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

作　　者：李月 魏玮 刘文平 赵凯旋 周泽扬[1] 朱朝东[2] 罗阿蓉[2] 黄敦元 LI Yue;WEI Wei;LIU Wen-Ping;ZHAO Kai-Xuan;ZHOU Ze-Yang;ZHU Chao-Dong;LUO A-Rong;HUANG Dun-Yuan(Key Laboratory of Conservation and Utilization of Pollinator Resources in the Upper Reaches of the Yangtze River of Ministry of Agriculture and Rural Affairs,College of Life Sciences,Chongqing Normal University,Chongqing 401331,China;Institute of Zoology,Chinese Academy of Sciences,Beijing 100101,China)

机构地区：[1]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 [2]中国科学院动物研究所,北京100101

出　　处：《应用昆虫学报》2022年第6期1336-1346,共11页Chinese Journal of Applied Entomology

基　　金：国家自然科学基金(31970484);科技部科技基础资源调查专项(2018FY100405);中国科学院动物进化与系统学重点实验室开放课题(O529YX5105)。

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Amegachilestrupigera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0.02mg。【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。[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.

关 键 词：传粉昆虫 混合花粉 粉源植物 DNA试剂盒 DNA宏条形码 

分 类 号：Q968[生物学—昆虫学]