耐辐射异常球菌dlp基因缺失突变株构建及其生物学功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2017.02.023

生物技术通报 ›› 2017, Vol. 33 ›› Issue (2): 155-163.doi: 10.13560/j.cnki.biotech.bull.1985.2017.02.023

耐辐射异常球菌dlp基因缺失突变株构建及其生物学功能研究

刘小利^1,2, 江世杰^2,3, 薛冬², 刘盈盈², 吴小丽^1,2, 冯帅^1,2, 韩佳慧², 汪雨舟⁴, 平淑珍², 王劲^1,2

1. 西南科技大学生命科学与工程学院,绵阳 621000;
2. 中国农业科学院生物技术研究所,北京 100081;
3. 四川大学生命科学学院,成都 610065;
4. 中国人民大学附属中学,北京 100080

收稿日期:2016-11-07 出版日期:2017-02-26 发布日期:2017-02-08
作者简介:刘小利,女,硕士研究生,研究方向：特殊环境微生物功能基因资源利用;E-mail：lxl615wzy@sina.com
基金资助:
国家重点基础研究发展计划（“973计划”）（2013CB733903）

Construction and Biological Characterization of Gene dlp Deletion Mutant of Deinococcus radiodurans R1

LIU Xiao-li^1,2, JIANG Shi-jie^2,3, XUE Dong², LIU Ying-ying², WU Xiao-li^1,2, FENG Shuai^1,2, HAN Jia-hui², WANG Yu-zhou⁴, PING Shu-zhen², WANG Jin^1,2

1. College of Life Science and Engineering,Southwest University of Science and Technology,Mianyang 621000;
2. Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081;
3. College of Life Sciences,Sichuan University,Chengdu 610065;
4. The High School Affiliated to Renmin University of China,Beijing 100080

Received:2016-11-07 Published:2017-02-26 Online:2017-02-08

摘要/Abstract

摘要： 耐辐射异常球菌（Deinococcus radiodurans,DR）因其具有对非生物胁迫超强的抵抗能力而备受关注。旨在了解该菌亲水蛋白Dlp在细胞耐受非生物胁迫中的作用,采用融合PCR和基因同源重组技术,获得了dlp基因缺失突变株Δdlp,对野生型DR及突变株Δdlp进行非生物胁迫。结果表明,dlp的缺失导致DR细胞对高盐和氧化胁迫敏感。体外检测氧化胁迫条件下Dlp蛋白对苹果酸脱氢酶（MDH）和乳酸脱氢酶（LDH）活性的保护,结果显示,Dlp蛋白的加入能缓解氧化胁迫条件下MDH、LDH酶活性的损失。由此表明,亲水蛋白Dlp增强了耐辐射异常球菌对非生物胁迫的抗性,并能以类似分子伴侣的形式保护胁迫条件下酶的活性。

关键词: 耐辐射异常球菌, 基因突变, 亲水蛋白Dlp, 非生物胁迫, 酶活性

Abstract: Deinococcus radiodurans（DR）has drawn growing attentions for its superior resistance to abiotic stress. To investigate the effect of the hydrophilic protein Dlp in cell tolerance to abiotic stress,the dlp deletion mutant strain（Δdlp）was obtained by fusion PCR and homologous recombination in vivo. The assays of wild strain DR and mutant Δdlp were analyzed under abiotic stress. The results showed that the deletion of dlp gene in D. radiodurans led cells to be more sensitive to high-salt and oxidative stresses. The in vitro protective effect of Dlp protein on the activities of MDH and LDH was measured under oxidative stress condition;and the data demonstrated that the addition of Dlp protein alleviated the loss of activities of MDH and LDH under oxidative stress. Our findings indicated that hydrophilic protein Dlp enhanced the resistance of D. radiodurans to abiotic stresses,and functioned as chaperone-like protein to protect enzyme activity under abiotic stresses.

Key words: Deinococcus radiodurans, gene mutant, hydrophilic protein Dlp, abiotic stress, enzyme activity

刘小利, 江世杰, 薛冬, 刘盈盈, 吴小丽, 冯帅, 韩佳慧, 汪雨舟, 平淑珍, 王劲. 耐辐射异常球菌dlp基因缺失突变株构建及其生物学功能研究[J]. 生物技术通报, 2017, 33(2): 155-163.

LIU Xiao-li, JIANG Shi-jie, XUE Dong, LIU Ying-ying, WU Xiao-li, FENG Shuai, HAN Jia-hui, WANG Yu-zhou, PING Shu-zhen, WANG Jin. Construction and Biological Characterization of Gene dlp Deletion Mutant of Deinococcus radiodurans R1[J]. Biotechnology Bulletin, 2017, 33(2): 155-163.

参考文献

[1] Goyal K, Walton LJ, Tunnacliffe A. LEA proteins prevent protein aggregation due to water stress[J]. Biochemical Journal, 2005, 388（1）：151-157.
[2] Caramelo JJ, Iusem ND. When cells lose water：Lessons from biophysics and molecular biology[J]. Progress in Biophysics and Molecular Biology, 2009, 99（1）：1-6.
[3] Potts M. Desiccation tolerance：a simple process?[J]. Trends in Microbiology, 2001, 9（11）：553-559.
[4] Hansen JM, Go YM, Jones DP. Nuclear and mitochondrial compartmentation of oxidative stress and redox signaling[J]. Annu Rev Pharmacol Toxicol, 2006, 46：215-234.
[5] Garay-Arroyo A, Colmenero-Flores JM, Garciarrubio A, et al. Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit[J]. Journal of Biological Chemistry, 2000, 275（8）：5668-5674.
[6] Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein[J]. Journal of Molecular Biology, 1982, 157（1）：105-132.
[7] Anchordoquy TJ, Carpenter JF. Polymers protect lactate dehydrogenase during freeze-drying by inhibiting dissociation in the frozen state[J]. Archives of Biochemistry and Biophysics, 1996, 332（2）：231-238.
[8] Soulages JL, Kim K, Walters C, et al. Temperature-induced extended helix/random coil transitions in a group 1 late embryogenesis-abundant protein from soybean[J]. Plant Physiology, 2002, 128（3）：822-832.
[9] Reyes JL, Campos F, Wei H, et al. Functional dissection of hydrophilins during in vitro freeze protection[J]. Plant, Cell & Environment, 2008, 31（12）：1781-1790.
[10] Grelet J, Benamar A, Teyssier E, et al. Identification in pea seed mitochondria of a late-embryogenesis abundant protein able to protect enzymes from drying[J]. Plant Physiology, 2005, 137（1）：157-167.
[11] Chakrabortee S, Tripathi R, Watson M, et al. Intrinsically disordered proteins as molecular shields[J]. Molecular BioSystems, 2012, 8（1）：210-219.
[12] Hara M, Terashima S, Fukaya T, et al. Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco[J]. Planta, 2003, 217（2）：290-298.
[13] Li RH, Liu GB, Wang H, et al. Effects of Fe 3+ and Zn 2+ on the structural and thermodynamic properties of a soybean ASR protein[J]. Bioscience, Biotechnology, and Biochemistry, 2013, 77（3）：475-481.
[14] Wu G, Zhang H, Sun J, et al. Diverse LEA（late embryogenesis abundant）and LEA-like genes and their responses to hypersaline stress in post-diapause embryonic development of Artemia franciscana[J]. Comparative Biochemistry and Physiology Part B：Biochemistry and Molecular Biology, 2011, 160（1）：32-39.
[15] Battaglia M, Olvera-Carrillo Y, Garciarrubio A, et al. The enigmatic LEA proteins and other hydrophilins[J]. Plant Physiology, 2008, 148（1）：6-24
[16] 刘洋, 邢鑫, 李德全. LEA蛋白的分类与功能研究进展[J]. 生物技术通报, 2011（8）：36-43.
[17] Tunnacliffe A, Wise MJ. The continuing conundrum of the LEA proteins[J]. Naturwissenschaften, 2007, 94（10）：791-812.
[18] Swire-Clark GA, Marcotte Jr WR. The wheat LEA protein Em func- tions as an osmoprotective molecule in Saccharomyces cerevisiae [J]. Plant Molecular Biology, 1999, 39（1）：117-128.
[19] Reyes JL, Rodrigo MJ, Colmenero-flores JM, et al. Hydrophilins from distant organisms can protect enzymatic activities from water limitation effects in vitro[J]. Plant, Cell & Environment, 2005, 28（6）：709-718.
[20] Nakayama K, Okawa K, Kakizaki T, et al. Arabidopsis Cor15am is a chloroplast stromal protein that has cryoprotective activity and forms oligomers[J]. Plant Physiology, 2007, 144（1）：513-523.
[21] Liu Y, Zheng Y. PM2, a group 3 LEA protein from soybean, and its 22-mer repeating region confer salt tolerance in Escherichia coli[J]. Biochemical and Biophysical Research Communications, 2005, 331（1）：325-332.
[22] 俞嘉宁, 张林生, 张劲, 等. 小麦耐逆基因-TaLEA3的克隆及在酵母中的功能分析[J]. 生物工程学报, 2004, 20（6）：832-838.
[23] Yu JN, Zhang JS, Shan L, et al. Two new group 3 LEA genes of wheat and their functional analysis in yeast[J]. Journal of Integrative Plant Biology, 2005, 47（11）：1372-1381.
[24] Makarova KS, Aravind L, Wolf YI, et al. Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics[J]. Microbiology and Molecular Biology Reviews, 2001, 65（1）：44-79.
[25] Omelchenko MV, Wolf YI, Gaidamakova EK, et al. Comparative genomics of Thermus thermophilus and Deinococcus radiodurans：divergent routes of adaptation to thermophily and radiation resistance[J]. BMC Evolutionary Biology, 2005, 5（1）：1.
[26] Khan F, Singh SP, Mishra BN. Conservation of the LexA repressor binding site in Deinococcus radiodurans[J]. J Integr Bioinform, 2008, 5：86-92.
[27] Browne J, Tunnacliffe A, Burnell A. Anhydrobiosis：plant desiccation gene found in a nematode[J]. Nature, 2002, 416（6876）：38.
[28] Park BJ, Liu Z, Kanno A, et al. Genetic improvement of Chinese cabbage for salt and drought tolerance by constitutive expression of a B. napus LEA gene[J]. Plant Science, 2005, 169（3）：553-558.
[29] Tompa P, Kovacs D. Intrinsically disordered chaperones in plants and animals[J]. Biochemistry and Cell Biology, 2010, 88（2）：167-174.
[30] Liu Y, Gao ZQ, She Z, et al. The structural basis of the response regulator DrRRA from Deinococcus radiodurans[J]. Biochemical and Biophysical Research Communications, 2012, 417（4）：1206-1212.
[31] Wang L, Xu G, Chen H, et al. DrRRA：a novel response regulator essential for the extreme radioresistance of Deinococcus radiodurans [J]. Molecular Microbiology, 2008, 67（6）：1211-1222.
[32] Wang L, Hu J, Liu M, et al. Proteomic insights into the functional basis for the response regulator DrRRA of Deinococcus radiodurans[J]. International Journal of Radiation Biology, 2016, 92（5）：273-280.
[33] Liu F, Pang SJ. Stress tolerance and antioxidant enzymatic activities in the metabolisms of the reactive oxygen species in two intertidal red algae Grateloupia turuturu and Palmaria palmate[J]. Journal of Experimental Marine Biology and Ecology, 2010, 382（2）：82-87.
[34] Fredrickson JK, Li SM, Gaidamakova EK, et al. Protein oxidation：key to bacterial desiccation resistance?[J]. The ISME Journal, 2008, 2（4）：393-403.
[35] Daly MJ, Gaidamakova EK, Matrosova VY, et al. Protein oxidation implicated as the primary determinant of bacterial radioresistance[J]. PLoS Biol, 2007, 5（4）：e92.
[36] Gebicki S, Gill KH, Dean RT, et al. Action of peroxidases on protein hydroperoxides[J]. Redox Report, 2002, 7（4）：235-242.

耐辐射异常球菌dlp基因缺失突变株构建及其生物学功能研究

Construction and Biological Characterization of Gene dlp Deletion Mutant of Deinococcus radiodurans R1

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