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目的评价3种不同的分子标记技术对植物属级的鉴定能力以选出最适合在中毒现场快速鉴定有毒植物的分子技术。方法选取毛茛科、大戟科共18种19份有毒植物叶片样品,用改良十六烷基三甲基溴化铵法提取基因组DNA,分别使用随机扩增多态性DNA标记(RAPD)、简单重复序列问标记(ISSR)和DNA条形码技术进行物种鉴定(属级),使用NYSTS、SPSS、PAUP、MEGA软件进行聚类分析,比较3种DNA分子标记技术的准确性、可靠性、时效性和可操作性。结果准确性:RAPD技术未能鉴定样品的属级,ISSR技术和DNA条形码技术的鉴定准确率分别为68%和100%。可靠性:RAPD技术与ISSR技术主观影响率分别为44%、26%,条带重复率分别为47%、45%;DNA条形码技术trnH-psbA片段扩增成功率与测序成功率均为100%。时效性:从总DNA提取开始至得到最终鉴定结果,RAPD、ISSR、DNA条形码技术耗时分别为8.5、9.0、42.2 h。可操作性:RAPD与ISSR技术的鉴定工作可在普通实验室完成,DNA条形码技术的鉴定工作则需要特殊的测序设备。结论初步认为DNA条形码技术是比较适用于突发有毒植物中毒事件中快速病因判定的分子鉴定技术。
Objective To evaluate the identification ability of three different molecular markers for plant genus level to select the molecular technique that is most suitable for the rapid identification of toxic plants in poisoning sites. Methods A total of 18 species of poisonous plant leaf samples from Ranunculaceae and Euphorbiaceae were selected and genomic DNA was extracted by modified cetyltrimethylammonium bromide method. Random amplified polymorphic DNA (RAPD) (ISSR) and DNA barcoding were used to identify the species (Genus), and the clustering analysis was performed using NYSTS, SPSS, PAUP and MEGA software. The accuracy, reliability and timeliness of the three DNA molecular markers were compared Operability. The accuracy of the results: RAPD technology failed to identify the genus of samples, and the accuracy of ISSR and DNA barcoding was 68% and 100%, respectively. Reliability: The subjective impact rates of RAPD and ISSR were 44% and 26% respectively, and the band repetition rates were 47% and 45% respectively. The success rate of trnH-psbA fragment amplification and DNA sequencing success rate were both 100% . Timeliness: From the beginning of total DNA extraction to the final identification results, RAPD, ISSR, DNA barcode technology time-consuming were 8.5,9.0,42.2 h. Operability: The identification of RAPD and ISSR technologies can be done in a common lab, and DNA barcode technology requires special sequencing equipment. Conclusion It is preliminarily believed that the DNA barcoding technique is a molecular identification technique that is more suitable for the rapid etiological determination of sudden toxic plant poisoning.