糙皮侧耳几丁质酶基因PoChi1的克隆及表达分析

Cloning and Expression of a Pleurotus ostreatus chitinase gene,PoChi1,in Escherichia coli

利用反转录PCR(reverse transcription PCR,RT-PCR)克隆糙皮侧耳(Pleurotus ostreatus)几丁质酶基因PoChi1,将其克隆至原核表达载体,得重组质粒pEASY-PoChi1,转化至大肠杆菌Escherichia coli BL21(DE3);通过半定量RT-PCR分析该基因在糙皮侧耳不同发育阶段的表达。糙皮侧耳几丁质酶基因PoChi1序列长度为1188bp,编码395个氨基酸,N端24个氨基酸为信号肽。经IPTG诱导后SDSPAGE检测,该基因编码产物的相对分子量约为29kDa。PoChi1在糙皮侧耳成熟子实体期的表达量最高。

Reverse transcription-PCR （RT-PCR）was used to clone a chitinase gene,PoChi1,from Pleurotus ostreatus,and the recombinant plasmid,pEASY-POCHI1,was constructed and transformed into Escherichia coli BL21 （DE3）.PoChi1 was 1188 bp in length,encoding 395 amino acid residues,and contained a 24 amino acid signal peptide at the N-terminal. Recombinant protein induced with IPTG had a relative molecular mass of approximately 29 kDa on SDS-PAGE.PoChi1expression levels at different developmental stages were determined by semi-quantitative RT-PCR and shown to be highest at the mature fruit body stage.