Method for sifting functional fungoid having specific affinity with non-leguminous plant by using agglutinin
A technology of legumes and lectins, applied in biochemical equipment and methods, microbial determination/inspection, biological testing, etc., can solve problems such as lack of
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Embodiment 1
[0020] Example 1: Screening of cotton rhizosphere-specific functional strains
[0021] 1 Extraction of cottonseed lectin
[0022] Cottonseed contains more lectins, but the seeds also contain more fat, so it is necessary to take measures to remove the fat in the material and then extract the lectins.
[0023] ① Peel 100g cottonseed into powder;
[0024] ②Add 500ml of anhydrous ether to degreasing for 10min, repeat twice, vacuum pump to drain excess ether;
[0025] ③Add 500ml PBS (0.01mol, pH7.2) for leaching at 4°C (the leaching solution has the strongest activity at 72h);
[0026] ④ Filter to remove coarse slag, and centrifuge the filtrate (4000rpm, 20min, normal temperature) to remove fine slag;
[0027] ⑤Put the supernatant in an ice bath, add (NH 4 ) 2 SO 4 to 40% to 90% saturation (add and stir to prevent protein denaturation);
[0028] ⑥ Let stand overnight, centrifuge (6000rpm, 30min, 4℃), discard the supernatant;
[0029] ⑦ Dissolve the precipitate with 40ml of ...
Embodiment 2
[0075] Embodiment 2: Screening of wheat rhizosphere-specific potassium-decomposing strains
[0076] 1 Extraction and purification of wheat germ agglutinin
[0077] A method for the extraction of lectins from materials containing a small amount of fat is used.
[0078] ① Wheat Germ 100g
[0079] ②Add 1000ml 0.05mol·L -1 Extract with HCl, stir for 1h, at room temperature, centrifuge at 2000×g for 10min, discard the residue, and the supernatant is the crude lectin extract;
[0080] ③Put the supernatant in an ice bath, add ammonium sulfate to 40%-90% saturation, stir for 1 hour; centrifuge at 10,000g at 4°C for 15 minutes, discard the supernatant;
[0081] ④Add 45ml 0.05mol·L to the precipitate -1 Dissolve in HCl, add 15ml of n-butanol dropwise, stir for 1h to degrease, centrifuge at 3000g for 30min, keep the water phase, (repeat the degreasing twice);
[0082] ⑤ Dilute the centrifuged supernatant to 0.05mol·L -1 HCl dialyzed overnight;
Embodiment 3
[0103] Example 3: Screening of rice rhizosphere-specific phosphorus-solubilizing strains
[0104] 1 Extraction and purification of rice lectins
[0105] The rice lectin was extracted and purified using the same "method of extraction of lectin in a small amount of fat-containing material" as in Example 2.
[0106] 2 Labeling Rice Lectin with Biotin
[0107] ① Dialyze the purified lectin (10ml) in labeling buffer (0.1Mol / LNaAc, pH 5.5, 0.1mol / LNaCl) at 4°C overnight;
[0108] ② Pipet 5ml into a centrifuge tube, add sodium periodate solution to a final concentration of 10mmol / L, and place it on an ice bath for 30min to avoid light to oxidize the sugar groups on the lectin molecule;
[0109] ③ Equilibrate the Sephadex G25 PD-10 prepacked column with PBS, pass the oxidized lectin through the column, and collect the lectin fraction with coagulation activity;
[0110] ④ Add biotin to the lectin tube to a final concentration of 5 mmol / L, and place it on a shaker for 1 hour at 25°C;...
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