羅志姣; 蔡為明; 李南羿; 吳坤
河南農業大學生命科學學院; 浙江省農業科學院園藝所

【中文摘要】 為建立適用于研究香菇(Lentinula edodes)分化中基因表達差異的銀染差異顯示方法,以香菇突變株fbd1及其出發菌株98411的菌絲體和子實體為材料,采用Trizol法提取總RNA,優化了反轉錄和PCR擴增中5項反應參數,再經6%變性聚丙烯酰胺凝膠電泳分離和銀染顯示差異條帶。試驗結果表明,適宜分析香菇mRNA差異的反轉錄體系中RNA投入量為0.51μg、dNTP濃度為20μmol/L,PCR擴增反應MgCl2、dNTP和Taq酶分別為2mmol/L、200μmol/L和1.0U,此條件下,目標條帶清晰且重復性好。

【英文摘要】 An effective and feasible silver staining-based display method for studying differentially expressed genes in Lentinula edodes is described.Five intrinsic factors(amount of template RNA,dNTP concentrations used in reverse transcription,and MgCl2,dNTP and Taq polymerase concentrations used in PCR amplification)affecting differential display were optimized using high-quality total RNA extracted from mycelia and fruiting bodies of L.edodes strain 98411 and its mutant strain fbd1.RNA extraction using the Trizol method,followed by separation of amplified cDNA fragments using 6%(w/v)denaturing polyacrylamide gel electrophoresis and visualization by silver staining,produced clear target bands and good reproducibility under the following optimal conditions:0.51 μg template RNA,20 μmol/L dNTP(reverse transcription),2 mmol/L MgCl2,200 μmol/L dNTP(PCR amplification)and 1 U Taq polymerase.

【中文關鍵詞】 香菇; 差異表達; 顯示體系; 銀染
【英文關鍵詞】 Lentinula edodes; differential expression; differential display; silver staining
【基金】浙江省自然科學基金“香菇子實體分化關鍵基因分離與克隆”(編號:Y305631)的部分研究內容

【文獻出處】 食用菌學報,Acta Edulis Fungi,編輯部郵箱,2009年01期 【DOI】CNKI:SUN:SYJB.0.2009-01-007