RT-PCR法探讨茉莉酸甲酯对Taxol生物合成影响.docVIP

RT-PCR法探讨茉莉酸甲酯对Taxol生物合成的影响中国农业大学 农学与生物技术学院 北京 100094 运用RT-PCR技术检测7个基因表达差异，结果表明MJ在处理后7种关键酶基因的表达活性第4天明显提高，其中GGPPS活性为对照的6.65倍，TBT活性为对照活性的941%。Methyl Jasmonate (MJ) Resorting to RT-PCR Chen Xiao-wen , Dong Xue-hui* ,Duan Liu-sheng College of Agricultural and Biotechnology, China Agricultural University, Beijing 100094, China Abstract: HPLC was used for content detection taxol in leaves of Taxus cuspidata, and the method of RT-PCR was applied for check of expression activities of the seven key genes in taxol biosysthesis. The results showed that the accumulation of taxol in treatment was notably higher than that in control, and the expression of the seven genes were more active on the fourth day after treatment, for example, the GGPPS activity was 6.65 times that in control and TBT activity was 941% of that in control. Keywords: Taxol GGPPS taxadiene synthase Methyl Jasmonate 前言 紫杉醇（taxol），从红豆杉属植物中提取的二萜类次生代谢物，是一种广普的抗癌药物，可应用于治疗乳腺癌、子宫癌和肺癌以及与爱滋病相关的Kaposi肉瘤[1，2]、Ⅲ和Ⅵ等紫杉烷类化合物的产量紫杉醇生物合成的诱导剂 [ 7-9 ] , B.H. Guo[10]等还对此作了详细的描述（见图1），但一些外源物质对紫杉醇合成的作用机理无明确报道。运用RT-PCR技术检测10-deacetylbaccatin III-10-O-acetyl transferase(DBAT)、taxoid 10-beta hydroxylase、taxadiene synthase (tasy)、taxadiene 5-alpha hydroxylase 、taxadien-5-alpha-ol-O-acetyltransferase、2-alpha-hydroxytaxane 2-O-benzoyltransferase (TBT)7个基因表达差异，。 Taxol biosynthetic pathway. ggpps: geranylgeranyl diphosphate synthase; ts: taxadiene synthase; h-5_: cytochrome P450 taxadiene 5_-hydroxylase; tat: taxa-4(20), 11(12)-dien-5a-ol-O-acetyltransferase; h- 10_: cytochrome P450 taxane 10_-hydroxylase; tbt: taxane 2a-O-benzoyltransferase; dbat: 10-deacetyl baccatin III-10-O-acetyltransferase. Multiple arrows indicate several as yet undefined steps. 图1 紫杉醇生物合成图（B.H. Guo et al, 2006） 材料和方法1.1 试验材料东北红豆杉扦插两年苗购于黑龙江省苇河林业局。选取生长健壮较一致的扦插苗于温室（白天20℃，晚上4℃）中缓苗18天后，结合试验三的结果，选取100μM.L-1茉莉酸甲酯进行喷施处理，对照喷施蒸馏水，平均每株4ml处理液。分别于第4、8、12、16、20天取当年生新叶，用纱布包好部分样品放到液氮中30min后置于-80℃低温冰箱中保存用于荧光定量PCR测定taxol合成中相关基因的活性表达，余下样品于65℃烘箱中烘干至恒重用于紫杉烷类物质检测，每个样品3次重复。1.2测定方法 1.