Journal of Agricultural Science and Technology ›› 2023, Vol. 25 ›› Issue (5): 77-86.DOI: 10.13304/j.nykjdb.2023.0226
• BIOTECHNOLOGY & LIFE SCIENCE • Previous Articles Next Articles
Xuemeng QIU(), Juan ZHENG, Wei XUE, Shaoyu WU, Chen QI, Yueyue HAN, Yongliang YAN(
), Yuhua ZHAN(
)
Received:
2023-03-24
Accepted:
2023-05-05
Online:
2023-05-20
Published:
2023-07-13
Contact:
Yongliang YAN,Yuhua ZHAN
邱雪萌(), 郑娟, 薛威, 毋少宇, 祁陈, 韩月月, 燕永亮(
), 战嵛华(
)
通讯作者:
燕永亮,战嵛华
作者简介:
邱雪萌E-mail: qiuxuemeng@163.com
基金资助:
CLC Number:
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.
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.
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 | |
nifH-F | CGCCTCGAGGCCACCATGAGACAAATTGCGTTTTAG | nifH |
nifH-R | TGCTCTAGACTACTGCCCGGACGCCTCAGCTT | |
nifD-F | TATCTCGAGGCCACCATGAGCAGTATTGTGGATAAGG | nifD |
nifD-R | AGCTCTAGATCATACCGGCACCTCCGCAGTCT | |
nifK-F | TATCTCGAGGCCACCATGGAGCCTGCTGCCCTCAC | nifK |
nifK-R | GGCTGTACATTAGCGCACCAGATCAAAGCTATGCCTTTGC | |
nifE-F | TATCTCCAGGCCACCATGGAGCCGGCTGTGTCTAACG | nifE |
nifE-R | GCGTCTAGATTAATCAGCAAATTCCGTCTCCTCC | |
nifN-F | TATCTCGAGGCCACCATGTATCGCTCAATGCCTTTGC | nifN |
nifN-R | TATTCTAGATCATCCTCCCCTCCCCCCTCTCGGCAA | |
nifX-F | TATCTCGAGGCCACCATGAAGGTTGCATTTGCGACGG | nifX |
nifX-R | GCGTCTAGATCATGATTCGGATTTGCCGCTGC | |
hesA-F | TATCTCGAGACCATGCTGAGGAGGGCAGCG | hesA |
hesA-R | GCGTCTAGATCACAAATATGCAACCGACTC | |
nifV-F | TATCTCGAGGCCACCATGAGTCGGTTGCATATTTGTG | nifV |
nifV-R | GCGTCTAGATTATTTCGGCAGCTCTGCCGGAT |
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 | |
nifH-F | CGCCTCGAGGCCACCATGAGACAAATTGCGTTTTAG | nifH |
nifH-R | TGCTCTAGACTACTGCCCGGACGCCTCAGCTT | |
nifD-F | TATCTCGAGGCCACCATGAGCAGTATTGTGGATAAGG | nifD |
nifD-R | AGCTCTAGATCATACCGGCACCTCCGCAGTCT | |
nifK-F | TATCTCGAGGCCACCATGGAGCCTGCTGCCCTCAC | nifK |
nifK-R | GGCTGTACATTAGCGCACCAGATCAAAGCTATGCCTTTGC | |
nifE-F | TATCTCCAGGCCACCATGGAGCCGGCTGTGTCTAACG | nifE |
nifE-R | GCGTCTAGATTAATCAGCAAATTCCGTCTCCTCC | |
nifN-F | TATCTCGAGGCCACCATGTATCGCTCAATGCCTTTGC | nifN |
nifN-R | TATTCTAGATCATCCTCCCCTCCCCCCTCTCGGCAA | |
nifX-F | TATCTCGAGGCCACCATGAAGGTTGCATTTGCGACGG | nifX |
nifX-R | GCGTCTAGATCATGATTCGGATTTGCCGCTGC | |
hesA-F | TATCTCGAGACCATGCTGAGGAGGGCAGCG | hesA |
hesA-R | GCGTCTAGATCACAAATATGCAACCGACTC | |
nifV-F | TATCTCGAGGCCACCATGAGTCGGTTGCATATTTGTG | nifV |
nifV-R | GCGTCTAGATTATTTCGGCAGCTCTGCCGGAT |
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
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 | ||
---|---|---|---|---|
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 |
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 | ||
---|---|---|---|---|
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 |
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.
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.
菌株 Bacteria | 条件 Condition | 绝对表达量 Absolute expression/ (copies·ng-1) |
---|---|---|
D. radiodurans R78 | 0 mmol·L-1 NH | 1.57 × 103 |
100 mmol·L-1 NH | 1.89 × 103 | |
P. stutzeri A1501 | 0 mmol·L-1 NH | 1.94 × 105 |
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 | 1.57 × 103 |
100 mmol·L-1 NH | 1.89 × 103 | |
P. stutzeri A1501 | 0 mmol·L-1 NH | 1.94 × 105 |
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.
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
1 | DIXON R, KAHN D. Genetic regulation of biological nitrogen fixation [J]. Nat. Rev. Microbiol., 2004, 2(8): 621-631. |
2 | MUTHUKUMARASAMY R, REVATHI G, SESHADRI S, et al.. Gluconacetobacter diazotrophicus (syn. Acetobacter diazotrophicus), a promising diazotrophic endophyte in tropics [J]. Current. Sci., 2002, 83(2): 137-145. |
3 | 林敏.转基因技术[M].北京: 中国农业科学技术出版社,2020:1-318. |
4 | RAYMOND J, SIEFERT J L, STAPLES C R, et al.. The natural history of nitrogen fixation [J]. Mol. Biol. Evol., 2004, 21(3): 541-554. |
5 | IVLEVA N B, GROAT J, STAUB J M, et al.. Expression of active subunit of nitrogenase via integration into plant organelle genome [J/OL]. PLoS ONE, 2016, 11(8): e0160951[2023-04-06]. . |
6 | DIXON R A, POSTGATE J R. Genetic transfer of nitrogen fixation from Klebsiella pneumoniae to Escherichia coli [J]. Nature, 1972, 237(5350): 102-103. |
7 | 燕永亮,田长富,杨建国,等.人工高效生物固氮体系创建及其农业应用[J].生命科学,2021, 33(12): 1532-1543. |
YAN Y L, TIAN C F, YANG J G, et al.. Establishment of artificial efficiency biological nitrogen fixation system and its agricultural application [J]. Chin. Bull. Life Sci., 2021, 33(12): 1532-1543. | |
8 | MINTON K W, DALY M J. A model for repair of radiation-induced DNA double-strand breaks in the extreme radiophile Deinococcus radiodurans [J]. Bioessays, 1995, 17(5): 457-464. |
9 | GERBER E, BERNARD R, CASTANG S, et al.. Deinococcus as new chassis for industrial biotechnology: biology, physiology and tools [J/OL]. J. Appl. Microbiol., 2015, 119(1): 12808 [2023-04-06]. . |
10 | WANG L, ZHANG L, LIU Z, et al.. A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli [J/OL]. PLoS Genet., 2013, (10): e1003865 [2023-04-06].. |
11 | 刘盈盈. 耐辐射异常球菌亲水蛋白DohL具有类分子伴侣和核酸内切酶功能并参与氧化胁迫保护[D]. 北京: 中国农业科学院, 2019. |
LIU Y Y. Chaperone-like and catalytic functions in Deinococcus radiodurans of hydrophilic protein DohL involved in protective against oxidative stress [D]. Beijing: Chinese Academy of Agricultural Sciences, 2019. | |
12 | 王莉瑛. 固氮类芽孢杆菌分离鉴定及固氮基因簇在大肠杆菌中的表达[D]. 北京: 中国农业大学, 2013. |
WANG L Y. Isolation and identification of nitrogen-fixing Paenibacillus spp. and expression of nitrogen fixation (nif) gene cluster in Escherichia coli [D]. Beijing: China Agricultural University, 2013. | |
13 | HERRIDGE D F, PEOPLES M B, BODDEY R M. Global inputs of biological nitrogen fixation in agricultural systems [J/OL]. Plant Soil, 2008, 311(1-2): 9668-3 [2023-04-06]. . |
14 | GEDDES B A, RYU M H, MUS F, et al.. Use of plant colonizing bacteria as chassis for transfer of N2-fixation to cereals [J]. Curr. Opin. Biotechnol., 2015, 32: 216-222. |
15 | ARNOLD W, RUMP A, KLIPP W, et al.. Nucleotide sequence of a 24 206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae [J]. J. Mol. Biol., 1988, 203(3): 715-738. |
16 | PAUL W, MERRICK M. The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis [J]. Eur. J. Biochem., 1989, 178(3): 675-682. |
17 | HERNANDEZ J A, IGARASHI R Y, SOBOH B, et al.. NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway [J]. Mol. Microbiol., 2007, 63(1): 177-192. |
18 | LI Q, CHEN S. Transfer of nitrogen fixation (nif) genes to non-diazotrophic hosts [J]. Chembiochem, 2020, 21(12): 1717-1722. |
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