赤芝LS基因酵母单杂交文库构建及其上游调控因子的筛选

Construction of yeast one-hybrid library and screening of transcription factors regulating LS expression in Ganoderma lucidum

赤芝羊毛甾醇合酶(lanosterol synthase,LS)是灵芝三萜生物合成甲羟戊酸途径(mevalonate pathway,MVA途径)中生成前体化合物--羊毛甾醇的关键酶,LS基因的转录调控可直接影响灵芝三萜含量的高低。为了进一步研究赤芝MVA途径关键酶基因的转录调控机制,该研究利用酵母单杂交技术,筛选出赤芝LS基因上游的转录调控因子。提取赤芝全基因组DNA,PCR扩增LS启动子序列。由LS启动子构建诱饵载体,转化Y1H酵母感受态细胞构建诱饵菌株。运用SMART技术构建灵芝酵母单杂交c DNA文库,再共转化诱饵菌株,经同源重组筛选LS基因上游的转录调控因子。该实验共筛选出23个阳性克隆,经测序后与全基因组序数据库行blast比对,筛选出LS基因上游的候选调控因子8个,包括转录起始因子TFIIB,α/β折叠水解酶超家族、醛脱氢酶超家族、60S核糖体蛋白L21,ATP合成酶β亚基、微管结合蛋白Cript、蛋白酶体亚基β-1和转醛醇酶。这8个调控因子在灵芝三萜生物合成路径中的调控作用尚未见报道,该研究为深入研究LS基因的表达调控机制提供了候选蛋白。

Lanosterol synthase(LS)is a key enzyme involving in the mevalonate pathway(MVA pathway)to produce lanosterol,which is a precursor of ganoderma triterpenoid.And the transcriptional regulation of LS gene directly affects the content of triterpenes in Ganoderma lucidum.In order to study the transcriptional regulation mechanism of LS gene,yeast one-hybrid technique was used to screen the transcription regulators which interact withthe promoter of LS.The bait vector was constructed by LS promoter,then the vector was transformed yeast cells to construct bait yeast strain.One-hybrid c DNA library was constructed via SMART technology.Then the c DNA and p GADT7-Rec vector were co-transformed into the bait yeast strain to screen the upstream regulatory factors of the promoter region of LS by homologous recombination.Total of 23 positive clones were screened.After sequencing,blast was performed against the whole-genome sequence of G.lucidum.As a result,8 regulatory factors were screened out including the transcription initiation TFIIB,the alpha/beta hydrolase super family,ALDH-SF superfamily,60 S ribosomal protein L21,ATP synthaseβ-subunit,microtubule associated protein Cript,prote asome subunitβ-1,and transaldolase.Until now,the regulation effect of these 8 regulatory factors in G.lucidum has not been reported.This study provides candidate proteins for in-depth study on the expression regulation of LS.