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

doi:10.13560/j.cnki.biotech.bull.1985.2022-0211

摘要/Abstract

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。

关键词: 柠条, nifH基因, 固氮菌群落, 多样性

Abstract:

Exploring the abundance information of nifH gene and the composition and diversity of nitrogen-fixing bacterial community in the rhizosphere soil of the desert sand-fixing plant may provide basic information for enriching the microbial resource pool in desert areas. Real-time quantitative PCR and high-throughput sequencing were applied to analyze the abundance of nifH gene and the community composition of nitrogen-fixing bacteria in the rhizosphere soil of 5 shrublands of Caragana spp. growing in the desert grassland of Ningxia. The method of isolation and culture were to screen nitrogen-fixing bacteria in the rhizosphere soil of Caragana spp. and to study their diversities. The results showed that the copy number of nifH gene in the 5 rhizosphere soil samples were significantly different, the highest in Luoshan（LS）and the lowest in Shapotou（SPT）. A total of 7 phyla, 12 classes, 26 families and 30 genera of nitrogen-fixing bacteria were detected in the rhizosphere soil of Caragana spp. At the family and genus level, the community composition of nitrogen-fixing bacteria in Shapotou sample was quite different. Unclassified Proteobacteria and Phyllobacteriaceae accounted for 25.54% and 22.77%, respectively, Rhodospirillaceae accounted for 17.43%, and Mesorhizobium were dominant genus, with a relative abundance of 22.77%; the other four soil samples were dominated by Rhodospirillaceae and Skermanella, with relative abundances of 23.71%-73.45% and 23.19%-71.14%, respectively, and it accounted for the highest proportion in Luoshan sample. The isolated nitrogen-fixing bacteria belong to 15 genera, in addition to the common nitrogen-fixing groups such as Pseudomonas, Rhizobium and Enterobacter, also including Leclercia, Chryseobacterium, Stenotrophomonas, etc., the diversity is abundant.

Key words: Caragana spp., nifH gene, nitrogen-fixing bacteria community, diversity

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.

图/表 9

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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	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

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

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	-	+	-	-	-	-	+