季节变化对聚果榕-榕小蜂互利共生系统生长与繁殖的影响

Triterpene biosynthesis in Euphorbia pekinensis induced by methyl jasmonate

关键词:  京大戟  茉莉酸甲酯  3-羟基-3-甲基戊二酰辅酶A还原酶  鲨烯合酶  法尼基焦磷酸合酶  实时定量PCR  总三萜

Key words:   Euphorbia pekinensis  methyl jasmonate  3-hydroxy-3-methylglutaryl coenzyme A reductase  squalene synthase  farnesyl pyrophosphate synthase  quantitative real-time PCR  total triterpenoids

摘要:

京大戟是多年生草本药用植物,入药部分是其干燥根,但可入药的京大戟资源由于生长缓慢以及环境污染的加剧而越发匮乏,因此解决大戟资源日益紧张的问题是当今药用植物资源开发与利用方向的重要课题。京大戟含有三萜类、二萜类、黄酮类等丰富的活性成分,一些常见药用植物的有效成分是三萜类化合物,其在抗病毒、抗肿瘤、免疫调节等方面具有很好的活性。对植物萜类物质代谢起重要作用的关键酶,如3-羟基,3-甲基戊二酰辅酶A还原酶(hmgr)、鲨烯合酶(sqs)、法尼基焦磷酸合酶(fps)的基因克隆及活性研究取得了进展和突破,但通过调控萜类物质代谢途径中关键酶基因的表达来诱导终产物合成的研究鲜有报道。通过研究大戟萜类物质代谢途径进而利用基因工程手段提升目的物质的产量来解决京大戟药源短缺问题具有重要意义。该研究以大戟愈伤组织为材料,使用茉莉酸甲酯分别按时间梯度和浓度梯度进行诱导,将诱导后的愈伤组织分为两部分:一部分提取其总RNA,以actin为内参基因进行反转录,实时定量RT-PCR分析大戟三萜类代谢途径中 hmgr、sqs与fps基因的相对表达差异; 另一部分用于提取其总三萜并使用分光光度法进行含量测定。实时定量RT-PCR分析结果表明,茉莉酸甲酯可诱导3个基因的表达,但其表达模式不一样。相应的京大戟愈伤组织中总三萜的含量明显提高,最高可较未处理样品增加27%。研究结果可为茉莉酸甲酯促进药用植物大戟三萜类物质积累的分子机制研究提供参考。

Abstract:

Euphorbia pekinensis is a perennial herb. The part of medicine is the dried root and is our traditional Chinese medicine. E. pekinensis have distributed in many provinces in China except for Tibet and Xinjiang. In recent years, medicine studies have showed that it can also be used on carbuncle swollen,sore-toxin,etc. So there are an increasing number of people who pay attention to it nowadays and later greatly exploit and unearth to it. But E. pekinensis with dried roots need many years cultivation and the deterioration of environmental pollution problem leads to the resources become more and more deficient. Therefore, many research workers faced the problem that how to relieve the resource tension of E. pekinensis. This is an important topic in development and utilization of the medicinal plant resources and it is also a huge challenge to science research personnel. Modern research shows that E. pekinensis mainly contains the active ingredient of three terpenoids, two terpenoids, flavonoids, alkaloids, organic acid, ect. The effective components of some commom medicinal plants are the three terpenoids and they have good activity in antiviral,antitumor,immune regulation and so on. So the terpenoids of Euphorbia pekinensis plays an important role in disease treatment. With the rapid development of molecular biology and the increasingly research in technology, the plant terpenoid metabolism pathway has been well studied. Now the key enzymes such as hmgr,sqs and fps play an important role in the metabolic flow. Now the researcher has made greatly progress in gene clone and activity research. But there are few reports in controlling the expression of the key enzyme of terpenoid metabolic pathway to induce the synthesis of terpenoids. Therefore,it is significantly important to study the metabolic pathway of the terpenoid synthesis in E. pekinensis and use the means of gene engineering and technology to greatly increase the output of target product in order to solve the problem of the shortage in medicinal resource of E. pekinensis. In this study, the callus of E. pekinensis was induced by methyl jasmonate with different concentrations in different treatment times. These treated calluses were respectively divided into two parts: one was used to extract total RNA,then reverse-transcribed to cDNA,transcript levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase(hmgr),farnesyl pyrophosphate synthase(fps)and squalene synthase(sqs)were determined by quantitative real-time PCR using actin gene as internal reference. The other part was used to extract triterpenoids whose content was detected by spectrophotometry. The results of quantitative real-time PCR showed that methyl jasmonate could induce the expression of these three genes,but the expression patterns were different. The result of total triterpenoids detection showed that methyl jasmonate induced the accumulation of total triterpenoids up to 27% compared with untreated sample. These results would afford the reference for the research on the molecular mechanism that methyl jasmonate promotes the accumulation of total triterpenoids.