【作者】 林涛；

【导师】 黄建忠；

【作者基本信息】 福建师范大学 ， 发酵工程， 2013， 硕士

【摘要】 通过里氏木霉转录谱分析(RNA-seq),并结合生物信息学方法,发现了1个具有锌指结构的基因fogl。对这个基因进行敲除(knock-out),分析在不同碳源条件下敲除株的生长情况和纤维素酶的表达特性,以期为寻找新的纤维素酶相关蛋白或转录因子提供证据。以里氏木霉T.reesei tu6△ku70作为对照菌株,比较敲除株T.reesei tu6△ku70Afogl和出发菌株的产孢情况,在乳糖和纤维素下的生长情况和纤维素酶活高低。通过SDS-PAGE初步分析敲除株的外分泌蛋白情况。结果显示：与出发菌株相比,fogl敲除株的产孢情况较差；在葡萄糖作为碳源条件下,敲除株与对照株的生长速度相当,没有明显差别,而在乳糖、纤维素诱导条件下,敲除株的菌丝生长速度较快；SDS-PAGE分析表明,在纤维素和乳糖诱导条件下,敲除株发酵液的外分泌蛋白较多；滤纸酶活(FPA)数据显示,在乳糖诱导下,fogl敲除株粗酶液的酶活分别比对照菌株高10.27%,5.29%。综上所述,初步确定锌指蛋白FOG1的敲除能够提高里氏木霉纤维素酶的分泌表达。为进一步研究FOG1的具体作用机理提供帮助,以及纤维素酶高产菌株的改造提供理论基础。从RNA-seq数据筛选到在葡萄糖下的高转录丰度的2个基因cdnal和tefl,分别克隆这2个基因的启动子序列。以pBluescriptIISK(+)为骨架,构建以PcDNAl和Ptefl为启动子,Pleurotus ostreatus漆酶基因poxl为报告基因的表达载体。转化里氏木霉T.reesei RUT-C30,以葡萄糖作为碳源,测定漆酶酶活。结果表明：cDNAl和tefl启动子均能够启动异源基因的组成型表达,且cDNAl启动子显示结果比tefl启动子更为优越。并且到目前为止,在里氏木霉中chbl启动子还是最强的诱导型启动子。更多还原

【Abstract】 A zinc-finger gene fogl was found via RNA-seq and bioinformatic softwares in Trichodema reesei. the fogl gene was knocked out from the genome of treesei.and the growth and cellulase expression of Afogl strain was analysed on different carbon source condition. This work might have contribution to the identification of new lignocellulose degradation related proteins or transcription factors of the cellulases. in this study,the T.reese tu6ku70was set as the control strain,the Sporulation,growth and cellulases activity of Afogl strain was observed and measured on different carbon source.the protein ex-secreted from the Afogl strain was analysed by SDS-PAGE-Result shows,the sporulation of Afogl strain was worse;the mycelium growth rate of△fogl strain was matched with the control strain on glucose,and a little faster than that of control strain on lactose and cellulose.SDS-PAGE analysis shows,the ex-secreted protein of Afogl strain was more than that of control strain on the induction of lactose and cellulose.FPA activity data shows,the cellulases activity of Afogl strain was10.27%and5.29%higher than that of control strain on the induction of lactose and cellulose respectively.conclude above,we preliminarily confirmed that the knockout of zinc-finger protein FOG1was able to improve the expression of cellulases.this might offer help for the further study on the specific mechanism of FOG1,and contribute to the improvement of cellulase production in T.reesei.the cdnal and tefl gene was screened from the RNA-seq data based on the high RPKM on glucose,the promoter region of this two genes was cloned by PCR.The expression vector was constructed by inserting the Pleurotus ostreatus pox1gene after the promoter cdnal and tefl respectively within the pBluescriptIISK(+) vector.the expression vector was transformed into the T.reesei RUT-C30,the laccase enzyme activity was measured on the growth of glucose.result shows,cdnal and tefl promoter can drive the constitutive expression of laccase in t.reesei bothly,and the cdnal promoter appear to be superior to the tefl promoter,so far,the cbhl promoter still is the strongest inductive promoter.更多还原