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

文献类型： 外文期刊

作者： Wan, Si-Bao 1 ; Wang, Wei 1 ; Luo, Mei 1 ; Huang, Wei-Dong 1 ; Yin, Jing-Yuan 2 ; Zhan, Ji-Cheng 1 ;

作者机构： 1.China Agr Univ, Coll Food Sci & Nutr Engn, Beijing 100083, Peoples R China

2.Shanghai Univ, Coll Life Sci, Shanghai 200444, Peoples R China

3.Beijing Acad Agr & Forestry Sci, Inst Forestry & Pomol, Beijing 100093, Peoples R China

关键词： amino acid sequence;prokaryotic expression;carboxyl-terminal sorting signal

期刊名称：PLANT MOLECULAR BIOLOGY REPORTER （ 影响因子：1.595； 五年影响因子：2.042 ）

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收录情况： SCI

摘要： 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-beta-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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