Study on cloning of diterpene synthase genes from plants

对油桐烃类组分的GC-MS分析表明，油桐中含有对映贝壳杉烯、松香烯和海松二烯，其光谱特征与大环二萜类化合物的光谱特征相似。类似的分析，这些大戟lachyris也进行了研究，以确定超过11个新的碳氢化合物。由于获得的样品量有限，因此采用从长穗马兜铃中的主要成分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-双脱氧化合物。以油桐cDNA为模板，利用设计的双萜合成酶基因简并引物进行RT-PCR，获得了多个PCR产物。目前，所获得的基因的表达和特性的研究。二萜合成负责生产的植物毒素在水稻植物。根据二萜烯合成酶基因设计简并引物，通过RT-PCR方法鉴定了经紫外线处理的水稻植株诱导表达的基因。将鉴定的基因克隆并在大肠杆菌中表达。GC-MS分析表明，OsCyc 1和OsCyc 2分别参与了syn-CDP和ent-CDP的合成。OsDTC 1、OsDTC 2、OsKS 4、OsKS 10分别产生对映-cassa-12，15-二烯、stemarene、ent-sandaracopimarradiene和ent-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.