灵芝漆酶基因转录Cu^(2+)诱导特性及其启动子的克隆与序列分析

Cu^(2+) inducing characteristics of the transcription of laccase gene from Ganoderma lucidum,and cloning,and analysis of the promoter of the gene

以Cu2+为诱导物检测了灵芝漆酶同工酶基因的转录特性,并对其启动子进行了克隆及序列分析。研究发现:灵芝漆酶基因在Cu2+的作用下表达量出现明显差异,其中以在发酵液中添加3.0 mmol.L-1Cu2+、诱导时间为6 d时,漆酶基因的mRNA表达量最高。根据GenBank中已报道的灵芝漆酶基因的序列信息,经PCR扩增获得了灵芝漆酶5′端长879bp的基因特异序列,进而通过self-formed adaptor PCR(SEFA-PCR)方法,扩增得到灵芝漆酶基因起始密码子上游长832bp的启动子序列。分析表明,该启动子区域除分布有TATA-box、CAAT-box及GC-box等基本的转录起始元件外,还存在多个潜在的顺式作用元件序列位点,包括4个MRE元件、4个STRE元件、11个HSE元件和5个氮因子结合位点等。

Copper was taken as an inducer to investigate the transcription characteristics of the laccase isozyme gene from Ganoderma lucidum, and the promoter sequence of the laccase gene was amplified from G. lucidum genomic DNA and analyzed. The expression of the laccase gene in the G. lucidum was significantly different under the effects of copper in this study. The expression of mRNA of the laccase gene was the highest after treated by adding 3.0 mmol.L^-1 copper into the liquid medium for 6 days. According to the sequence of the laccase gene of the G. lucidum from GenBank, an 879 bp sequence, which was the 5＇terminal sequence of the laccase gene of G. lucidum HG, was obtained. Then an 832 bp promoter sequence on the upstream of the initiation codon in laccase gene of the G. lucidum HG was amplified through a self-formed adaptor PCR （SEFA-PCR） method. The promoter was analyzed using the Softberry software. The result showed that, besides some basic transcription initiation elements, such as TATA-box, CAAT-box and GC-box, some other potential cis-acting element sequence sites also existed, including four MRE, four STRE, eleven HSE and five nitrogen factor binding sites, etc.