关键词: 枯斑三生烟SKP1基因, 克隆, 序列分析, 表达

Abstract:

To investigate the ability of oligochitosan to affect gene transcription, the messenger RNA (mRNA) differential display technique was applied to the identification and isolation of the genes whose transcription were altered in cultured Nicotiana tabacum (var. Samsun NN) plants treated with oligochitosan. Four differential displayed cDNA were subcloned and confirmed by reverse Northern blot analysis. Then the 3’ end cDNA of gene SKP1 was obtained by sequence, the 5’ end and full-length cDNA of gene SKP1 from Nicotiana tabacum var. samsun NN was cloned using the SMART-RACE technique. BLAST analysis revealed that the sequence of full-length cDNA showed 81% identity to that of Nicotiana benthamiana. Analysis of domains showed there was one predicted Skp1 domain between 4 and 105 amino acids whose E-value is 1.25e-52, so the gene was named SKP1 of Nicotiana tabacum var. samsun NN, and submitted to GenBank (accession number: AY702087). To express protein of the gene, cDNA of SKP1 from Nicotiana tabacum var. samsun NN was ligated with the expression vector pET23b. SDS-PAGE indicated that one fused protein was expressed efficiently. These results are useful for studying the function of SKP1.

Key words: Gene SKP1 from Nicotiana tabacum var. samsun NN, Clone, Sequence analysis, Expression