Abstract

In this study, the cDNA of a NOA (nitric oxide associated factor) was isolated from potato (Solanum tuberosum L.) for the first time (named StNOA1). The StNOA1 bears a centrally positioned GTPase-binding domain. Sequence alignment and phylogenetic analysis of the deduced StNOA1 protein with other known NOA family protein indicates that StNOA1 is highly homologenous with NbNOA1 (NOA fromNicotiana benthamiana). The cDNA was cloned into prokaryotic expression vector, pET-30a (+) and expressed in Escherichia coli BL21 (DE3) after induction with IPTG. The recombinant protein was dissolved by 8 M urea and recovered by dialysis due to most of them were in inclusion bodies. Then the recovered recombinant protein was purified by Ni-NTA and analyzed by SDS-PAGE. The results of SDS-PAGE showed that the StNOA1 was successfully expressed with the pET prokaryotic expression system and purified. The present study is the basis for further elucidating the biochemical characteristics of StNOA1 and is very significant for elucidating the nature of plant NOA and its action mechanisms in endogenous NO synthesis in plant species.

Key words: NOS, Solanum tuberosum L., AtNOA1.

Abbreviation

NOA, Nitric oxide associated factor; NOS, nitric oxide synthase;NR, nitrite reductase; EST, expression sequence tag; cDNA, complementary DNA;RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase- polymerase chain reaction; StNOA1, NOA from Solanum tuberosum L.; NbNOA1,NOA from Nicotiana benthamiana; AtNOS1, NOS gene from Arabidopsis thaliana;OsNOA, NOA from Oryza sativa; VvNOA, NOA from Vitis vinifera; ZmNOA, NOA from Zea may; IPTG, isopropyl thiogalactoside; SDS-PAGE, sodium dodesyl sulfate-polyacrylamide gel electro-phoresis; GTP, guanidine 5’-triphosphate;GTPase, guanidine 5’-triphosphatase