短花针茅荒漠草原单条土壤线虫分子鉴定方法研究

Molecular Identification of Soil Nematodes in Stipa breviflora Desert Steppe

土壤线虫在土壤中具有重要的生态作用,目前大多通过形态学鉴定方法研究土壤线虫,但该方法有很多弊端。为了更好地在分子水平上研究土壤线虫群落及其多样性,对内蒙古短花针茅荒漠草原土壤线虫进行了单条线虫的分子鉴定,通过比较4种不同的DNA提取方法(试剂盒法、冻融法、切割法、切割冻融法),确定了土壤线虫最佳的DNA提取方法为切割冻融法。同时,选出10对土壤线虫的通用引物经过重复试验,从10对引物中筛选出3对引物(分别为线虫rDNA的18S区引物G18SU/R18Tyl1、28S区引物D2A/D3B、ITS区引物TW81/AB28)能达到较高的成功率,其中适用于内蒙古短花针茅荒漠草原土壤线虫的最佳通用引物是:D2A/D3B(5‘-ACAAGTACCGTGAGGGAAAGTTG-3’;5‘-TGCGAAGGAACCAGCTACTA-3’)。本研究通过明确短花针茅荒漠草原土壤线虫最佳DNA提取方法和最佳通用引物,为进一步通过高通量测序技术在分子水平上进行土壤线虫研究奠定基础。

Soil nematodes play an important ecological role in soil.At present,the study of soil nematodes mostly uses morphological methods,but this method has many disadvantages.In order to better study the community characteristics and diversity of soil nematodes at the molecular level,the researchers carried out molecular identification of single nematodes on soil nematodes in the desert steppe of Inner Mongolia.We compared a total of four different DNA extraction methods:kit,freeze-thaw,cutting and cutting freezethaw,and determined that the best DNA extraction method for soil nematodes is the cutting freeze-thaw method.At the same time,the researchers selected a total of 10 pairs of universal PCR primers for soil nematodes.After repeated verification experiments,3 pairs of optimal primers were selected from the 10 pairs of primers(18S region primers G18SU/R18Tyl1 and 28S region primer D2A of the rDNA of the nematode,and D3B,ITS region primer TW81/AB28 respectively).These 3 pairs of PCR primers can achieve a high success rate of amplification,and the best universal primers for soil nematodes in the Stipa breviflora desert steppe of Inner Mongolia are:D2A/D3B(5'-ACAAGTACCGTGAGGGAAAGTTG-3';5'-TGCGAAGGAACCAGCTACTA-3').In this study,we identified the best DNA extraction method and the best universal primer for soil nematodes in the Stipa breviflora desert steppe,laying a foundation for the further study of soil nematodes at the molecular level by using high-throughput sequencing technology.