目的 分析内生真菌菌株GXRz2、GXRz3和GXRz10对铁皮石斛多糖含量和多糖合成途径关键酶尿苷二磷酸葡萄糖焦磷酸化酶（UDP-glucose pyrophosphorylase，UGPase）基因表达以及生物碱含量和生物碱合成途径中关键酶3-羟基-3-甲基戊二酸单酰辅酶A还原酶（3-hydroxy-3-methylglutaryl coenzyme-A reductase，HMGR）、法尼基焦磷酸合酶（farnesyl pyrophosphate synthase，FPS）基因表达的影响。方法 铁皮石斛幼苗施加内生真菌菌液，采用分光光度法测定铁皮石斛多糖和生物碱含量。以18 S rRNA为内参照基因，荧光定量PCR（qRT-PCR）法检测多糖、生物碱关键酶基因表达的变化。结果 铁皮石斛活性成分多糖含量较高，主要集中于茎，根中最低；而生物碱含量较低，主要存在于叶，根中最低，3株内生真菌处理均能在一定程度上促进铁皮石斛多糖及生物碱的积累。qRT-PCR技术检测UGPase、HMGR和FPS基因在不同内生真菌处理下的相对表达，结果显示，内生真菌GXRz3和GXRz10均可显著提高铁皮石斛UGPase、HMGR和FPS基因的表达量；其中，多糖合成途径中，UGPase基因在茎中的相对表达量最高，叶次之，根中最少；生物碱合成途径中，HMGR基因在茎中的相对表达量最高，叶次之，根中最少，而FPS基因在叶中的相对表达量最高，茎次之，根中最少。结论 内生真菌可能通过调控UGPase的表达，影响多糖的合成，结合多糖积累情况，推测UGPase可能是铁皮石斛多糖合成途径中的关键酶；内生真菌可能通过调控HMGR、FPS的表达，影响生物碱的合成，而结合生物碱积累情况，推测FPS可能是生物碱合成途径中的关键酶。

Objective To analyze the effect of endophytic fungi GXRz2, GXRz3 and GXRz10 on content of poly saccharide and alkaloid, and the expression of key enzyme genes UGPase, HMGR, and FPS in Dendrobium officinale. Methods The endophytic fungi liquid were added to D. officinale seedlings. Polysaccharide and alkaloid content were measured by spectrophotometry method. With 18S rRNA as internal control gene, the expression of key enzyme genes was detected by real-time quantitative PCR method. Results It was found that the content of polysaccharide in D. officinale was higher, mainly concentrated in stem, but the lowest in root. And the content of alkaloid in D. officinale was lower, mainly accumulated in leaf, but the lowest in root. In addition, the three endophytic fungi strains could promote accumulation of polysaccharide and alkaloid in D. officinale to a certain extent. The expression of UGPase, HMGR and FPS genes in D. officinale induced by different strains was detected by real-time quantitative PCR. The results showed that endophytic fungi GXRz3 and GXRz10 could significantly increase the expression of UGPase, HMGR and FPS genes in D. officinale. In the polysaccharide synthesis pathway, the UGPase gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. In the alkaloid synthesis pathway, HMGR gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. However, FPS gene had the highest relative expression in the leaf, followed by the stem, and the least in the root. Conclusion Endophytic fungi may affect the synthesis of polysaccharide by regulating the expression of UGPase. Considering the accumulation of polysaccharide, it is speculated that UGPase may be a key enzyme in the polysaccharide synthesis pathway of D. officinale. Endophytic fungi may affect the synthesis of alkaloid by regulating the expression of HMGR and FPS. Considering the accumulation of alkaloid, it is speculated that FPS may be a key enzyme in the alkaloid synthesis pathway of D. officinale.

R282.12

浙江省医药卫生科研基金项目（2019KY469）；浙江中医药大学校级科研基金项目（2019ZY22）