Cloning and sequence analysis of laccase gene fragments from Climacocystis borealis
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摘要: 根据报道的10几种真菌漆酶氨基酸序列的保守区域设计了一对简并引物,以北方梭囊孔菌总DNA为模板,通过PCR扩增到一个1201bp的基因片段.将片段序列在NCBI的BLAST软件上进行同源性搜寻,显示99个序列,全部为漆酶基因及其类似基因,因而认为克隆到的片段就是北方梭囊孔菌漆酶基因片段.经过与同源性最高的粗毛革孔菌（laccaselcc1）同一区段编码区序列进行ClustalW比较,其中有6个间隔区,确定为内含子区.去除内含子后,推导的氨基酸序列为286个残基.经与其他几种真菌漆酶的氨基酸序列进行ClustalW比对,同源性均在51%以上.Abstract: Two degenerate oligonucleotide primers were designed,coding for two conservative amino acid regions in fungi laccase protein family.A PCR amiplification was performed on Climacocystis borealis DNA template and a fragment of 1201 bp was obtained.By using the program of BLAST on NCBI GenBank database,the sequence presented a very high match with the laccase gene from other fungi and 99 searched sequences were all laccase genes.It is concluded that the cloned sequence is Climacocystis borealis laccase gene fragment. The sequence data have been submitted to the GenBank database under accession number AY333124. According to the ClustalW comparison analysis with Coriolopsis gallica(laccase lcc 1)cds,six introns are included and the sequence codes 286 amino acids after excluding the introns.Through ClustalW analysis with other fungi laccase genes,it is found that the Climacocystis borealis laccase amino acid fragment shares considerable homology of above 51%.
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Keywords:
- Climacocystis borealis /
- laccase /
- degenerate primer /
- gene cloning
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