Abstract:
A class-III chitinase promoter was isolated from Lupinus albus. The region 5′ to the coding
sequence of the IF3 gene was amplified by gene walking and sequenced. The proximal 2.0 kb
sequence contains a predicted promoter site, including a TATA box, near the ATG start site.
To test for minimal sequences needed for promoter activity, the region was restricted into
fragments of 1.81, 1.51 and 1.13 kb and cloned into the pDM327 vector, upstream from the
bar-gus fusion gene for Biolistic™ transformation. Transformation of lupin embryos, bean
callus tissue, maize embryos and Ornithogalum callus demonstrated promoter activity for all
fragments. In silico analysis identified putative cis-acting elements in the 1.81 kb fragment
that could be important in controlling gene expression. Fungal elicitor activated-, woundinducible-
and ethylene responsive elements were present in the 1.51 kb fragment. Myb
elements and CAAT boxes that regulate responses to environmental factors and modulate
promoter efficiency were identified in the 1.81 kb fragment. The 1.51 and 1.81 kb fragments
were inserted upstream of the gus gene into the pBI121 vector for Agrobacterium
tumefaciens transformation of tobacco. Quantitative GUS assays indicated that the promoter
fragments are functional in planta and inducible by defense-related signals, wounding, as
well as chemical elicitation. All important elements essential for Bion inducibility are present
on the shorter (1.51 kb) promoter fragment, but both 5′ distal and proximal cis-elements are
required for full functionality. The IF3 promoter is, thus, suitable for use in defense gene
constructs prepared for the production of anthracnose resistant lupin.
