西瓜炭疽菌核糖体基因ITS区段的克隆及序列分析

Cloning and Sequencing of Ribosomal RNA Gene ITS in Colletotrichum orbiculare

采用真菌核糖体基因转录间隔区域(ITS)通用引物,PCR扩增4个来自不同地方的西瓜炭疽病菌核糖体基因的ITS1和ITS2,并对PCR产物进行了克隆和序列分析。结果表明:西瓜炭疽病菌的ITS全长481 bp,其中ITS1长163 bp,5.8 S长153 bp,ITS2长165 bp,各碱基A、T、G、C的个数分别为1101、061、181、47,GC含量为55.1%。用Mega3软件对此4个序列以及GeneBank中登录的炭疽属其他24个种的ITS1和ITS2聚类分析,结果显示:Colletotrichum属可以分为13个聚类组,各聚类组间存在着一定的遗传多样性,其中Colletotrichum orbicu-lare、Colletotrichum lindemuthiana在ITS1I、TS2上都聚为一组,这就为用ITS作为靶序列来设计特异性引物将Colletotrichum orbiculare、Colletotrichum lindemuthiana与其他种分开提供了分子依据。

The internal transcribed spacer regions（ITS1 and ITS2） of the ribosomal RNA gene from Colletotrichum orbicular were amplified with the polymerase chain reaction through the universal primers and sequenced.The size of total length was 481 bp;5.8S was 153 bp and ITS2 was 165 bp.The number of A,T, G and C was 110,106,118 and 147 respectively and the CC content was 55.1%. Based on the four sequences and another 24 species Of published sequences of Colletotrichum spp in GeneBank database, the ITS1 and ITS2 phylogenetic trees were produced with the soft of Mega3. The Colletotrichum spp was divided into 13 groups. Colletotrichum orbiculare and Colletotrichum lindemuthiana were formed in a single group both in ITS1 and ITS2.So,it provided the molecule proof to design specialties primers to distinguish Colletotrichum orbiculare and Colletotrichum lindemuthiana from other species with the sequence of ITS.