Prokaryotic Expression and Enzyme Activity Determination of C4 Key Enzyme Pyruvate Phosphate Dikinase Gene in Amaranth hypochondriacus

doi:10.7668/hbnxb.2017.02.010

Abstract

Abstract: To further explore the mechanism of action of AhPPDK protein,the prokaryotic expression vector of AhPPDK gene was constructed.The plasmid was extracted by alkaline lysis,digested with restriction endonuclease,detected with 1.0% Agarose gel.The correct recombinant plasmid pEASY-E1-AhPPDK was transformed into E. coli Transset (DE3),and the recombinant protein was induced by IPTG.SDS-PAGE analysis showed that recombinant AhPPDK could be highly expressed in E. coli Transset (DE3).The expressed recombinant protein had a molecular weight of 108 kDa,which was consistent with the expected molecular weight and was soluble protein.Pyruvate formed by pyruvate phosphodiesterase (PPDK)catalyzes the formation of lactate by excess lactate dehydrogenase and oxidizes reduced coenzyme I (NADH).Using ultraviolet spectrophotometry,the amount of NADH could be calculated according to the change of OD value at 340 nm,and then the activity of PPDK could be deduced.Spectrophotometric method showed that the prokaryotic expression of AhPPDK had the activity of enzyme.These results provide a basis for the further study on the mechanism of action of AhPPDK and the use of transgenes.

Key words: Amaranth hypochondriacus, AhPPDK, Prokaryotic expression, Enzyme activity determination

CLC Number:

S330
Q78

HE Feiyan, YAN Jianjun, BAI Yunfeng, FENG Ruiyun, ZHANG Weifeng. Prokaryotic Expression and Enzyme Activity Determination of C₄ Key Enzyme Pyruvate Phosphate Dikinase Gene in Amaranth hypochondriacus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(2): 61-65. doi: 10.7668/hbnxb.2017.02.010.

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Prokaryotic Expression and Enzyme Activity Determination of C₄ Key Enzyme Pyruvate Phosphate Dikinase Gene in Amaranth hypochondriacus

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