When barley (or wheat) seeds germinate, the seed embryo produces GA3 that diffuses into the endosperm's pasteurized cells (known as the "target cells" of the GA3 reaction), stimulating them to synthesize or activate α-amylase, which then enters the endosperm and hydrolyzes the stored starch into reductase; therefore, embryo-free seeds do not release GA3, nor do they form or activate α-amylase. α-amylase. The externally added GA3 can also replace the releasing action of the embryo, thus inducing α-amylase synthesis. The amount of reducing sugars produced by de-embryonated suckling barley was, within a certain range, proportional to the logarithm of the concentration of externally added GA3, thus indicating the induction of α-amylase formation by GA3.
Operation method
Experiments on the induction, extraction and activity determination of amylase
Principle
When barley (or wheat) seeds germinate, the seed embryo produces GA3 that diffuses into the endosperm's pasteurized cells (known as the "target cells" of the GA3 reaction), stimulating them to synthesize or activate α-amylase, which then enters the endosperm and hydrolyzes the stored starch into reductase; therefore, embryo-free seeds do not release GA3, nor do they form or activate α-amylase. α-amylase. The externally added GA3 can also replace the releasing action of the embryo, thus inducing α-amylase synthesis. The amount of reducing sugars produced by de-embryonated suckling barley was, within a certain range, proportional to the logarithm of the concentration of externally added GA3, thus indicating the induction of α-amylase formation by GA3. Move I. Materials and supplies For more product details, please visit Aladdin Scientific website.
1. Material: Barley (or wheat) seeds;
2. Instruments: 721 spectrophotometer; ultra-clean bench (or sterilizer); temperature chamber; shaker; constant temperature water bath; autoclave; cotton plugs; kraft paper; razor blades; tweezers; beakers; petri dishes; test tubes; pipettes; glass rods.
3. Drugs: 10-3 mol/L acetate buffer (pH 4.8) [1 mg of streptomycin sulfate (or 40 μg of chloramphenicol) per ml]. 10 mg/L GA310 mg, dissolved with a small amount of 95% ethanol, and fixed to 1000 ml with distilled water. Starch solution (weigh 0.67 g of soluble starch and KH2PO40.82 g dissolved in 20 ml of distilled water and added to 70 ml of boiling water with constant stirring, and finally added with water and fixed to 100 ml). I2- KI solution (I2 0.06g and KI 0.6g were weighed and dissolved in 0.05 mol/L HCL solution 1000ml). 5% bleach solution (W/V) . 5% H2SO4 solution (W/V). Sterilized water . Quartz sand.
II. Methods and steps
1. Material preparation
Selection of GA3 sensitive. The germination was high. Wheat seeds of uniform size were soaked in 50% H2SO4 for 2h, removed, rinsed with tap water for about 20 times, and then rubbed vigorously to remove the glumes; the seeds were cut in two halves of nearly equal length with a razor blade, so that they were made into the non-embryonic and embryonic halves, each of which was divided into 150 grains and two small beakers, and sterilized in 5% bleach solution for 15min, and then poured off the bleach solution and washed with sterile water for 5 times in aseptic conditions. 5 times. Then, the embryo-free and embryonic half seeds were placed in a sterile petri dish with a layer of quartz sand, poured into the sterile water that just submerged the seeds, and placed the petri dish in a 25-degree incubator for 24-48 h. The seeds were then washed with sterile water for 5 times.
2. Configuration of GA3 series concentration standard solution
Take 5 dry and clean test tubes (numbered), each with 9 ml of distilled water, add 1 ml of GA3 mother liquor to No. 1 tube, mix well and then suck out 1 ml and add it to No. 2 tube; No. 2 tube mix well and then suck out 1 ml and add it to No. 3 tube; and dilute them in turn, and then formulate them into GA3 series concentration standard solution of 0.10-1 mg/L, 10-2 mg/L, 10-3 mg/L, 10-4 mg/L, 10-5 mg/L in turn. standard solution. Then take 8 dry clean test tubes (0-7), according to the table to add a variety of test liquids and materials (beaker in the absorption of half a grain of barley seeds), and 25 degrees under the oscillation insulation for 24 h, filtration (or centrifugation), filtrate (or the supernatant) standby.
3. Determination of α-amylase activity
Take 8 dry and clean test tubes (0-7), add distilled water 0.8 ml and starch solution 1 ml, and then add half of the seed holding filtrate (or supernatant) 0.2 ml mixing according to the number, and then keep warm for 10 min in a constant temperature water bath at 25 degrees (the appropriate time is determined by the preparatory test, i.e., the reaction time is appropriate for the reaction solution with iodine reagent with 1mg/L GA3 to reach OD value of 0.4-0.5), and then immediately hold it for 24 hours with shaking at 25 degrees. The reaction time was determined by the preparatory test, i.e., the reaction solution of 1 mg/L GA3 with iodine reagent was used to reach 0.4-0.5 OD value.), the test tube was immediately taken out and put into cold water, and the reaction was terminated by adding 1 ml of I2- KI reagent, and then 2 ml of distilled water was added, and then the OD value was determined at 620 nm after mixing. The standard curve was plotted with the negative logarithm of GA3 concentration as the horizontal coordinate and OD value as the vertical coordinate. 