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.