Full Length Research Paper

Cloning and characterization of a nitrite reductase gene related to somatic embryogenesis in Gossypium hirsutum

G. Y. Han^#, J. N. Chi^#, X. F. Wang, G. Y. Zhang and Z. Y. Ma*

Key Laboratory of Crop Germplasm Resources of Hebei Province, Agricultural University of Hebei, Baoding, 071001, People’s Republic of China.

*Corresponding author. E-mail: mzhy@hebau.edu.cn. Tel: +86 312 7528401.

Fax: +86 312 7528400.

Abbreviations: cDNA, Complementary deoxyribonucleic acid; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; bp, base pair; NR, nitrate reductase; NiR,  nitrite reductase enzyme; NiR, nitrite reductase gene; GhNiR, a nitrite reductase gene cloned from Gossypium hirsutum; IBA, indolebutyric acid; KT, kinetin; 2,4-D, 2,4-dichloro-phenoxyacetic acid; MS, murashige and skoog medium; CTAB, cetyltrimethyl ammonium bromide; ORF, open reading frame; EST, expressed sequence tag; EtBr, ethidium bromide; IPTG, isopropylthiogalactoside; SDS-PAGE, sodium dodesyl sulfate- polyacrylamide gel electrophoresis; SSH, suppression subtractive hybridization.

^#These authors contributed equally to this paper

Accepted 29 January, 2010

   Abstract

A nitrite reductase gene related to somatic embryogenesis was first cloned from Gossypium hirsutum. The cDNA sequence of the gene, named GhNiR, is 2,257 bp in length, with 254 bp of the 5’ untranslated region and 236 bp of the 3’ untranslated region. The open reading frame is 1,767 bp in length, encoding a deduced amino acid sequence of 588 residues with a molecular weight of 65.722 kDa and an isoelectric point of 7.07. Semi-quantitative RT-PCR analysis showed that the expression level of GhNiR was higher in embryogenic calli and somatic embryoids than in nonembryogenic calli among different somatic embryogenesis stages, and that the level of GhNiR mRNA was also higher in the cultivar with higher somatic embryogenesis ability. The catalytic GhNiR was verified by transformation in E. coli BL21 (DE3) strain with the recombinant expression vector pET-28A-GhNiR. NiR activity assay showed that the crude GhNiR protein had obvious activity to NaNO₂ substrate.

Key words: Cotton, nitrite reductase, prokaryotic expression, semi-quantitative RT-PCR, GenBank Accession No: GQ389691.