Mildew Resistance Locus O Gene Cloning, Characterization, and Expression Pattern in Mulberry (Morus multicaulis) and Its Prokaryotic Expression in E. coli

Abstract

MLO (mildew resistance locus O), which encodes a transmembrane protein 7TM, is considered to be a model plant gene suitable for studying broad-spectrum resistance. It is a negative regulator of powdery mildew resistance and thus has potential applications in plant breeding. In the present paper, a full cDNA sequence encoding MLO was cloned from the leaves of mulberry (Morus multicaulis) based on mulberry expressed sequence tags (EST), homologous cloning technology, and 5′-RLM-RACE using RT-PCR;the sequence was designated MMLO (GenBank accession no. KX683296). The full cDNA was 1943 bp in length with 5′-untranslated region (UTR) of 106 bp, 3′-UTR of 160 bp, and an open reading frame (ORF) of 1677 bp encoding a protein of 558 amino acids. The estimated molecular weight and isoelectric point (pI) of the putative protein were 62.48 kDa and 9.03, respectively. The MMLO protein had Mlo domain and belonged to the Mlo superfamily. Phylogenetic analysis based on the amino acid sequences encoded by the MLO gene from various species showed that mulberry was closely related to Eucalyptus grandis, Ziziphus jujube, and Juglansregia. Quantitative real-time PCR (qRT-PCR) analysis revealed that MMLO was expressed in all the tissues tested, including leaf, bud, fruit, stem, phloem, and xylem in mulberry with the highest expression in the phloem. The expression level of the mRNA increased and significantly changed under drought, cold, and salt stress treatments compared to the normal growth environment. The ORF segment of the MMLO was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. SDS-PAGE result revealed that fusion protein was successfully expressed. Overall, these results provide a better understanding of the molecular basis for the signal transduction mechanism during the stress responses in mulberry trees.