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The molecular identification and study on properties of the ammonium-excreting mutants of Alcaligenes faeccalis

Author: WuWei
Tutor: LinMin
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: Alcaligenes faecalis Secretion of ammonia Tn5 mutants Molecular Identification
CLC: Q945.13;S188
Type: Master's thesis
Year: 2000
Downloads: 88
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Abstract


By engaging the experiment will be carrying the suicide plasmid vector pSUP1011 Tn5 transposon into the wild-type Alcaligenes faecalis A1501 selection plates containing kanamycin and the A15-limiting selection of resistant single colonies constructed Alcaligenes faecalis Tn5 insertion mutation Library. Screened from the mutant library of more than 3,000 strains zygote the three secretion of ammonia mutant named A15M1 A15M2, A15M3. A15-limiting culture medium, and nitrate-containing culture broth, the mutant and wild-type growth curve trend is consistent, but the mutant secretin ammonia amount higher than that of wild-type, in the liquid containing nitrate culture cultured to logarithmic growth the late secretion amount of ammonia up to 3.96mM, the secretion of ammonia in the wild-type A1501 almost undetectable. Experiments will carry through conjugation have the Alcaligenes faecalis nifH-lacZ fusion gene transfer vector pSK6 of and carry Azospirillum brasilense nifH-lacZ fusion gene the transfer vector pAB358 transferred to wild-type A1501 and mutant A15M1, A15M2 A15M3 in in containing homologous and heterologous nifH-lacZ fusion gene mutant and wild-type β-half-galactosidase activity of different ammonium concentrations were measured. The result: in nitrogen-free conditions homologous nifH-lacZ fusion gene expression activity in the three mutants were higher than wild the A1501 (pSK6), A1501 (pSK6) 168.5 vitality units (units) mutant A15M3 ( pSK6) the highest activity for 1600 activity unit (units). With the increase of the concentration of ammonium, A1501, (pSK6), β-galactosidase enzyme activity is drastically decreased, but the mutant strain remains high activity. This shows nifH-lacZ expression in the wild-type A1501 inducible highly expressed low levels of expression under conditions of high ammonium without ammonium conditions; nifH-lacZ fusion gene in the mutant strain constitutive from NH 4 concentration inhibition. NifH-lacZ heterologous and homologous nifH-lacZ expression is consistent description of Alcaligenes faecalis nitrogenase gene expression and regulation mechanisms may be similar with Azospirillum brasilense. A15 nitrogen restrictive semi-solid medium, mutant A15M1 of nitrogenase activity 22.75nmol C 2 H 4 / hour. Bottles, A15M3 23.27n mol C 2 H 4 / hour. bottle, were higher than the wild-type A1501 (18.33n mol C 2 H 4 / hour. bottle). 15 N tracer experiment results show that, the mutant A15M1 nitrogen fixation efficiency 17.8gN/10 7 bacteria / day, the mutant A15M2 the nitrogen fixation efficiency 18.96gN/10 the 7 a bacteria / day, A15M3 nitrogen fixation efficiency 19.14gN/10 7 the fungus / day, respectively, compared with the wild-type (15.36μgN/10 7 bacteria / day) increased by 15.9%, 23.4% and 24.6%. Root surface colonization experiments showed that the mutant rice root colonization ability is slightly stronger than the wild type. Mutant and wild-type mixing equal amounts of inoculated into cultured on sterile rice seedlings, three mutant bacteria colonize the root surface, respectively, accounting for 61.53% of the total bacterial colonization, 70.2% and 60%, and only a small portion at the root of the internal colonization. Sterile rice seedlings inoculated with nifH-lacZ fusion gene mutant, the concept of sacrifice in situ X-gal staining showed that the mutant rice root surface [JJ \the junction and lateral efficient expression. scanning electron microscopy showed that the the inoculated mutant rice root tip of root hairs than inoculated with the wild-type rice root thin and long, more the number of root hairs per unit area relative. Tns core area card that neomycin resistance gene *) a pair of specific primers for PCR amplification of wild-type and mutant DNA, respectively, can from mutant A15MI, A15MZ, A15M3, amplified an approximately 500hp knock t 11 characteristics with prove * transposable sequence is indeed inserted to the secretion of ammonia mutant women chromophores, its insertion site characteristics associated with the secretion of ammonia, the sites may be involved in the process of nitrogen fixation negative regulator. DIG labeled nptll fragment Southern hybridization probes mutant AI SMI ^ 15M3 total DNA hybridization showed that: in mutation punish AI SMI in secretion of ammonia Tns insertion site is located in the size of about 10kb of the EcoR ISac of! DNA fragment; in mutant A15M3 and bleeding ammonia Tns insertion site is located in the size of approximately 18kb the EcoR!-RL DNA fragment,.

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