Cloning, prokaryotic expression, purification and interaction with ABI5 of Arabidopsis thaliana AFP4

doi:10.3969/j.issn.1004-1524.2018.12.12

›› 2018, Vol. 30 ›› Issue (12): 2072-2080.DOI: 10.3969/j.issn.1004-1524.2018.12.12

• Plant Protection • Previous Articles Next Articles

Cloning, prokaryotic expression, purification and interaction with ABI5 of Arabidopsis thaliana AFP4

DENG Zibing, QIU Liangkun, MA Jianzhong^*

School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China

Received:2018-03-14 Online:2018-12-25 Published:2018-12-28

Abstract

Abstract: Arabidopsis thaliana ABI5 interacting protein AFP4 (ABI five binding protein 4) is a small molecule protein in plants, and its expression characteristics was similar with ABI5, and was induced by ABA. In this paper, Arabidopsis thaliana L. cv. Columbia was used as a material to amplify the complete coding sequence of AFP4 gene by PCR. The amplified fragment was inserted into the multiple cloning site of prokaryotic expression vector pET-32a(+) and yeast two-hybrid vector pGBKT7. The coding sequence of the ABI5 gene was inserted into the multiple cloning site of the yeast two-hybrid vector pGADT7. The results of recombinant plasmid showed that the cloned AFP4 and ABI5 coding sequences were 100% identical to the AFP4 gene (GenBank accession number NM_111081.2) and ABI5 gene (GenBank accession number NM_129185.3) included in NCBI database. The recombinant molecular weight of recombinant protein containing AFP4 fragment was 53.4 ku. The recombinant plasmids were transformed into Escherichia coli BL21 Star (DE3) and induced, and then analyzed by Ni-NTA affinity chromatography, SDS-PAGE and Western blot. Analysis of yeast two hybridization was carried out as recombinant plasmids containing AFP4 and ABI5 were co-transformed into Saccharomyces cerevisiae AH109. The results suggested that the suitable conditions for the expression of recombinant fusion protein in E. coli BL21 Star (DE3) were as follows: IPTG concentration was 0.4 mmol·L^-1,induction at 25 ℃ for 6 h. Under the above conditions, the recombinant fusion protein could account for 41.6% of the E. coli total proteins. After purification by Ni-NTA affinity chromatography column, AFP4 fusion protein showed a single band when analyzed by SDS-PAGE electrophoresis. The band which contained 6×His tag peptide was verified by Western blot. The growth status of colony and the color reaction showed that AFP4 and ABI5 could interact in yeast cells.

Key words: Arabidopsis thaliana, AFP4, prokaryotic expression, Western blot, yeast two hybrid, ABI5

CLC Number:

DENG Zibing, QIU Liangkun, MA Jianzhong. Cloning, prokaryotic expression, purification and interaction with ABI5 of Arabidopsis thaliana AFP4[J]. , 2018, 30(12): 2072-2080.

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Cloning, prokaryotic expression, purification and interaction with ABI5 of Arabidopsis thaliana AFP4

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