Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (5): 149-158.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1067
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ZHAO Ming-ming1(), TANG Yin1, GUO Lei-zhou1, HAN Jia-hui1, GE Jia-ming1, MENG Yong2, PING Shu-zhen1, ZHOU Zheng-fu1, WANG Jin1()
Received:
2021-08-09
Online:
2022-05-26
Published:
2022-06-10
Contact:
WANG Jin
E-mail:82101182064@caas.cn;wangjin@caas.cn
ZHAO Ming-ming, TANG Yin, GUO Lei-zhou, HAN Jia-hui, GE Jia-ming, MENG Yong, PING Shu-zhen, ZHOU Zheng-fu, WANG Jin. Function Analysis of Lon1 Protease Involved in High Temperature Stress and Cell Division of Deinococcus radiodurans R1[J]. Biotechnology Bulletin, 2022, 38(5): 149-158.
菌株及质粒Strains and plasmids | 特性Relevant characteristic | 来源Source | |
---|---|---|---|
菌株 | Deinococcus radioduransR1 | 耐辐射异常球菌野生型 | 中科院微生物所菌种保藏中心 |
△lon1 | 缺失lon1基因的突变株(Kanr) | 本研究构建 | |
comlon1 | lon1回补株(Kanr+ Cmr) | 本研究构建 | |
Z3-△lon1 | △lon1中导入质粒pRADZ3(Cmr) | 本研究构建 | |
质粒 | pRAD Z3 | 构建回补菌株所用质粒 | 本实验室保存 |
Table 1 Strains and Plasmids
菌株及质粒Strains and plasmids | 特性Relevant characteristic | 来源Source | |
---|---|---|---|
菌株 | Deinococcus radioduransR1 | 耐辐射异常球菌野生型 | 中科院微生物所菌种保藏中心 |
△lon1 | 缺失lon1基因的突变株(Kanr) | 本研究构建 | |
comlon1 | lon1回补株(Kanr+ Cmr) | 本研究构建 | |
Z3-△lon1 | △lon1中导入质粒pRADZ3(Cmr) | 本研究构建 | |
质粒 | pRAD Z3 | 构建回补菌株所用质粒 | 本实验室保存 |
扩增产物 Product for PCR amplification | 引物名称 Primer name | 序列Sequence(5'-3') | 片段长度Size/bp |
---|---|---|---|
lon1-Up | lon1-UP-F | AGCTGCTGACCCGTGACACCGAGCCATGGAGACCGAGGGCCC | 613 |
lon1-up-R | GGGCCCTCGGTCTCCATGGCTCGGTGTCACGGGTCAGCAGCT | ||
lon1-Kan | Kan-lon1-F | AGCTGCTGACCCGTGACACCGAGCCATGGAGACCGAGGGCCC | 965 |
Kan-Down-R | TGGTCCACGCCAGGCCCTGCGTTAGAAAAACTCATCGAGCAT | ||
lon1-Down | lon1-Down-F | ATGCTCGATGAGTTTTTCTAACGCAGGGCCTGGCGTGGACCA | 614 |
lon1-Down-R | TTATGCGCCCGGCTGCACCGA | ||
Kan内部序列 | YZ-Km-F | CGATTGTATGGGAAGCCCGAT | 591 |
YZ-Km-R | CTCACCGAGGCAGTTCCATAG | ||
敲除的lon1序列 | YZ-lon1-F | ACCGTGGTTCGCAACTACAT | 400 |
YZ-lon1-R | CGATCTCGACCTTCTCCTGC |
Table 2 Sequences of Primers
扩增产物 Product for PCR amplification | 引物名称 Primer name | 序列Sequence(5'-3') | 片段长度Size/bp |
---|---|---|---|
lon1-Up | lon1-UP-F | AGCTGCTGACCCGTGACACCGAGCCATGGAGACCGAGGGCCC | 613 |
lon1-up-R | GGGCCCTCGGTCTCCATGGCTCGGTGTCACGGGTCAGCAGCT | ||
lon1-Kan | Kan-lon1-F | AGCTGCTGACCCGTGACACCGAGCCATGGAGACCGAGGGCCC | 965 |
Kan-Down-R | TGGTCCACGCCAGGCCCTGCGTTAGAAAAACTCATCGAGCAT | ||
lon1-Down | lon1-Down-F | ATGCTCGATGAGTTTTTCTAACGCAGGGCCTGGCGTGGACCA | 614 |
lon1-Down-R | TTATGCGCCCGGCTGCACCGA | ||
Kan内部序列 | YZ-Km-F | CGATTGTATGGGAAGCCCGAT | 591 |
YZ-Km-R | CTCACCGAGGCAGTTCCATAG | ||
敲除的lon1序列 | YZ-lon1-F | ACCGTGGTTCGCAACTACAT | 400 |
YZ-lon1-R | CGATCTCGACCTTCTCCTGC |
Fig. 3 Construction and identification of the lon1 mutant strain A:Schematic representation of the Δlon1mutant. B:Electrophoretogram of the lon1 mutant strain(M:Trans 2K PlusⅡ DNA marker. 1, 4, 7:PCR products amplified from wild type D. radiodurans. 2, 5, 8:PCR products amplified from the lon1mutant. 3, 6, 9:Negative control). C:Effects of lon1 deleted on adjacent genes in transcriptional level
UniProtKB | Locus | Predicted function | Protein name | FC |
---|---|---|---|---|
Protein fate(heat shock responsive chaperones and protease) | ||||
Q9RV58 | DR_1172 | Chaperone-like protein | DosH | 13.37 |
Q9RWQ9 | DR_0607 | 60 kD chaperone | GroL | 7.08 |
Q9RVI3 | DR_1046 | Chaperone protein ClpB | ClpB | 4.70 |
Q9RXG4 | DR_0349 | Lon protease | Lon2 | 4.52 |
Q9RWR0 | DR_0606 | 10 kD chaperone | GroS | 4.46 |
Q9RY24 | DR_0128 | Chaperone protein GrpE | GrpE | 3.24 |
DNA metabolism(repair and recombination) | ||||
Q9RXI7 | DR_0326 | DNA damage response protein D | DdrD | 2.59 |
Q9RS64 | DR_2263 | DNA protection during starvation protein 1 | Dps1 | 2.88 |
Q9RY51 | DR_0099 | Single-stranded DNA-binding protein | Ssb | 3.25 |
Q9RSJ6 | DR_2128 | DNA-directed RNA polymerase subunit alpha | RpoA | 2.06 |
Q9RRE4 | DR_2548 | Transcriptional regulatory protein | 7.75 | |
Membrane functions and transport | ||||
Q9RWU0 | DR_0575 | Protein translocase subunit SecA | SecA | 2.18 |
P56867 | DR_2508 | Hexagonally packed intermediate-layer surface protein | Hpi | 3.48 |
Q9RWH3 | DR_0695 | V-type ATP synthase subunit | AtpI | 1.57 |
Translation | ||||
Q9RST0 | DR_2043 | 50S ribosomal protein L7/L12 | RplL | 0.14 |
Central intermediary metabolism(synthesis of nitrogen,sulfur,polyamine compounds) | ||||
Q9RR70 | DR_2627 | Fumarate hydratase class II | FumC | 7.59 |
Q9RWB2 | DR_0757 | Citrate synthase | GltA | 3.06 |
Q9RZ06 | DR_A0147 | Histidine ammonia-lyase | HutH | 2.66 |
Q9RTN7 | DR_1720 | Aconitate hydratase A | Acn | 2.10 |
P56861 | DR_A0014 | Adenylyl-sulfate kinase | CysC | 0.23 |
Amino acid biosynthesis and energy metabolism | ||||
Q9RRT0 | DR_2405 | Demethylmenaquinone methyltransferase | MenG | 0.51 |
Q9RV69 | DR_1161 | 5-hydroxyisourate hydrolase | 0.33 | |
Q9RV98 | DR_1131 | Ferrochelatase | HemH | 2.04 |
Q9RRC4 | DR_2568 | Arginine--tRNA ligase | ArgS | 0.54 |
Cell morphogenesis | ||||
Q9RRJ4 | DR_2496 | UDP-N-acetylmuramoylalanine--D-glutamate ligase | MurD | 0.65 |
Q9RWN8 | DR_0628 | UDP-N-acetylenolpyruvoylglucosamine reductase | MurB | 0.59 |
Q9RXF1 | DR_0362 | D-alanine--D-alanine ligase | Ddl | 0.46 |
Q9RWN9 | DR_0627 | UDP-N-acetylmuramate--L-alanine ligase | MurC | 0.42 |
Q9RXL3 | DR_0297 | UDP-N-acetylmuramyl-tripeptide synthetase | MurE | 0.40 |
Table 3 Differentially expressed proteins associated with lon1 in response to high temperature stress
UniProtKB | Locus | Predicted function | Protein name | FC |
---|---|---|---|---|
Protein fate(heat shock responsive chaperones and protease) | ||||
Q9RV58 | DR_1172 | Chaperone-like protein | DosH | 13.37 |
Q9RWQ9 | DR_0607 | 60 kD chaperone | GroL | 7.08 |
Q9RVI3 | DR_1046 | Chaperone protein ClpB | ClpB | 4.70 |
Q9RXG4 | DR_0349 | Lon protease | Lon2 | 4.52 |
Q9RWR0 | DR_0606 | 10 kD chaperone | GroS | 4.46 |
Q9RY24 | DR_0128 | Chaperone protein GrpE | GrpE | 3.24 |
DNA metabolism(repair and recombination) | ||||
Q9RXI7 | DR_0326 | DNA damage response protein D | DdrD | 2.59 |
Q9RS64 | DR_2263 | DNA protection during starvation protein 1 | Dps1 | 2.88 |
Q9RY51 | DR_0099 | Single-stranded DNA-binding protein | Ssb | 3.25 |
Q9RSJ6 | DR_2128 | DNA-directed RNA polymerase subunit alpha | RpoA | 2.06 |
Q9RRE4 | DR_2548 | Transcriptional regulatory protein | 7.75 | |
Membrane functions and transport | ||||
Q9RWU0 | DR_0575 | Protein translocase subunit SecA | SecA | 2.18 |
P56867 | DR_2508 | Hexagonally packed intermediate-layer surface protein | Hpi | 3.48 |
Q9RWH3 | DR_0695 | V-type ATP synthase subunit | AtpI | 1.57 |
Translation | ||||
Q9RST0 | DR_2043 | 50S ribosomal protein L7/L12 | RplL | 0.14 |
Central intermediary metabolism(synthesis of nitrogen,sulfur,polyamine compounds) | ||||
Q9RR70 | DR_2627 | Fumarate hydratase class II | FumC | 7.59 |
Q9RWB2 | DR_0757 | Citrate synthase | GltA | 3.06 |
Q9RZ06 | DR_A0147 | Histidine ammonia-lyase | HutH | 2.66 |
Q9RTN7 | DR_1720 | Aconitate hydratase A | Acn | 2.10 |
P56861 | DR_A0014 | Adenylyl-sulfate kinase | CysC | 0.23 |
Amino acid biosynthesis and energy metabolism | ||||
Q9RRT0 | DR_2405 | Demethylmenaquinone methyltransferase | MenG | 0.51 |
Q9RV69 | DR_1161 | 5-hydroxyisourate hydrolase | 0.33 | |
Q9RV98 | DR_1131 | Ferrochelatase | HemH | 2.04 |
Q9RRC4 | DR_2568 | Arginine--tRNA ligase | ArgS | 0.54 |
Cell morphogenesis | ||||
Q9RRJ4 | DR_2496 | UDP-N-acetylmuramoylalanine--D-glutamate ligase | MurD | 0.65 |
Q9RWN8 | DR_0628 | UDP-N-acetylenolpyruvoylglucosamine reductase | MurB | 0.59 |
Q9RXF1 | DR_0362 | D-alanine--D-alanine ligase | Ddl | 0.46 |
Q9RWN9 | DR_0627 | UDP-N-acetylmuramate--L-alanine ligase | MurC | 0.42 |
Q9RXL3 | DR_0297 | UDP-N-acetylmuramyl-tripeptide synthetase | MurE | 0.40 |
Fig. 6 Electron microscopy images of WT D. radiodurans DR,△lon1 and comlon1 A:Transmission electron microscopy(TEM). B:Scanning electron microscopy(SEM)
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