枣疯病植原体的分子检测与同源性鉴定

【目的】采用植原体16S通用引物建立枣疯病植原体PCR检测体系。【方法】采用植原体16S rDNA通用引物R16mF1/R16mR1,对陕西和山西具有枣疯病的枣树植株总DNA进行PCR扩增,获得了相关的枣疯病植原体序列。采用DNAMAN软件分析其同源性,绘制系统进化树。运用pDRAW32软件对17种具有16S rDNA分组的典型的限制性内切酶进行计算机模拟RFLP,确定枣疯病植原体同源性关系。【结果】从陕西和山西的枣疯病的植株中,分别得到1 433 bp和1 432 bp的条带,与已报道的枣疯病植原体比较,同源性达到99%以上,而与其他植物的植原体16S rDNA同源性多低于93%,并且与16S rDNA V-B组的遗传距离最近。pDRAW32软件模拟RFLP结果表明它们和已报道的JWB的RFLP图谱相似。【结论】枣疯病植原体归属于16SrDNA V-B,为枣疯病植原体PCR检测体系提供了理论基础。RFLP分析进一步验证了枣疯病植原体属于16SrDNA V-B,具有随地域变异性较小的特性,这将有利于不同地区枣疯病检测技术的建立。

Molecular Detection and Homologous Identification of Jujube Witches, Broom Phytoplasma

【Objective】To establish a detected system of the Jujube Witches,Broom Phytoplasma by PCR through 16S universal primer.【Method】The symptomatic leaves which were used to isolate total DNA were collected from Shaanxi and Shanxi provinces and universal primers of phytoplasma 16S rDNA R16mF1/R16mR1 were used for PCR amplification.The sequences of 16S rDNA with published sequences were calculated using Blastn,and 27 on the 16S rDNA with a clear sequence of phytoplasma grouped were selected.The simulated RFLP mapping among 17 restriction enzymes which are the typical of 16S rDNA group were analyzed by pDRAW32.【Result】The 1 433 bp and 1 432 bp DNA fragement were amplified in DNA samples from Shaanxi and Shanxi provinces,and the two isolates shared approximately 99% identity with Jujube Witches,Broom Phytoplasma which have been reported,but the phytoplasma 16S rDNA homology of other plants were obviously under 93%. The results of blast showed that the genetic distance of two isolates was closely to the 16S rDNA V-B group and computer-simulated RFLP showed that the two isolates were similarity to the reported RFLP patterns of JWB,improved that the two isolates have little geographical variability in further,which was conducive to establishing the detection system of JWB.【Conclusion】The result of PCR and RFLP showed that Jujube Witches,Broom Phytoplasm belonged to 16S rDNA V-B group and had little geographical variability.All of these results would provide atheoretical basis for molecular detection of Jujube Witches,Broom Phytoplasm in future.