棉花杜松烯合成酶基因的克隆及其表达分析

Cloning and Expression Analysis of Cotton GhCdn Gene

杜松烯合成酶是棉酚合成途径中的关键酶,催化(E,E)-法呢基焦磷酸(FPP)环化形成(+)-δ-杜松烯。从陆地棉Y18R中克隆分离了杜松烯合成酶基因(GhCdn),该基因的基因组序列为2700bp,具有6个内含子,剪切后其ORF为1665bp,编码554个氨基酸,该基因属于杜松烯合成酶C亚家族。应用Overlap PCR方法将其上的2个HindⅢ酶切位点钝化后,将GhCdn基因连接到表达载体pBI121上,构建出分别由组成型启动子CaMV 35S和绿色组织高效启动子Psbp驱动的2个植物表达载体pGBI-CaMV 35S-GhCdn和pGBI-Psbp-GhCdn。通过农杆菌介导法转化棉花下胚轴并进行组织培养,获得了9个35S转基因阳性愈伤系和2个P转基因阳性愈伤系。经检测,阳性愈伤组织中GhCdn基因的mRNA表达量和棉酚含量均有所增加,但35S系普遍高于P系。本研究为通过基因工程手段提高棉花组织器官中的棉酚含量提供了依据。

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 HindⅢ 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.