Study on cloning of diterpene synthase genes from plants

广泛的GC-MS分析表明，Aleurites fordii hemsl中存在未鉴定的对位高岭烯、松二烯和海松二烯，它们的光谱与大环二萜C膜相似。对大戟藻的类似分析也被调查，以确定11种以上的新碳氢化合物。由于所获得的样品数量有限，因此从E.lachyris中的主要成分L3合成了预期的生物合成前体，得到了具有正确的立体化学和C-C双键位置的5，10-二脱氧化合物。用Fordii的cDNA和针对植物二萜合成酶基因设计的简并引物进行RT-PCR，得到了几种不同的扩增产物。目前，对这些基因的表达和特性进行了研究，并对水稻产生植物毒素的二萜类化合物进行了研究。利用针对二萜烯合成酶基因设计的简并引物，利用RT-PCR技术对紫外光处理水稻植株诱导表达的基因进行鉴定。克隆鉴定出的基因，并在大肠杆菌中表达。酶促反应产物与GGDP的GC-MS分析表明，OsCyc1和OsCyc2分别负责合成syn-CDP和ent-CDP。另一方面，与syn-CDP和ent-CDP的类似反应表明，OsDTC1、OsDTC2、OsKS4和OsKS10分别生成ent-Cassa-12、15-二烯、球茎芳烃、ent-andaracopimaradiene和ent-pimara-8(14)、15-diene。

Extensive GC-MS analysis of hydrocarbon fractions in Aleurites fordii Hemsl showed the presence of ent-kaurene, abietadiene and pimaradiene unidentified constituent whose spectrum resembled to that of macrocyclic diterpene cembrane. Similar analysis of those of Euphobia lachyris was also investigated to identify more than 11 novel hydrocarbons. Since limited amounts of samples were obtained, synthesis of an expected biosynthetic precursor from major constituent L3 in E.lachyris was employed to obtain the 5,10-dideoxy compound which has correct stereochemistry and location of C-C double bonds. Several PCR products were obtained when we performed RT-PCR with cDNA from A.fordii and with degenerated primers designed for plant diterene synthase genes. Currently, expression and characterization of the resultant genes are investigated.Diterpene syntheses responsible for production of phytotoxins in rice plant were investigated. Genes induced by the rice plant treated with UV-irradiation were identified by RT-PCR with degenerated primers designed for diterene synthase genes. The identified genes were cloned and expressed in Esherichia coli. GC-MS analysis of enzymatic reaction products with GGDP showed that OsCyc1 and OsCyc2 were responsible for production of syn-CDP and ent-CDP, respectively. On the other hand, similar enzymatic reactions with syn-CDP and ent-CDP established that OsDTC1, OsDTC2, OsKS4, OsKS10 produced ent-cassa-12,15-diene, stemarene, ent-sandaracopimarradiene and ent-pimara-8(14),15-diene, respectively.