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cDNA Cloning, Prokaryotic Expression, Polyclonal Antibody Preparation of the Auxin-Binding Protein 1 Gene from Grape Berry

Author:
Wan, Si-Bao, Wang, Wei, Luo, Mei, Huang, Wei-Dong, Yin, Jing-Yuan, Zhan, Ji-Cheng
Source:
Plant molecular biology reporter 2010 v.28 no.3 pp. 373-380
ISSN:
0735-9640
Subject:
molecular cloning, gene expression, polyclonal antibodies, plant proteins, reverse transcriptase polymerase chain reaction, Vitis vinifera, small fruits, prokaryotic cells, recombinant fusion proteins, rabbits, Escherichia coli, antiserum, Western blotting
Abstract:
Auxin-binding protein 1 (ABP1) plays an important role in the growth and development of plants. In this study, a novel grape ABP1 cDNA was isolated by reverse transcription polymerase chain reaction, using in silico cloning strategy based on grape dbEST. The obtained grape ABP1 cDNA is 756 bp containing a 567 bp open reading frame, which encodes a 188 amino acid protein. The deduced amino acid sequence possessed all of the three typical domains of plant ABP1s, an amino-terminal signal peptide and a carboxyl-terminal sorting signal, KDEL. A full-length ABP1 cDNA was introduced into an expressed plasmid pET-30a(+) vector at the EcoR I and Xho I restriction sites. The pET-ABP1 was found to be highly expressed in Escherichia coli BL21(DE3) pLysS cells with isopropyl-β-d-thiogalactoside induction. A fusion protein was purified and used as the antigen to immunize a New Zealand rabbit. The resulting antiserum was then further purified by HiTrap rProtein A FF Affinity Purification Kit to obtain the immunoglobulin G fraction. Western blot analysis indicated that ABP1 was regulated in fruits depending on the developmental stage. Our work represented a first step towards a better understanding of function analysis of grape ABP1.
Agid:
781608