Cloning of Cotton CBF Gene for Cold Tolerance and Its Expression in Transgenic Tobacco

Abstract

C-repeat binding factor (CBF) is a kind of transcription factor that regulates expression of a number of genes related to abiotic stresses. Three CBF genes were isolated from the genomic DNA of Gossypium hirsutum L. cultivars Gh12 and Gh36 and G. barbadense L. cultivar Gb7124. Cotton CBF gene encodes 184 amino acids, containing the CBF-family signature PKRRAGRKKFQETRHP and FADSAW. Southern blotting result showed that CBF genes were present as the form of gene family in the genome of cotton. Northern blotting result indicated that GbCBF1 was induced by low temperature, drought, salt, and abscisic acid. This gene was constructed into plant expression vector pCambia2301, in which the gene was driven by 35S and NOS promoters separately. Plant expression vectors were then transferred into Nicotiana tabacum L. cultivar NC89 using Agrobacterium-mediated transformation method. Twenty-six transgenic tobacco lines were obtained after kanamycin screening and PCR detection. After PCR and reverse tanscription PCR analyses in partial T₁ transgenic plants, the GbCBF1 gene was confirmed to be transcripted and inherit in offspring normally. Under low temperature stress, the electrolytic leakage rate of transgenic tobacco was lower than that of the wild type tobacco; however, free proline and soluble sugar contents of transgenic tobacco were higher than those of the wild type tobacco. This result indicated that GbCBF1 enhances cold tolerance in transgenic tobacco.