花脸香蘑荧光定量PCR内参基因筛选及多糖合成代谢相关基因表达量测定

Screening of Internal Reference Genes by Fluorescence Quantitative PCR and Determination of Gene Expression Related to Polysaccharide Synthesis and Metabolism in Lepista sordida

对适用于花脸香蘑(Lepista sordida)荧光定量PCR内参基因进行筛选,并检测花脸香蘑高产胞外多糖诱变菌株LS01_10_9及其出发菌株LS01_10多糖合成途径关键基因和多糖代谢途径基因表达量。结果表明:核糖体蛋白L4(ribosomal protein L4,rpl4)基因和3-磷酸甘油脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,gapdh)基因表达丰度高、扩增效率较好且稳定,是花脸香蘑荧光定量PCR理想的内参基因。与出发菌株LS01_10比较,高产胞外多糖诱变菌株LS01_10_9中5个参与多糖合成途径的关键基因GK、GMD-3、UGE、UGDG、PFK表达量显著增加,4个参与多糖代谢途径的基因GT-1、GT-2、NOX-3、NOX-4表达量显著增加。

Using fluorescence quantitative PCR(qPCR),internal reference genes for Lepista sordida were screened,and the expression of genes involved in polysaccharide biosynthesis and metabolism in the high exopplysaccharide yield mutant strain LS01_10_9 and its starting strain LS01_10 were determined.The results showed that the ribosomal protein L4(rpl4)and glyceraldehyde-3-phosphate dehydrogenase(gapdh)had a high expression abundance and amplification efficiency,and thus were selected as ideal internal reference genes for L.sordida.Compared with LS01_10_9,five key genes(GK,GMD-3,UGE,UGDG and PFK)involved in polysaccharide synthesis and four genes(GT-1,GT-2,NOX-3 and NOX-4)involved in polysaccharide metabolism were significantly up-regulated in LS01_10_9.