Rhizobiaceae gene and polypeptide and application
A rhizobia and gene technology, applied in the fields of application, bacterial peptides, genetic engineering, etc., can solve problems such as limitations, complex signal recognition gene regulation relationship, etc., and achieve energy-saving and environment-friendly effects
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Embodiment 1
[0032] 1. Acquisition of novel polypeptide genes
[0033] (1) Gene fragment amplification
[0034] Design forward and reverse primers:
[0035] Forward primer: HnifF, 5′-TGA CCC CAA GGC CGA CC-3′;
[0036] Reverse primer: HnifR, 5'-GTG CCA TCA TCT CGC C-3'.
[0037] The present inventor isolated a strain of nitrogen-fixing bacteria from the Psammochloa villosa plant in the desert of Xiangshuiwan, Inner Mongolia. The total DNA was extracted by conventional phenol-chloroform extraction method, ethanol precipitation and washing, and the reagents used were conventional molecular biology reagents. , performing PCR amplification on the genomes of strains of the Rhizobiaceae family.
[0038] PCR reaction system:
[0039] 10×PCR Buffer 2.5μL
[0040] dNTPmix (2.5mmol.L -1 ) 0.5μL
[0041] HnifF (25μM) 0.5μL
[0042] HnifR (25 μM) 0.5 μL
[0043] DNA template 0.5 μL
[0044] Taq enzyme (4U / μL) 0.5μL
[0045] dd H 2 O 20 μL
[0046]
...
Embodiment 2
[0154] Example 2 Determination of Nitrogenase Activity by Acetylene Reduction Method
[0155] Taking Escherichia coli BL21 as the control sample, take 3 mL each of the control sample and the transformant 3-4, respectively, and put them into a test tube with a rubber stopper, inject 1 / 10 of the volume of acetylene gas into the test tube, cultivate it in a 37°C incubator for 24 hours, and use a gas phase Chromatographic determination of nitrogenase activity, the transformant can reduce acetylene to ethylene; use gas chromatography to measure the generation of ethylene in each reaction solution, and the reduction results of the control sample and sample 3-4 are shown in the appendix respectively. figure 2 And attached image 3 , of which, attached figure 2 For the reduction results of the control sample, attached image 3 It is the reduction result of sample 3-4, attached figure 2 And attached image 3 Among them, curve 1 represents methane, curve 2 represents ethylene, an...
Embodiment 3
[0157] Take 3mL of the control and transformant 3-4 samples respectively, put them into the test tubes with rubber stoppers, and seal them with rubber stoppers. After culturing in a 37°C incubator for 24 hours, take samples and use a flow injection analyzer to directly inject samples to determine NH by Nash reagent colorimetry. 4 + concentration, the results are shown in Table 1 below:
[0158] Table 1 NH in control and sample 4 + concentration
[0159]
[0160] Note: The values in the table are mean ± standard deviation; lowercase English letters in the same row indicate significant differences in Duncan’s multiple comparisons (P<0.05); letters appearing in the following tables have the same meaning.
[0161] It can be seen from Table 1 that the NH in the transformant samples 3-4 4 + The concentration increased significantly, indicating that transformants 3-4 could convert N in the air 2 , converted to NH 4 + .
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