摘要
运用PCR方法扩增了卡氏住白细胞虫rDNA的ITS 2及部分 5 .8S和 2 8S序列 (ITS2 +) ,并对该序列进行了克隆和测序。采用巨型裂殖体为材料 ,并根据SaccharomycescerevisiaerDNA中的 5 .8S、2 8S保守序列所设计的通用引物ITS3、ITS4 ,进行了虫体ITS2 +的PCR扩增 ,将所扩增片段纯化后成功地克隆于 pGEM Teasy和PMD18 T载体的T T窗口。经双向自动测序显示 ,该ITS2 +片段的大小为 2 70bp ;经NCBIBlast联机检索 ,结果显示 ,所扩片段中 5 .8S序列与S .cerevisiae的 5 .8S序列有部分相同 ,而ITS 2序列为虫体所特有 ;同时经wDNA SIS软件分析 ,显示该ITS 2序列与S .cerevisiae间同源性相对较远 ,而与柔嫩艾美耳球虫间同源性相对较近 ,故而本试验中所测序列为卡氏住白细胞虫的ITS 2特有序列。
ITS2+ gene of Leucocytozoon caulleryi was amplified by PCR using a pair of conservative primers and cloned into the T T windows of plasmid pGEM T easy and PMD18 T carrier. The inserts were successfully sequenced and the results revealed that the ITS2+ gene was composed of 270 nucleotides while the ITS 2 gene was species specific with 113 nucleotides. The ITS 2 of L.caulleryi genes were sorted by NCBI Blast, and the degree of homology of the ITS 2 gene of L.caulleryi was compared with Eimeria tenella, Candida tropicalis, Saccharomyces kluyveri and others and analyzed by wDNASIS software. The results showed that the ITS 2 of L.caulleryi is characteristic and has the greatest similarity with E.tenella (21.2%).
出处
《中国农业科学》
CAS
CSCD
北大核心
2003年第5期573-576,共4页
Scientia Agricultura Sinica
基金
国家自然科学基金资助项目 ( 3 9870 5 49)
广东省自然科学基金资助项目 ( 980 13 4)
