利用CODEHOP设计简并引物克隆镰刀菌果胶酶基因片段

利用CODEHOP(Consensus-degenerate hybrid oligonucleotide primers)设计真菌果胶酶基因片段的简并引物,并对设计的多对引物进行筛选,比较了普通PCR和Touchdown-PCR(TD-PCR)程序的扩增效果,并对产物进行了测序、比对和分析。结果表明:利用CODEHOP设计简并引物可信性强,阳性率高,能够从供试菌株中获得与目的片段大小相近的产物。利用TD-PCR程序扩增比普通PCR扩增效果好。扩增产物序列BLASTX比对和分析结果表明,产物片段编码的氨基酸序列与镰刀菌属来源的果胶酶氨基酸片段相似性均超过90%,说明所扩增的序列即为镰刀菌果胶酶基因片段。

The cloning of Fusarium polygalacturonase gene fragments using degenerate primers designed by CODEHOP

With the help of CODEHOP,degenerate primers were designed for cloning the fungal polygalacturonase gene fragment.The proper primer was screened from many pairs of primers.TD-PCR and the popular PCR reaction were compared to determine the good reaction system.The result showed that a 700 bp PCR product with a length close to the target gene fragment was obtained,the TD-PCR with the same reaction system has higher specificity than the popular PCR reaction system,and the amino acid fragments encoded by the products of the cloned DNA had high similarity with those of polygalacturonase amino acid sequence from GenBank.The cloned sequence should be polygalacturonase gene fragment from the tested Fusarium.This study indicated that the degenerate primers designed by the CODEHOP software are practicable and high in positive rates.Cloning of pectinase gene fragment of the tested Fusarium would afford a scientific warrant and possibility for the research of pathogenic mechanism of Fusarium.