An efficient method for rapid amplification of Arisaema heterophyllum agglutinin gene using a genomic walking technique.

The genomic sequence of Arisaema heterophyllum agglutinin (AHA), a mannose-binding lectin (MBL), was cloned through a novel genomic walking technique. Adaptor ligation reactions and subsequent amplifications with adaptor primer and multiple specific primers were used to generate specificity in this method. The method allowed for the amplification of over 1 kb of genomic DNA sequence immediately upstream and downstream from the 5' and 3' ends of full-length cDNAs. For aha gene, the upstream regions contained a putative transcription initiation start site and other sequences commonly found in eukaryotic promoters. The downstream regions of aha contained two polyadenylation signals. Our study demonstrated that aha had no intron like mannose-binding lectin genes cloned from other plant species so far. This efficient method, based on a genomic walking technique, was useful for the cloning of promoters, insertion sites, and other sequences of interest without constructing and screening genomic libraries.