Characterization and expression patterns of mannose-binding lectin (MMBL) gene in mulberry (Morus multicaulis) and its prokaryotic expression in E. coli
2017
Li, R. | Wang, T. | Chen, D. | Dominic, K. | Li, R. | Wang, Y. | Liu, L. | Zhao, W.
Mannose-binding lectin (MBL) is an important natural immune molecule which plays a significant role in plant self-defense and shows a broad spectrum resistance on plant pathogens, viral pathogens, and fungi. In the present paper, a cDNA sequence encoding MBL, which was designated MMBL (GenBank accession NO: KY348866), was cloned from the leaves of mulberry (Morus multicaulis) based on mulberry expressed sequence tags (EST) and homologous cloning technology using RT-PCR. The cDNA was 978bp in length with a 5′ untranslated regions (UTR) of 189bp, a 3′ UTR of 303bp and an open reading frame (ORF) of 486bp encoding a protein of 161 amino acids. The estimated molecular weight and isoelectric point (pI) of the putative protein were16.97kDa and 6.07, respectively. The MMBL had Jacalin domain and six sugar binding sites, and belonged to Jacalin-like superfamily. Phylogenetic analysis based on the amino acid sequences encoded by the MBL gene from various species showed that mulberry was closely related to Morus notabilis, Ipomoea nil, Capsicum annuum, Solanum lycopersicum and Solanum tuberosum. Quantitative real-time PCR (qRT-PCR) analysis revealed that MMBL was expressed in all the tested tissues, including leaf, bud, fruit, stem, phloem and xylem of the mulberry with the highest expression in the bud and leaf. The expression level of the mRNA has changed significantly under drought, cold, salt and mechanical damage stress treatments compared to the normal growth environment. The ORF segment of the MMBL was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. SDS-PAGE and western blot results revealed that His-tagged fusion protein was successfully expressed. Overall, these results showed a better understanding of the molecular basis for the signal transduction mechanism during the stress responses in mulberry trees.
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