Submitted to: National Cotton Council Beltwide Cotton Conference
Publication Type: Abstract Only
Publication Acceptance Date: 1/7/2005
Publication Date: 5/25/2005
Citation: Liu, J., Stipanovic, R.D., Bell, A.A. 2005. Terpenoid pathway in cotton: Desoxyhemigossypol-4-hydroxylase [abstract]. In: Proceedings of the Beltwide Cotton Conferences, January 4-7, 2005, New Orleans, Louisiana. 2005 CDROM. Interpretive Summary:
Technical Abstract: The cotton sesquiterpenoid, hemigossypolone and its further down stream metabolites heliocides are found exclusively in the foliage. Hemigossypolone is biosynthesized from farnesyl diphosphate via the intermediates '-cadinene, 8-hydroxy-'-cadinene, desoxyhemigossypol, and 4-hydroxydesoxyhemigossypol. The conversion of desoxyhemigossypol to 4-hydroxydesoxyhemigossypol constitutes the first committed step of the foliage specific biosynthesis of hemigossypolone and heliocides. The enzyme responsible for the conversion of desoxyhemigossypol to 4-hydroxydesoxyhemigosspol is expected to be expressed only in the green tissue. Therefore, the identification of desoxyhemigossypol-4-hydroxylase (dHG-4-H) gene promoter will be of great value as it can be used as a green tissue specific promoter in cotton. Hemigossypolone and heliocides play a major role in defending cotton plants against insects. Therefore, cloning of the dHG-4-H will provide a tool to manipulate the biosynthesis of these defense compounds through genetic engineering in cotton plants. Cloning of dHG-4-H is accomplished by RT-PCR to selectively amplify P450 hydroxylase and Cu-containing hydroxylase using consensus primers. The amplified P450 hydroxylase cDNAs are cloned into cloning vector pUC 18 and individual clones are screened by differential hybridization with cDNA probes derived from glanded vs. glandless leaves. Using this approach, we have identified 13 different partial P450 clones. Among them, only two clones are only expressed in glanded leaves but not in the glandless leaves. One clone, GHC4 is 99% identical to Gossypium arboreum '-cadinene 8-hydroxylase, therefore, is not further pursued. The other clone, GHC28 is 59% identical to a soybean cytochrome P450 82 A4. Using this partial clone as a probe, we have detected a 1.9 kb message in the glanded leaves but not in the glandless leaves as expected for dHG-4-H gene. Full-length clone of GHC28 is obtained by repeated PCR screening of a cDNA library made from glanded leaves. The 1935 bp clone contained a 34 bp 5'-leader and a 322 bp 3'-untranslated region. It coded 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. Expression of GHC28 in E. Coli is attempted with pET-28a (+) expression vector. In the first two constructs, full-length protein sequence is fused with vector sequence containing His- and T7-tags for easy 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 is removed in the next two constructs. The expected 57.1 kDa and 58.2 kDa fusion proteins are detected for these two constructs.