基因工程根瘤菌表达融合蛋白GST-HAL1条件的优化及耐盐水平的提高

Optimization of Inducing Parameters to Express GST Fusion Protein in Engineered Rhizobia and the Enhancement of Salt Resistance

利用原核表达载体pGEX-4T-1构建重组质粒载体pGEX-HAL1,三亲本杂交转化根瘤菌,表达融合蛋白GST-HAL1。结果表明,与空白菌和未诱导工程根瘤菌相比,诱导后工程根瘤菌的耐盐水平有明显提高。通过检测不同温度、不同诱导时间和不同IPTG诱导浓度条件下融合蛋白的表达量,优化各个参数。在28℃的温度条件下,融合蛋白GST-HAL1表达量明显增加;在IPTG浓度为1.0 mmol.L-1,诱导时间为3 h的条件下,融合蛋白的表达量最高,培养基产生的融合蛋白为3.02 mg.L-1,占可溶性蛋白质的39.34%。本研究对今后利用基因工程技术研制新型生物肥料做了有益的尝试。

Recombined plasmid pEGX-HAL1 was constructed using prokaryotic expression vector pGEX-4T-1, and transformed to Rhizobia by tri-parental mating to express fusion protein GST-HAL1. The results revealed that the salt resistance of engineered Rhizobia induced by IPTG was obviously improved, compared with blank and engineered Rhizobia without inducemeat. Under different temperatures, different inducing times and different IPTG inducement concentration, the expressing quantities of fusion protein were investigated to optimize the inducing parameters. The expressing quantities of fusion protein GST-HAL1 obviously increased under 28℃. Moreover, GST fusion protein can be induced effectively in engineered Rhizobia, and the product is up to 3.02 mg·L^-1liquid medium, which is 39.34% of total soluble protein, under the inducing conditions of 3 h and 1.0 mmol·L^-1 IPTG concentration. The study may contribute to bio-fertilizer production by utilizing gene engineering technology in the future.