利用PCR技术快速检测根际产ACC脱氨酶细菌

doi:10.13560/j.cnki.biotech.bull.1985.2017-0370

摘要/Abstract

摘要： 通过产生ACC脱氨酶降低胁迫乙烯水平并缓解盐胁迫危害,是植物根际促生菌（PGPR）促进宿主生长和抗逆的重要机制。本研究提出了利用PCR技术快速检测产ACC脱氨酶细菌的快捷方法。以编码ACC脱氨酶的acdS基因为标记,分别使用acdSf3/acdSr4、DegACCf/DegACCr和F1936f/F1938r三对引物,对多种盐生植物和美洲黑杨（Populus deltoids）的根部及根际土中分离得到的细菌菌株进行检测。结果表明,结合acdSf3/acdSr4引物和递减PCR（touchdown-PCR）方法时,能获得单一的特异性扩增条带且扩增成功率高;但DegACCf/DegACCr和F1936f/F1938r两对引物特异性较差。从247个菌株中检测到25株含有acdS基因,旨为今后研究植物根际细菌acdS基因遗传性及储备丰富的功能性菌株奠定基础。

关键词: 盐生植物, 杨树, 植物促生长根际细菌, 内生细菌, 产ACC脱氨酶细菌

Abstract: Producing ACC（1-aminocyclopropane-1-carboxylate）deaminase to reduce ethylene levels and eliminate salt stress is the key mechanism of plant-growth-promoting rhizobacteria（PGPR）in improving plant growth and stress tolerance. This work aims to establish an expeditious approach to screen bacteria containing ACC deaminase based on polymerase chain reaction（PCR）. We set the acdS gene encoding the ACC deaminase as the probe,then selected three pairs of primers（acdSf3/acdSr4,DegACCf/DegACCr and F1936f/F1938r）respectively,and detected the various bacterial strains isolated from the root and rhizosphere soil of diverse halophytes and Populus deltoids. The results showed that single specific amplified bands were produced and the rate of amplification was high when using the primers acdSf3/acdSr4 combined with touch-down PCR methods;however,DegACCf/DegACCr or F1936f/F1938r was in poor specificity. Finally,we acquired 25 strains containing acds gene from 257 bacterial isolates,aiming at laying a foundation for reserving rich and functional microbial resources and further studying the genetic diversity and function of acdS in PGPR.

Key words: halophytes, poplar, plant growth promoting rhizobacteria, endophytic bacteria, ACC deaminase-producing bacterium

秦媛, 潘雪玉, 袁志林. 利用PCR技术快速检测根际产ACC脱氨酶细菌[J]. 生物技术通报, 2017, 33(11): 112-122.

QIN Yuan, PAN Xue-yu, YUAN Zhi-lin. PCR-based Method for the Rapid Detection of ACC Deaminase-producing Rhizosphere Bacteria[J]. Biotechnology Bulletin, 2017, 33(11): 112-122.

参考文献

[1] Hyodo H. Stress/wound ethylene[M]// Mattoo AK, Suttle JC. The Plant Hormone Ethylene. Boca Raton：CRC Press, 1991：65-80.
[2] Hasegawa PM, Bressan RA, Zhu JK, et al. Plant cellular and molecular responses to high salinity[J]. Annual Review of Plant Biology, 2000, 51（1）：463-499.
[3] Mahajan S, Tuteja N. Cold, salinity and drought stresses：an overview[J]. Archives of Biochemistry and Biophysics, 2005, 444（2）：139-158.
[4] Morgan PW, Drew MC. Ethylene and plant responses to stress[J]. Physiologia Plantarum, 1997, 100（3）：620-630.
[5] Glick BR. Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase[J]. FEMS Microbiology Letters, 2005, 251（1）：1-7.
[6] Nadeem SM, Zahair ZA, Naveed M, et al. Rhizobacteria capable of producing ACC-deaminase may mitigate salt stress in wheat[J]. Soil Science Society of America Journal, 2010, 74：533-542.
[7] Deepti B, Nidhi B, Deepamala M, et al. ACC deaminase containing Arthrobacter protophormiae induces NaCl stress tolerance through reduced ACC oxidase activity and ethylene production resulting in improved nodulation and mycorrhization inPisum sativum[J]. Journal of Plant Physiology, 2014, 171：884-894.
[8] Singh RP, Shelke GM, Kumar A, et al. Biochemistry and genetics of ACC deaminase：a weapon to “stress ethylene” produced in plants[J]. Frontiers in Microbiology, 2015, 6：937.
[9] Duan J, Müller KM, Charles TC, et al. 1-aminocyclopropane-1-carboxylate（ACC）deaminase genes in rhizobia from southern Saskatchewan[J]. Microbial Ecology, 2006, 57（3）：423-436.
[10] Glick BR, Cheng Z, Czarny J, et al. Promotion of plant growth by ACC deaminase-producing soil bacteria[J]. European Journal of Plant Pathology, 2007, 119（3）：329-339.
[11] Brotman Y, Landau U, Cuadros-Inostroza Á, et al. Trichoderma-plant root colonization：escaping early plant defense responses and activation of the antioxidant machinery for saline stress tolerance[J]. PLoS Pathogens, 2013, 9：e1003221.
[12] Nascimento FX, Rossi MJ, Soares CR, et al. New insights into 1-aminocyclopropane-1-carboxylate（ACC）deaminase phylogeny, evolution and ecological significance[J]. PLoS One, 2014, 9：e99168.
[13] Glick BR, Cheng Z, Czarny J, et al. Promotion of plant growth by ACC deaminase-producing soil bacteria[J]. European Journal of Plant Pathology, 2007, 119（3）：329-339.
[14] Glick BR, Penrose DM, Li J. A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria[J]. Journal of Theoretical Biology, 1998, 190（1）：63-68.
[15] Penrose DM, Glick BR. Methods for isolating and characterizing ACC deaminase-containing plant growth promoting rhizobacteria [J]. Physiologia Plantarum, 2003, 118（1）：10-15.
[16] 刘琛, 赵宇华, 傅庆林, 等. ACC 脱氨酶活性菌株的筛选, 鉴定及其对茄子耐盐性的影响[J]. 浙江大学学报：农业与生命科学版, 2008, 34（2）：143-148.
[17] 田磊, 姜云, 陈长卿, 等. 一株人参内生1-氨基环丙烷-1-1羧酸（ACC）脱氨酶活性细菌的筛选, 鉴定及其对宿主生长的影响[J]. 微生物学报, 2014, 54（7）：760-769.
[18] 方芳, 刘佳莉, 史煦涵, 等. 石油污染土壤中植物根际促生菌的筛选及特性分析[J]. 生物技术通报, 2012, 6：154-158.
[19] 窦雅静, 康丽华, 陆俊锟, 等. 黑木相思根瘤菌ACC脱氨酶活性的研究[J]. 中南林业科技大学学报, 2014, 34（11）：77-83.
[20] 谢明杰, 程爱华. 我国微生物肥料的研究进展及发展趋势[J]. 微生物学杂志, 2010, 20（4）：42-45.
[21] 姚军朋, 姚拓, 王小利. ACC 脱氨酶的应用研究进展与评述[J]. 生物技术, 2010, 20（2）：87-91.
[22] Jogaiah S, Abdelrahman M, Tran LS, et al. Characterization of rhizosphere fungi that mediate resistance in tomato against bacterial wilt disease[J]. Journal of Experimental Botany, 2013, 64：3829-3842.
[23] Blaha D, Prigent-Combaret C, Mirza MS, et al. Phylogeny of the 1-aminocyclopropane-1-carboxylic acid deaminase-encoding gene acdS in phytobeneficial and pathogenic Proteobacteria and relation with strain biogeography[J]. FEMS Microbiology Ecology, 2006, 56（3）：455-470.
[24] Nikolic B, Schwab H, Sessitsch A. Metagenomic analysis of the 1-aminocyclopropane-1-carboxylate deaminase gene（acdS）operon of an uncultured bacterial endophyte colonizing Solanum tuberosum L[J]. Archives of Microbiology, 2011, 193（9）：665-676.
[25] Li Z, Chang S, Ye S, et al. Differentiation of 1-aminocyclopropane-1-carboxylate（ACC）deaminase from its homologs is the key for identifying bacteria containing ACC deaminase[J]. FEMS Microbiology Ecology, 2015, 91（10）：fiv112.