Cloning and Prokaryotic Expression of Apple stem pitting virus CP Gene in Ya Pear in E. coli  

Na Liu , Jianxin Niu
Department of Horticulture, Agricultural College of Shihezi University, Shihezi, 832003, P.R. China
Both authors contributed equally
Author    Correspondence author
Molecular Pathogens, 2011, Vol. 2, No. 5   doi: 10.5376/mp.2011.02.0005
Received: 25 Oct., 2011    Accepted: 30 Nov., 2011    Published: 13 Dec., 2011
© 2011 BioPublisher Publishing Platform
This article was first published in Molecular Plant Breeding in Chinese, and here was authorized to translate and publish the paper in English under the terms of Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Liu et al., 2011, Cloning and Prokaryotic Expression of Apple stem pitting virus CP Gene in Ya Pear in E. coli, Molecular Plant Breeding, 9(6): 722-727 (doi: 10.3969/mpb.009.000722)

Abstract

Abstract In order to clarify evolutionary relationship and genetic diversity of apple stem pitting virus (ASPV), and preparation the corresponding special antiserum. In this study, total RNA was extracted from phloem tissue of Ya (Y) pear which infected by ASPV and used as template for cDNA synthesis. The coat protein (CP) gene of ASPV was cloned and sequenced. The CP gene of Ya pear was cloned into expression vector PET-28a, and transformed into E. coli BL21 (DE3), and SDS-PAGE electrophoresis analysis. The results showed that the complete CP gene consisted of 1 194 nucleotides and encodes a polypeptide of 397 amino acid (aa). Comparison of the amino acid sequences of Ya pear CP gene with other ASPV isolates showed approximately 70% similarity. Phylogenetic tree showed that all isolates of the coat protein (CP) genes of ASPV isolates at AA sequence revealed three groups. All ASPV isolates from apple were clustered to group I, whereas pear were clustered to groups â…¡ (except NC_003462) and the Ya pear were clustered into group â…¢. Results of SDS-PAGE showed that specific expression of a 42 kD fusion protein was achieved by the inducing of 1 mmol/L IPTG. The complete CP gene and prokaryotic expression vector of Ya pear provided additional baseline data for preparation recombinant polyclonal antibody of ASPV and further study of ASPV molecular biology.

Keywords
Apple stem pitting virus (ASPV); Coat protein; Prokaryotic expression
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