Cloning and Characterization of Cyclophilin Gene from Sorghum bicolor (L.) Moench and Its Transformation into Tobacco Plant

Abstract

Cyclophilins are ubiquitous proteins present in all subcellular compartments, which are involved in a variety of processes such as immunosuppression and major biotic and abiotic stresses. The research was made at the laboratory of Biotechnology Research and Development Office, Department of Agriculture, Pathum Thani province during October 2008 – September 2010. In this study, two full-length genomic DNA sequences of sorghum (Sorghum bicolor) encoding cyclophillin (SbCyP) have been isolated from two sorghum varieties named U-Thong 1 (UT1) and Supanburi 60 (SPR 60) via PCR – based method with CyP4 (forward) + CyP4 (reverse). Amino acid sequence of SbCyP disclosed similarity to each other and with that of cyclophillins of various organisms. The gene sequence contains a fragment of 1,062 kb (genbank accession no EU722309), including a 519 bp complete ORF, the 5’UTR of 130 bp, 3’UTR of 413 bp and a typical AATAA motif, encoding the 172 amino acid polypeptide. A 519-bp fragment of the SbCyP gene was further amplified by PCR-based method with CyPXbal (forward) + CyPKpnl (forward), addition of XbaI and KpnI restriction sites for protein translation purpose and then ligated into plant expression vector pCAMBIA2300 containing 35SCaMV promoter and NOS terminator to obtain 10 kb - pCAMBIA2300 - CyP expression cassette. The expression cassette was then transferred into Nicotiana tobacum (tobacco) plant by Agrobacterium-mediated transformation with kanamycin antibiotic selection. A total of twenty-five transformed plants were randomly selected and screened for the presentation of transgenes by PCR-based method with two pairs of CyPXbaI (forward) + CyPKpnI (reverse) and NOS (forward) + 35SCaMV (reverse). Ten out of twenty-five selected plants were found to be transgenic tobacco plants.