生物技术通报 ›› 2022, Vol. 38 ›› Issue (12): 252-262.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0211
出版日期:
2022-12-26
发布日期:
2022-12-29
作者简介:
刘爽,女,硕士研究生,研究方向:微生物资源开发与利用;E-mail:基金资助:
LIU Shuang(), YAO Jia-ni, SHEN Cong, DAI Jin-xia()
Published:
2022-12-26
Online:
2022-12-29
摘要:
探索荒漠固沙植物根际土壤nifH基因丰度信息和固氮菌群落结构组成和多样性,可为丰富荒漠区微生物资源库提供基础资料。通过实时荧光定量PCR和高通量测序方法,分析了宁夏荒漠区5个柠条林地根际土壤nifH基因丰度和固氮菌群落结构组成,并结合分离培养方法对柠条根际固氮菌进行筛选和多样性研究。结果表明,5个柠条群落根际土壤nifH基因的拷贝数存在较大的差异,罗山林地(LS)nifH基因拷贝数最高,沙坡头林地(SPT)最低。柠条根际土壤共检测到固氮菌7门12纲26科30属。在科属水平上,沙坡头林地固氮菌群落组成差异较大,未分类的变形杆菌和叶杆菌科分别占25.54%和22.77%,红螺菌科占17.43%,中慢生根瘤菌属是其优势属,相对丰度为22.77%;其余4个林地均以红螺菌科、斯科曼氏球菌属为优势类群,相对丰度分别为23.71%-73.45%和23.19%-71.14%,在罗山林地中占比最高。分离出的固氮菌隶属于15个属,除常见的假单胞菌属、根瘤菌属和肠杆菌属等固氮类群外,还包括勒克氏菌属、金黄杆菌属和寡养单胞菌属等,多样性十分丰富。
刘爽, 姚佳妮, 沈聪, 代金霞. 荒漠植物柠条根际土壤nifH基因荧光定量及固氮菌多样性分析[J]. 生物技术通报, 2022, 38(12): 252-262.
LIU Shuang, YAO Jia-ni, SHEN Cong, DAI Jin-xia. Fluorescent Quantitative PCR of nifH Gene and Diversity Analysis of Nitrogen-fixing Bacteria in the Rhizosphere Soil of Caragana spp. of Desert Grassland[J]. Biotechnology Bulletin, 2022, 38(12): 252-262.
样地Sampling site | Ct值Ct value | 拷贝数Number of copies/(copies·μL-1 DNA) | 称重Weight/g | 拷贝数Number of copies/(copies·g-1 ) |
---|---|---|---|---|
LS | 22.76 | 3.7298E+03 | 0.27 | 1.5922E+06a |
HSP | 25.67 | 6.3276E+02 | 0.38 | 1.7188E+05b |
SPT | 25.36 | 7.4268E+02 | 0.48 | 1.5550E+05b |
ZQ | 25.41 | 7.1586E+02 | 0.28 | 2.6036E+05b |
TSH | 24.47 | 1.4555E+03 | 0.30 | 5.2318E+05b |
表1 nifH基因Ct值和定量结果
Table 1 Ct values and quantitative results of nifH gene
样地Sampling site | Ct值Ct value | 拷贝数Number of copies/(copies·μL-1 DNA) | 称重Weight/g | 拷贝数Number of copies/(copies·g-1 ) |
---|---|---|---|---|
LS | 22.76 | 3.7298E+03 | 0.27 | 1.5922E+06a |
HSP | 25.67 | 6.3276E+02 | 0.38 | 1.7188E+05b |
SPT | 25.36 | 7.4268E+02 | 0.48 | 1.5550E+05b |
ZQ | 25.41 | 7.1586E+02 | 0.28 | 2.6036E+05b |
TSH | 24.47 | 1.4555E+03 | 0.30 | 5.2318E+05b |
样地 Sampling site | 有效序列数No. of valid sequences | 操作分类单元数目 No. of OTU | 覆盖度Coverage | 香农指数 Shannon index | 辛普森指数 Simpson index | Ace 指数 Ace index | Sobs指数 Sobs index | Chao1指数 Chao1 index |
---|---|---|---|---|---|---|---|---|
LS | 32 736 | 546 | 0.98864 | 3.2507b | 0.076655a | 546.53b | 320.00b | 462.98b |
SPT | 56 020 | 2 320 | 0.98389 | 4.9285a | 0.031739b | 1 459.70a | 1 244.70a | 1 415.70a |
ZQ | 62 271 | 2 703 | 0.98551 | 4.6876a | 0.047986ab | 1 468.50a | 1 266.70a | 1 454.50a |
TSH | 52 969 | 1 546 | 0.98491 | 4.0688ab | 0.040206b | 981.44ab | 710.67ab | 942.75ab |
HSP | 60 767 | 2 462 | 0.98696 | 4.3394a | 0.049855ab | 1 281.00a | 1 077.70a | 1 249.90a |
表2 不同样地固氮菌OTUs数目及 Alpha 多样性指数
Table 2 Number of OTUs and Alpha diversity index of nitrogen-fixing bacteria at different sample sites
样地 Sampling site | 有效序列数No. of valid sequences | 操作分类单元数目 No. of OTU | 覆盖度Coverage | 香农指数 Shannon index | 辛普森指数 Simpson index | Ace 指数 Ace index | Sobs指数 Sobs index | Chao1指数 Chao1 index |
---|---|---|---|---|---|---|---|---|
LS | 32 736 | 546 | 0.98864 | 3.2507b | 0.076655a | 546.53b | 320.00b | 462.98b |
SPT | 56 020 | 2 320 | 0.98389 | 4.9285a | 0.031739b | 1 459.70a | 1 244.70a | 1 415.70a |
ZQ | 62 271 | 2 703 | 0.98551 | 4.6876a | 0.047986ab | 1 468.50a | 1 266.70a | 1 454.50a |
TSH | 52 969 | 1 546 | 0.98491 | 4.0688ab | 0.040206b | 981.44ab | 710.67ab | 942.75ab |
HSP | 60 767 | 2 462 | 0.98696 | 4.3394a | 0.049855ab | 1 281.00a | 1 077.70a | 1 249.90a |
图2 柠条根际土壤固氮菌在科水平的Venn图(A)及群落组成柱形图(B)
Fig. 2 Venn diagram(A)and column diagram(B)of community composition of nitrogen-fixing bacteria in the rhizosphere soil of Caragana spp. at family level
图3 柠条根际土壤固氮菌在属水平的Venn图(A)及群落组成柱形图(B)
Fig. 3 Venn diagram(A)and column diagram(B)of community composition of nitrogen-fixing bacteria in rhizosphere soil of Caragana spp. at genus level
速效氮 Availliable N /(mg·kg-1) | 速效磷 Availliable P /(mg·kg-1) | 速效钾 Availliable K /(mg·kg-1) | 有机质 Soil organic Matter/(g·kg-1) | 总氮 Total N /(g·kg-1) | 总磷 Total P /(g·kg-1) | 总钾 Total K /(g·kg-1) | pH | |
---|---|---|---|---|---|---|---|---|
nifH | 0.102 | 0.942* | 0.832 | 0.965** | 0.977** | 0.225 | 0.388 | 0.455 |
表3 nifH基因拷贝数与土壤理化性质的Pearson相关分析
Table 3 Pearson correlation analysis between nifH gene copy number and soil physicochemical properties
速效氮 Availliable N /(mg·kg-1) | 速效磷 Availliable P /(mg·kg-1) | 速效钾 Availliable K /(mg·kg-1) | 有机质 Soil organic Matter/(g·kg-1) | 总氮 Total N /(g·kg-1) | 总磷 Total P /(g·kg-1) | 总钾 Total K /(g·kg-1) | pH | |
---|---|---|---|---|---|---|---|---|
nifH | 0.102 | 0.942* | 0.832 | 0.965** | 0.977** | 0.225 | 0.388 | 0.455 |
图4 固氮菌群落与环境因子相关性分析Heatmap图 AN:速效氮;AP:速效磷;AK:速效钾;SOM:有机质;TN:总氮;TP:总磷;TK:总钾
Fig. 4 Heatmap chart of correlation analysis between nitr-ogen-fixing bacteria and environmental factors AN:Availliable N;AP:availliable P;AK:availliable K;SOM:soil organic matter;TN:total N;TP:total P;TK:total K
菌株编号 Strain No. | 革兰氏染色 Gram stain | H2O2酶实验 Catalase test | 淀粉水解 Starch hydrolysis | V.P实验 V.P test | 甲基红实验 Methyl red test | 吲哚实验 Indole test | 硝酸盐还原 Nitrate reduction |
---|---|---|---|---|---|---|---|
LG-H2 | - | + | - | - | - | - | - |
LG-H3 | + | - | - | - | - | - | - |
LG-H4 | + | + | - | - | + | + | + |
LG-H5 | + | + | - | - | + | + | + |
LG-L3 | - | + | - | + | - | - | - |
LG-T2 | - | + | - | - | - | - | + |
LG-T3 | - | + | - | - | - | - | + |
LG-T6 | - | + | - | - | + | - | + |
LG-Z1 | - | + | - | - | - | - | + |
LG-Z3 | - | + | - | - | - | - | - |
LG-Z5 | - | + | - | + | - | - | + |
LG-Z7 | + | + | + | - | - | - | + |
GY-T1 | - | + | - | - | - | - | + |
GY-T4 | - | + | - | - | - | - | + |
GY-T5 | + | + | - | - | + | - | + |
GY-L1 | - | + | - | - | - | - | + |
GY-L5 | + | + | - | + | - | - | + |
GY-H3 | - | + | - | + | - | - | - |
GY-H8 | + | + | - | - | + | + | - |
GY-Z2 | - | + | - | - | - | - | + |
WD-L1 | - | - | - | - | - | - | - |
WD-L2 | - | + | - | - | - | - | - |
WD-Z1 | - | - | - | - | - | - | - |
WD-T3 | - | + | - | - | - | + | - |
WD-H1 | - | - | - | - | - | - | - |
YMA-T2 | - | + | - | - | - | - | + |
表4 菌株的生理生化特征
Table 4 Physiological and biochemical characteristics of strains
菌株编号 Strain No. | 革兰氏染色 Gram stain | H2O2酶实验 Catalase test | 淀粉水解 Starch hydrolysis | V.P实验 V.P test | 甲基红实验 Methyl red test | 吲哚实验 Indole test | 硝酸盐还原 Nitrate reduction |
---|---|---|---|---|---|---|---|
LG-H2 | - | + | - | - | - | - | - |
LG-H3 | + | - | - | - | - | - | - |
LG-H4 | + | + | - | - | + | + | + |
LG-H5 | + | + | - | - | + | + | + |
LG-L3 | - | + | - | + | - | - | - |
LG-T2 | - | + | - | - | - | - | + |
LG-T3 | - | + | - | - | - | - | + |
LG-T6 | - | + | - | - | + | - | + |
LG-Z1 | - | + | - | - | - | - | + |
LG-Z3 | - | + | - | - | - | - | - |
LG-Z5 | - | + | - | + | - | - | + |
LG-Z7 | + | + | + | - | - | - | + |
GY-T1 | - | + | - | - | - | - | + |
GY-T4 | - | + | - | - | - | - | + |
GY-T5 | + | + | - | - | + | - | + |
GY-L1 | - | + | - | - | - | - | + |
GY-L5 | + | + | - | + | - | - | + |
GY-H3 | - | + | - | + | - | - | - |
GY-H8 | + | + | - | - | + | + | - |
GY-Z2 | - | + | - | - | - | - | + |
WD-L1 | - | - | - | - | - | - | - |
WD-L2 | - | + | - | - | - | - | - |
WD-Z1 | - | - | - | - | - | - | - |
WD-T3 | - | + | - | - | - | + | - |
WD-H1 | - | - | - | - | - | - | - |
YMA-T2 | - | + | - | - | - | - | + |
图5 基于 16S rRNA 基因序列构建的柠条根际固氮菌的系统发育树
Fig. 5 Phylogenetic tree of nitrogen-fixing bacteria in the rhizosphere soil of Caragana spp. based on the 16S rRNA gene sequences
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