外显子拼接法合成真菌灵芝漆酶基因cDNA

Cloning of a Laccase Gene cDNA from Fungi Ganoderma lucidum by the Method of Exon Spicing

为真菌漆酶基因cDNA的克隆提供了一种简便快速的新方法．从含有内含子的灵芝漆酶基因出发，采用外显子拼接PCR方法，合成了灵芝漆酶的cDNA．设计了10对引物分别经PCR扩增该基因的10个外显子，引物设计使前一个外显子的下游引物与下一个外显子的上游引物都有一段同源匹配序列，然后通过两两片段混合作为模板PCR扩增得到两两拼接的外显子片段，再将得到的5个片段前两个混合，后3个混合分别作为模板扩增获得拼接好的外显子1～4大片段和外显子5～10大片段，最后将这两个大片段混合作为模板扩增得到拼接好的全长cDNA序列．与常规方法相比，该方法既避免了RNA的操作，又不用摸索酶表达的条件，可以和基因组步行技术结合快速获得真菌漆酶基因的cDNA．

Laccases of white-rot fungi attract considerable attention due in the transformation of a wide variety of phenolic compounds including th to their possible involvement e polymeric lignin and humic substances. In this research, a new simple method was developed for laccase eDNA cloning devoid RNA manipulation. After the fungi Ganoderma lucidum laccase gene that contain introns was cloned by the method of genome walking PCR. 10 pair of primers were designed to amplify 10 exons. When these primers were designed, reward primer sequence of last exon must have 20 base homologous with forward primer sequence of next exon. Then, 10 exons were amplied and recovered, respectively. Two exons in sequence were mixed as template to obtain 5 double-exon fragments. 2 double-exon fragments were mixed as template to obtain large fragment exonl-4, and 3 double-exon fragments to obtain large fragment exon 5-10, respectively. At last, these two large fragments were mixed as template to obtain total eDNA sequence. Comparing with usual used, it is unnecessary to manipulate RNA and explore enzyme expression condition in this method. If this method combined with genomewakling PCR method can clone laccase gene and eDNA rapidly.