松材线虫rDNA的测序和 PCR-SSCP分析

SEQUENCING AND PCR-SSCP ANALYSIS OF RIBOSOMAL DNA OF Bursaphelenchus NEMATODES

本文为克服形态鉴定的不足 ,用分子生物学的方法鉴别松材线虫。线虫核糖体 DNA(r DNA)的内部转录间隔区 (ITS1)区 (约 30 8bp)的测序结果显示 :松材线虫种内区别很小 ,不超过 1bp;拟松材线虫种内区别较大 ,最大达 7bp;这 2种线虫的种间区别为 32～ 39bp。根据以上测序结果 ,本文结合单条线虫 DNA的提取技术 ,对 14个松材线虫和拟松材线虫样本进行了单链构象多态性 (PCR-SSCP)分析 ,结果表明 PCR- SSCP分析技术可明确区分这 2种线虫 ,该技术可为单条松材线虫的鉴定提供一套灵敏而可靠的方法。

To overcome limitations in the morphological identification of Bursaphelenchus species, a molecular approach was used. The internal transcribed spacer 1 (ITS1) of the nuclear ribosomal DNA (rDNA) region was amplified and sequenced. A pairwise comparison of this sequence data revealed that the differences in the ITS1 region (approximately 308 bp) between four B. xylophilus isolates were slight (only 1 bp), but there are marked differences (up to 7 bp) in this region between the B. mucronatus isolates from China (Guangdong) and France. The data also revealed that the differences in the ITS1 region between the two Bursaphelenchus species range from 32 39 bp. Based on these data, the method of extracting the genomic DNA of the individual pinewood nematode and PCR SSCP (single strand conformation polymorphism) analysis were employed for the unequivocal differentiation of the Bursaphelenchus variant from B. xylophilus and B. mucronatus. These methods should provide a valuable tool for sensitive and accurate identification of individual worm of B. xylophilus.