Molecular Cloning And Analysis Of CBF1and BADH Genes In Mulberry(Morus L.)

In this paper,we designed primers by reference to conserved sequence of homologous genes ofother plants. We cloned MCBF1,and MBADH genes, then carried out the analysis of their sequences.Furthermore,we conducted the expression and functional verification of MCBF1.The main resultswere summarized briefly as follows:In this experiment, we cloned cDNA sequence of the CBF1transcription factor gene of Morusalba by using RT-PCR and RACE techniques, designed it MCBF1.(GenBank accession number:JX186750). The results showed that the length of the whole sequence was1134bp, which includedan open reading frame of693bp, the5’UTR(untranslated region)and3’UTR of87bp and354bprespectively. Bioinformatics predicts that its deduced amino acid sequence consists the conservativeAP2domain, which has two short unique polypeptide sequence.Its amino acid sequence has highhomology with CBF from other plant species.The constructed pET28a-MCBF1prokaryoticexpression vector was transformed into Escherichia coli BL21.The expressed protein had a molecularweight of33kD which was close to the deduced molecular weight of MCBF1. A phylogeneticanalysis revealed that mulberry had close relationships with Citrusjambhiri，Betula pendula and Rosahybrid cultivar Semi-quantitative RT-PCR analysis showed MCBF1was not expressed in the controlmulberry but expressed in mulberry with low temperature treatment..It has the highest relativeexpression level in the mulberry under low temperature for6days.In this experiment, we cloned cDNA sequence of the BADH gene of Morus alba by usingRT-PCR and RACE techniques, designed it MBADH.(GenBank accession number:KC244769).MBADH gene is1883bp in length with an open reading frame(ORF) of501aminoacids,corresponding to a protein of predicted molecular mass54.6KD,and an isoelectric point of5.19.Its amino acid sequence has high homology with BADH from other plant species.A phylogeneticanalysis revealed that mulberry had close relationships with Corylus heterophylla,Camelliasinensis,and Panax ginseng.