Cloning Of CYP450 C DNA Of Psammoilene Tunicoides W.C.Wu Et C.Y.Wu

Psammoilene tunicoides W.C.Wu et C.Y.Wu(Caryophyllaceae)is one kind of perennial herb from monotypic genus nd is mainly distributing at southwestern China.It is one of the important medicinal materials in Chinese patent medicine “Yunnan Baiyao" and "Yunnan Hongyao capsules". Psammoilene tunicoides has low rate of natural reproduction, grows slowly. In addition, the source of its medicinal materials mainly depends on the wild digging, so the resource of Psammoilene tunicoides declines rapidly. Now it has been listed as national second-class protective plant. At present, the amount of Psammoilene tunicoides from digging is far away from the market requirement. The Psammoilene tunicoides is urgent to be researched for protection and it is also urgent to find and expand new medicinal herbs resource.The main effective constituent of Psammoilene tunicoides, Psammosilene saponins, is oleanane type pentacyclic triterpenoid saponin which is mainly synthesized through mevalonate pathway. After the triterpenes skeleton is synthesized,it is structurally modified by cytochrome P450 monooxygenase and glycosyl transferase. Different products of triterpene saponins are obtained at last. So the cytochrome P450 monooxygenase is the key enzyme gene in the synthesis of Psammosilene saponins. Obtaining the gene of cytochrome P450 monooxygenase related to the synthesis of Psammosilene saponins is very important to the guarantee of sustainable utilization of Psammoilene tunicoides resource.In the present study, the c DNA of the gene of cytochrome P450 monooxygenase in Psammoilene tunicoides was separated. Through RT-PCR and the rapid-amplification of c DNA ends(RACE) of c DNA, the c DNA with total length was cloned. The results are as follows:1 The total RNA in the root of Psammoilene tunicoides is extracted. The core sequences of five CYP450 genes, CYP85A1,CYP88A3,CYP88 A,CYP716A12 and CYP716A53v2, were obtained through RT-PCR.2 The c DNAs with total length of two CYP450 genes, CYP88A4 and CYP85A1,were obtained through RACE.3. Successful piece together contains a complete open reading frame CYP90A1 gene from the transcriptome data, and design primers success amplification the gene.