酵母提取物对颠茄毛状根氮代谢及次生代谢影响的机制探究

Mechanism exploration on nitrogen metabolism and secondary metabolism in Atropa belladonna hairy roots treated with yeast extract

以颠茄毛状根为试验材料,向培养基中添加酵母提取物（yeast extract,YE）,测定颠茄毛状根氮代谢关键酶的活性,各形态氮的含量,次生代谢前体物质含量,次生代谢途径中关键酶基因相对表达量以及主要托品烷类生物碱（tropane alkaloids,TAs）莨菪碱和东莨菪碱的产量,初步探究YE诱导合成颠茄毛状根次生代谢产物的机制。结果表明：较对照组而言,氮代谢关键酶硝酸还原酶（NR）和谷氨酰胺合成酶（GS）活性显著升高,谷氨酸脱氢酶（GDH）活性变化不显著。硝态氮,铵态氮含量显著下降,而可溶性蛋白,游离氨基酸,总氮含量均显著升高。此外,YE处理颠茄毛状根提高了次生代谢途径中的前体氨基酸（鸟氨酸和精氨酸）的含量以及代谢前体腐胺的含量,3个关键酶基因1,4-丁二氨-氮-甲基转移酶基因（putrescine N-methyl transferase,pmt）、托品酮还原酶-Ⅰ基因（tropinone reductase-Ⅰ,tr I）和莨菪碱6-β-羟化酶基因（hyoscyamine 6-β-hydroxylase,h6h）的相对表达量也呈现不同幅度的上升,最终导致莨菪碱和东莨菪碱产量的增多,在处理16 d后分别是对照组的3.09,1.85倍。这说明YE诱导氮代谢关键酶活性升高使氮代谢加速,为次生代谢中含氮化合物的合成提供更多原料,同时通过调控次生代谢途径中关键酶基因的表达,使莨菪碱和东莨菪碱产量提高。

In order to study the mechanism of nitrogen metabolism and secondary metabolism in Atropa belladonna hairy roots treated with yeast extract,yeast extract（ YE） was added to the culture medium. Then the changes of physiological and biochemical indexes of A. belladonna hairy roots after treatment with YE were detected. The results are as follows,the activity of key enzymes of nitrogen metabolism changed differently. Compared with the control group（ CK）,the activity of nitrate reductase（ NR） and glutamine synthetase（ GS） were significantly increased,while the activity of glutamate dehydrogenase（ GDH） was not changed significantly. The content of nitrate nitrogen,ammonium nitrogen had a significant decrease,but the content of soluble protein,free amino acid,total nitrogen are significantly more than CK. Moreover,YE treatment led to the increase of the content precursor amino acids（ ornithine and arginine） and precursor putrescine in secondary metabolic pathways of A. belladonna. The expression level of gene putrescine N-methyl transferase（ pmt）,tropinone reductase-I（ tr I） and hyoscyamine 6-β-hydroxylase（ h6 h） all increased in a different rate caused by YE treatment,which eventually led to the increase of the yield of tropane alkaloids. The yield of hyoscyamine and scopolamine were 3. 09 and 1. 85 folds than that of CK after 16 days treatment time. The results indicated that YE can induce more synthesis of tropane alkaloids by increasing the activity of key enzymes in nitrogen metabolism to provide more synthetic materials for secondary metabolism,meanwhile it regulated the expression level of some genes of key metabolic enzyme to accelerate secondary metabolism.