Tudzynski B, Kawaide H, Kamiya Y
Institut für Botanik, Westfälische Wilhelms-Universität Münster, Schlossgarten 3, D-48149 Münster, Germany.
Curr Genet. 1998 Sep;34(3):234-40. doi: 10.1007/s002940050392.
The gene coding for copalyl diphosphate synthase (CPS), which represents the first gene of the gibberellin pathway, was isolated from the rice pathogen Gibberella fujikuroi. This fungus is used commercially for the production of gibberellic acid and related gibberellins. CPS is a terpene cyclase which catalyzes the first specific step of the gibberellin (GA) pathway as it branches off from the general isoprenoid (biosynthetic) pathway at geranylgeranyl disphosphate (GGDP). A cDNA fragment of the cps gene from the fungus G. fujikuroi was amplified by RT-PCR using oligonucleotides based on amino-acid sequences which were conserved between the plant CPSs and the bifunctional CPS/KS of the fungus Phaeosphaeria sp. L487. A 588-bp fragment obtained with nested PCR was used to isolate the corresponding genomic clone of the cps gene from the wild-type lambda-library. This gene consists of three exons and two introns. The three exons are 2877 bp long and encode 959 amino-acid residues. The protein shares 48% identity with the bifunctional Phaeosphaeria sp. L487 FCPS and between 16% and 18% identity to the corresponding plant CPSs. Expression of the G. fujikuroi cps gene is strongly enhanced under conditions optimized for gibberellin biosynthesis and is reduced when high amounts of ammonium are present in the medium. Gene disruption, followed by gibberellin assays and Southern-blot analysis of the transformants, demonstrated clearly that the cloned gene has the expected function in the biosynthesis of fungal gibberellins.
编码柯巴基焦磷酸合酶（CPS）的基因是赤霉素途径的首个基因，它是从水稻病原菌藤仓赤霉菌中分离得到的。这种真菌在商业上用于生产赤霉酸及相关赤霉素。CPS是一种萜烯环化酶，它催化赤霉素（GA）途径从牻牛儿基牻牛儿基二磷酸（GGDP）处从通用类异戊二烯（生物合成）途径分支出来后的首个特定步骤。利用基于植物CPS与真菌球腔菌属L487的双功能CPS/KS之间保守氨基酸序列的寡核苷酸，通过RT-PCR扩增了藤仓赤霉菌cps基因的cDNA片段。用巢式PCR获得的一个588 bp片段用于从野生型λ文库中分离cps基因的相应基因组克隆。该基因由三个外显子和两个内含子组成。三个外显子长2877 bp，编码959个氨基酸残基。该蛋白与双功能球腔菌属L487 FCPS的同一性为48%，与相应植物CPS的同一性在16%至18%之间。藤仓赤霉菌cps基因的表达在针对赤霉素生物合成优化的条件下显著增强，而当培养基中存在大量铵时则降低。通过基因破坏，随后对转化体进行赤霉素测定和Southern杂交分析，清楚地表明克隆的基因在真菌赤霉素生物合成中具有预期功能。
