Molecular cloning, structural analysis and expression of a zinc binding protein in cotton

Abstract

The full-length zinc-binding protein (ZnBP) gene was cloned from a normalized cDNA library constructed from a cotton mutant (Xiangmian-18) during the gland-forming stage. The clone was sequenced and analysed. BLASTP analysis showed that the deduced amino acid sequence of ZnBP in Xiangmian-18 is similar to that in Arabidopsis thaliana (GenBank accession no. EFH46337.1) with an overall similarity of 77%. The cDNA insert comprises 654 base pairs (bp) and 217 amino acid residues. Its molecular weight is 24.6 kDa, and the theoretical pI is 9.33. The cotton ZnBP gene was cloned from the gDNA from Xiangmian-18 leaves. After sequencing the two fragments, a 1731 bp cotton ZnBP gene with three introns was identified. Using pET-28a(+) as a prokaryotic expression vector, the gene was expressed in Escherichia coli BL21(DE3). The conditions for achieving optimal ZnBP expression were 37°C, IPTG 1 mmol/L, 8 h and a shaker speed of 150 rpm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis confirmed the correct expression of the protein. pCAMBIA2300-35S-OCS was used as a eukaryotic expression vector. The recombinant plasmid pCAMBIA2300-ZnBP was used to transform competent Agrobacterium GV3101 by the freeze-thaw method. Then, A. thaliana plants were transformed by the floral dipping method. Transformed plants were grown to maturity in a growth chamber. After screening on kanamycin-resistant half-strength Murashige and Skoog plates and polymerase chain reaction (PCR) analysis, two transgenic plant strains were obtained. Northern blot analysis showed that ZnBP expression was higher in homozygous plants than in wild-type plants. The differences between the phenotypes of homozygous and wild-type plants indicate that the ZnBP gene affects the growth and development of A. thaliana. The results of prokaryotic expression of ZnBP and overexpression of the ZnBP gene in A. thaliana improve our understanding of the function of this gene. Future studies should investigate the molecular mechanisms involved in gland morphogenesis in cotton.

Key words: Gossypium hirsutum, pigment gland, zinc binding protein, prokaryotic expression, overexpression.