生物技术通报 ›› 2021, Vol. 37 ›› Issue (8): 162-175.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0273
刘传和1(), 贺涵1, 何秀古2, 刘开1, 邵雪花1, 赖多1, 匡石滋1, 肖维强1
收稿日期:
2021-03-10
出版日期:
2021-08-26
发布日期:
2021-09-10
作者简介:
刘传和,男,博士,副研究员,研究方向:菠萝育种及高效种植技术;E-mail: 基金资助:
LIU Chuan-he1(), HE Han1, HE Xiu-gu2, LIU Kai1, SHAO Xue-hua1, LAI Duo1, KUANG Shi-zi1, XIAO Wei-qiang1
Received:
2021-03-10
Published:
2021-08-26
Online:
2021-09-10
摘要:
为探讨菠萝连作对土壤代谢物和细菌群落结构的影响,本研究采用非靶向代谢组学和细菌16S高通量测序技术,以菠萝园边角地未耕作过的土壤为对照(T0),对连续种植了5 a(T5)和15 a(T15)菠萝的土壤进行了代谢物与细菌群落差异比较分析。研究结果表明,与T0比较,T5和T15土壤中分别检测到120种和80种差异代谢物;T0-T5和T0-T15组间共筛选出11种具有显著性差异(VIP>1,P<0.05)的代谢物,包括有机酸4种,糖类3种,氨基酸及其衍生物2种,醇类2种;KEGG代谢通路分析表明差异代谢物主要富集在不饱和脂肪酸的生物合成途径等11条关键代谢通路中。细菌16S高通量测序结果表明,与T0相比,T5中土壤细菌群落多样性、丰富度指数均升高;T15中土壤细菌群落多样性指数升高,但丰富度指数下降。T5和T15的土壤细菌群落结构存在明显差异,在门、属水平上T5和T15土壤细菌群落的相对丰度明显高于T0。与T0相比,菠萝园土壤的两个优势菌群变形细菌门(Proteobacteria)和酸杆菌门(Acidobacteriota)在T5中所占比例升高,但在T15中所占比例下降。CCA分析结果表明,土壤pH与连作年限呈负相关。本结果可为菠萝连作对土壤环境的影响相关研究提供参考,对提高菠萝园土壤管理水平有指导意义。
刘传和, 贺涵, 何秀古, 刘开, 邵雪花, 赖多, 匡石滋, 肖维强. 不同连作年限菠萝园土壤差异代谢物和细菌群落结构分析[J]. 生物技术通报, 2021, 37(8): 162-175.
LIU Chuan-he, HE Han, HE Xiu-gu, LIU Kai, SHAO Xue-hua, LAI Duo, KUANG Shi-zi, XIAO Wei-qiang. Analysis of Differential Metabolites and Bacterial Community Structure in Soils of a Pineapple Orchard in Different Continuous-cropping Years[J]. Biotechnology Bulletin, 2021, 37(8): 162-175.
项目Item | 参数Parameter |
---|---|
进样量Sample volume | 1 μL |
分流模式Front inlet mode | 不分流模式Splitless mode |
隔垫吹扫流速 Front inlet septum purge flow | 3 mL/min |
载气Carrier gas | 氦Helium |
色谱柱Column | DB-5MS(30 m×250 μm×0.25 μm) |
柱流速Column flow | 1 mL/min |
柱箱升温程序Oven temperature ramp | 50℃保持1 min,以10℃/min的速度升至 310℃,并保持8 min 50℃ Stay at 50℃ for 1 min,raise to 310℃ at a rate of 10℃/min,Stay at 50℃ for 8 min |
前进样口温度 Front injection temperature | 280℃ |
传输线温度 Transfer line temperature | 280℃ |
离子源温度 Ion source temperature | 250℃ |
电离电压Electron energy | -70 eV |
质量范围Mass range | m/z:50-500 |
扫描速率Acquisition rate | 12.5光谱/秒Spectra per second |
溶剂延迟Solvent delay | 6.35 min |
表1 仪器参数
Table 1 Instrument parameters
项目Item | 参数Parameter |
---|---|
进样量Sample volume | 1 μL |
分流模式Front inlet mode | 不分流模式Splitless mode |
隔垫吹扫流速 Front inlet septum purge flow | 3 mL/min |
载气Carrier gas | 氦Helium |
色谱柱Column | DB-5MS(30 m×250 μm×0.25 μm) |
柱流速Column flow | 1 mL/min |
柱箱升温程序Oven temperature ramp | 50℃保持1 min,以10℃/min的速度升至 310℃,并保持8 min 50℃ Stay at 50℃ for 1 min,raise to 310℃ at a rate of 10℃/min,Stay at 50℃ for 8 min |
前进样口温度 Front injection temperature | 280℃ |
传输线温度 Transfer line temperature | 280℃ |
离子源温度 Ion source temperature | 250℃ |
电离电压Electron energy | -70 eV |
质量范围Mass range | m/z:50-500 |
扫描速率Acquisition rate | 12.5光谱/秒Spectra per second |
溶剂延迟Solvent delay | 6.35 min |
图2 差异代谢物火山图 A为T0-T5比较组,B为T0-T15比较组。火山图中每个点代表一个代谢物,横坐标代表该组对比各物质的倍数变化(取以2为底的对数),纵坐标表示t检验的P-value(取以10为底的对数),散点大小代表OPLS-DA模型的VIP值。图中绿色的点代表下调差异表达代谢物,红色的点代表上调差异表达代谢物,黑色代表差异不显著的代谢物
Fig.2 Volcano plot of differential metabolites A and B are the comparison groups of T0-T5 and T0-T15,respectively. Each point from the volcano plot refers to a metabolite,the abscissa refers to the multiple change of each sample in the group(take the logarithm based on 2),and the ordinate refers to the P-value of the t test(take the logarithm based on 10),the size of the scatter points refers to the VIP value of the OPLS-DA model. The green point refers to the down-regulated differential metabolites,the red point refers to up-regulated the differential metabolites,and the black point refers to the differential metabolites with no significant difference
代谢物序号 Metabolite ID | 代谢物名称 Metabolite name | 分类Classification | 表达差异倍数 log2Fold Change | VIP值 VIP value | ||||
---|---|---|---|---|---|---|---|---|
T0-T5 | T0-T15 | T0-T5 | T0-T15 | |||||
meta_395 | (2R,3S)-2-羟基-3-异丙基丁二酸 (2R,3S)-2-hydroxy-3-isopro pylbutanedioic acid | 有机酸Organic acid | 1.924 | 2.099 | 1.504 | 1.685 | ||
meta_423 | 4-羟基苯甲酸4-Hydroxybenzoic acid | 有机酸Organic acid | 1.538 | 2.191 | 1.524 | 1.635 | ||
meta_465 | 4-羟基-3-甲氧基苯甲酸 4-hydroxy-3-methoxybenzoic acid | 有机酸Organic acid | 1.569 | 1.879 | 1.496 | 1.665 | ||
meta_484 | 3,4-二羟基苯甲酸3,4-dihydroxybenzoic acid | 有机酸Organic acid | 1.740 | 1.746 | 1.430 | 1.642 | ||
meta_304 | N-乙酰基-β-丙氨酸N-Acetyl-beta-alanine | 氨基酸衍生物 Amino acid derivatives | 2.211 | 3.186 | 1.491 | 1.726 | ||
meta_489 | N-氨基甲酰谷氨酸N-Carbamylglutamate | 氨基酸衍生物 Amino acid derivatives | 2.327 | 3.440 | 1.506 | 1.686 | ||
meta_504 | 葡萄糖Glucose | 糖类Carbohydrates | 4.878 | 1.580 | 2.840 | 1.634 | ||
meta_706 | 槐糖Sophorose | 糖类Carbohydrates | 4.201 | -3.892 | 1.471 | 1.112 | ||
meta_789 | 松三糖Melezitose | 糖类Carbohydrates | 1.292 | -1.386 | 1.368 | 1.156 | ||
meta_783 | (3β,5α,6β)-胆固醇3,5,6-三醇 (3β,5α,6β)-Cholestane-3,5,6-triol | 醇类Alcohols | 3.071 | 1.568 | 1.428 | 1.469 | ||
meta_513 | 山梨糖醇Sorbitol | 醇类Alcohols | 2.244 | 1.901 | 1.493 | 1.607 |
表2 不同比较组间差异代谢物
Table 2 Differential metabolites among the comparison groups
代谢物序号 Metabolite ID | 代谢物名称 Metabolite name | 分类Classification | 表达差异倍数 log2Fold Change | VIP值 VIP value | ||||
---|---|---|---|---|---|---|---|---|
T0-T5 | T0-T15 | T0-T5 | T0-T15 | |||||
meta_395 | (2R,3S)-2-羟基-3-异丙基丁二酸 (2R,3S)-2-hydroxy-3-isopro pylbutanedioic acid | 有机酸Organic acid | 1.924 | 2.099 | 1.504 | 1.685 | ||
meta_423 | 4-羟基苯甲酸4-Hydroxybenzoic acid | 有机酸Organic acid | 1.538 | 2.191 | 1.524 | 1.635 | ||
meta_465 | 4-羟基-3-甲氧基苯甲酸 4-hydroxy-3-methoxybenzoic acid | 有机酸Organic acid | 1.569 | 1.879 | 1.496 | 1.665 | ||
meta_484 | 3,4-二羟基苯甲酸3,4-dihydroxybenzoic acid | 有机酸Organic acid | 1.740 | 1.746 | 1.430 | 1.642 | ||
meta_304 | N-乙酰基-β-丙氨酸N-Acetyl-beta-alanine | 氨基酸衍生物 Amino acid derivatives | 2.211 | 3.186 | 1.491 | 1.726 | ||
meta_489 | N-氨基甲酰谷氨酸N-Carbamylglutamate | 氨基酸衍生物 Amino acid derivatives | 2.327 | 3.440 | 1.506 | 1.686 | ||
meta_504 | 葡萄糖Glucose | 糖类Carbohydrates | 4.878 | 1.580 | 2.840 | 1.634 | ||
meta_706 | 槐糖Sophorose | 糖类Carbohydrates | 4.201 | -3.892 | 1.471 | 1.112 | ||
meta_789 | 松三糖Melezitose | 糖类Carbohydrates | 1.292 | -1.386 | 1.368 | 1.156 | ||
meta_783 | (3β,5α,6β)-胆固醇3,5,6-三醇 (3β,5α,6β)-Cholestane-3,5,6-triol | 醇类Alcohols | 3.071 | 1.568 | 1.428 | 1.469 | ||
meta_513 | 山梨糖醇Sorbitol | 醇类Alcohols | 2.244 | 1.901 | 1.493 | 1.607 |
代谢通路ID Metabolic pathway ID | 代谢通路名称 Pathway name | -lgp | 代谢物序号 Metabolite ID | |
---|---|---|---|---|
T0-T5 | T0-T15 | |||
ko00051 | 果糖和甘露糖代谢 Fructose and mannose metabolism | 5.5963 | 4.6088 | 山梨糖醇meta_513 Sorbitol meta_513 |
ko00100 | 类固醇生物合成 Steroid biosynthesis | 2.3240 | 7.2183 | 胆固醇meta_762 Cholesterol meta_762 |
ko00940 | 苯丙烷生物合成 Phenylpropanoid biosynthesis | 3.3804 | 3.2558 | 4-羟基肉桂酸meta_517 4-Hydroxycinnamic acid meta_517 |
ko00130 | 泛醌和其他萜类醌的生物合成 Ubiquinone and other terpenoid-quinone biosynthesis | 3.3901 | 3.8455 | 4-羟基苯甲酸meta_423;4-羟基肉桂酸meta_517 4-Hydroxybenzoic acid meta_423;4-Hydroxycinnamic acid meta_517 |
ko00350 | 酪氨酸代谢 Tyrosine metabolism | 3.3733 | 1.6328 | 4-羟基肉桂酸meta_517 4-Hydroxycinnamic acid meta_517 |
ko00360 | 苯丙氨酸代谢 Phenylalanine metabolism | 2.7754 | 2.7181 | 4-羟基苯甲酸meta_423;4-羟基肉桂酸meta_517 4-Hydroxybenzoic acid meta_423;4-Hydroxycinnamic acid meta_517 |
ko00400 | 苯丙氨酸,酪氨酸和色氨酸的生物合成 Phenylalanine,tyrosine and tryptophan biosynthesis | 3.4807 | 1.9702 | 3,4-二羟基苯甲酸meta_484 3,4-Dihydroxybenzoic acid meta_484 |
ko00591 | 亚油酸代谢 Linoleic acid metabolism | 2.3376 | 4.1084 | 亚油酸meta_594 Linoleic acid meta_594 |
ko00790 | 叶酸生物合成 Folate biosynthesis | 6.7045 | 3.6467 | 4-羟基苯甲酸meta_423 4-Hydroxybenzoic acid meta_423 |
ko00950 | 异喹啉生物碱的生物合成 Isoquinoline alkaloid biosynthesis | 3.0892 | 4.2224 | 4-羟基肉桂酸meta_517 4-Hydroxycinnamic acid meta_517 |
ko01040 | 不饱和脂肪酸的生物合成 Biosynthesis of unsaturated fatty acids | 2.7263 | 2.9874 | 亚油酸meta_594;山嵛酸meta_681;木质酸meta_714 Linoleic acid meta_594. Behenic acid meta_681.Lignoceric acid meta_714 |
表3 差异代谢物代谢通路分析
Table 3 Metabolic pathway analysis of the differential metabolites
代谢通路ID Metabolic pathway ID | 代谢通路名称 Pathway name | -lgp | 代谢物序号 Metabolite ID | |
---|---|---|---|---|
T0-T5 | T0-T15 | |||
ko00051 | 果糖和甘露糖代谢 Fructose and mannose metabolism | 5.5963 | 4.6088 | 山梨糖醇meta_513 Sorbitol meta_513 |
ko00100 | 类固醇生物合成 Steroid biosynthesis | 2.3240 | 7.2183 | 胆固醇meta_762 Cholesterol meta_762 |
ko00940 | 苯丙烷生物合成 Phenylpropanoid biosynthesis | 3.3804 | 3.2558 | 4-羟基肉桂酸meta_517 4-Hydroxycinnamic acid meta_517 |
ko00130 | 泛醌和其他萜类醌的生物合成 Ubiquinone and other terpenoid-quinone biosynthesis | 3.3901 | 3.8455 | 4-羟基苯甲酸meta_423;4-羟基肉桂酸meta_517 4-Hydroxybenzoic acid meta_423;4-Hydroxycinnamic acid meta_517 |
ko00350 | 酪氨酸代谢 Tyrosine metabolism | 3.3733 | 1.6328 | 4-羟基肉桂酸meta_517 4-Hydroxycinnamic acid meta_517 |
ko00360 | 苯丙氨酸代谢 Phenylalanine metabolism | 2.7754 | 2.7181 | 4-羟基苯甲酸meta_423;4-羟基肉桂酸meta_517 4-Hydroxybenzoic acid meta_423;4-Hydroxycinnamic acid meta_517 |
ko00400 | 苯丙氨酸,酪氨酸和色氨酸的生物合成 Phenylalanine,tyrosine and tryptophan biosynthesis | 3.4807 | 1.9702 | 3,4-二羟基苯甲酸meta_484 3,4-Dihydroxybenzoic acid meta_484 |
ko00591 | 亚油酸代谢 Linoleic acid metabolism | 2.3376 | 4.1084 | 亚油酸meta_594 Linoleic acid meta_594 |
ko00790 | 叶酸生物合成 Folate biosynthesis | 6.7045 | 3.6467 | 4-羟基苯甲酸meta_423 4-Hydroxybenzoic acid meta_423 |
ko00950 | 异喹啉生物碱的生物合成 Isoquinoline alkaloid biosynthesis | 3.0892 | 4.2224 | 4-羟基肉桂酸meta_517 4-Hydroxycinnamic acid meta_517 |
ko01040 | 不饱和脂肪酸的生物合成 Biosynthesis of unsaturated fatty acids | 2.7263 | 2.9874 | 亚油酸meta_594;山嵛酸meta_681;木质酸meta_714 Linoleic acid meta_594. Behenic acid meta_681.Lignoceric acid meta_714 |
图4 差异代谢物KEGG富集网络图 A:T0-T5比较组,B:T0-T15比较组。图中淡黄色节点为通路,与之相连的小节点是注释到该通路的具体代谢物,颜色的深浅表示差异倍数取log2的对数值,圆圈的大小代表差异代谢物对通路的影响值。通常,颜色越深、圆圈越大表明代谢物差异越显著、对通路的影响越大,该图最多显示5个通路
Fig. 4 KEGG pathway enrichment map of differential metabolites A and B are the comparison group of T0-T5 and T0-T15,respectively. The light yellow nodes in the figures refer to pathways,and the small nodes connected to the pathways are the specific metabolites remarked on them. The shade of the color refers to the different multiple based on the log2fold change,the size of circle refers to the value of the effects of differential metabolites on the pathways. Usually,the darker of the color and bigger of the circle refer to the differences of metabolites are more significant and effects on pathways bigger. The figure shows at most 5 pathways
图6 不同连作年限菠萝园土壤细菌群落NMDS分析
Fig. 6 NMDS analysis of different soil bacterial community in the different continuous-cropping years of the pineapple orchard
图7 不同连作年限菠萝园土壤细菌群落在门、属、目水平上的相对丰度 图中A、B、C分别为细菌门水平、属水平和目水平
Fig. 7 Relative abundance of different soil bacterial community at phyla,genus and order level in the different continuous-cropping years of the pineapple orchard Picture A,B and C are refer to the levels of bacterial phyla,genus and order,respectively
连作年限 Continuous-cropping year | ACE指数 ACE index | Chao1指数 Chao1 index | Simpson指数 Simpson index | Shannon指数 Shannon index |
---|---|---|---|---|
T5 | 802.89±22.72a | 805.23±20.92a | 0.01±0.00b | 5.39±0.11a |
T15 | 577.27±17.43b | 579.76±16.38b | 0.04±0.00b | 4.66±0.03b |
T0 | 643.25±32.15b | 610.45±39.27b | 0.12±0.03a | 3.90±0.18c |
表4 不同连作年限菠萝园土壤细菌丰度与多样性
Table 4 Diversity of relative abundance of different bacterial of soil under different continuous-cropping years of the pineapple orachard
连作年限 Continuous-cropping year | ACE指数 ACE index | Chao1指数 Chao1 index | Simpson指数 Simpson index | Shannon指数 Shannon index |
---|---|---|---|---|
T5 | 802.89±22.72a | 805.23±20.92a | 0.01±0.00b | 5.39±0.11a |
T15 | 577.27±17.43b | 579.76±16.38b | 0.04±0.00b | 4.66±0.03b |
T0 | 643.25±32.15b | 610.45±39.27b | 0.12±0.03a | 3.90±0.18c |
图8 不同连作年限菠萝园土壤中细菌群落与pH值的相关性CCA分析
Fig. 8 CCA analysis correlating bacterial community to pH value of soil under different continuous-cropping years of the pineapple orchard
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