<em>Sinorhizobium fredii</em> WGF03胞外多糖分泌相关基因<em>exoD</em>功能初探

doi:10.13523/j.cb.20140607

中国生物工程杂志

2014, Vol. 34

Issue (06): 47-54 DOI: 10.13523/j.cb.20140607

研究报告

Sinorhizobium fredii WGF03胞外多糖分泌相关基因exoD功能初探

马婷婷^1,2, 张健^1,2, 粟月萍^1,2, 宋张杨^1,2, 唐咸来⁴, 申佩弘¹, 武波³

1. 广西大学生命科学与技术学院南宁 530005;
2. 微生物及植物遗传工程教育部重点实验室南宁 530005;
3. 亚热带农业生物资源保护与利用国家重点实验室南宁 530005;
4. 广西壮族自治区科学技术厅南宁 530012

A Preliminary Study of exoD Gene Relating Exopolysaccharides Secretion in Sinorhizobium fredii WGF03

MA Ting-ting^1,2, ZHANG Jian^1,2, SU Yue-ping^1,2, SONG Zhang-yang^1,2, TANG Xian-lai⁴, SHEN Pei-hong¹, WU Bo³

1. College of Life Science and Technology of Guangxi University, Nanning 530005, China;
2. The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, Nanning 530005, China;
3. State Key Laboratory for Conservation and Utilization of Agricultural Bioresources in the Subtropics, Nanning 530005, China;
4. The Science and Technology Department of Guangxi, Nanning 530012, China

全文: PDF(982 KB) HTML

摘要：

从费氏中华根瘤菌WGF03的基因组中克隆出与胞外多糖分泌密切相关的基因exoD，研究该基因对胞外多糖合成、菌株共生结瘤能力和固氮效率的影响。利用自杀性质粒pK18mobsacB，通过同源双交换法构建exoD基因的缺失突变体ΔexoD。实验发现：与野生菌株相比，突变株在YMA培养基平板上胞外多糖产量明显减少、运动能力也有所减弱；在NaCl浓度小于350 mmol/L范围内，菌体均能维持稳定生长；接种于大豆幼苗后，产生的根瘤数量较多，但个体小、形状不一，且固氮酶活也显著下降。研究结果说明exoD基因参与S.fredii WGF03 胞外多糖的合成并影响菌株共生结瘤能力和固氮效率。

关键词： 费氏中华根瘤菌WGF03; 胞外多糖; exoD; 突变; 植株实验

Abstract:

An extracellular polysaccharide secretion related gene exoD was cloned from genome of S.fredii WGF03 and the influence of genes on extracellular polysaccharide synthesis as well as nodulation,nitrogen fixation with host plant was investigated. ΔexoD mutant was constructed through homologous double-crossover using suicide plasmid pk18mobsacB as a vector. Compared with the wile type strain, the mutant strain produced less exopolysaccharides (EPS) on YMA medium plate and the motility was decreased.Nothing changes in the growth situation on the medium containing less than 350 mmol/L of NaCl. The plant test showed that the number of nodules was more, but small, varying shapes, and nitrogen-fixing enzyme activity was also significantly decreased after inoculation mutant strain. This demonstrated that exoD gene affect EPS synthesis in S.fredii WGF03, and involve in nodulation and nitrogenase activity.

Key words: S. fredii WGF03 EPS Deletion mutation exoD Plant tests

收稿日期: 2014-03-27 出版日期: 2014-06-25

ZTFLH:

Q789

基金资助:

国家重点基础研究发展计划资助项目（2010CB126502）

通讯作者: 武波 E-mail: wubogx@gxu.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

引用本文:

马婷婷, 张健, 粟月萍, 宋张杨, 唐咸来, 申佩弘, 武波. Sinorhizobium fredii WGF03胞外多糖分泌相关基因exoD功能初探[J]. 中国生物工程杂志, 2014, 34(06): 47-54.

MA Ting-ting, ZHANG Jian, SU Yue-ping, SONG Zhang-yang, TANG Xian-lai, SHEN Pei-hong, WU Bo. A Preliminary Study of exoD Gene Relating Exopolysaccharides Secretion in Sinorhizobium fredii WGF03. China Biotechnology, 2014, 34(06): 47-54.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140607 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I06/47

[1] 陈文新, 汪恩涛. 中国根瘤菌. 北京:科学出版社,2011.29-31. Chen W X,Wang N T. China Rhizobium. Beijing: Science Press,2011.29-31.

[2] Downie,J A. The roles of extracellular proteins, polysaccharides and signals in the interactions of rhizobia with legume roots. FEMS Microbiol Rev,2010,34:150-170.

[3] Alisa P L,Sharon R L. Exopolysaccharides from Sinorhizobium meliloti can protect against H₂O₂-dependent damage. J Bacteriol,2013, 195(23):5362-5369.

[4] Quelas J I,López-García S L, Casabuono A,et al. Effects of N-starvation and C-source on Bradyrhizobium japonicum exopolysaccharide production and composition and bacterial infectivity to soybean roots. Arch Microbiol,2006,186:119-128.

[5] Rinaudi L V, Giordano W. An integrated view of biofilm formation in rhizobia. FEMS Microbiol Lett,2010,30:1-11.

[6] Amelia D T, Bronwyn R H, Travis W D,et al. Agrobacterium tumefaciens ExoR represses succinoglycan biosynthesis and is required for biofilm formation and motility. Microbiol,2010, 156(9):2670-2681.

[7] Fraysse N, Couderc F, Poinsot V. Surface polysaccharide involvement in establishing the rhizobium-legume symbiosis. Eur J Biochem,2003,270:1365-1380.

[8] Laus M C, Van B A, Kijne J W. Role of cellulose fibrils and exopolysaccharides of Rhizobium leguminosarum in attachment to and infection of Vicia sativa root hairs. Mol Plant Microbe Interact, 2005, 18:533-538.

[9] Monika J. Environmental signals and regulatory pathways that influence exopolysaccharide production in Rhizobia. J Mo Sci, 2011,12:7898-7933.

[10] Anna S,Monika J, Magorzata M,et al. Rhizobial exopolysaccharides: genetic control and symbiotic functions. Microbial Cell Factories, 2006, 5:7.

[11] Yao S Y,Luo L, Har K J,et al. Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production. J Bacteriol,2004,186:6042-6049.

[12] Zhan H, Leigh J A. Two genes that regulate exopolysaccharide production in Rhizobium meliloti. J Bacteriol,1990,172:5254-5259.

[13] Becker A, Küster H, Niehaus K,et al. Extension of the Rhizobium meliloti succinoglycan biosynthesis gene cluster: Identification of the exsA gene encoding an ABC transporter protein, and the exsB gene which probably codes for a regulator of succinoglycan biosynthesis. Mol Gen Genet,1995,249:487-497.

[14] Keller M,Roxlau A,Wenig W M, et al. Molecular analysis of the Rhizobium meliloti mucR gene regulating the biosynthesis of the exopolysaccharides succinoglycan and galactoglucan. Mol Plant Microbe Interact. 1995, 8, 267-277.

[15] Hoang H H,Becker A,Gonzalez J E. The LuxR homolog ExpR, in combination with the Sinquorum sensing system, plays a central role in Sinorhizobium meliloti gene expression. J Bacteriol,2004, 186:5460-5472.

[16] Dusha I, Olah B, Szegletes Z,et al. syrM is involved in the determination of the amount and ratio of the two forms of the acidic exopolysaccharide EPS I in Rhizobium meliloti. Mol Plant Microbe Interact, 1999, 12:755-765.

[17] Bahlawane C,Baumgarth B, Serrania J,et al. Fine-tuning of galactoglucan biosynthesis in Sinorhizobium meliloti by differential WggR (ExpG)-, PhoB-, and MucR-dependent regulation of two promoters. J Bacteriol,2008,190: 3456-3466.

[18] Reed J W,Walker G C. The exoD gene of Rhizobium meliloti encodes a novel function needed for alfalfa nodule invasion. J Bacteriol,1991,173:664-677.

[19] Reed J W,Walker G C. Acidic conditions permit effective nodulation of alfalfa by invasion-deficient Rhizobium meliloti exoD mutants. Genes Dev,1991,5:2274-2287.

[20] Helinski D R, Figurski D. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA,1979,76:1648-1652.

[21] Tang J L,Gough C L,Barber C E,et al. Molecular cloning of protease gene(s) from Xanthomonas campestris pv. campestris:Expression in Escherichia coli and role in pathogenicity. Mol Gen Genet,1987,210:443-448.

[22] 罗贤安,凉安千.生物固氮研究中乙炔还原法的应用. 微生物学通报,1979,2:37-40. Luo X A,Liang A Q. The application of acetylene reduction method in biological nitrogen-fixing research. Microbiology,1979,2:37-40.

[23] Lai H C,Soo P C, Wei J R,et al. The RssAB two-component signal transduction system in Serratia marcescens regulates swarming motility and cell envelope architecture in response to exogenous saturated fatty acids. J Bacteriology,2005,187(10):3407-3414.

[24] Derek H W,Esther J C,Robeert F F. ExoR is genetically coupled to the ExoS-ChvI two-component system and located in the periplasm of Sinorhizobium meliloti. Mol Microbiol,2007,64(3):647-664.

[25] 罗利,刘芳华,朱家壁,等. 苜蓿中华根瘤菌(Sinorhizobium meliloti)LuxR家族转录因子ExpR调节motC操纵子的表达. 微生物学报,2006,46(3):474-477. Luo L,Liu F H,Zhu J B,et al. A LuxR family regulator,ExpR regulates the expression of motC operon from Sinorhizobium meliloti. Acta Microbiolgica Sinica,2006,46(3):474-477.

[26] Crespo-Rivas J C,Margaret I,Hidalgo A,et al.Sinorhizobium fredii HH103cgs mutants are unable to nodulate determinate and indeterminate-nodule forming legumes and overproduce an altered EPS. Mol Plant-Microbe Interact,2009,22:575-588.

[27] Isabel M,Juan C,Crespo R,et al. Sinorhizobium fredii HH103 rkp-3Genes are required for K-antigen polysaccharide,biosynthesis,affect lipopolysaccharide structure and are essential for infection of legumes forming determinate nodules. Molecular Plant-Mircrobe Interactions,2012,25(6):825-838.

[28] Margaret I,Lucas M M,Acosta J S,et al. The Sinorhizobium fredii HH103 lipopolysaccharide is not only relevant at early soybean nodulation stages but also for symbiosome stability in mature nodules. PLoSONE,2013,8(10):e74717.doi:10.137/journal.pone.0074717.

[29] 方宣钧,尤崇杓,固氮菌酸性胞外多糖生物合成的基因调控.农业生物技术学报,1995,3(1):21-27. Fang X J,You C B. Gene regulation of biosynthesis of acidic exoploysaccharides in diazotrophs. J Agric Biotechnol,1995,3(1):21-27.

[30] Leigh J A,Lee C C. Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules. J Bacteriol,1988,170(8):3327-3332.

[31] Unni S,Rao K K. Protein and lipopolysaccharide profiles of a salt-sensitive Rhizobium sp. and its exopolysaccharide-deficient mutant. Soil Biol Biochem,2001,33(1):111-115.

[32] Kan F L,Chen Z Y,Wang E T, et al. Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai-Tibet plateau and in other zones of China. Archives of Microbiology, 2007, 188(2):103-115.

[33] Esther J C,Erich A S,Sharon R L. The periplasmic regulator ExoR inhibits ExoS/ChvI two-component signalling in Sinorhizobium meliloti. Mol Microbiol,2008,69(5):1290-1303.

[34] 王正荣,生吉萍,申琳. 细菌胞外多糖的生物合成与基因控制. 生物技术通报,2010,11:48-55. Wang Z R,Sheng J P,Shen L. Biosynthesis of bacterial exopolysaccharides and gene cluster. Biotech Bulletin,2010,11:48-55.

[1]	郭芳,张良,冯旭东,李春. 植物源UDP-糖基转移酶及其分子改造^*[J]. 中国生物工程杂志, 2021, 41(9): 78-91.
[2]	李开秀,司维. 间充质干细胞来源的外泌体治疗炎症性肠病研究进展^*[J]. 中国生物工程杂志, 2021, 41(7): 66-73.
[3]	冯宝琪,冯娇,张苗,刘洋,曹睿,尹涵之,齐凤仙,李子龙,尹守亮. 利用Tn5型转座突变系统筛选高产阿维菌素菌株^*[J]. 中国生物工程杂志, 2021, 41(7): 32-41.
[4]	郭广超,周于用,曹三杰,武耀民,伍锐,赵勤,文心田,黄小波,文翼平. 乙型脑炎病毒NS2A-C60A位点突变对其生物学特性影响研究^*[J]. 中国生物工程杂志, 2020, 40(9): 1-10.
[5]	陈东,李程程,史仲平. 植物乳杆菌胞外多糖包覆的高稳定性硒纳米颗粒的制备及其抗氧化活性的研究^*[J]. 中国生物工程杂志, 2020, 40(9): 18-27.
[6]	彭向雷,王烨,王丽男,苏彦斌,付远辉,郑妍鹏,何金生. 单引物PCR法引入定点突变 ^*[J]. 中国生物工程杂志, 2020, 40(8): 19-23.
[7]	赵晓艳,陈允妲,章雅倩,吴晓玉,王飞,陈金印. **Myxococcus sp.V11海藻糖合酶TreS II分子改造 ^***[J]. 中国生物工程杂志, 2020, 40(3): 79-87.
[8]	苏永君,胡蝶,胡博淳,李闯,文正,章晨,邬敏辰. 定点突变提高环氧化物水解酶AuEH2催化对甲基苯基缩水甘油醚的对映选择性^*[J]. 中国生物工程杂志, 2020, 40(3): 88-95.
[9]	王谦,陈苏宁. 混合表型急性白血病的细胞及分子遗传学研究进展[J]. 中国生物工程杂志, 2019, 39(9): 91-97.
[10]	杨林,王柳月,李慧美,陈华波. 改进的多片段重叠延伸PCR制作基因多位点突变 ^*[J]. 中国生物工程杂志, 2019, 39(8): 52-58.
[11]	阚婷婷,宗迅成,苏永君,王婷婷,李闯,胡蝶,邬敏辰. 定点突变改善PvEH1对邻甲基苯基缩水甘油醚的催化特性 ^*[J]. 中国生物工程杂志, 2019, 39(6): 9-16.
[12]	王越,李江华,堵国成,刘龙. L-氨基酸脱氨酶的分子改造及其用于全细胞催化法生产α-酮戊二酸条件的优化 ^*[J]. 中国生物工程杂志, 2019, 39(3): 56-64.
[13]	王兆官,吴洋,齐浩. 人工合成多样性突变文库研究进展^*[J]. 中国生物工程杂志, 2019, 39(11): 113-122.
[14]	杨雅楠,孙超,崔浩琳,赵欣. M145F/F146M突变对光受体蛋白细菌视紫红质和古紫质4光循环的影响 ^*[J]. 中国生物工程杂志, 2019, 39(1): 21-30.
[15]	陈弘远,陈红岩,乔纯,李建勇,卢大儒. 华氏巨球蛋白血症相关突变MYD88 L265P新型检测体系的建立 ^*[J]. 中国生物工程杂志, 2018, 38(9): 35-40.

Viewed

Full text

Abstract

Cited

Shared

Discussed