基因envZ、iscS、asnC调控根瘤菌胞外多糖产量和环境适应性

doi:10.13560/j.cnki.biotech.bull.1985.2024-0388

摘要/Abstract

摘要：

【目的】 Rhizobium yanglingense CCBAU 01603可与豆科锦鸡儿属植物形成高效固氮根瘤，前期研究在含碱培养基中连续进化500代得到进化菌株，发现与其他进化菌株相比，Alk_40中基因envZ、iscS、asnC发生特异SNPs变化导致胞外多糖产量大幅降低，进而探究关键基因调控根瘤菌胞外多糖产量和环境适应性。【方法】 采用同源重组法构建根瘤菌envZ、iscS、asnC单基因缺失突变株，测定突变株的胞外多糖产量和不同环境条件下的生长情况。【结果】 根瘤菌envZ、iscS、asnC基因缺失突变株在YMA培养基中胞外多糖产量显著下降，酸性环境适应性降低，65℃热处理的耐受能力减弱，且胞外多糖产量与根瘤菌耐酸性和耐热性具有一定相关性；根瘤菌envZ和asnC基因缺失突变株对NaCl十分敏感，在含NaCl培养基中的生长繁殖严重受限，在弱碱性环境中适应能力显著降低。【结论】 根瘤菌胞外多糖产量受基因iscS、envZ、asnC的调控。

关键词: 根瘤菌, envZ, iscS, asnC, 胞外多糖, 环境适应性

Abstract:

【Objective】 Rhizobium yanglingense CCBAU 01603 form efficient nitrogen-fixing nodules with Caragana legumes. Alk_40, one of the offspring of CCBAU 01603, was obtained through experimental evolution in alkaline medium for 500 generations. It was discovered that a significant decrease in exopolysaccharide（EPS）yields of Alk_40 with specific SNPs changes in the gene envZ, iscS, and asnC. EPS yields and environmental adaptability of rhizobia regulated by these key genes were investigated. 【Method】 Three mutants of rhizobia were created by deleting gene envZ, iscS, and asnC, respectively. EPS yields and growth under different environmental conditions of mutants were assessed. 【Result】 In comparison to CCBAU 01603, the envZ, iscS, and asnC mutants presented significantly lower EPS yields in YMA medium, demonstrated diminished fitness in acidic environments, and had a weakened tolerance to high temperatures of 65℃. This observation suggests a correlation between the EPS yield of rhizobia and their resistances to acidic or heat stress. In addition, the mutants lacking the envZ and asnC genes showed the sensitivity to NaCl, resulting in severely limited growth and reproduction in NaCl-containing media. Furthermore, their adaption in alkalescent environments significantly reduced as well. 【Conclusion】 It is discovered that EPS yields of rhizobia can be regulated by gene envZ, iscS, and asnC.

Key words: Rhizobium, envZ, iscS, asnC, exopolysaccharide, environmental adaptation

杨冰洁, 袁晓霞, 高梦哲, 申奥龙, 李华, 冀照君. 基因envZ、iscS、asnC调控根瘤菌胞外多糖产量和环境适应性[J]. 生物技术通报, 2024, 40(11): 277-284.

YANG Bing-jie, YUAN Xiao-xia, GAO Meng-zhe, SHEN Ao-long, LI Hua, JI Zhao-jun. Exopolysaccharide Yield and Environmental Adaptation of Rhizobia Regulated by Gene envZ, iscS and asnC[J]. Biotechnology Bulletin, 2024, 40(11): 277-284.

图/表 8

表1 供试菌株列表

Table 1 Strains in this study

菌株 Strain	特征 Description	来源 Source
R. yanglingense CCBAU 01603	野生型菌株 Wild-type strain NA^r	实验室保藏 Preserved in Lab
Alk_40	CCBAU 01603的进化菌株 Evolved clone of CCBAU 01603	实验室保藏 Preserved in Lab
ΔenvZ	envZ基因敲除突变株 Gene envZ deletion mutant NA^r	已构建 Established in Lab
ΔiscS	iscS基因敲除突变株 Gene iscS deletion mutant NA^r	已构建 Established in Lab
ΔasnC	asnC基因敲除突变株 Gene asnC deletion mutant NA^r	已构建 Established in Lab

图1 R. yanglingense CCBAU01603与进化菌株Alk_40关键基因序列及翻译的氨基酸序列比对分析 A为envZ基因序列及氨基酸组成比对结果；B为iscS基因序列及氨基酸组成比对结果；C为asnC基因序列及氨基酸组成比对结果

Fig. 1 Comparative analysis of key gene sequences and translated amino acid sequences of R. yanglingense CCBAU01603 and evolved Alk_40 A is from the comparison of the envZ gene sequence and amino acid composition; B is from the comparison of the iscS gene sequence and amino acid composition; C is from the comparison of the asnC gene sequence and amino acid composition

图2 YMA固体培养基上根瘤菌生长情况

Fig. 2 Growth of each rhizobia on YMA solid medium

图3 根瘤菌胞外多糖产量

Fig. 3 Exopolysaccharide yield among different rhizobia *: 0.01<P≤0.05; **: 0.001<P≤0.01; ***: 0.0001<P≤0.001; ****: P ≤0.0001, the same below

图4 根瘤菌在含不同浓度NaCl培养基中生长情况 A：含0.1% NaCl的液体培养基；B：含0.2% NaCl的液体培养基；C：含0.3% NaCl的液体培养基；D：含0.4% NaCl的液体培养基

Fig. 4 Growth of rhizobia in the media with different concentrations of NaCl A: Liquid medium containing 0.1% NaCl. B: Liquid medium containing 0.2% NaCl. C: Liquid medium containing 0.3% NaCl. D: Liquid medium containing 0.4% NaCl

表2 胞外多糖产量与菌液OD600值的相关性分析

Table 2 Correlation between exopolysaccharide yield and OD600 values

环境条件Environmental conditions		皮尔逊相关系数Pearson’s correlation coefficient	显著性（双尾） Sig（2-tailed）（P）
盐浓度NaCl concentrations	0.1%	0.734ns	0.158
	0.2%	0.613ns	0.271
	0.3%	0.508ns	0.382
	0.4%	-0.211ns	0.733
pH 值 pH Value	4.02	0.981**	0.003
	5.01	-0.087ns	0.89
	8.99	0.389ns	0.517
	9.98	0.138ns	0.825
热处理温度Heat treatment temperature	28℃	0.976**	0.004
	37℃	0.840ns	0.075
	65℃	0.979^**	0.004

图5 根瘤菌在不同pH值的培养基中生长情况 A：pH=4.02的液体培养基；B：pH=5.01的液体培养基；C：pH=6.99的液体培养基；D：pH=8.99的液体培养基；E：pH=9.98的液体培养基

Fig. 5 Growth of rhizobia in the medium with different pH A: Liquid medium with pH=4.02; B: liquid medium with pH=5.01; C: liquid medium with pH=6.99; D: liquid medium with pH=8.99; E: liquid medium with pH=9.98

图6 不同温度处理的根瘤菌生长情况 A：28℃处理10 min的阴性对照组；B：37℃处理10 min；C：65℃处理10 min

Fig. 6 Growth of rhizobia after treatment at different temperature A: Negative control group treated at 28℃ for 10 min; B: treated at 37℃ for 10 min; C: treated at 65℃ for 10 min

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