Construction of Binary Vector for TAP and Protoplast Expression of CPK10 in Arabidopsis

doi:10.7668/hbnxb.2015.04.008

Abstract

Abstract: In order to study the molecular mechanism of Arabidopsis CPK10,the gene was amplified by PCR,and constructed into the binary vector with FLAG and HA tags for tandem affinity purification(TAP).Then the recombinant plasmid pCM1307-3FLAG-3HA-CPK10 was transformed into Arabidopsis wild-type protoplast mediated with PEG,about 10 hours expression,the CPK10 fusion protein with FLAG and HA tags was detected by Western Blotting.Immunoblot analysis performed with FLAG and HA antibodies showed a distinct cross-reaction with fusion protein at expected molecular weight.The successful expression of fusion protein in Arabidopsis protoplast laid the foundation for screening CPK10 interaction proteins through tandem affinity purification technique.

Key words: TAP, Binary expression vector, CPK10, Protoplast transformation

CLC Number:

LIANG Yun, WEI Feng-ju. Construction of Binary Vector for TAP and Protoplast Expression of CPK10 in Arabidopsis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(4): 43-46. doi: 10.7668/hbnxb.2015.04.008.

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