不同试剂盒对传粉昆虫携带混合花粉DNA提取效果比较
Comparison of different kits for the extraction of mixed pollen DNA carried by pollinators
李月，魏玮，刘文平，赵凯旋，周泽扬，朱朝东，罗阿蓉，黄敦元
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DOI：10.7679/j.issn.2095-1353.2022.135
作者单位：农业农村部长江上游传粉昆虫资源保护与利用重点实验室（部省共建），重庆师范大学生命科学学院，重庆 401331
中文关键词：传粉昆虫；混合花粉；粉源植物；DNA试剂盒；DNA宏条形码
英文关键词：insect pollinator; mixed pollen; pollen source plants; DNA kits; DNA metabarcoding
中文摘要：【目的】 传粉昆虫所携带花粉的准确鉴定是构建传粉网络的重要环节。本研究通过比较不同植物试剂盒在微量混合花粉DNA提取中的差异，以阐明不同试剂盒的提取效果，并明确满足DNA提取的混合花粉最低需求量。【方法】 将不同地区中华蜜蜂Apis cerana cerana、凹唇壁蜂Osmia excavata和白斑切叶蜂Amegachile strupigera体表携带的花粉进行混合。利用上海生工磁珠法植物基因组DNA抽提试剂盒（CZ）、北京BioTeke（百泰克）离心柱型微量样品基因组DNA提取试剂盒（BT）、美国Omega E.Z.N.A.®MicroElute基因组DNA提取试剂盒（EZ）、北京天根微量样品基因组DNA提取试剂盒（TG）及江苏吉锐Genloci®TNA抽提试剂盒（GL）提取混合花粉DNA。基于ITS2宏条形码技术获取测序数据，通过方差、线性判别和层次聚类分析，比较5种试剂盒提取效果。最后，调整试剂盒BT的提取方法，降低混合花粉的需求量。【结果】 试剂盒TG与GL提取混合花粉DNA浓度较高，试剂盒CZ、BT和EZ提取混合花粉DNA浓度较低，前者浓度约为后者的5-15倍。试剂盒EZ和TG提取混合花粉样品的DNA纯度与其它试剂盒存在显著差异。5种试剂盒检测到植物种类分别为（143 ± 12）（CZ）、（167 ± 8）（BT）、（160 ± 10）（EZ）、（160 ± 6）（TG）和（161 ± 16）种（GL），且不同试剂盒在目、科和属水平上具有显著提取优势的植物，部分植物只能由特定试剂盒提取到DNA模板。此外，试剂盒BT通过花粉所鉴定的植物种类最多，且能提取到合格DNA模板的最低混合花粉质量为0.02 mg。【结论】 不同试剂盒提取微量混合花粉效果不同且各有优势，混合花粉质量最低可以降至0.02 mg。本研究为选择提取微量混合花粉DNA试剂盒继而用于传粉网络研究提供参考。
英文摘要：[Objectives] To compare the performance of different kits
in extracting DNA from micro mixed pollen and determine which kits meet the
minimum demands of DNA extraction from mixed pollen samples. [Methods] Pollen carried on the body surface of Apis
cerana cerana, Osmia excavata and Amegachile strupigera from
different regions was mixed. Genomic DNA was extracted using the Shanghai
BioTeke Magnetic Bead Extraction Kit (CZ), the Beijing BioTeke Centrifugal
Column Genomic DNA Extraction Kit (BT), the Omega E.Z.N.A.® MicroElute Genomic
DNA Extraction Kit (EZ), the Beijing Tiangen Genomic DNA Extraction Kit (TG)
and the Jiangsu Jirui Genloci® TNA Extraction Kit (GL). Sequencing data were
obtained based on ITS macro-barcoding and the results obtained from the
five kits were compared in terms of variance and analyzed using linear
discrimination and hierarchical clustering analysis. The extraction method of
the BT kit was adjusted to reduce the amount of mixed pollen required. [Results] The concentration of DNA obtained using the
TG and GL kits was 5-15 times higher than that obtained using the CZ, BT and EZ
kits. The purity of the DNA obtained using the EZ and TG kits was significantly
different from that obtained using the other kits. The number of plant species
detected by each kit was as follows: (167 ± 8) (BT), (161 ± 16) (GL), (160 ±
10) (EZ), (160 ± 6) (TG), (143 ± 12) (CZ). Some kits were significantly better
at extracting DNA from specific plant orders, families and genera. Indeed, some
plant DNA could only be extracted to templates by specific kits. The smallest quantity of mixed pollen that
could be extracted to a qualified DNA template was 0.02 mg. [Conclusion] Different kits varied in their ability to
extract the DNA of different plant orders, families and genera. DNA could be
extracted from mixed pollen samples as low as 0.02 mg. These findings provide
information to guide the selection of kits for extracting DNA from mixed pollen
samples.