Enzyme spectrum method for determining two-dimensional spatial distribution of activity of aquatic plant rhizosphere phosphatase
A technology for phosphatase activity and aquatic plants, applied in the field of zymography to determine the two-dimensional spatial distribution of phosphatase activity in the rhizosphere of aquatic plants in sediments, can solve problems such as large measurement errors, improve spatial resolution, and improve experimental efficiency , the effect of uniform excitation light conditions
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Embodiment 1
[0031] Example 1: Construction of zymography imaging system
[0032] Such as figure 1 As shown, in order to standardize the zymogram imaging steps, a set of visual experiment brackets including a camera bracket and a light source bracket were used to fix the positions of the camera and the light source respectively. The light source was a strip of 375nm LEDs and combined into a square to obtain uniform excitation light. The nylon film of the fluorescent substance can be excited evenly; the light source and the camera can be simultaneously triggered to acquire images by using the trigger, and the operation steps are simplified by combining with the computer control system.
Embodiment 2
[0033] Embodiment 2: the drafting of zymogram calibration curve specifically comprises the following steps:
[0034] S1: Dissolve 4-methylumbelliferone in methanol to prepare calibration solutions of different concentrations of fluorescently labeled substrates: 0, 0.01, 0.1, 0.5, 1, 2, 5, and 10 mM, and then soak nylon membranes with a diameter of 2 cm To the above solutions for 10min;
[0035] S2: After the nylon membrane was soaked for 10 minutes, the nylon membrane was taken out and dried for 10 minutes, and then the fluorescence image was obtained using the above-mentioned zymography imaging system;
[0036] S3: Use Matlab software to extract the blue channel of the fluorescence image, and calculate the accumulation of 4-methylumbelliferone per unit area according to the solution volume absorbed by the membrane and the membrane size, and then convert the fluorescence image acquired at each concentration Curve fitting is carried out between the intensity of the blue channe...
Embodiment 3
[0039] Embodiment 3: actually measure the two-dimensional distribution of aquatic plant rhizosphere acid and alkaline phosphatase activity in the sediment, the specific steps are as follows:
[0040] M1: Prepare a root box with a detachable surface. Plant the seedlings of the aquatic plants bitter grass and reed in the root box containing sediment. The plants are cultivated close to the detachable surface. During the cultivation process, the root box is inclined 30-45°, so that Plant roots grow close to the removable face.
[0041] M2: Rapidly inject the mixture of polyacrylamide, ammonium persulfate and tetramethylethylenediamine into two glass plates with a 0.4mm interlayer to prepare a polyacrylamide gel diffusion layer, and store the polyacrylamide gel in Reserve in deionized water.
[0042] M3: Prepare 12mM acid phosphatase and alkaline phosphatase substrate stock solutions by dissolving fluorescently labeled substrate 4-methylumbelliferyl phosphate in MES at pH=6.5 and ...
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