氮素形态对颠茄生物碱积累及相关基因表达的影响

Effects of nitrogen form on accumulation of alkaloids and expression of relative genes in Atropa belladonna

莨菪碱和东莨菪碱是颠茄含有的2种主要生物碱,具有重要的药用价值。该研究以颠茄实生苗为材料,设置不同铵硝配比处理,分析不同形态氮素对颠茄莨菪碱和东莨菪碱的含量和生物碱合成的上游产物、信号分子以及关键酶基因表达量的影响。结果显示,铵硝比50∶50更有利于颠茄生物碱的积累和莨菪碱向东莨菪碱的转化。对Put及其合成关键酶的检测发现,腐胺的含量与腐胺合成的关键酶活性变化基本一致,它们都随铵态氮比例的增加而升高,在铵硝比75∶25达到最高;对信号分子NO的检测发现,NO浓度随处理液中硝态氮比例的减小而减小;对PMT,TRⅠ,H6H表达量的检测发现,少量铵态氮增加了根部PMT,H6H基因表达量,铵硝比为50∶50时叶片和根部PMT,TRⅠ,H6H基因都有较高的表达水平。以上结果表明,氮素形态对颠茄莨菪碱和东莨菪碱合成的调控主要通过影响关键酶基因的表达量起作用,铵硝等比供氮条件提高了颠茄叶片中TRⅠ和根部PMT与H6H表达量,因此促进了腐胺向莨菪碱的合成以及莨菪碱向东莨菪碱的转化,使颠茄能够积累更多的莨菪碱和东莨菪碱。

Hyoscyamine and scopolamine are two main alkaloids in Atropa belladonna with great medicinal value.In this paper,the contents of hyoscyamine and scopolamine,the upstream products in alkaloid synthesis,and the expression levels of key enzyme genes PMT,TRⅠ and H6H in secondary metabolism of A.belladonna seedlings were measured to clarify the mechanism of nitrogen forms regulating alkaloids synthesis.The results showed that the 50/50（NH_4-＋/NO_3--） treatment was more favorable for the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine.The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine,they both increased with the rise of ammonium ratio,reaching the highest at 75/25（NH_4-＋/NO_3--）.The detection of signaling molecule nitric oxide（NO） showed that the NO concentration decreased with the decrease of nitrate proportion.Further detection of gene expression levels of PMT,TRⅠ and H6H in TAs synthesis pathway showed that a certain amount of ammonium promoted the expression of PMT and H6H in roots.When the ratio of ammonium to nitrate was 50/50,PMT,TRⅠ and H6H in leaves and roots had higher expression levels.It can be speculated that the regulation of the formation of hyoscyamine to scopolamine by nitrogen forms mainly through affecting the expression of key enzyme genes.50/50（NH_4-＋/NO_3--） treatment increased the gene expression of TRⅠ in both leaves and roots as well as PMT and H6H in roots,promoting the synthesis of putrescine to hyoscyamine and the conversion of hyoscyamine to scopolamine.