转BpGLK白桦土壤酶活及根际土壤细菌、真菌群落组成分析

doi:10.12302/j.issn.1000-2006.202208001

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南京林业大学学报（自然科学版） ›› 2024, Vol. 48 ›› Issue (6): 129-137.doi: 10.12302/j.issn.1000-2006.202208001

转BpGLK白桦土壤酶活及根际土壤细菌、真菌群落组成分析

曹俐¹(), 金冬雪¹, 姜静¹, 李天芳²^,^*()

1.林木遗传育种全国重点实验室(东北林业大学),黑龙江哈尔滨 150040
2.黑龙江省林业科学研究院,黑龙江哈尔滨 150081

收稿日期:2022-08-01 修回日期:2023-03-19 出版日期:2024-11-30 发布日期:2024-12-10
通讯作者: *李天芳(18646079568@126.com),高级工程师。
作者简介:
曹俐(cl08182022@163.com)。
基金资助:
转基因杨树中间试验安全性检测(KJZXSA202002)

Analysis of bacterial and fungal community composition and soil enzyme activities in the rhizosphere of transgenic Betula platyphylla

CAO Li¹(), JIN Dongxue¹, JIANG Jing¹, LI Tianfang²^,^*()

1. State Key Laboratory of Forest Genetics and Breeding,Northeast Forestry University,Harbin 150040,China
2. Heilongjiang Academy of Forestry, Harbin 150081, China

Received:2022-08-01 Revised:2023-03-19 Online:2024-11-30 Published:2024-12-10

摘要/Abstract

摘要：

【目的】白桦(Betula platyphylla)BpGLK转录因子参与调控叶绿体发育及叶色,BpGLK抑制表达白桦在生长期内叶色呈黄绿色,在园林绿化中极具观赏价值。但作为转基因植物,大面积的推广应用是否会对环境产生不利影响引起研究者的重视。研究转BpGLK白桦对土壤酶活及根际土壤细菌、真菌群落组成的影响,为后续环境释放及商业化应用提供理论依据。【方法】以3年生转BpGLK白桦(OE株系及RE株系)及对照野生型(WT)白桦为材料,采用分光光度法测定土壤蔗糖酶、脲酶、中性蛋白酶、过氧化氢酶及纤维素酶的活性,利用 Illumina-Miseq 高通量测序平台对根际土壤微生物进行 16S rRNA 和 ITS 测序分析,对根际土壤细菌和真菌群落丰富度和多样性变化、结构差异性、群落组成进行分析,了解转基因活动对白桦土壤酶活性及根际土壤微生物组成的影响。【结果】在测定的4个时间点(6月15日、7月15日、8月15日和9月15日)转BpGLK白桦与WT株系的土壤脲酶、蔗糖酶、纤维素酶、中性蛋白酶活性均存在显著差异(P<0.05),而过氧化氢酶活性则呈现WT株系与部分转基因株系间存在显著差异,土壤脲酶和纤维素酶活性在8月中旬和9月中旬明显降低,而土壤中性蛋白酶活性在9月中旬明显升高,OE株系的土壤中性蛋白酶活性在4个时期均高于或显著高于WT株系(P<0.05)。根际土壤细菌群落属水平上,具有促生功能的伯克霍尔德菌属(Burkholderia)是转基因株系与WT株系的优势菌,该菌属在转基因株系根际土壤中的相对丰度显著提高;在根际真菌群落属水平上,棉革菌属(Tomentella)是转基因RE株系根际的优势类群,锁瑚菌属(Clavulina)是WT及转基因OE株系根际的优势类群,蜡蘑菌属(Laccaria)的相对丰度在OE株系和RE株系中显著降低(P<0.05);Alpha多样性分析显示,在细菌群落组成方面,转基因白桦的可观测物种数(observed species)、Chao1、香农(Shannon)及辛普森(Simpson)指数与WT株系间未达到显著差异;在真菌群落组成方面,RE株系的群落丰度、多样性均显著高于或高于WT株系,而OE与WT株系间差异未达到显著水平。韦恩图结果显示,转基因株系与野生型株系间的特征序列(ASV)差异显著;主成分分析结果显示,在细菌群落组成上,RE与WT株系的群落组成差异较小,而OE与WT株系差异相对较大;在真菌群落组成上,OE与WT株系的群落组成差异较小,而RE与WT株系差异相对较大。【结论】目标BpGLK基因的导入对白桦根际细菌、真菌群落丰度及多样性产生一定影响,并可能利于植物生长并提升自身抗逆性,但这种影响是否长期存在仍需后续试验验证。

关键词: 白桦, 转基因, 土壤酶活性, 根际, 细菌, 真菌, 群落组成

Abstract:

【Objective】The BpGLK transcription factor of Betula platyphylla is involved in regulating chloroplast development and leaf color. The leaf color of B. platyphylla with inhibited BpGLK expression is yellow-green during the growth period, which holds great ornamental value in landscaping. However, as a genetically modified crop, concerns exist regarding the potential adverse environmental effects of widespread use. This study aims to examine the impact of transgenic BpGLK B. platyphylla on soil enzyme activity and the composition of rhizosphere soil bacterial and fungal communities, providing theoretical data for future environmental release and commercialization.【Method】Three-year-old transgenic B. platyphylla (OE and RE strains) and wild-type (WT) B. platyphylla are used as materials. Soil sucrase, urease, neutral protease, catalase, and cellulase activities were measured using the spectrophotometric method. In addition, 16S rRNA and ITS sequencing analyses of rhizosphere soil microorganisms were conducted using the Illumina-Miseq high-throughput sequencing platform. The richness and diversity of bacterial and fungal communities, structural differences, and community composition in rhizosphere soil were analyzed to understand the effects of transgenic activity on soil enzyme activity and microbial composition in the rhizosphere soil of B. platyphylla.【Result】Significant differences (P < 0.05) are observed in soil urease, sucrase, cellulase and neutral protease activities between BpGLK-transformed B. platyphylla and WT strains at four time points (June 15, July 15, August 15 and September 15). However, catalase activity showed significant differences between WT and some transgenic strains. Soil urease and cellulase activities decrease significantly in mid-August and mid-September. The activity of soil neutral protease in OE strains was higher than that of the WT strain at all four periods (P < 0.05). At the rhizosphere bacterial community level, Burkholderia, a growth-promoting function, was the dominant genus in transgenic and WT strains. The relative abundance of Burkholderia in the rhizosphere soil of transgenic strains significantly increased. At the rhizosphere fungal community level, Tomentella dominated the rhizosphere of transgenic RE strains, while Clavulina dominated the rhizosphere of WT and OE strains. The relative abundance of Laccaria was significantly lower in OE and RE strains (P < 0.05). The Observed, Chao1, Shannon and Simpson indices of transgenic B. platyphylla did not differ significantly from those of WT strains, while the community abundance and diversity of RE strains were significantly higher than those of WT strains. The difference between OE and WT strains was not significant. Venn diagram analysis revealed significant differences in ASV composition between transgenic and WT strains. In contrast, principal component analysis showed slight differences in community composition between RE and WT strains, while the difference between OE and WT strains was relatively larger. However, the difference between OE and WT strains remainsed smaller than that between RE and WT strains.【Conclusion】The results indicated that the introduction of the exogenous BpGLK gene had a specific effect on the abundance and diversity of rhizosphere bacterial and fungal communities in B. platyphylla. These changes can promote plant growth and enhance resistance. However, whether these effects persist over the long term remains to be confirmed in future experiments.

Key words: birch(Betula platyphylla), transgenic, soil enzyme activities, rhizosphere, bacteria, fungi, community composition

中图分类号:

S718
S68

曹俐,金冬雪,姜静,等. 转BpGLK白桦土壤酶活及根际土壤细菌、真菌群落组成分析[J]. 南京林业大学学报（自然科学版）, 2024, 48(6): 129-137.

CAO Li, JIN Dongxue, JIANG Jing, LI Tianfang. Analysis of bacterial and fungal community composition and soil enzyme activities in the rhizosphere of transgenic Betula platyphylla[J].Journal of Nanjing Forestry University (Natural Science Edition), 2024, 48(6): 129-137.DOI: 10.12302/j.issn.1000-2006.202208001.

图/表 6

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微生物 microbial	样品 sample	可观测物种数 observed species	Chao1指数 Chao1 index	香农指数 Shannon index	辛普森指数 Simpson index	测序深度指数 coverage
细菌 bacteria	WT	4 868.37±170.75 a	5 491.06±122.26 a	10.51±0.14 a	0.997 6±0.000 3 a	0.985 7±0.000 4 ab
	OE1	4 107.43±537.31 a	4 724.57±559.73 a	9.47±0.51 b	0.989 6±0.004 7 a	0.998 59±0.00 1 ab
	OE2	4 743.73±94.58 a	5 363.12±109.37 a	9.95±0.19 ab	0.991 5±0.004 2 a	0.984 0±0.000 6 ab
	OE3	4 188.23±350.80 a	4 742.88±475.25 a	9.25±0.39 b	0.985 3±0.010 4 a	0.985 6±0.002 1 ab
	RE1	4 996.20±162.15 a	5 747.51±127.58 a	10.27±0.08 ab	0.996 5±0.000 8 a	0.982 2±0.000 2 b
	RE2	4 290.53±379.71 a	4 731.19±417.10 a	9.73±0.39 ab	0.990 4±0.004 8 a	0.986 9±0.001 7 a
	RE7	4 763.50±121.07 a	5 412.76±177.10 a	10.07±0.14 ab	0.994 6±0.001 0 a	0.983 9±0.000 8 ab
真菌 fungi	WT	256.10±26.30 bc	261.17±31.06 bc	2.68±0.31 bc	0.69±0.06 bc	0.999 7±0.000 2 a
	OE1	195.57±22.35 c	197.59±23.65 c	3.33±0.42 ab	0.82±0.06 ab	0.999 8±0.000 08 a
	OE2	262.75±33.45 bc	267.20±30.09 bc	1.89±0.01 c	0.42±0.04 d	0.999 7±0.000 12 a
	OE3	313.80±40.90 bc	319.76±37.89 bc	2.90±0.10 bc	0.63±0.10 c	0.999 7±0.000 07 a
	RE1	356.27±19.35 b	384.18±27.02 b	3.58±0.27 ab	0.82±0.02 ab	0.999 0±0.000 20 b
	RE2	488.43±3.00 a	515.52±3.72 a	4.39±0.23 a	0.87±0.02 a	0.999 1±0.000 14 b
	RE7	331.00±98.70 b	347.15±111.00 b	3.32±0.49 ab	0.77±0.04 ab	0.999 4±0.000 34 ab

转BpGLK白桦土壤酶活及根际土壤细菌、真菌群落组成分析

Analysis of bacterial and fungal community composition and soil enzyme activities in the rhizosphere of transgenic Betula platyphylla

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