring broad-spectrum disease resistance, worms disperse ability, stress resistance and stimulate plants growth and development through activating multiple-gene expression and regulation of plants heredity system. During the experiment of screening for cheap inducer in high cell density fermentation of recombinant Escherichia coli, the TB medium without adding any exogenous inducer was used as a negative control, surprisingly the negative control showed high expression of HrpNEcc protein. The aim of this study was to find the factors inducing synthesis of the HrpNEcc protein, and study the influence of the sources and concentration of the factors on synthesis of the HrpNEcc protein. 【Method】 The recombinant E. coli was cultured by flask fermentation, and cell pellets was harvested by centrifugation. Total protein was determined by the method of Coomassie brilliant blue, using bovine serum albumin as the standard. The qualitative analysis of protein was performed by SDS-PAGE, the Coomassie-stained protein bands were scanned by Bandscan software to estimate the percentage of HrpNEcc protein in total cellular proteins. And then the concentration of HrpNEcc protein was calculated. 【Result】 The highest yield of HrpNEcc protein of the recombinant E.coli BL21(DE3)/pET30a(+)hrpNEcc reached about 301.45 mg?L-1, when the recombinant E. coli was cultured in TB liquid medium without adding any exogenous inducer. The yield of HrpNEcc protein is 72.43% higher than that of the recombinant E. coli which was cultured in LB liquid medium and was induced with IPTG. Further studies indicated that high content of yeast extract in TB medium was responsible for the high-level synthesis of HrpNEcc protein , probably there are some inducing factors in yeast extract. And it also was found that the expression levels of hrpNEcc gene varied greatly with the concentrations and sources of the yeast extract used in the media. 【Conclusion】 High content of yeast extract is found to be responsible for high-level synthesis of HrpNEcc protein without any exogenous inducer, but the inducing mechanism needs to be further studied.

Key words: induction, hrpNEcc, protein synthesis, biopesticide