Optimization of Prokaryotic Expression Conditions of Cotton GhPDP Gene

doi:10.7668/hbnxb.20191119

Abstract

Abstract: Pyruvate dehydrogenase phosphatase GhPDP is closely related to the regulation of cotton male sterility energy. To obtain a large number of active GhPDP proteins, the prokaryotic expression conditions the target protein were optimized in this experiment. The cDNA sequence encoding the GhPDP gene was inserted into pET-22b to construct a recombinant expression vector pET-22b-GhPDP, which was transformed into Escherichia coli Transetta(DE3) and induced expression by IPTG. Single factor test and orthogonal experiment were used to optimize the induction temperature, induction time, final IPTG concentration and bacterial cell density OD₆₀₀, using SDS-PAGE and GeneTools gel analysis software to analyze the expression of the target protein and explore the best conditions for soluble protein expression. The results showed that successfully constructed the recombinant expression vector pET-22b-GhPDP. The target protein was expressed in Transetta(DE3) strain, mainly in the form of inclusion body. The results of single factor experiments showed that the soluble expression level of the target protein was the highest under the conditions of induction temperature of 20℃, induction time of 5 h, IPTG final concentration of 0.10 mmol/L and cell density of OD₆₀₀ of 0.8. Through orthogonal analysis, it was concluded that the intensity of the factors affecting the soluble expression of the target protein from high to low as induced temperature, final IPTG concentration, induction time, and bacterial cell density OD₆₀₀. Based on the results of single factor experiment and orthogonal experiment, the optimal induction conditions were determined to be a bacterial cell density of OD₆₀₀ of 0.8, an induction temperature of 20℃, an induction time of 5 h, and IPTG final concentration of 0.10 mmol/L.

Key words: Cotton, GhPDP genes, Prokaryotic expression, Condition optimization

CLC Number:

S562.03

LIU Dong, LAN Yanping, ZHANG Li, ZHAO Yan, QU Yunfang, HUANG Jinling. Optimization of Prokaryotic Expression Conditions of Cotton GhPDP Gene[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(5): 55-61. doi: 10.7668/hbnxb.20191119.

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