Braun 7526

Braun Linear

Molecular analysis of the gene by PCR Azotobacter Nif H

Introduction
Nitrogen fixation is the reduction of N2 (atmospheric nitrogen) for NH3 (ammonia). free-living prokaryotes with the ability to fix atmospheric nitrogen (diazotrophs) are ubiquitous in soil. But our consciousness products of its importance and their diversity remains incomplete. In natural ecosystems, biological N2 fixation is the most important source of N. The binding capacity nitrogen is widespread among bacteria and archaea. The estimated contribution of free nitrogen-fixing soil N input varies from prokaryotes 00-60 kg / ha / year (Burgmann et al., 2003). Dinitrogen (N2)-fixing microorganisms (diazotrophs) play an important role in ocean biogeochemistry plankton and productivity (Church et al., 2005).

nitrogen fixation may be an important source of nitrogen for biological productivity in the marine environment. biological fixation of nitrogen is catalyzed by the enzyme nitrogenase, which is the property of various micro-organisms that represent almost all phylogenetic groups. The interest in fixing nitrogen in the sea has been mainly concentrated in the fixation rate nitrogen, but information on the types of species present in the capacity of nitrogen fixation may be important for predicting the rate of nitrogen fixation in situ (and Zehr al., 1998).

Nitrogenase catalyzes the reduction of nitrogen to ammonia in a reaction depends ATP and reductive. It is one of the best characterized metalloenzymes and is an excellent model for elucidating metalloprotein assembly. Nitrogenase is composed of two oxygen-labile proteins metallo; dinitrogen and dinitrogen reductase. Dinitrogen is 240 kDa alpha2-beta2 tetramer of the nifD NIFK and gene products. dinitrogen reductase is a dimer of 60 kDa alpha2 nifH gene product that contains a single coordinating center 4Fe-4S between the two subunits (Rubio et al. 2005). Understanding how nitrogenase fixed N regulates the availability is necessary for the development of strategies to increase amount of synthesized ammonia from nitrogen-fixing bacteria with the potential to be used in agriculture (Kennedy et al., 2004).

Molecular tools for detection and characterization of nitrogenase (NIF) genes, and immunological protein nitrogenase can provide new information on factors that regulate the distribution and activity of various nitrogen fixing organisms in the marine environment. Amplification and characterization of the nifH sequences identified the type (s) of the organism responsible for nitrogen fixation, such as aggregate and the cyanobacterium Trichodesmium. Differences in patterns of nitrogen fixation have been associated with Trichodesmium differences genetic differences between strains. The development of these methods provide powerful new ways to connect the genetic potential for nitrogen fixation rates of nitrogen fixation in the ocean (Zehr et al., 1998)

nitrogenase gene (nifH) sequences amplified directly from ocean waters have shown the ocean contains more diverse diazotrophic microbial populations and diverse habitats for nitrogen fixers previous techniques by classical microbiological (Zehr et al., 1998). Understanding how to regulate the availability of fixed N nitrogenase is necessary for developing strategies to increase the amount of ammonia synthesized by nitrogen-fixing bacteria with the potential to be used in agriculture (Kennedy et al., 2004).

The commercial biofertilizer history began with the release of "Nitrogin" by Nobbe and Hiltner, a laboratory culture of rhizobia in 1895, followed by the discovery Azotobacter and blue-green algae and a number of other micro-organisms. biofertilizer Azotobacter is used as the cultivation of most crops. Azotobacter is an aerobic diazotrophs requires living on the ground with a variety of metabolic capabilities, including the ability to fix atmospheric nitrogen and convert it into ammonia. Natural Azotobacter fixes atmospheric nitrogen in the rhizosphere. It there are different strains of Azotobacter each approach has chemical, biological and other characters. However, some strains have a capacity of nitrogen fixation higher than others (Burgmann et al., 2003). In addition to nitrogen fixation, Azotobacter also produces thiamine, riboflavin, indole acetic and gibberellins. When Azotobacter is applied to seeds, seed germination was greatly improved, control of diseases plants due to these substances produced by Azotobacter (Kader et al., 2002.)

The nifH gene has been extensively studied by culture methods independent. These approaches provide a more complete picture of the diazotrophic community than culture-based approaches. Various techniques, such as cloning PCR, electrophoresis, denaturing gradient gel, PCR-restriction polymorphism length (RFLP), and fluorescently labeled terminal (FLT)-RFLP, were used to analyze the composition of nifH gene pools in different environments. These studies showed that the nifH gene is present in environments different forest soil, the rhizosphere of native wetland species such as Spartina or grown, including rice, water or other Polar cyanobacteria, and the bacteria found in the guts of termites. All these studies describe a large number of unknown sequences that match to various unidentified diazotrophs. Some are characteristic of genes nifH ecological niche (Shaffer et al., 2000) discussed the link possible between land habitats nitrogen fixing bacteria and the structure of nifH gene pools (Poli et al., 2001).

nitrogen fixation in the A. vinelandii is complicated by the presence of three nitrogenase enzymes, biochemical and genetic distance, which is synthesized under conditions different metal supply. Regulation of nitrogenase molybdenum classic whose subunits are encoded by genes Nif-HDK which is similar to the enzyme purified from several other nitrogen fixing organisms. (Al Sabra et al., 2000). Nif-HDK genes in a large group nif genes, which includes, in order, NifHDKTYENXUSVWZMF (Bali et al., 1992). Molecular methods based on PCR detection of marker genes Universal nifH has been successfully applied to describe the population diazotrophs in the environment (Burgmann et al., 2003).

Materials and Methods

Take samples
Samples were collected at different locations in the area of the sea Rameshwaram (Gulf of Mannar) at a depth of 1.5 m. The samples taken at random from the sterile plastic bags (soil sample) and bottled water sample (water sample) the bottles are kept in an ice box and transported safely to a laboratory for further analysis within 12 hours. The sampling tubes were packed with media and safely transported to the laboratory.

Isolation of Azotobacter water samples and sediments (Mary et al., 1985)
Different selective media are used for the isolation of Azotobacter sp marine source as described above. Azotobacter strains used in this study were maintained and grown in Burk as described previously (Joerger et al., 1988). Since the isolates are of marine origin, the media were prepared by the chloride Sodium 3.5% (NaCl). The media used for isolation of nitrogen fixing organism (Azotobacter) from marine sources have been Agar Jensen agar Azotobacter, half Burk and marine agar.

characteristics of culture (Bagwell et al., 1988)
staining characteristics Gram stain and cell morphology was determined by standard methods (Gerhardt et al., 1981). Motility was observed under the microscope Phase contrast costs. Preliminary physiological characterization of the catalase test, starch hydrolysis test were also performed.

DNA extraction and purification (Kelly et al., 1990)
Azotobacter genomic DNA was isolated as described previously (Robson et al. 1980). Linear DNA fragments were analyzed by agarose gel electrophoresis in TEB buffer (Maniatis et al., 1982). Purity DNA found that the spectrophotometric method using the formula OD 260 nm / OD at 280 nm (Wilfinger et al., 1997).

amplification PCR fragment of nifH genes

nitrogenase Fe protein gene (nifH) were amplified from genomic DNA derived Azotobacter sp, using the primary diagnosis OPERON Ltd., USA. The samples were amplified by PCR in a reaction mixture containing 5.0 ml of buffer, 1.0 ml 10 mM dNTP, Primary 1 (25 Wed) 1.0 ml primer 2 (24 Sea) 1.0 ml, 1.0 ml DNA template, Taq polymerase 1.0 ml for 35 cycles (1 min 94 ° C 1 min at 54 ° C and 1 min at 72 ° C) (Zehr et al., 1988).

Results and discussion
Totally 100 samples were collected the area of marine waters and sediments at intervals of about 20 days. Of the 70 samples of seawater collected every 70 samples reveal the presence of Azotobacter, but only 23 of the 30 marine sediments show the presence of Azotobacter. These samples were treated by procedures commonly used as selective media. Gram stain, phase contrast observation of motility, test starch hydrolysis and catalase test for the identification of non-living diazotrophs Azotobacter body from the examples above, and can be processed, the result shows Azotobacter sp that are mobile, Gram-negative, catalase and hydrolysis starch positve.

Colony morphology of Azotobacter strains were variable during isolation in selective media. The colonies were very clear decreases, large, slimy, watery, because, as a source of first Marine said. The stock culture was under cultivation in the same colony morphology is different from say small, circular, convex in nature. All strains Azotobacter were numbered for easy identification and convenience. Among these isolates, defined as the pure culture strains of Azotobacter (1, 16, 27, 82, 101, 103, 108, 115, 125 and 132) were selected for analysis of fatty acids Nucleic.

Link (Standard), crops such as Azotobacter beijerinckii (123), A. chroococcum (446), and A. vinelandii (124) obtained MTCC, (Chandigarh, India) is also used in association with the Navy isolates for nucleic acid extraction and purification. The DNA of selected strains were isolated and found OD at 260 nm ranges from 0.141 to 0.177. The estimated value of the extracted DNA will 0.88.The 0.70 to the purity of DNA was analyzed by the spectrophotometric method with OD 260 and OD at 280 nm. The presence and purity of DNA was checked by OD 260nm / OD 280 nm, the value varies from 1.13 to 2.21. If the estimated value of 1.8 forms of the presence of pure DNA. If the estimated value is lower / higher 1.8 corresponds to the presence of DNA to protein / RNA contamination, according to the respective value of the DNA was purified by proteases and RNase enzymes. The purified form of the DNA was separated by agarose gel electrophoresis on a comparison between the profiles Tape samples randomly selected marine strains and standard. There is no substantial difference between the banding patterns of chromosomal DNA in the gel, as shown in the plates. This result confirms that the molecular weight chromosomal DNA in all strains is similar.

One l of DNA was used as template in PCR. primers selected primer1 :5-3-GGAATTCCTGYGAYCCNAARGCCNA
Adhesive 2.3 :5-CGGATCCGDNGCCATCATYTCNCC acquis diagnosis OPERON LTD, USA, respectively, were used to amplify a sequence of 324-bp region between positions 336 and 660. All clips lead to N2 nifH gene, which encodes the Fe protein of nitrogenase (Poli et al., 2001). The results of the PCR products were compared with 2% agarose gel. selective primers nifH strain PCC7120 Anabaena sp was used for amplification of SP Azotobacter, the primers used in my study corresponding exactly the genome of Azotobacter. Free-nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically, physiologically diverse. Molecular methods based on PCR detection universal nifH marker gene have been successfully applied to describe diazotrophic populations in the environment. However, the use of degenerate primers and amplification conditions of low stringency of these methods are prone to amplification bias, while less degenerate primers do not amplify all nifH genes (Burgmann et al., 2003).

References

Bagwell, CE, Piceno, YM, Lucas, AM and Lovell, CR, 1988. Diversity of diazotrophic rhizosphere communities physiological salt selected the marsh grass. Appl. Environ. Microbiol., 64 (11): 4276-4282.
Bali, A., Gonzalo Blanco, Susan Hill, Christina and Kennedy 1992. Execretion management ammonium Azotobacter vinelandii mutant from nitrogen fixation. Appl. Environ. Microbiol., 58 (5): 1711-1718.
Burgmann H, Franco Widmer, William Von Sigler, and Josef Zeyer. New screening tools for molecular analysis of free-living diazotrophs in the field. Environmental Microbiology, vol.70, p. 240 # 1-247.1999.
Burgmann H, Manuel Pesaro, Franco Widmer and Josef Zeyer strategy to optimize the quality and quantity of DNA extracted from soil. Bacteriological Reviews, Vol.36, No. 2 p.295-341. 2003.
Church MJ, Cindy M. Short, D. Bethany Jenkins, David Karl, Jonathan P. Zehr, temporal trends of nitrogenase Gene (nifH) Expression in the Oligotrophic North Pacific Ocean with "Marine Environment Department of Microbiology, Vol 134, No. 1 p.155-193, 1999.
Gerhard P. RGEMurray, RNCostilow, EWNester, WAWood, NRKrieg, the method of generalbacteriology.American andG.B.phillips.1981.Manual Society for Microbiology, Washington, DC
Joerger, RD, Jacobson, MR, Premakumar, R., Wolfinger, EW and Bishop, PE, 1989. The nucleotide sequence and analysis of the structural gene mutation (AnfHDGK) for the second variant of Azotobacter vinelandii nitrogenase. J. Bacteriol., 171 (2): 1075-1086.
Kader.MA, Mian.MH, Effect Hoque.MS or Azotobacter inoculant on yield and nitrogen uptake by wheat. Department of Soil Science, Bangladesh Agricultural University, Mymensigh, Bangladesh. Vol 2 (4) p 251-261.2002.
Kelly, MJS, Poole RK, Yates, MG and Kennedy, C., 1990. Cloning and mutagenesis of genes encoding cytochrome terminal oxidase mutants Azotobacter vinelandii complex BD: deficient in cytochrome d complex are unable to fix nitrogen in the air. J. Bacteriol., 172 (10): 6010-6019.
Kennedy.C, RKPoole, and MGYates MJSKelly. Cloning and mutagenesis of genes encoding the cytochrome oxidase complex terminal BD Azotobacter vinelandii: mutants deficient in cytochrome d complex are unable to fix nitrogen in Air.Journal of Bacteriology, Vol.172, No. 10 p.6010-6019. 1990
Maniatis, T. and J.Sambrook.1982 EFFritsch. Cloning Molecular: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Mary. L. G and R. Rita Colwell, Enumeration, isolation and characterization of N2-fixing bacteria of seawater Department of Microbiology at the University of Maryland. Vol 50 No 0,2. 1985
Poli. M Lionel Ranjard, Sylvie Nazaret, François and Lucile Gourbiere Jocteur .. Comparison of nifH Gene Monrozier pools in soils and soil microenvironments contrasting properties in Applied and Environmental Microbiology Vol. 67 p. 2255-2262. 2001
Robson, RL, JAChesshyre, C. Wheller, R. Jones, PRWoodley and JRPostgate. 1984.Genomic size and complexity of Azotobacter chroococcum.J.Gen Microbial.130 :1603-1612.
Rubio. LM, and PW Ludden singer.SW purification and characterization of Azotobacter nafy vinelandii. Plants and resources of the Department of Microbiology of the University, the University of native California, California. 2005
Sabra, W., Zeng, AP, Lunsdorf, H. and Deckwer, WD, 2000. Effect of oxygen in the formation and structure of alginate Azotobaceter vinelandii and its role in protection of nitrogenase. Appl. Environ. Microbiol., 66 (9): 4037-4044.
Shaffer, BT, F. Widmer, LA Porteous and RJ Seidler. genes in time and the distribution spatial nifH N2-fixing bacteria in forests and clear in western Oregon. Microb. Ecol. 39 P.12-21. 2000.
W. Mackey K. Wilfinger Chomczynski P.1997 and effect of pH and ionic strength on the spectrophotometric assessment of the purity of nucleic acids. Bio Technology ,22,474-481.
Zehr. J. P T. Mark Mellon, and Sabino Zani. New micro-organisms fixing nitrogen in the oligotrophic oceans detected by amplification of nitrogenase (nifH) gene. 1998.
Zehr. Sara Braun JP, Chen and Mark Yibu Mellon nitrogen fixation in the marine environment: the relationship between the genetic potential to nitrogenase activity. Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA. 1999.

About the Author

Karthick.A* and Jayashree, V.S
Dr.G.R. Damodaran College of Science
Coimbatore

* Corresponding address

Dr.G.R. Damodaran College of Science,
Avanashi road ,civil aerodrome post,
coimbatore 14

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