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

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

Abstract

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.

References

[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.