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背景:辣椒素类物质,包括辣椒素及其类似物,是导致辣椒果实辛辣的原因。尽管辣椒素为人熟知并且每天都会用到,但是对于辣椒素合成途径中的相关基因并不是完全了解。已有研究表明,p AMT和Pun1编码的蛋白分别催化辣椒素合成途径中的第二步和最后一步反应,并且Pun1编码的蛋白具有辣椒素合酶活性。然而,并没有直接的证据证明Pun1具有辣椒素合酶活性。结果:为了证明Pun1蛋白在辣椒素合成中的作用,我们利用大肠杆菌合成了抗Pun1蛋白抗体,利用该抗体拮抗内源的Pun1活性。同时通过病毒介导的基因沉默技术靶定了Pun1的m RNA,以确认抗Pun1抗体的特异性。在Pun1下调表达的胎座组织中,利用该抗体进行western blot,检测到Pun1蛋白的积累减少,同时辣椒素在胎座中的积累量也降低。从胎座组织中分离得到原生质体,在体外进行辣椒素的从头合成,加入抗Pun1抗体之后,辣椒素的合成受到抑制。通过分析不同辣椒品种的p AMT和Pun1的表达,我们发现辣椒素的高水平积累总是伴随着p AMT和Pun1的高水平表达,也就是说这两个基因对辣椒素合成很重要。比较有辣味辣椒和无辣味辣椒的香草基胺(辣椒素合成的前体物质)和辣椒素的积累水平,结果表明:在辛辣味辣椒中香草基胺的含量很低,推测可能是香草基胺合成之后,Pun1把香草基胺快速转化为辣椒素;而在无辣味辣椒中由于缺乏Pun1,香草基胺的含量积累到很高的水平。结论:对辣椒素从头合成途径和抗Pun1抗体进行原生质体试验,证明了Pun1基因及其产物参与了辣椒素的合成。与辣椒素的积累相比较,分析香草基胺的积累过程,揭示了Pun1是辣椒素和香草基胺积累水平的决定因素。
Background: Capsaicinoids, including capsaicin and its analogs, contribute to the spiciness of pepper fruits. Although capsaicin is well-known and is used daily, it is not fully understood about the genes involved in the capsaicin synthesis pathway. It has been reported that proteins encoded by p AMT and Pun1 catalyze the second and final reactions in the capsaicin synthesis pathway, respectively, and that the Pun1-encoded protein has capsaicin synthase activity. However, there is no direct evidence that Pun1 has capsaicin synthase activity. RESULTS: In order to demonstrate the role of Pun1 in the synthesis of capsaicin, we synthesized an anti-Pun1 antibody using E. coli to antagonize endogenous Pun1 activity. Pun1 m RNA was also targeted by virus-mediated gene silencing technology to confirm the specificity of the anti-Pun1 antibody. In the downregulated expression of Pun1 in the placental tissue, Western blotting with this antibody detected a decrease in the accumulation of Pun1 protein and a decrease in the accumulation of capsaicin in the placenta. Protoplasts were isolated from the placenta tissue and de novo synthesis of capsaicin was performed in vitro. After the anti-Pun1 antibody was added, the synthesis of capsaicin was inhibited. By analyzing the expression of p AMT and Pun1 in different pepper varieties, we found that the high level of capsaicin accumulation is always accompanied by the high level expression of p AMT and Pun1, which means that these two genes are important for the synthesis of capsaicin. The results showed that the content of vanillylamine in spicy pepper was very low, which may be the result of vanillin (vanillin) After the synthesis of nicotine, Pun1 rapidly converted vanillylamine to capsaicin, whereas the vanillin content accumulated to a very high level due to the lack of Pun1 in hot pepper. Conclusion: The protoplast test of capsaicin de novo synthesis and anti-Pun1 antibody proves that Pun1 gene and its product are involved in the synthesis of capsaicin. Compared with the accumulation of capsaicin, the accumulation of vanillylamine was analyzed, revealing that Pun1 is the determining factor for the accumulation of capsaicin and vanillylamine.