长春花叶片三种生物碱含量及合成基因表达的初步研究

Preliminary Studies on Dynamic Change of Three Alkaloids and Expression of The Associated Biosynthetic Genes In Catharathus Roseus Leaves

为探明长春花叶片中生物碱的动态变化和生物碱合成途径中的关键酶基因的表达量,利用高效液相色谱技术(HPLC)测定了5对真叶期、开花前期、出现花蕾期、出现开花期、成熟期1和成熟期2等6个生长阶段的长春花幼嫩、中等成熟、成熟三种叶片的生物碱(文多灵、长春质碱、长春碱)的含量。幼嫩叶片中文多灵、长春质碱和成熟叶片中长春碱的含量最高。长春花叶片的最佳采集期是花蕾期到开花期。利用荧光定量PCR技术检测了长春花叶片中生物碱合成途径中10个相关基因的相对表达量,GGPP,G10 H,SLS,STR,D4 H及DAT这6个基因对生物碱含量贡献较大,ASA及TDC基因对生物碱的增加的作用不明显。

In order to clarify the dynamic change of three alkaloids（TIAs including vindoline,catharanthine and vinblastine） and the expression of the associated biosynthetic genes in the Catharathus Roseus leaves（young,medium and old） at different growing stages,high performance liquid chromatography was applied to determine the contents of TIAs at the leaf period,pre-floweringperiod,bud stage,flowering period,fully-mature period 1 and fully-mature period 2 in five pairs of leaves from Catharathus Roseus.In addition,the expression of the associated biosynthetic genes was analyzed by molecular biological technique（real time PCR,FQ-PCT）.The results showed that highest contents of vindoline and catharanthine were presented in young leaves while the content of vinblastine was highest in old leaves.The best time for collecting the leaves of Catharathus Roseus was between the stage of alabastrum and flowering since the maximum contents of TIAs could be extracted in this period.The data from the FQ-PCR determination indicated that the expression of TIAs contents in C.Roseus leaves was more affected by the six genes including GGPP,G10H,SLS,STR,D4H and DAT while two genes（ASA,TDC） showed less influence on the production TIAs.