Construction of TaGAPC Site-directed Mutagenesis Gene Vector and Prokaryotic Expression Analysis from Wheat

doi:10.7668/hbnxb.2016.01.008

Abstract

Abstract: In order to elucidate the roles of catalytic active Cys¹⁵⁴,Cys¹⁵⁸ in Triticum aestivum L.cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (TaGAPC),these two cysteins were site-directed mutated into serine by overlap-extension PCR.Then recombinant prokaryotic expression vectors of TaGAPC and its mutants TaCys154S/TaCys158S were constructed and transformed into E.coli BL21.The recombinant protein were induced by 0.25 mmol/L IPTG at 25℃ and subsequently purified by Ni²⁺-IDA column.These researches could lay the foundation for the enzyme activity assay of TaGAPC and its mutants TaCys154S/TaCys158S and cysteine active sites.

Key words: TaGAPC, Overlap-extension PCR, Site-directed mutagenesis, Prokaryotic expression, Protein purification

CLC Number:

Q78
S512.03

GUO Ziping, ZHAI Qinghua, ZENG Lingfeng, DENG Xiping, YANG Shushen. Construction of TaGAPC Site-directed Mutagenesis Gene Vector and Prokaryotic Expression Analysis from Wheat[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(1): 46-50. doi: 10.7668/hbnxb.2016.01.008.

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http://www.hbnxb.net/EN/Y2016/V31/I1/46

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