氯虫苯甲酰胺胁迫下二化螟中肠细菌类微生物的多样性

doi:10.16819/j.1001-7216.2020.0101

摘要/Abstract

摘要： 目的明确氯虫苯甲酰胺处理下二化螟中肠细菌类微生物多样性的变化。方法综合运用宏基因组测序以及传统微生物分离纯化的方法分析不同浓度氯虫苯甲酰胺处理的二化螟种群中肠微生物的多样性。结果不同浓度(100、200、400µg/mL)氯虫苯甲酰胺处理后二化螟中肠细菌类微生物丰度及多样性降低,处理种群中肠细菌类微生物的OTU数值、特有的OTU数值均低于对照;摩根氏菌属(Morganella)、普罗威登斯菌属(Providencia)以及变形菌属(Proteus)在处理种群中的比例明显升高;同时培养结果显示氯虫苯甲酰胺处理种群获得的菌株除肠杆菌属(Enterobacter)外,还存在超压莱略特氏菌(Lelliottianimipressuralis)、非脱羧勒菌(Leclerciaadecarboxylata)、成团泛菌(Pantoeaagglomerans)等其他菌属。COG数据库对比与KEGG分析显示,对照与处理二化螟种群的各功能分类基因数量及相对丰度无明显差异。结论解析了氯虫苯甲酰胺胁迫下二化螟中肠细菌类微生物的多样性。这些结果为进一步研究中肠细菌在二化螟对氯虫苯甲酰胺的抗药性奠定基础。

关键词: 二化螟, 氯虫苯甲酰胺：宏基因组, 中肠细菌, 微生物多样性

Abstract:

【Objective】We aim toilluminatethestructural changes in the midgut microbial community of Chilosuppressalis exposed to chlorobenzamide.【Method】Macrogenomic sequencing and in vitro culturewere used to analyze the diversity of midgut microbiotaof Chilosuppressalistreatedwith different concentrations (100, 200, 400µg/mL) of chlorobenzamide.【Result】The results showed that chlorobenzamide treatmentdecreased the abundance anddiversityof the midgut bacteria of Chilosuppressalis,and the OTU number and specific OTU number of the intestinal bacteria of treated population were lower than those of the control;the proportion of Morganella, Provincia and Proteus in the chlorobenzamide treated population increased significantly. Meanwhile, the result of in vitro culture indicated besides enterobacter,Lelliottinimipressuralis, Leclerciadecarboxylata, Pantoeaagglomeranswere isolated from chlorobenzamide treated Chilosuppressalis population. COG classification and KEGG analysis didn’t show significant difference in number and relative abundance of functional genes among control and three chlorobenzamide treated populations.【Conclusion】The study had clarified the diversity of intestinal bacteria and the relationshipbetween gutbacteria of Chilosuppressalis andthe formation of chlorobenzamide resistance, and will lay a scientific basis for the role of intestinal bacteria in resistance of Chilosuppressalistochlorobenzamide.

Key words: Chilosuppressalis, chlorobenzamide, macrogenomic sequencing, midgut bacteria, microbial diversity

中图分类号:

张珏锋, 张琴, 李芳, 钟海英, 陈建明. 氯虫苯甲酰胺胁迫下二化螟中肠细菌类微生物的多样性[J]. 中国水稻科学, 2020, 34(6): 586-594.

Juefeng ZHANG, Qin ZHANG, Fang LI, Haiying ZHONG, Jianming CHEN. Diversity of Midgut Microbial Community of ChilosuppressalisExposed to Chlorobenzamide[J]. Chinese Journal OF Rice Science, 2020, 34(6): 586-594.

图/表 6

表1 不同处理二化螟试虫中肠细菌群落多样性指数

Table 1 Bacterial community richness, diversity indices of midgut in Chilosuppressalis under different treatments.

样本 Sample	OUT (97%)	Ace指数 Ace index	Chao指数 Chao index	测序深度指数 Sequencing depth	香农指数 Shannon index	辛普森指数 Simpson index
100_24h	333	442(407,494)	405(377,452)	0.99	2.19(2.18, 2.21)	0.20(0.20, 0.20)
100_48h	432	758(693,838)	640(571,742)	0.99	2.39(2.37, 2.40)	0.17(0.17, 0.17)
100_72h	580	718(680,771)	745(690,827)	0.99	2.98(2.96, 2.99)	0.13(0.13, 0.13)
200_24h	412	512(481,557)	500(467,553)	0.99	2.02(2.00, 2.04)	0.32(0.32, 0.32)
200_48h	357	415(394,448)	407(385,447)	0.99	2.49(2.47, 2.50)	0.16(0.16, 0.16)
200_72h	639	742(713,782)	730(698,778)	0.99	3.02(3.01, 3.04)	0.13(0.12, 0.13)
400_24h	474	578(547,623)	569(535,623)	0.99	2.70(2.68, 2.71)	0.12(0.12, 0.12)
400_48h	751	901(863,952)	893(849,956)	0.99	3.31(3.29, 3.33)	0.09(0.09, 0.09)
400_72h	321	539(473,634)	557(473,690)	0.99	2.22(2.20, 2.23)	0.16(0.15, 0.16)
CK_24h	1003	1127(1096,1169)	1123(1085,1177)	0.99	3.91(3.89, 3.93)	0.07(0.07, 0.07)
CK_48h	718	782(763,811)	780(757,817)	0.99	3.38(3.36, 3.40)	0.08(0.09, 0.09)
CK_72h	650	791(754,842)	769(730,827)	0.99	3.38(3.36, 3.39)	0.08(0.08, 0.08)

图1 不同处理二化螟试虫中肠细菌类微生物OUT韦恩图分析图中红色为对照处理二化螟种群中肠细菌类微生物群落OUTs,绿色为100μg/mL氯虫苯甲酰胺处理二化螟种群中肠细菌类微生物群落OUTs,蓝色为200μg/mL氯虫苯甲酰胺处理二化螟种群中肠细菌类微生物群落OUTs,黄色为400μg/mL氯虫苯甲酰胺处理二化螟种群中肠细菌类微生物群落OUTs。

Fig. 1. Venn analysis of the number and community of intestinal bacteria of Chilosuppressalis under different treatments. The red circle is the outs of the intestinal bacterial community in the control population; the green circle, the outs of the intestinal bacterial community in the 100µg/mL chlorobenzamide treated population, the blue circle, the outs of the intestinal bacterial community in the 200µg/mL chlorobenzamide treated population, the yelloow, the outs of the intestinal bacterial community in the 400µg/mL chlorobenzamide treated population.

图2 基于属水平不同处理二化螟体内中肠细菌群落结构 Serratia–沙雷氏菌;Morganella–摩根氏菌;Enterobacteriaceae_unclassified–肠杆菌科(未确定);Providencia–普罗维登斯菌属;Enterococcus–肠球菌属;Lactococcus–乳球菌属;Bacteroides–拟杆菌属;Proteus–变形杆菌属;Lactobacillus–乳酸菌属;Bacillus–芽孢杆菌属;Dysgonomonas–营发酵单胞菌属;Bacteroidales_unclassified–拟杆菌目(未确定);Escherichia-Shigella–大肠杆菌属-志贺氏菌属;Anoxybacillus–无氧芽胞杆菌属;Pantoea–泛菌属;Stenotrophomonas–寡养单胞菌属;Halomonas–嗜盐单胞菌属;Cyanobacteria_norank–蓝细菌(未分类);Bacteroidales_S24-7_group_norank–拟杆菌目S24-7(未分类);Faecalibaculum–粪杆菌属;Burkholderia-Paraburkholderia–伯克霍尔德菌-拟伯克氏菌属;Desulfovibrio–脱磷孤菌属;Comamonas–丛毛单胞菌属;Aliihoeflea–砷氧化菌;Paracoccus–副球菌属;Tolumonas–甲苯单胞菌属;Sphaerochaeta–螺旋菌属;Others–其他。

Fig. 2. Genera of intestinal bacteria found in different treated Chilosuppressalis population.

图3 不同处理二化螟试虫COG功能预测 A–RNA加工与修饰;B–染色质结构与动力学;C–能量产生与转化;D–细胞周期控制、细胞分裂、染色体分区;E–氨基酸运输和代谢;F–核苷酸运输和代谢;G–碳水化合物运输和代谢;H–辅酶运输和代谢;I–脂质运输和代谢;J–翻译、核糖体结构和生物起源;K–转录;L–复制、重组和修复;M–细胞壁/膜/信封生源论;N–细胞活性;O–翻译后修饰、蛋白质转换、分子伴侣;P–无机离子运输和代谢;Q–次级代谢产物生物合成、运输和分解代谢;R–通用功能预测;S–未知功能;T–信号转导机制;U–胞内运输、分泌和膜泡运输;V–防御机制;W–细胞外结构;Y–核结构;Z–细胞骨架。图中a为CK_24h,b为100_24h,c为200_24h,d为400_24h。

Fig. 3. COG classificationof intestinal bacteria of Chilosuppressalis in different treatments. A, RNA processing and modification; B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism;G, Carbohydrate transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, Replication, recombination and repair; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction only; S, Function unknown; T, Signal transduction mechanisms; U, Intracellular trafficking, secretion, and vesicular transport; V, Defense mechanisms; W, Extracellular structures; Y, Nuclear structure; Z, Cytoskeleton. In the figure, a is CK_24h,b, 100_24h,c, 200_24h,d is 400_24h.

图4 基于宏基因组测序KEGG功能预测 Membrane transport–膜运输,Carbohydrate metabolism–水化合物代谢,Amino acid metabolism–氨基酸代谢,Replication and repair–复制和修复,Poorly characterized–缺乏特征,Cellular processes and signaling–细胞过程和信号,Energy metabolism–能量代谢,Translation–转译,Metabolism of cofactors and vitamins–辅酶因子和维生素代谢,Nucleotide metabolism–核苷酸代谢,Lipid metabolism–脂质代谢,Metabolism–代谢,Transcription–转录,Genetic information processing–遗传信息处理,Glycan biosynthesis and metabolism–多糖合成和代谢,Signal transduction–信号转导,Cell motility–细胞运动性,Folding, sorting and degradation–折叠,排序和退化,Xenobiotics biodegradation and metabolism–外源性生物降解与代谢,Enzyme families–酶家族,Metabolism of other amino acids–其他氨基酸代谢,Metabolism of terpenoids and polyketides–萜类和多酮类化合物代谢,Biosynthesis of other secondary metabolites–其他次生代谢物的生物合成,Infectious diseases–传染性疾病,Cell growth and death–细胞生长与死亡,Transport and catabolism–运输和分解代谢,Endocrine system–内分泌系统,Neurodegenerative diseases–神经退行性疾病,Signaling molecules and interaction–信号分子和相互作用,Cancers–癌症,environmental adaptation–环境适应,Metabolic diseases–代谢疾病,Others–其他。

Fig.4. KEGG function prediction based on macrogenomic sequencing.

表2 二化螟中肠细菌类微生物离体培养测序结果

Table 2 Isolation and identification of bacteria from the midgut ofChilosuppressalis.

分类 Classification	对照CK		100µg/mL		200µg/mL		400µg/mL
分类 Classification	菌株名称 Strain	数量 Quantity	菌株名称 Strain	数量 Quantity	菌株名称 Strain	数量 Quantity	菌株名称 Strain	数量 Quantity
肠杆菌属 Enterobacter	霍氏肠杆菌 Enterobacter hormaechei	4					霍氏肠杆菌 Enterobacter hormaechei	3
	阿氏肠杆菌YT Enterobacter asburiae strain YT	1					阿氏肠杆菌YT Enterobacter asburiaestrain YT	2
	肠杆菌属 CZBSY4Enterobacter sp. CZBSY4	4					肠杆菌属 CZBSY4 Enterobacter sp. CZBSY4	1
	肠杆菌属 09-M1 Enterobacter sp. 09-M1	2
	肠杆菌属 XM Enterobacter sp. strain XM	3
	阴沟肠杆菌 Enterobacter cloacaether	1
	路氏肠杆菌 Enterobacter ludwigii	2
			肠杆菌属 S4(2015) Enterobacter sp. S4(2015)	4			肠杆菌属 S4(2015) Enterobacter sp. S4(2015)	1
			产气肠杆菌	2	产气肠杆菌	1	产气肠杆菌	1
			Enterobacter roggenkampii Enterobacter aerogenes	1	Enterobacter roggenkampii Enterobacter aerogenes	3	Enterobacter roggenkampii Enterobacter aerogenes	1
			森肠杆菌 Enterobacter mori	1	森肠杆菌 Enterobacter mori	2	森肠杆菌 Enterobacter mori	1
			河生肠杆菌 Enterobacter amnigenus	3	河生肠杆菌 Enterobacter amnigenus	4
			神户肠杆菌 Enterobacter kobei	1	神户肠杆菌 Enterobacter kobei	1
			阿氏肠杆菌 Enterobacter asburiae	3
其他 Others	非脱羧埃希氏菌 Leclerciaadecarboxylata	2	非脱羧埃希氏菌 Leclerciaadecarboxylata	1	非脱羧埃希氏菌 Leclerciaadecarboxylata	2	非脱羧埃希氏菌 Leclerciaadecarboxylata	5
			成团泛菌 Pantoeaagglomerans	1	成团泛菌 Pantoeaagglomerans	3	成团泛菌 Pantoeaagglomerans	2
			泛菌属DAP16 Pantoea sp. DAP16	1	泛菌属DAP16 Pantoea sp. DAP16	2	泛菌属DAP16 Pantoea sp. DAP16	2
			克雷伯氏菌属 Klebsiella sp.	1	克雷伯氏菌属 Klebsiella sp.	1
			超压莱略特氏菌 Lelliottianimipressuralis	1	超压莱略特氏菌 Lelliottianimipressuralis	1
			ɤ-变形菌门细菌 Gammaproteobacteria bacterium strain	3	ɤ-变形菌门细菌 Gammaproteobacteria bacterium strain	1
			不可培养细菌 Uncultured bacterium	3	不可培养细菌 Uncultured bacterium	3	不可培养细菌 Uncultured bacterium	1

表2 二化螟中肠细菌类微生物离体培养测序结果

Table 2 Isolation and identification of bacteria from the midgut ofChilosuppressalis.

分类 Classification	对照CK		100µg/mL		200µg/mL		400µg/mL
分类 Classification	菌株名称 Strain	数量 Quantity	菌株名称 Strain	数量 Quantity	菌株名称 Strain	数量 Quantity	菌株名称 Strain	数量 Quantity
肠杆菌属 Enterobacter	霍氏肠杆菌 Enterobacter hormaechei	4					霍氏肠杆菌 Enterobacter hormaechei	3
	阿氏肠杆菌YT Enterobacter asburiae strain YT	1					阿氏肠杆菌YT Enterobacter asburiaestrain YT	2
	肠杆菌属 CZBSY4Enterobacter sp. CZBSY4	4					肠杆菌属 CZBSY4 Enterobacter sp. CZBSY4	1
	肠杆菌属 09-M1 Enterobacter sp. 09-M1	2
	肠杆菌属 XM Enterobacter sp. strain XM	3
	阴沟肠杆菌 Enterobacter cloacaether	1
	路氏肠杆菌 Enterobacter ludwigii	2
			肠杆菌属 S4(2015) Enterobacter sp. S4(2015)	4			肠杆菌属 S4(2015) Enterobacter sp. S4(2015)	1
			产气肠杆菌	2	产气肠杆菌	1	产气肠杆菌	1
			Enterobacter roggenkampii Enterobacter aerogenes	1	Enterobacter roggenkampii Enterobacter aerogenes	3	Enterobacter roggenkampii Enterobacter aerogenes	1
			森肠杆菌 Enterobacter mori	1	森肠杆菌 Enterobacter mori	2	森肠杆菌 Enterobacter mori	1
			河生肠杆菌 Enterobacter amnigenus	3	河生肠杆菌 Enterobacter amnigenus	4
			神户肠杆菌 Enterobacter kobei	1	神户肠杆菌 Enterobacter kobei	1
			阿氏肠杆菌 Enterobacter asburiae	3
其他 Others	非脱羧埃希氏菌 Leclerciaadecarboxylata	2	非脱羧埃希氏菌 Leclerciaadecarboxylata	1	非脱羧埃希氏菌 Leclerciaadecarboxylata	2	非脱羧埃希氏菌 Leclerciaadecarboxylata	5
			成团泛菌 Pantoeaagglomerans	1	成团泛菌 Pantoeaagglomerans	3	成团泛菌 Pantoeaagglomerans	2
			泛菌属DAP16 Pantoea sp. DAP16	1	泛菌属DAP16 Pantoea sp. DAP16	2	泛菌属DAP16 Pantoea sp. DAP16	2
			克雷伯氏菌属 Klebsiella sp.	1	克雷伯氏菌属 Klebsiella sp.	1
			超压莱略特氏菌 Lelliottianimipressuralis	1	超压莱略特氏菌 Lelliottianimipressuralis	1
			ɤ-变形菌门细菌 Gammaproteobacteria bacterium strain	3	ɤ-变形菌门细菌 Gammaproteobacteria bacterium strain	1
			不可培养细菌 Uncultured bacterium	3	不可培养细菌 Uncultured bacterium	3	不可培养细菌 Uncultured bacterium	1

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