Submitted to: National Cotton Council Beltwide Cotton Conference
Publication Type: Abstract Only
Publication Acceptance Date: December 15, 2006
Publication Date: January 10, 2007
Citation: Liu, J., Stipanovic, R.D., Bell, A.A. 2007. Cloning and heterologous expression of a cytochrome P450 hydroxylase involved in the terpenoid biosynthetic pathway in cotton [abstract]. Proceedings of Beltwide Cotton Conferences, January 7-13, 2007, New Orleans, Louisiana. 2007 CDROM. 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 limits 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. A terpenoid compound, hemigossypolone, is found only in the foliage of glanded plant. It is absent in the rest of the plant or in the glandless plant. We hypothesized that the gene responsible for the synthesis of this compound is a P450 hydroxylase. We have screened for the differentially expressed P450 genes, and identified and cloned a 1.9 kb P450 gene. The gene coded for a 522 amino acid protein and is 48% identical to a soybean cytochrome P450 82 A3. Northern analysis indicated that 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. For the functional characterization of this cloned P450 gene, this gene was heterologously expressed in yeast Saccharomyces cereviae. P450 hydroxylase function depends on its electron transfer partner cytochrome P450 reductase (CPR). We used a genetically modified yeast Saccharomyces cereviae strain W(R) which over-expresses microsomal CPR when galactose is added to the media. The open reading frame of the cloned P450 gene was inserted into the yeast expression vector pYeDP60 which is also inducible by the addition of galactose. A construct containing an insert with an additional T7 tag and His tag was also made in order to facilitate purification and detection in case the expressed protein fails the functional test. The yeast W(R) strain was transformed with expression constructs by lithium acetate method. The transformed yeast strains were induced with galactose for the expression of cloned P450 hydroxylase. The expressed proteins were characterized by western blot analysis, spectroscopic analysis, and enzymatic functional analysis.