生物技术通报 ›› 2022, Vol. 38 ›› Issue (11): 202-209.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1604
武林辉(), 耿必苗, 王艳杰, 周国伟, 孙庆业, 赵琼()
收稿日期:
2021-12-28
出版日期:
2022-11-26
发布日期:
2022-12-01
作者简介:
武林辉,男,硕士研究生,研究方向:森林土壤磷循环;E-mail:基金资助:
WU Lin-hui(), GENG Bi-miao, WANG Yan-jie, ZHOU Guo-wei, SUN Qing-ye, ZHAO Qiong()
Received:
2021-12-28
Published:
2022-11-26
Online:
2022-12-01
摘要:
为研究氮添加影响森林土壤有机磷矿化的微生物调控机制,分析了10年的野外氮添加(100 kg N ha-2year-1)对沙地樟子松人工林土壤微生物中编码酸性磷酸单酯酶、碱性磷酸单酯酶和植酸酶的功能基因(phoC、phoD和appA)丰度及相关酶活性和土壤理化性质的影响。结果表明,氮添加使樟子松人工林土壤中酸性和碱性磷酸单酯酶活性分别下降了18.09%和55.29%,植酸酶活性下降了41.88%。氮添加使土壤微生物中各基因拷贝数分别下降40.97%(16S-rRNA)、78.38%(phoD)、67.92%(phoC)、74.37%(appA)。各基因拷贝数占总细菌基因拷贝数的比例显著下降了61%(phoD)、44%(phoC)、55%(appA)。土壤微生物量碳、微生物量磷含量与酸性磷酸单脂酶、碱性磷酸单脂酶、植酸酶活性及16S rRNA、phoD、phoC、appA基因丰度显著正相关。土壤铵态氮含量与酸性磷酸单脂酶、碱性磷酸单脂酶活性及16S rRNA、phoC、appA基因丰度显著负相关。酸性磷酸单酯酶活性与其基因丰度显著正相关,其他两种酶活性与其基因丰度无显著相关性。以上结果表明,氮添加不仅降低了土壤解磷微生物丰度,还降低了解磷细菌在总细菌中的比例,从而大大降低了磷酸酶活性,抑制了有机磷的矿化。
武林辉, 耿必苗, 王艳杰, 周国伟, 孙庆业, 赵琼. 氮添加对樟子松人工林土壤细菌磷酸酶编码基因丰度的影响[J]. 生物技术通报, 2022, 38(11): 202-209.
WU Lin-hui, GENG Bi-miao, WANG Yan-jie, ZHOU Guo-wei, SUN Qing-ye, ZHAO Qiong. Effects of Nitrogen Addition on the Abundance of Bacterial Phosphatase Encoding Genes in the Soil of Pinus sylvestris var. mongolica Plantation[J]. Biotechnology Bulletin, 2022, 38(11): 202-209.
引物Primer | 引物名称Primer name | 引物长度Primer length/bp | 引物序列Primer sequence(5'-3') | 参考文献Reference |
---|---|---|---|---|
phoD | ALPS-F730 | 370 | CAGTGGGACGA CCACGAGGT | [ |
ALPS-1101 | GAGGCCGATCG GCATGTCG | |||
phoC | phoC-A-F1 | 155 | CGGCTCCTATCCGTCCGG | [ |
phoC-A-R1 | CAACATCGCTTTGCCAGTG | |||
appA | appA-F | 247 | CAGATACGTCCAGTCCCGAT | |
appA-R | AGTTCCGATGGTAATGCCTG | |||
16S rRNA | 338F | 468 | ACTCCTACGGGAGGCAGCAG | [ |
806R | GGACTACHVGGGTWTCTAAT |
表1 功能基因扩增引物
Table 1 Amplification primers for functional gene
引物Primer | 引物名称Primer name | 引物长度Primer length/bp | 引物序列Primer sequence(5'-3') | 参考文献Reference |
---|---|---|---|---|
phoD | ALPS-F730 | 370 | CAGTGGGACGA CCACGAGGT | [ |
ALPS-1101 | GAGGCCGATCG GCATGTCG | |||
phoC | phoC-A-F1 | 155 | CGGCTCCTATCCGTCCGG | [ |
phoC-A-R1 | CAACATCGCTTTGCCAGTG | |||
appA | appA-F | 247 | CAGATACGTCCAGTCCCGAT | |
appA-R | AGTTCCGATGGTAATGCCTG | |||
16S rRNA | 338F | 468 | ACTCCTACGGGAGGCAGCAG | [ |
806R | GGACTACHVGGGTWTCTAAT |
引物Primer | 扩增体系Amplification system | 扩增程序Amplification procedure |
---|---|---|
phoD | 10 μL体系:Bestar Sybr Green qPCR Master Mix 5 μL,引物0.4 μL,DNA样品2 μL(10 ng·μL-1),灭菌水2.6 μL | 预变性95℃ 300 s,变性 95℃ 15 s,退火58℃ 30 s,延伸 72℃ 20 s,40 个循环 |
phoC | 预变性 95℃ 300 s,变性 95℃ 15 s,退火57℃ 30 s,延伸 72℃ 20 s,40 个循环 | |
appA | 预变性 95℃ 300 s,变性 95℃ 15 s,退火60℃ 30 s,延伸 72℃ 20 s,40 个循环 | |
16S rRNA | 预变性 95℃ 300 s,变性 95℃ 15 s,退火55℃ 30 s,延伸 72℃ 20 s,40 个循环 |
表2 荧光定量PCR反应条件
Table 2 Fluorescence quantitative PCR reaction conditions
引物Primer | 扩增体系Amplification system | 扩增程序Amplification procedure |
---|---|---|
phoD | 10 μL体系:Bestar Sybr Green qPCR Master Mix 5 μL,引物0.4 μL,DNA样品2 μL(10 ng·μL-1),灭菌水2.6 μL | 预变性95℃ 300 s,变性 95℃ 15 s,退火58℃ 30 s,延伸 72℃ 20 s,40 个循环 |
phoC | 预变性 95℃ 300 s,变性 95℃ 15 s,退火57℃ 30 s,延伸 72℃ 20 s,40 个循环 | |
appA | 预变性 95℃ 300 s,变性 95℃ 15 s,退火60℃ 30 s,延伸 72℃ 20 s,40 个循环 | |
16S rRNA | 预变性 95℃ 300 s,变性 95℃ 15 s,退火55℃ 30 s,延伸 72℃ 20 s,40 个循环 |
处理Treatment | 测定指标Measurement indicators | ||||||
---|---|---|---|---|---|---|---|
pH | 铵态氮 NH4+-N/(mg·kg-1) | 硝态氮 NO3--N/(mg·kg-1) | 有效磷 AP/(mg·kg-1) | 总磷 TP/(g·kg-1) | 总氮 TN/(g·kg-1) | 有机质 SOM/(g·kg-1) | |
ZCK | 6.58±0.14 | 2.62±0.18 | 0.09±0.01 | 2.06±0.36 | 0.16±0.02 | 0.38±0.04 | 10.66±0.72 |
ZN | 5.78±0.05** | 17.89±1.09** | 0.60±0.12* | 1.12±0.05* | 0.10±0.02* | 0.45±0.04 | 12.18±0.55 |
表3 氮添加对土壤基本理化性质的影响
Table 3 Effects of nitrogen addition on the basic physical and chemical properties of soil
处理Treatment | 测定指标Measurement indicators | ||||||
---|---|---|---|---|---|---|---|
pH | 铵态氮 NH4+-N/(mg·kg-1) | 硝态氮 NO3--N/(mg·kg-1) | 有效磷 AP/(mg·kg-1) | 总磷 TP/(g·kg-1) | 总氮 TN/(g·kg-1) | 有机质 SOM/(g·kg-1) | |
ZCK | 6.58±0.14 | 2.62±0.18 | 0.09±0.01 | 2.06±0.36 | 0.16±0.02 | 0.38±0.04 | 10.66±0.72 |
ZN | 5.78±0.05** | 17.89±1.09** | 0.60±0.12* | 1.12±0.05* | 0.10±0.02* | 0.45±0.04 | 12.18±0.55 |
图1 对照(ZCK)和氮添加(ZN)处理下土壤微生物量磷(MBP)和微生物量碳(MBC)的变化
Fig.1 Changes in soil microbial biomass phosphorus(MBP)and microbial biomass carbon(MBC)under control(ZCK)and nitrogen addition(ZN)treatments
图2 对照(ZCK)和氮添加(ZN)处理下土壤有机磷转化的3种酶活性的变化
Fig. 2 Changes in activities of three enzymes involved in soil organic phosphorus conversion under control(ZCK)and nitrogen addition(ZN)treatments
图3 对照(ZCK)和氮添加(ZN)处理下土壤16S rRNA、phoD、phoC、appA的基因拷贝数
Fig. 3 Gene copy numbers of soil 16S rRNA,phoD,phoC,and appA under control(ZCK)and nitrogen addition(ZN)treatments
pH | MBP | MBC | NH4+-N | NO3--N | AP | TP | TN | SOM | 16S rRNA | phoD | phoC | appA | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ACP | 0.890** | 0.864** | 0.877** | -0.995** | -0.918** | 0.692 | 0.582 | -0.465 | -0.528 | 0.883** | 0.595 | 0.764* | 0.735* |
ALP | 0.883** | 0.725* | 0.764* | -0.860** | -0.739* | 0.438 | 0.346 | -0.382 | -0.220 | 0.799* | 0.267 | 0.597 | 0.646 |
PHY | 0.692 | 0.811* | 0.757* | -0.642 | -0.698 | 0.361 | 0.484 | -0.095 | -0.589 | 0.383 | 0.567 | 0.344 | 0.396 |
16S rRNA | 0.762* | 0.755* | 0.796* | -0.910** | -0.791* | 0.709* | 0.550 | -0.430 | -0.524 | 1 | 0.671 | 0.880** | 0.923** |
phoD | 0.461 | 0.731* | 0.727* | -0.631 | -0.582 | 0.856** | 0.857 | -0.500 | -0.884** | 0.671 | 1 | 0.865** | 0.796* |
phoC | 0.596 | 0.713* | 0.781* | -0.782* | -0.674 | 0.935** | 0.770 | -0.653 | -0.775* | 0.880** | 0.865** | 1 | 0.902** |
appA | 0.676 | 0.772* | 0.821* | -0.781* | -0.698 | 0.702 | 0.608 | -0.464 | -0.677 | 0.923** | 0.796* | 0.902** | 1 |
表4 土壤酶活性、基因拷贝数、土壤理化性质间的相关性
Table 4 Correlation among soil enzyme activities,gene copy numbers and soil physicochemical properties
pH | MBP | MBC | NH4+-N | NO3--N | AP | TP | TN | SOM | 16S rRNA | phoD | phoC | appA | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ACP | 0.890** | 0.864** | 0.877** | -0.995** | -0.918** | 0.692 | 0.582 | -0.465 | -0.528 | 0.883** | 0.595 | 0.764* | 0.735* |
ALP | 0.883** | 0.725* | 0.764* | -0.860** | -0.739* | 0.438 | 0.346 | -0.382 | -0.220 | 0.799* | 0.267 | 0.597 | 0.646 |
PHY | 0.692 | 0.811* | 0.757* | -0.642 | -0.698 | 0.361 | 0.484 | -0.095 | -0.589 | 0.383 | 0.567 | 0.344 | 0.396 |
16S rRNA | 0.762* | 0.755* | 0.796* | -0.910** | -0.791* | 0.709* | 0.550 | -0.430 | -0.524 | 1 | 0.671 | 0.880** | 0.923** |
phoD | 0.461 | 0.731* | 0.727* | -0.631 | -0.582 | 0.856** | 0.857 | -0.500 | -0.884** | 0.671 | 1 | 0.865** | 0.796* |
phoC | 0.596 | 0.713* | 0.781* | -0.782* | -0.674 | 0.935** | 0.770 | -0.653 | -0.775* | 0.880** | 0.865** | 1 | 0.902** |
appA | 0.676 | 0.772* | 0.821* | -0.781* | -0.698 | 0.702 | 0.608 | -0.464 | -0.677 | 0.923** | 0.796* | 0.902** | 1 |
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