【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为...【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为诱饵来筛选酵母双杂交文库,确定与BsTup1相互作用的蛋白。【结果】1)利用SMART(switching mechanism at 5′end of the RNA transcript)技术首次成功构建了麦根腐平脐蠕孢(B.sorokini-ana)分生孢子和菌丝体的混合cDNA文库。文库鉴定结果表明,构建的cDNA文库库容为4.8×10^(7) cfu·mL^(-1),文库插入片段重组率达100%且平均大小为1000 bp。2)构建了pGBKT7-BsTup1诱饵载体,无自激活活性。3)使用诱饵蛋白载体pGBKT7-BsTup1对麦根腐平脐蠕孢(B.sorokiniana)酵母双杂交cDNA文库进行筛选,经测序、序列比对和酵母回转验证,获得38个与BsTup1相互作用的候选蛋白。【结论】成功构建了麦根腐平脐蠕孢(B.sorokiniana)的cDNA文库,并鉴定出38个与BsTup1相互作用的候选蛋白。展开更多
Total RNA was separated from the abdomen of house fly,after which had been challenged for 24 hours with E. coli and Staphylococcus aureus . Then,the mRNA was purified from it. After that,ds cDNA was synthesized by rev...Total RNA was separated from the abdomen of house fly,after which had been challenged for 24 hours with E. coli and Staphylococcus aureus . Then,the mRNA was purified from it. After that,ds cDNA was synthesized by reverse transcriptase from mRNA,and small cDNA which was lower than 400?bp was removed by Sepharose CL 4B spun column. After Eoc RⅠ/ Not Ⅰ Adaptors were added to cDNA,those that weren’t ligated to cDNA were removed by another Sepharose CL 4B spun column. The cDNA was inserted into λgtll,the recombined vector packaged in vitro ,infected a host strain Y1090. Thus,the cDNA library was constructed. The titer of the newly constructed cDNA library was 3 46×10 5 pfu/mL,and its recombination rate was 99 6%. The library would provide basis for the cloning of the antimicrobial peptide genes of house fly.展开更多
基金supported by Modern Agro-industry Technology Research System (nycytx-19)National High-Tech Research and Development Plan of China (2006AA10A114+1 种基金 2007AA10Z189)National Project of Scientific and Technical Sup-porting Program (2008BAD97B04)
文摘【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为诱饵来筛选酵母双杂交文库,确定与BsTup1相互作用的蛋白。【结果】1)利用SMART(switching mechanism at 5′end of the RNA transcript)技术首次成功构建了麦根腐平脐蠕孢(B.sorokini-ana)分生孢子和菌丝体的混合cDNA文库。文库鉴定结果表明,构建的cDNA文库库容为4.8×10^(7) cfu·mL^(-1),文库插入片段重组率达100%且平均大小为1000 bp。2)构建了pGBKT7-BsTup1诱饵载体,无自激活活性。3)使用诱饵蛋白载体pGBKT7-BsTup1对麦根腐平脐蠕孢(B.sorokiniana)酵母双杂交cDNA文库进行筛选,经测序、序列比对和酵母回转验证,获得38个与BsTup1相互作用的候选蛋白。【结论】成功构建了麦根腐平脐蠕孢(B.sorokiniana)的cDNA文库,并鉴定出38个与BsTup1相互作用的候选蛋白。
文摘Total RNA was separated from the abdomen of house fly,after which had been challenged for 24 hours with E. coli and Staphylococcus aureus . Then,the mRNA was purified from it. After that,ds cDNA was synthesized by reverse transcriptase from mRNA,and small cDNA which was lower than 400?bp was removed by Sepharose CL 4B spun column. After Eoc RⅠ/ Not Ⅰ Adaptors were added to cDNA,those that weren’t ligated to cDNA were removed by another Sepharose CL 4B spun column. The cDNA was inserted into λgtll,the recombined vector packaged in vitro ,infected a host strain Y1090. Thus,the cDNA library was constructed. The titer of the newly constructed cDNA library was 3 46×10 5 pfu/mL,and its recombination rate was 99 6%. The library would provide basis for the cloning of the antimicrobial peptide genes of house fly.
