生物技术通报 ›› 2022, Vol. 38 ›› Issue (3): 256-263.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0550
张雨函1(), 范熠1, 李婷婷1, 庞爽1, 刘为1, 白可喻2, 张西美1()
收稿日期:
2021-04-25
出版日期:
2022-03-26
发布日期:
2022-04-06
作者简介:
张雨函,女,硕士研究生,研究方向:农业水土环境;E-mail: 基金资助:
ZHANG Yu-han1(), FAN Yi1, LI Ting-ting1, PANG Shuang1, LIU Wei1, BAI Ke-yu2, ZHANG Xi-mei1()
Received:
2021-04-25
Published:
2022-03-26
Online:
2022-04-06
摘要:
获得高质量的微生物基因组DNA是进行复杂微生物群落宏基因组学研究的基础和难点。植物叶表是一个微生物多样性丰富的复杂生态系统,这些微生物群落可以调节叶片功能性状,影响植物的适应性。深入了解叶表微生物群落的基本结构和功能原理,有助于在促进植物生长和植物保护方面发挥重要的应用价值。由于叶表严苛的环境,导致富集叶表微生物难度较大,严重限制了高质量叶表微生物基因组DNA的提取。基于现有DNA提取方法,加入表面活性剂Silwet L-77进行前处理,同时循环利用洗脱液,加强叶表微生物的富集,以提高叶表微生物的获取量。结合商业试剂盒方法进行提取得到高纯度、高浓度的基因组DNA。经过质量控制和建库测序验证,DNA质量达到宏基组建库的要求。通过此方法可以提高叶表微生物分离和收集效率的方法,提高叶表微生物DNA提取成功率,为应用高通量测序技术研究叶表微生物组成和其他植物分子生物学研究提供参考。
张雨函, 范熠, 李婷婷, 庞爽, 刘为, 白可喻, 张西美. 基于宏基因组测序的植物叶表微生物富集及DNA提取方法[J]. 生物技术通报, 2022, 38(3): 256-263.
ZHANG Yu-han, FAN Yi, LI Ting-ting, PANG Shuang, LIU Wei, BAI Ke-yu, ZHANG Xi-mei. Microbial Enrichment on Leaf Surface and DNA Extraction Method Based on the Metagenomics Sequencing[J]. Biotechnology Bulletin, 2022, 38(3): 256-263.
样品名称 Sample name | 无菌水Sterile water | PBS+Silwet L-77 | ||||||
---|---|---|---|---|---|---|---|---|
DNA浓度DNA concentration/(ng·µL-1) | A260/A280 | 质量 Mass/µg | DNA浓度DNA concentration/(ng·µL-1) | A260/A280 | 质量 Mass/ µg | |||
LC | 14.43±2.79 | 1.86 | 2.94 | 40.08±10.29 | 1.82 | 19.30 | ||
PD | 13.61±7.52 | 1.91 | 1.44 | 74.25±5.77 | 1.96 | 19.85 | ||
AF | 11.51±2.33 | 1.84 | 1.15 | 49.55±0.21 | 1.93 | 7.44 | ||
IL | 19.51±8.13 | 1.89 | 1.95 | 64.42±13.03 | 1.93 | 34.97 | ||
TL | 18.75±3.61 | 1.99 | 1.78 | 35.45±7.42 | 1.81 | 22.28 | ||
AR | 18.14±3.87 | 1.97 | 1.81 | 44.93±31.67 | 1.96 | 30.31 | ||
All | 15.99±4.95 | 1.93 | 1.63 | 51.44±18.10 | 1.86 | 22.36 |
表1 无菌水和PBS缓冲液+Silwet L-77表面活性剂对叶表微生物基因组DNA的提取效果
Table 1 Extraction effect of sterile water and PBS buffer solution +Silwet L-77 surfactant on microbial genomic DNA from leaf surface
样品名称 Sample name | 无菌水Sterile water | PBS+Silwet L-77 | ||||||
---|---|---|---|---|---|---|---|---|
DNA浓度DNA concentration/(ng·µL-1) | A260/A280 | 质量 Mass/µg | DNA浓度DNA concentration/(ng·µL-1) | A260/A280 | 质量 Mass/ µg | |||
LC | 14.43±2.79 | 1.86 | 2.94 | 40.08±10.29 | 1.82 | 19.30 | ||
PD | 13.61±7.52 | 1.91 | 1.44 | 74.25±5.77 | 1.96 | 19.85 | ||
AF | 11.51±2.33 | 1.84 | 1.15 | 49.55±0.21 | 1.93 | 7.44 | ||
IL | 19.51±8.13 | 1.89 | 1.95 | 64.42±13.03 | 1.93 | 34.97 | ||
TL | 18.75±3.61 | 1.99 | 1.78 | 35.45±7.42 | 1.81 | 22.28 | ||
AR | 18.14±3.87 | 1.97 | 1.81 | 44.93±31.67 | 1.96 | 30.31 | ||
All | 15.99±4.95 | 1.93 | 1.63 | 51.44±18.10 | 1.86 | 22.36 |
Df | F value | Pr(>F) | |
---|---|---|---|
富集方法Enrichment method | 1 | 59.11 | P<0.05 |
植物种类Plant species | 5 | 1.71 | 0.21 |
富集方法:植物种类 Enrichment method:Plant species | 5 | 1.96 | 0.16 |
残差Residual | 12 | NA | NA |
表2 植物种类与不同富集方法对DNA浓度的影响
Table 2 Effects of plant species and different enrichment methods on DNA concentration
Df | F value | Pr(>F) | |
---|---|---|---|
富集方法Enrichment method | 1 | 59.11 | P<0.05 |
植物种类Plant species | 5 | 1.71 | 0.21 |
富集方法:植物种类 Enrichment method:Plant species | 5 | 1.96 | 0.16 |
残差Residual | 12 | NA | NA |
图2 六种植物叶表微生物DNA电泳图 1-6和7-12分别是羊草、叉分蓼、冷蒿、马蔺、披针叶黄华和野韭,1-6以PBS+ Silwet L-77为洗脱液,7-12以无菌水为洗脱液
Fig. 2 Microbial DNA electrophoresis patterns on the leaf surfaces of 6 plants 1-6 and 7-12 were L. chinensis(Trin.)Tzvel.,P. divaricatum L.,A. frigida Willd.,I. lactea Pall. var. chinensis(Fisch.)Koidz.,T. lanceolata R. Br. and A.ramosum L.. PBS+ Silwet L-77 as eluent for 1-6,and sterile water as eluent for 7-12,respectively
样品名称Sample name | Clean reads | Clean base/bp | Percent in raw reads/% | Percent in raw bases/% |
---|---|---|---|---|
LC | 84554592 | 12743415315 | 99.08±0.12 | 98.89±0.26 |
PD | 76636682 | 11558890595 | 98.97±0.04 | 98.86±0.09 |
AF | 70985020 | 10682348648 | 98.70±0.10 | 98.36±0.14 |
IL | 77764022 | 11727532109 | 99.11±0.08 | 98.97±0.11 |
TL | 87048063 | 13081919500 | 98.93±0.08 | 98.47±0.24 |
AR | 80930005 | 12202864242 | 98.82±0.69 | 98.67±0.74 |
表3 宏基因组测序统计
Table 3 Metagenomics sequencing statistics
样品名称Sample name | Clean reads | Clean base/bp | Percent in raw reads/% | Percent in raw bases/% |
---|---|---|---|---|
LC | 84554592 | 12743415315 | 99.08±0.12 | 98.89±0.26 |
PD | 76636682 | 11558890595 | 98.97±0.04 | 98.86±0.09 |
AF | 70985020 | 10682348648 | 98.70±0.10 | 98.36±0.14 |
IL | 77764022 | 11727532109 | 99.11±0.08 | 98.97±0.11 |
TL | 87048063 | 13081919500 | 98.93±0.08 | 98.47±0.24 |
AR | 80930005 | 12202864242 | 98.82±0.69 | 98.67±0.74 |
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