冠突曲霉莽草酸形成相关基因表达量及酶活检测

通过对冠突曲霉Aspergillus cristatus 5个生长阶段的3-脱氧-阿拉伯庚酮糖酸-7-磷酸合成酶（aroF）、奎尼酸脱氢酶（quiB）、脱氢奎宁酸脱水酶（aroD）酶活及其对应基因表达量进行检测，推测莽草酸、奎宁酸途径在该菌不同生长阶段中的运行情况。分别采用硫代巴比妥法、NAD+法、免疫酶联法测定冠突曲霉5个生长阶段中aroF、quiB、aroD的酶活性，并利用qRT-PCR及相对定量2-ΔΔCt法分析对应基因的表达量。结果显示:在冠突曲霉的有性生长阶段，随着菌体闭囊壳的形成，aroF、quiB、aroD的酶活均出现升高的趋势，分别由（62.08±0.43）U/L升至（68.60±0.19）U/L，（5.37±0.58）U/L升至（19.56±0.67）U/L，（33.76±0.03）U/L升至（34.30±0.10）U/L，随着子囊和子囊孢子的形成，aroF和aroD的酶活呈现下降（P>0.05）的趋势，但quiB的酶活仍呈现上升（P<0.05）的趋势；无性生长阶段，随着菌体的生长及分生孢子的形成，aroF、quiB、aroD的酶活均显著（P<0.05）下降，分别由（64.37±0.53）U/L降至（60.65±0.33）U/L，（36.74±1.42）U/L降至（18.87±0.75）U/L，（31.42±0.70）U/L降至（26.59±3.13）U/L。在整个有性生长阶段，随着菌体的生长，aroF和quiB基因相对表达量呈显著（P<0.05）上升的趋势，至子囊和子囊孢子形成阶段，分别升高了114.53倍、95.48倍，但aroD出现下降（P＜0.05）的趋势，下降了99.34%；在无性阶段，随着分生孢子的形成，aroF、quiB、aroD对应基因的相对表达量均也呈下降（P＜0.05）趋势，分别下降了54.46%、52.83%、98.03%。相关性分析可得，quiB（R2=0.91）的表达量与对应酶活的相关性较强，aroF（R2=0.43）与aroD（R2=0.06）的表达量和酶活之间相关性较弱；aroF与aroD在冠突曲霉不同时期的酶活变化表现出较强的协同性，但在对应基因表达量的变化上aroF与quiB表现出较强的协同性。该研究揭示了莽草酸、奎宁酸途径关键酶酶活及其对应基因在冠突曲霉不同生长阶段的活性差异及表达规律。 

Detection of Gene Expression and Enzyme Activity Related to Shikimic Acid Formation in Aspergillus cristatus

Activities of 3-deoxy-7-phosphate synthase（aroF），quinic dehydrogenase（quiB），dehydroquinic acid dehydratase（aroD）and their corresponding gene expression in five growth stages of Aspergillus cristatus were detected to speculate operation of shikimic acid and quinic acid pathway in different growth stages of Aspergillus cristatus.The enzyme activities of aroF，quiB and aroD in five growth stages of Aspergillus cristatus were determined by thiobarbituric method，NAD+method and immune enzyme-linked method，respectively.qRT-PCR and the method of 2-ΔΔCt were used to determine relative expression levels of corresponding genes.Results showed that: in the sexual growth stage of Aspergillus cristatus，enzyme activities of aroF，quiB and aroD increased from（62.08±0.43）U/L to（68.60±0.19）U/L，（5.37±0.58）U/L to（19.56±0.67）U/L，（33.76±0.03）U/L to（34.30±0.10）U/L.With formation of ascospores，enzyme activities of aroF and aroD decreased（P＞0.05），but the enzyme activities of quiB still increased（P＜0.05）.In the asexual development stage，enzyme activities of aroF，quiB and aroD decreased significantly（P＜0.05），from（64.37±0.53）U/L to（60.65±0.33）U/L，（36.74±1.42）U/L to（18.87±0.75）U/L，（31.42±0.70）U/L to（26.59±3.13）U/L.In the sexual development stage，expression levels of aroF and quiB upregulated 114.53 times and 95.48 times，meanwhile，but aroD downregulated 99.34%.In the asexual development stage，expression levels of aroF，quiB and aroD were obvious decline（P＜0.05），they were downregulated with 54.46%，52.83% and 98.03%，respectively.These results indicated that enzyme activities of quiB（R2=0.91）was related with its expression level，but aroF（R2=0.43）and aroD（R2=0.06）had weak correlation with expression corresponding genes.The relevance from enzyme activities of aroF and aroD was stronger，and relevance of expression level of aroF and quiB were also more accordant.This study revealed activity differences and expression patterns of shikimic acid，quinic acid pathway key enzymes and their corresponding genes in different growth stages of Aspergillus cristatus.