固氮模块在耐辐射异常球菌底盘中的表达特性分析

doi:10.13304/j.nykjdb.2023.0226

摘要/Abstract

摘要：

利用生物技术方法将固氮模块转入真核细胞实现真核生物自主固氮具有重大科学意义和应用价值，迄今为止始终未能获得具有自主固氮能力的真核生物。为了探究固氮模块在真核底盘中的表达适配性，以进化地位介于原核生物和真核生物之间的极端微生物耐辐射异常球菌（Deinococcus radiodurans ）R1为底盘，将类芽孢杆菌（Paenibacillus polymyxa）WLY78的固氮模块导入耐辐射异常球菌中获得重组耐辐射异常球菌R78，并利用qPCR （quantitative real-time PCR）和Western Blot等方法，同时结合转录组分析，对重组耐辐射异常球菌的表达特性进行探究。结果表明，重组耐辐射异常球菌固氮模块中的9个固氮基因均能够正常转录，但是固氮酶铁蛋白的翻译受到影响；转录组测序结果显示，参与能量传递、氮代谢以及铁硫转运的相关基因的表达变化可能是影响重组菌株中固氮酶表达的限制因子。以上研究结果为进一步的固氮模块设计和微生物底盘优化，并最终构建人工高效固氮装置奠定了理论基础。

关键词: 生物固氮, 耐辐射异常球菌, 固氮模块, 底盘适配

Abstract:

Transferring the nitrogen fixation module into a eukaryotic cell to accomplish autonomous nitrogen fixation in eukaryotes via biotechnology is of enormous scientific significance and application benefit. So far， no eukaryotes have been discovered that can fix nitrogen on their own. In order to explore the expression adaptability of nitrogen fixation modules in eukaryotic chassis， the nitrogen-fixing module of Paenibacillus polymyxa WLY78 was introduced into the Deinococcus radiodurans R1， which was a gram-positive model bacterium with an evolutionary position between prokaryotes and eukaryotes， to obtain the Deinococcus radiodurans R78. The expression characteristics of the recombinant Deinococcus radiodurans were investigated by qPCR（quantitative real-time PCR）and Western Blot analysis. The results showed that the genes related to the nine genes in nitrogen fixation module of the recombinant Deinococcus radiodurans could be transcribed normally， but the translation of nitrogenase ferritin genes was affected. Transcriptome results showed that the expression changes of related genes involved in energy transfer， nitrogen metabolism， iron and sulfur transport might be the limiting factors affecting the expression of nitrogenase in the recombinant strain. Above results laid a theoretical foundation for further design of nitrogen fixation module and chassis optimization， and finally built artificial high-efficiency nitrogen fixation device.

Key words: biological nitrogen fixation, Deinococcus radiodurans, nitrogen-fixing module, chassis adaptation

中图分类号:

Q935

邱雪萌, 郑娟, 薛威, 毋少宇, 祁陈, 韩月月, 燕永亮, 战嵛华. 固氮模块在耐辐射异常球菌底盘中的表达特性分析[J]. 中国农业科技导报, 2023, 25(5): 77-86.

Xuemeng QIU, Juan ZHENG, Wei XUE, Shaoyu WU, Chen QI, Yueyue HAN, Yongliang YAN, Yuhua ZHAN. Analyses of Deinococcus radiodurans Chassis Expressing a Nitrogen-fixing Module[J]. Journal of Agricultural Science and Technology, 2023, 25(5): 77-86.

图/表 11

表 1 试验所用引物

Table 1 Primers used in this study

引物名称 Name of primer	序列 Sequence （5’→3’）	扩增基因 Amplified gene
nifB-F	TATCTCGAGGCCACCATGGACTCTTTAGCTGATCTC	nifB
nifB-R	CGCTGTACATCAGCCTCCAAAATGTTCAATG	nifB
nifH-F	CGCCTCGAGGCCACCATGAGACAAATTGCGTTTTAG	nifH
nifH-R	TGCTCTAGACTACTGCCCGGACGCCTCAGCTT	nifH
nifD-F	TATCTCGAGGCCACCATGAGCAGTATTGTGGATAAGG	nifD
nifD-R	AGCTCTAGATCATACCGGCACCTCCGCAGTCT	nifD
nifK-F	TATCTCGAGGCCACCATGGAGCCTGCTGCCCTCAC	nifK
nifK-R	GGCTGTACATTAGCGCACCAGATCAAAGCTATGCCTTTGC	nifK
nifE-F	TATCTCCAGGCCACCATGGAGCCGGCTGTGTCTAACG	nifE
nifE-R	GCGTCTAGATTAATCAGCAAATTCCGTCTCCTCC	nifE
nifN-F	TATCTCGAGGCCACCATGTATCGCTCAATGCCTTTGC	nifN
nifN-R	TATTCTAGATCATCCTCCCCTCCCCCCTCTCGGCAA	nifN
nifX-F	TATCTCGAGGCCACCATGAAGGTTGCATTTGCGACGG	nifX
nifX-R	GCGTCTAGATCATGATTCGGATTTGCCGCTGC	nifX
hesA-F	TATCTCGAGACCATGCTGAGGAGGGCAGCG	hesA
hesA-R	GCGTCTAGATCACAAATATGCAACCGACTC	hesA
nifV-F	TATCTCGAGGCCACCATGAGTCGGTTGCATATTTGTG	nifV
nifV-R	GCGTCTAGATTATTTCGGCAGCTCTGCCGGAT	nifV

图 1 重组耐辐射异常球菌R78构建及验证A：pRADZ3-78质粒构建示意图；B：重组耐辐射异常球菌R78中固氮基因的PCR验证。M—Trans2K Plus II DNA marker；1~9—nifB， nifH， nifD， nifK， nifE， nifN， nifX， hesA， nifV基因PCR扩增产物；10—阴性对照，以H2O为模板

Fig. 1 Construction and verification of recombinant D. radiodurans R78 strainA：Schematic representation of constructed recombinant plasmid pRADZ3-78； B：PCR verification of nif genes in recombinant strain D. radiodurans R78. M—Trans2K Plus II DNA marker. 1~9—PCR product of nifB， nifH， nifD， nifK， nifE， nifN， nifX， hesA and nifV genes；10—Negative control， with H2O as template

图 2 菌株的固氮酶活性A：重组大肠杆菌E. coli Z3；B：野生型施氏假单胞菌A1501

Fig. 2 Nitrogenase activities of bacteriaA： Recombinant E. coli Z3；B：Wild type P. stutzeri A1501

图3 野生型D. radiodurans R1和重组菌株D. radiodurans R78在TGY培养基的生长曲线

Fig. 3 Growth curves of the wild-type strain D. radiodurans R1and recombinant strain D. radiodurans R78 in TGY medium

表2 不同氧含量下野生型D. radiodurans R1和重组菌株D. radiodurans R78在TGY培养基中的生长

Table 2 Growth of wild-type D. radiodurans R1 and recombinant strain D. radiodurans R78 in TGY medium under different oxygen contents

氧气含量 Oxygen content/%	耐辐射异常球菌 R78 D. radiodurans R78		耐辐射异常球菌 R1 D. radiodurans R1
氧气含量 Oxygen content/%	初始OD₆₀₀ Initial OD₆₀₀	最终OD₆₀₀（24 h后） Final OD₆₀₀（after 24 h）	初始OD₆₀₀ Initial OD₆₀₀	最终OD₆₀₀（24 h后） Final OD₆₀₀（after 24 h）
0.0	0.10	0.20±0.00	0.10	0.20±0.03
0.5	0.10	0.41±0.01	0.10	0.42±0.01
1.0	0.10	0.61±0.04	0.10	0.69±0.01
5.0	0.10	3.20±0.09	0.10	3.20±0.10
21.0	0.10	7.33±0.05	0.10	7.62±0.63

图4 重组耐辐射异常球菌R78和野生型施氏假单胞菌A1501的固氮酶活

Fig. 4 Nitrogenase activities of recombinant strain D. radiodurans R78 and wild type P. stutzeri A1501

图5 不同铵氧条件下耐辐射异常球菌R78中固氮相关基因的转录水平表达注：M—Trans2K Plus II DNA marker；1—阳性对照，基因组DNA；2~7—0 mmol·L-1 NH4++0.0% O2、0 mmol·L-1 NH4++0.5% O2、0 mmol·L-1 NH4++1.0% O2、0 mmol·L-1 NH4++21.0% O2、100 mmol·L-1 NH4++0.5% O2、100 mmol·L-1 NH4++21.0% O2条件下相应基因PCR扩增产物；8—阴性对照，去除基因组且不加入逆转录酶的RNA。

Fig. 5 Transcription level expression of nif genes in D. radiodurans R78 under different NH4+ and O2 conditionsNote：M—Trans2K Plus II DNA marker；1—Positive controls， genomic DNA； 2~7—PCR amplification products of corresponding genes under the conditions of 0 mmol·L-1 NH4++0.0% O2， 0 mmol·L-1 NH4++0.5% O2， 0 mmol·L-1 NH4++1.0% O2， 0 mmol·L-1 NH4++21.0% O2， 100 mmol·L-1 NH4++0.5% O2， 100 mmol·L-1 NH4++21.0% O2； 8—Negative control， RNA without genome and reverse transcriptase.

图6 耐辐射异常球菌R78中nifH基因在不同铵氧条件下的相对表达量注：*和***分别表示与100 mmol·L-1 NH4++21.0% O2处理相比在P<0.05和P<0.001水平差异显著；ns表示与100 mmol·L-1 NH4++21.0% O2处理相比差异不显著。

Fig. 6 Relative expression of nifH of the D. radiodurans R78 in different NH4+ and O2 conditionsNote：* and *** mean significantly differences with 100 mmol·L-1 NH4++21.0% O2 treatment at P<0.05 and P<0.001 levels，respectively； ns indicates no significant differences with 100 mmol·L-1 NH4++21.0% O2 treatment.

表3 数字PCR研究铵和氧对不同菌株固氮酶编码基因nifH转录的影响

Table 3 Effects of NH4+and O2 on nifH gene transcription in different strains by dPCR

菌株

Bacteria

条件

Condition

绝对表达量

Absolute expression/ （copies·ng^-1）

D. radiodurans R78

0 mmol·L^-1 NH

4 +

+0.5% O₂

1.57 × 10³

100 mmol·L^-1 NH

4 +

4 +

+0.5% O₂

1.94 × 10⁵

表3 数字PCR研究铵和氧对不同菌株固氮酶编码基因nifH转录的影响

Table 3 Effects of NH4+and O2 on nifH gene transcription in different strains by dPCR

菌株

Bacteria

条件

Condition

绝对表达量

Absolute expression/ （copies·ng^-1）

D. radiodurans R78

0 mmol·L^-1 NH

4 +

+0.5% O₂

1.57 × 10³

100 mmol·L^-1 NH

4 +

+21.0% O₂

1.89 × 10³

P. stutzeri A1501

0 mmol·L^-1 NH

4 +

+0.5% O₂

1.94 × 10⁵

图7 重组耐辐射异常球菌R78碳代谢途径中的差异表达基因注：红色标记为表达量上调基因，绿色标记为表达量下调基因。

Fig. 7 Differentially expressed genes in carbohydrate metabolism of recombinant strain D. radiodurans R78Note：Red marks are genes with up-regulated expression and green marks are genes with down-regulated expression.

图8 NifH蛋白表达情况A： Western Blot 检测固氮条件下NifH的表达； M—标准蛋白分子量Marker； 1~4—分别为野生型DR1、无氮厌氧条件下重组耐辐射异常球菌R78、无氮0.5%微好氧条件下重组耐辐射异常球菌R78和野生型A1501细胞破碎后上清。 B： SDS-PAGE分析蛋白质量；M—标准蛋白分子量Marker；7~10—分别为野生型DR1、无氮厌氧条件下重组耐辐射异常球菌R78、无氮0.5%微好氧条件下重组耐辐射异常球菌R78和野生型A1501细胞破碎后沉淀

Fig. 8 Expression of NifH proteinA： Western blot analysis of NifH detected with antiserum to NifH；M—Standard protein molecular weight marker； 1~4—Supernatant after ultrasonication of wild-type strain DR1， recombinant Deinococuus radiodurans R78 in 0 mmol·L-1 NH4+ and 0.0% O2，recombinant Deinococuus radiodurans R78 in 0 mmol·L-1 NH4+ and 0.5% O2 and wild-type strain A1501. B： SDS-PAGE analysis of protein quality； M—Standard protein molecular weight marker； 7~10—Precipitation after ultrasonication of wild-type strain DR1，recombinant Deinococuus radiodurans R78 in 0 mmol·L-1 NH4+ and 0.0% O2，recombinant Deinococuus radiodurans R78 in 0 mmol·L-1 NH4+ and 0.5% O2 and wild-type strain A1501

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