芸薹根肿菌(Plasmodiophora brassicae Wor.)DNA提取方法的研究与应用

Plasmodiophora brassicae Wor. DNA Extraction Method Improvement and Application

芸薹根肿菌是专一的活体寄生菌,通过分子技术对其群体遗传结构研究的过程中,根肿菌DNA的提取是基础之一。利用硅藻土法和改良CTAB法从根肿菌孢子中直接提取芸薹根肿菌DNA,使用PCR技术对所得到的DNA用大白菜特异引物TCR01、检测根肿菌特异引物TC01进行检测,对2种根肿菌DNA的提取方法进行比较,采用EST-SSR分子标记对纯单孢菌系4号、10号生理小种及混合菌1,2,3,4,7,10,11,14,16号生理小种进行分析。结果表明:硅藻土法操作步骤少,较改良CTAB法操作简单;且硅藻土法提取获得DNA的PCR结果优于CTAB法,能够隔离寄主DNA。EST-SSR标记能对单孢菌4号、10号生理小种特异扩增,片段大小分别约为200bp,混合菌4号、10号生理小种扩增结果相符。H2的带型与H1中的1条带型一致,而H2、H7、H10、H11、H14和H16主带型基本一致,不同生理小种的根肿菌的变异性和致病性可能是相似的。

Plasmodiophora brassicae is an obligate biotroph that causes clubroot, one of the most damaging diseases of crucifers.Molecular technology is used to study their population genetic structure and DNA extraction is one of basic methods. In this research, two different DNA extraction methods, diatomaceous earth and CTAB, were used to directly extract DNA of Plasmodiophora brassicae from spore. The PCR detections were carried out by a primer TCR01 of Chinese Cabbage and primer TC01 of Plasmodiophora brassicae. Both extracted DNA methods were compared and analyzed. But in the progress of DNA extraction, operation step of diatomaceous earth method was less and simpler than CTAB. The DNA of the single spore plasmodiophora brassicae of 4 and 10 race and the field plasmodiophora brassicae of 1, 2, 3, 4, 7, 10, 11, 14, 16 race were extracted and analysed by EST-SSR molecular marker. The results showed that DNA of Plasmodiophora brassicae extracted by diatomaceous earth method could isolate host DNA, so diatomaceous earth method is the best method to extracted DNA of Plasmodiophora brassicae. Specific amplification sequence of the field plasmodiophora brassicae 4 and 10 is similar to the single spore plasmodiophora brassicae of 4 and 10 race, specific amplification sequence is 200 bp. Specific amplification sequence of the field plasmodiophora brassicae 2 is similar to the one of 1 race. The main specific amplification sequence is identical of the field plasmodiophora brassicae 2, 7, 10, 11, 14, 16 race. The variability and pathogenic bacteria may be similar of the Plasmodiophora brassicae of different race.