不同试剂盒对烟丝DNA提取及其微生物群落多样性分析效果的比较

doi:10.11924/j.issn.1000-6850.casb19010133

中国农学通报 ›› 2019, Vol. 35 ›› Issue (21): 110-117.doi: 10.11924/j.issn.1000-6850.casb19010133

所属专题：生物技术；烟草种植与生产

不同试剂盒对烟丝DNA提取及其微生物群落多样性分析效果的比较

何沛¹, 杨俊誉², 苏代发², 肖炜², 刘志华¹, 崔晓龙²

1.云南中烟工业有限责任公司技术中心;2.云南大学生命科学学院

收稿日期:2019-01-27 修回日期:2019-07-06 接受日期:2019-05-20 出版日期:2019-08-13 发布日期:2019-08-13
通讯作者: 崔晓龙
基金资助:
云南省科技厅项目“烟草本香成分强化云产卷烟风格关键技术研究”(2017ZE027)；云南中烟工业有限责任公司重大专项“基于微生物技术的卷烟品质提升关键技术研究”(2017CP01)。

Three Kits on Extracting Cut Tobacco DNA and Their Analysis Effect on Microbial Community Diversity

Received:2019-01-27 Revised:2019-07-06 Accepted:2019-05-20 Online:2019-08-13 Published:2019-08-13

摘要/Abstract

摘要： 筛选能高效提取烟丝DNA，并能获取其微生物群落多样性较高的试剂盒。选择3种不同的试剂盒提取烟丝DNA，比较DNA的纯度和浓度，并将提取的DNA进行原核生物16S rRNA基因V3~V4区和真菌ITS2区的PCR扩增及扩增子高通量测序，进而比较烟丝样品微生物群落多样性和结构差异。结果表明：（1）DNeasy? PowerPlant? Pro Kit植物基因组提取试剂盒(P)提取的烟丝DNA浓度为(103.67±1.7) ng/μL，显著高于其他试剂盒(P<0.01)，且3种试剂盒提取的DNA纯度均达到合格水平。（2）3种试剂盒提取的DNA样本均能反映样本中原核生物群落多样性与结构，从门分类水平看，试剂盒P检测到11个门类，E.Z.N.A.TM HP Plant DNA Kit植物基因组提取试剂盒(M)检测到8个门类，DNeasy? PowerSoil? Kit土壤基因组提取试剂盒(S)检测到5个门类；从属分类水平看，试剂盒P检测到83个属，试剂盒S检测到71个属，试剂盒M检测到69个属。（3）3种试剂盒均能检测到Unclassified类群、子囊菌门Ascomycota、接合菌门Zygomycota和Others等4个真菌门类，但试剂盒P检测到的类群比例最高；在属分类水平上，试剂盒P检测出30个属，试剂盒S检测出20个属，试剂盒M检测出11个属。DNeasy? PowerPlant? Pro Kit植物基因组提取试剂盒(P)能有效提取烟丝及其所含微生物的总DNA，并能检测出更高的烟丝微生物群落多样性，推荐该试剂盒用于烟丝样品中微生物群落多样性与结构的分析。

关键词: 连平县, 连平县, 生态农业, 自然资源, 可持续发展

Abstract: Abstract: The paper aims to select a kit that efficiently extracted cut tobacco DNA and obtained a higher diversity of the microbial community. The cut tobacco DNAs were extracted by using three different kits, and these extracted DNAs were used for amplicon high-throughput sequencing of the prokaryotic 16S rRNA gene V3-V4 region and the fungal ITS2 region. We compared their purities and concentrations and their microbial community diversity and structures. The results showed that: (1) the concentration of cut tobacco DNA from DNeasy? PowerPlant? Pro Kit (P) was (103.67±1.7) ng/μL significantly higher than those of other kits (P<0.01), and these DNA purities from three different kits reached the qualified level. (2) All DNA samples extracted by using the three different kits could reflect the prokaryotic community diversity and structures. At the phylum level, the kit P detected 11 phyla, E.Z.N.A.TM HP Plant Kit (M) detected 8 phyla, and DNeasy? PowerSoil? Kit (S) detected 5 phyla. And at the genus level, 83 genera were detected by using the kit P, 71 genera were detected by using the kit S, and 69 genera were detected by using the kit M. (3) These three kits could detect four phyla of fungi such as Unclassified group, Ascomycota, Zygomycota and Others, but the kit P detected the highest diversity of microbial community at the phylum level. And at the genus level of fungi, 30 genera were detected by using the kit P, 20 genera were detected by using the kit S, and 11 genera detected by using the kit M. DNeasy? PowerPlant? Pro Kit (P) can effectively extract the total DNAs of microbes in cut tobacco and cut tobacco, and it can detect more microbial community diversity. So the kit P is recommended for the analysis of tobacco microbial community diversity and structure.

何沛,杨俊誉,苏代发,肖炜,刘志华,崔晓龙. 不同试剂盒对烟丝DNA提取及其微生物群落多样性分析效果的比较[J]. 中国农学通报, 2019, 35(21): 110-117.

参考文献

[1] 李珊辉. 卷烟加工及储存过程中微生物群落结构与其品质关系的研究 [D]; 云南大学, 2015.
[2] 祝明亮, 雷丽萍. 烟草微生物学 [M]. 北京: 科学出版社. 2008: 1-15.ZHU Mingliang, LEI Liping. Tobacco microbiology [M]. Beijing: Science Press. 2008: 1-15.
[3] 邓国宾, 李雪梅, 李成斌, 等. 降果胶菌改善烟叶品质研究[J]. 烟草科技, 2003(11):17-18.
[4] 贺兆伟, 奚家勤, 邓宾玲, 等. 烟叶中高产果胶酶菌株的筛选及酶学性质[J]. 烟草科技, 2013(5):000072-76.
[5] 李梅云. 烟碱的微生物降解研究进展[J]. 微生物学杂志, 2006, 26(3):94-97.LI Meiyun. Advancement of nicotine degrading microorganisms for flue-cured tobacco [J]. Journal of microbiology, 2006, 26(3):94-97.
[6] 张彦东, 罗昌荣, 王辉龙, 等. 微生物降解烟碱研究应用进展[J]. 烟草科技, 2003(12):3-7.
[7] 夏炳乐, 颜春雷. 生物酶制剂提高烟叶醇化质量 [J]. 烟草科技, 2007, 49(11): 7-13.
[8] CHOPYK J, CHATTOPADHYAY S, KULKARNI P, et al. Temporal variations in cigarette tobacco bacterial community composition and tobacco-specific nitrosamine content are influenced by brand and storage conditions [J]. Frontiers in microbiology, 2017, 8: 358.
[9] 刘春奎, 蔡佳, 杨靖, 等. 烤烟细胞壁物质与烟叶质量的关系研究 [J]. 广东农业科学, 2012, 39(12): 33-35.
[10] 宋鹏, 陈五岭. 烟草发酵中施加微生物及制剂研究进展 [J]. 安徽农业科学, 2008, 36(2): 588-589.
[11] WANG F, MEN X, ZHANG G, et al. Assessment of 16S rRNA gene primers for studying bacterial community structure and function of aging flue-cured tobaccos [J]. AMB Express, 2018, 8(1): 182.
[12] WANG F, ZHAO H, XIANG H, et al. Species Diversity and Functional Prediction of Surface Bacterial Communities on Aging Flue-Cured Tobaccos [J]. Current microbiology, 2018, 1-10.
[13] HEALEY A, FURTADO A, COOPER T, et al. Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species [J]. Plant methods, 2014, 10(1): 21.
[14] 陆铮铮, 李芳. 3种试剂盒提取烤后烟叶DNA的效果比较 [J]. 云南农业大学学报, 2018, 33(1): 172-175.
[15] MACHUCA L L, JEFFREY R, MELCHERS R E. Microorganisms associated with corrosion of structural steel in diverse atmospheres [J]. International Biodeterioration Biodegradation, 2016, 114:234-243.
[16] TOJU H, TANABE A S, YAMAMOTO S, et al. High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples [J]. PloS one, 2012, 7(7): e40863.
[17] TEDERSOO L, ANSLAN S, BAHRAM M, et al. Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi [J]. MycoKeys, 2015(10): 1-43.
[18] 陈兴, 杨莹, 李珊辉, 等. 卷烟加工及储存过程中细菌群落组成及变化研究 [J]. 安徽农业科学, 2018, 46(08): 15-18.
[19] 施河丽,向必坤,谭军, 等. 烟草青枯病发病烟株根际土壤细菌群落分析 [J]. 中国烟草学报, 2018, 24(05): 57-65.
[20]常安然, 李佳, 张耸, 等. 基于宏基因组学 16S rDNA 测序对烟草根际土壤细菌群落组成分析 [J]. 中国农业科技导报, 2017, 19(2): 43-50.
[21]李小龙, 李红丽, 曾强, 等. 钙镁磷肥对青枯病发病烟株根际土壤微生物区系的影响 [J]. 中国烟草学报, 2016, 22(1): 71-75.

不同试剂盒对烟丝DNA提取及其微生物群落多样性分析效果的比较

Three Kits on Extracting Cut Tobacco DNA and Their Analysis Effect on Microbial Community Diversity

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