Molecular Cloning, Expression And Function Research Of A Zinc Binding Protein In Gossypium

Gossypol is not only the important resistant substance of Gossypium, but also a natural product which is of great value in medicine. Gossypol has the function of male antifertility, and anti-cancer and anti-virus. In order to control this secondary metabolism substance effectively, People has long been engaged in the studies of gossypol and its storage organ pigment gland.The full-length zinc-binding protein (ZnBP) gene was cloned from a normalised 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-18is similar to that in Arabidopsis thaliana (GenBank accession no: EFH46337.1)(overall similarity77%). The cDNA insert comprises654base pairs (bp) and217amino acid residues. Its molecular weight is24.6kDa, and the theoretical pI is9.33. The cotton ZnBP gene was cloned from gDNA from Xiangmian-18leaves. After sequencing the two fragments, a1731bp 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 were37℃, IPTG1mmol/L,8h, and a shaker speed of150rpm. SDS-PAGE and Western blot analysis confirmed 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 GV3101by 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 function of the gene involved in gland morphogenesis and gosspol metabolism in cotton.