炭疽芽胞杆菌基因组中串联重复序列拷贝数的分析

Analysis of variable number of tandem repeat copy in Bacillus anthracis geoome

目的研究炭疽芽胞杆菌基因组中多位点串联重复序列遗传标记的遗传变异规律。方法根据文献上发表的串联重复序列位点,利用已发表的13对引物,对88株炭疽芽胞杆菌基因组DNA进行PCR扩增,扩增产物进行琼脂糖和聚丙烯酰胺凝胶电泳,利用凝胶影像分析软件对PCR产物DNA片段的碱基含量进行测算,计算出串联重复拷贝数。对部分PCR扩增产物DNA片段进行测序,利用DNAStar软件进行序列比对,分析不同菌株间的串联重复序列遗传变异规律。结果(1)炭疽芽胞杆菌基因组中的串联重复序列位置,同一菌株具有相对稳定的串联重复拷贝数。(2)88株炭疽芽胞杆菌DNA上的13个串联重复序列遗传标记中,有的串联重复序列拷贝数变异较小,有的却存在着复杂的多态性;(3)有些串联重复序列单位内的核苷酸数目不变,但核苷酸的排列组合有差异,不过其重复单位内都包含着一个相同的稳定不变的核心核苷酸序列。结论炭疽芽胞杆菌基因组DNA中串联重复序列可以精确定位,测定结果可以数值化,并且串联重复序列在菌株DNA中具有相对的稳定性,因此,串联重复序列是研究炭疽芽胞杆菌遗传特征的较好指标,具有鉴别炭疽芽胞杆菌菌株间差别的能力。

Objective To study the heredity variation character of variable-number tandem repeats（VNTR） loci in the Bacillus anthracis genome. Methods We selected 13 VNTR loci to study 88 strains of B. anthracis isolated from China. The primers from published papers were at the both sides of the tandem repeat loci, which were amplified by PCR. The PCR products were detected by agarese gel and polyacrylamide electrepboresis. The molecular mass was reckoned by the software of gel images instrument. The repeat numbers and the VNTR character were analyzed by sequencing PCR fragments and alignment comparing. Results The location of the VNTR in B. anthracis is immovable and exclusive, and the VNTR copies of the same strain DNA genome is invariable. In the 13 VNTR loci, the numbers of tandem repeat were different. And the nucleotide numbers consist of each tandem repeat unit were different. They presented complicated polymorphism. The number and array of the nucleotides in most VNTR unit was invariable. But some VNTR unit has changes lightly in the number and army of the nucleotide. However there was a core sequence in tandem repeat unit of each VNTR loci, which was intrinsic invariably. Conclusion The VNTR in the B. anthracis genome can be located accurately, and the result of detection may turn into numerical. The characters of VNTR are good targets to study B. anthracis heredity vaxiafion. VNTR possess the polymorphism of the type and copy number, which acts as good index to discriminate B. anthracis isolates.