羅志姣; 蔡為明; 李南羿; 吳坤
河南農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院; 浙江省農(nóng)業(yè)科學(xué)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(dá)差異的銀染差異顯示方法,以香菇突變株fbd1及其出發(fā)菌株98411的菌絲體和子實(shí)體為材料,采用Trizol法提取總RNA,優(yōu)化了反轉(zhuǎn)錄和PCR擴(kuò)增中5項(xiàng)反應(yīng)參數(shù),再經(jīng)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(yàn)結(jié)果表明,適宜分析香菇mRNA差異的反轉(zhuǎn)錄體系中RNA投入量為0.51μg、dNTP濃度為20μmol/L,PCR擴(kuò)增反應(yīng)MgCl2、dNTP和Taq酶分別為2mmol/L、200μmol/L和1.0U,此條件下,目標(biāo)條帶清晰且重復(fù)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(guān)鍵詞】 香菇; 差異表達(dá); 顯示體系; 銀染
【英文關(guān)鍵詞】 Lentinula edodes; differential expression; differential display; silver staining
【基金】浙江省自然科學(xué)基金“香菇子實(shí)體分化關(guān)鍵基因分離與克隆”(編號(hào):Y305631)的部分研究?jī)?nèi)容
【文獻(xiàn)出處】 食用菌學(xué)報(bào),Acta Edulis Fungi,編輯部郵箱,2009年01期 【DOI】CNKI:SUN:SYJB.0.2009-01-007