Study on cloning of diterpene synthase genes from plants

广泛的气相色谱-质谱分析表明，福地阿莱莱的烃类组分中存在与大环二萜膜相似的对烯、二烯和海桃二烯等未识别成分。类似的分析也被用于对Euphobia lachyris进行研究，鉴定出超过11种新的碳氢化合物。由于获得的样品数量有限，因此采用了从E.lachyris的主要成分L3合成预期的生物合成前体来获得具有正确立体化学和C-C双键位置的5,10-二脱氧化合物。用fordii的cDNA和为植物二萜合成酶基因设计的退化引物进行RT-PCR，得到了多个PCR产物。目前，对合成基因的表达和特性进行了研究。对水稻植物毒素产生的二萜合成进行了研究。利用设计的二萜合成酶基因的退化引物，采用RT-PCR技术对紫外辐照诱导的水稻植株进行了基因鉴定。鉴定的基因被克隆并在大肠杆菌中表达。与GGDP酶促反应产物的GC-MS分析表明，OsCyc1和OsCyc2分别负责生成syn-CDP和ent-CDP。另一方面，通过与syn-CDP和ent-CDP的类似酶促反应，OsDTC1、OsDTC2、OsKS4、OsKS10分别生成了对-cassa-12,15-二烯、系统芳烃、对-sandaracopimarradiene和对-pimara-8(14)，15-二烯。

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.