线虫寄生菌巴斯德杆菌遗传多样性分析

Genetic Diversity of Nematode Parasitic Bacteria Pasteuria spp.

摘要:

应用16S rDNA-PCR技术快速检测不同根系样品中的巴斯德杆菌(Pasteuria spp.), 并采用PCR-RFLP和PCR-SSCP法初步分析这些样品中巴斯德杆菌群体的遗传多样性, 结果表明, 来自福建、广东的30份感染根结线虫病的根系中, 有9份样品含有巴斯德杆菌; PCR-RFLP分析表明, 克隆子的EcoHⅠ酶切带型分为5类, 其中2类占相对优势, PCR-SSCP带型分类的情况与PCR-RFLP的基本一致, 但表现出更大的差异性。选取12个克隆子进行测序分析, 结果显示, 克隆子的16S rDNA序列与穿刺巴斯德杆菌(Pasteuria penetrans)的相应序列具有较高的同源性(97.8%~99.7%); 系统进化关系分析进一步表明, 不同根系样品中的巴斯德杆菌(Pasteuria spp.)16S rDNA序列与GenBank已收录的穿刺巴斯德杆菌(P. penetrans)序列形成1个主要分支和 7个独立分支, 具有一定的遗传差异。

Abstract:

Pasteuria spp. was one of the highly potential biocontrol agents of plant-parasitic nematodes. Based on their 16S rDNA sequences, we developed simple PCR, PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) methods for rapid detecting the genetic diversities of Pasteuria spp. in root samples. Using simple PCR, we detected Pasteuria spp. from nine out of thirty nematode-infected samples collected from different crops in Fujian and Guangzhou of China. The genetic diversities of Pasteuria spp. in root samples were examined by PCR-RFLP and PCR-SSCP. Two of the five variant RFLP patterns were predominant among the clones when they were digested by restriction enzyme EcoHI. The results from PCR-SSCP analysis were consistent with those of PCR-RFLP and showed greater gene diversities. Twelve clones were chosen and sequenced. The results indicated the sequences of clones shared 97.8%~99.7% similar identity with those of previously reported P. penetrans, and the clones were further divided into one major clades and seven individual clades in the phylogenetic tree.