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

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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.

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Abbreviations

ABP1:

Auxin-binding protein 1

IPTG:

Isopropyl-β-d-thiogalactoside

ER:

Endoplasmic reticulum

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant nos. 30571279 and 30871699) and China Postdoctoral Science Foundation (20070420095).

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Authors and Affiliations

  1. College of Life Science, Shanghai University, Shanghai, 200444, People’s Republic of China

    Si-Bao Wan & Jing-Yuan Yin

  2. College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, 100083, People’s Republic of China

    Si-Bao Wan, Wei Wang, Mei Luo, Wei-Dong Huang & Ji-Cheng Zhan

  3. Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Science, Beijing, 100093, People’s Republic of China

    Wei Wang

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  1. Si-Bao Wan
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  2. Wei Wang
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  3. Mei Luo
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  4. Wei-Dong Huang
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  5. Jing-Yuan Yin
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  6. Ji-Cheng Zhan
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Correspondence to Ji-Cheng Zhan.

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Si-Bao Wan and Wei Wang contributed equally to this work.

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Wan, SB., Wang, W., Luo, M. et al. cDNA Cloning, Prokaryotic Expression, Polyclonal Antibody Preparation of the Auxin-Binding Protein 1 Gene from Grape Berry. Plant Mol Biol Rep 28, 373–380 (2010). https://doi.org/10.1007/s11105-009-0161-9

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