关键词: 杜松烯合成酶基因, 表达载体, CaMV 35S启动子, Psbp启动子, 棉酚

Abstract: Cadinene synthase is a key enzyme in the biosynthesis of gossypol, which catalyze (E, E)-farnesyl pyrophosphate (FPP) cyclization to form (+)-δ-cadinene. In this study, a gene of cadinene synthase(GhCdn) was cloned from the upland Y18R. The genome sequences of GhCdn gene are 2 700 bp, including six introns. Its sheared open reading frame(ORF) are 1 665 bp, encoding 554 amino acid residues. This gene belongs to cadinene synthase C subfamily. The Overlap PCR method was applied to making two Hind Ⅲ restriction sites of GhCdn gene passivated. The GhCdn gene was ligated to plant expression vector pBI121, with the constitutive promoter CaMV 35S and green tissue-specific promoter Psbp, and two plant expression vector pGBI-CaMV 35S-GhCdn and pGBI-Psbp-GhCdn were constructed successfully. By Agrobacterium-mediated transformation of cotton hypocotyls and tissue culture, nine 35S transgenic callus lines and two P transgenic callus lines were botained. The mRNA expression of GhCdn gene and gossypol content increased in transgenic callus lines by experimental testing, however, 35S lines are higher than P lines generally. This research provided theoretical basis to improve gossypol content in vegetative organs through genetic engineering.

Key words: cadinene synthase gene, expression vector, CaMV 35S promoter, Psbp promoter, gossypol