Cloning and Characterization of OsSKIPa gene from rice (Oryza sativa Linn. Var. KDML 105)

Abstract

SKIPa gene plays role as critical and necessary gene affecting cell viability and enhances cell’s ability to survive from abiotic stress such as drought and salinity. This research study aimed to clone OsSKIPa gene from KDML 105 rice and to invent expression cassette for applying to plant variety improvement particularly against abiotic stress by gene transformation technique. The research was done at the Biotechnology Research and Development Office, Department of Agriculture, Pathumthani Province during May – September 2011. In this study, full-length genomic DNA sequences of rice (Oryza Sativa Linn.) encoded to OsSKIPa was isolated from rice via PCR – based method. The gene sequence contains a fragment of 2,353 bp, including a 1,824 bp complete ORF, the 5’UTR of 130 bp, 3’UTR of 399 bp and a polyadenylation signal ATAAA motif. OsSKIPa gene can be encoded to 607 amino acids polypeptide with molecular weight of 81.2 kDa and has putative function as SKIP / SNW domain containing protein. The highly conserved region of the gene is SNW/SKI-interacting protein-like, SNW/SKI-interacting protein, transcript variant X1, transcript variant X2 and transcript variant X3 which are found in Oryza sativa L. (NM001054719.1), Oryza brachyantha (XM006649000.1) and Brachypodium distachyon L. (XM010237787.2, XM010237788.2, XM010237789.2) with 99, 94 and 86% of homology, respectively. A 1,824 bp fragment of OsSKIPa gene was inserted into plant expression vector pCAMBIA2300 containing 35SCaMV promoter and NOS terminator. NPTII was used as a selectable marker in this selection. It was found that the total size of derived over-expression cassette (pCAMBIA2300 – OsSKIPa) was 11.5 kb.