Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 6/4/2005
Publication Date: 12/12/2005
Citation: Liu, J., Stipanovic, R.D., Bell, A.A. 2005. Terpenoid biosynthetic pathway in cotton: Cloning, expression and characterization of a cytochrome P450 [abstract]. In: Proceedings of the 14th International Conference on Cytochromes P450. p. 149.
Technical Abstract: Lysigenous glands in cotton plants contain terpenoid compounds. These compounds act as a defense mechanism against disease and insects. However, the presence of these toxic compounds in the seed limit the utilization of cottonseed as a feed/food source. The elucidation of the terpenoid biosynthetic pathway and cloning of the associated genes will provide a tool to manipulate the biosynthesis of these defense terpenoids through genetic engineering. Our long-term goal is to increase resistance of cotton to insects and pathogens and to expand utilization and thus commercial value of cottonseed. Toward that goal, we have identified and cloned a 1.9 kb P450 coding for a 522 amino acid protein. It is 48% identical to a soybean cytochrome P450 82 A3 and contained a conserved heme-binding motif and a consensus oxygen-binding pocket sequence. This P450 is expressed in the leaves of glanded cotton G. hirsutum, but not in the leaves of glandless cotton. The leaves of glandless plants are devoid of glands and the associated terpenoids. This suggests that this P450 enzyme is involved in the terpenoid biosynthetic pathway in cotton. Expression of the cloned P450 in E. Coli was attempted with pET-28a (+) expression vector. In the first two constructs, full-length protein sequence was fused with vector sequence containing His- and T7-tags for ready purification and detection. However, both constructs failed to express the fusion proteins. In order to increase the expression of the GHC28 P450 hydroxylase in E. Coli, the membrane-anchoring region located in the N-terminal region was removed in the next two constructs. The expected 57.1 kDa and 58.2 kDa fusion proteins were detected for these two constructs.