野油菜黄单胞菌分泌组双向电泳样品制备方法的建立

野油菜黄单胞菌野油菜致病变种（Xanthomonas campestris pv.campestris,Xcc）是十字花科植物黑腐病的病原细菌。我们建立了Xcc的蛋白质组学研究平台,用于分离、鉴定该菌的致病相关蛋白。为了减少胞外多糖（exopolysaccharide,EPS）对蛋白材料质量的影响,我们构建了EPS缺陷的Xcc8004ΔgumB突变体。本研究以Xcc8004ΔgumB出发菌株,用诱导培养液培养,取细胞上清通过超滤浓缩得到蛋白质粗提物,分别采用丙酮沉淀法、试剂盒纯化法和丙酮沉淀-试剂盒联用法来纯化蛋白质粗提物,通过比较双向电泳的结果优选最佳的样品制备方法。结果证明丙酮沉淀-试剂盒联用法较为理想,所得双向电泳图片清晰,分辨率高。因此,此方法可以用于制备野油菜黄单胞菌分泌组双向电泳样品,并可以满足进一步研究的需要。

Establishment of a Method for Preparation of the Secretome Samples from Xanthomonas campestris pv.campestris for Two Dimensional Electrophoresiss

Xanthomonas campestris pv.campestris（Xcc） is the causal agent of the black rot disease of cruciferous plants.We have established a proteomics research platform of Xcc for the separation and identification of pathogenic bacteria associated protein.In preliminary work,the gumB mutant of Xcc 8004 was constructed in the 8004 background,named as Xcc 8004ΔgumB,for reduing the influences of exopolysaccharide（EPS） on the quality of isolated protein.Xcc 8004ΔgumB strains were cultured in inducing medium and the protein extracts were yielded by ultrafiltration of the cell supernatants.The protein samples were prepared for 2-DE by acetone precipitation,2D clean up kit or acetone precipitation-2D clean up kit methods,respectively.The optimized method was determined by comparing the generated 2-DE results from different preparations.The results indicated that the 2-DE images generated from acetone precipitation-2D clean up kit method were clearer with highest resolution.Therefore,this method could be chosen for preparation of the Xcc secretome 2-DE samples and meet the need for further research.