Experimental results of the experiment on the induction of ?α?-?amylase by gibberellins

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The mobilization of stored substances during seed germination requires the catalysis of a series of enzymes. Some of these enzymes are already present in dry seeds, and some need to be resynthesized after the seeds absorb water. The decomposition of starch during seed germination is mainly completed under the catalysis of amylase. Amylase exists in many forms in plants, including α-amylase, β-amylase, etc. β-amylase already exists in dry seeds, while α-amylase does not exist or rarely exists in dry seeds and needs to be resynthesized after the seeds absorb water. Experiments have shown that the chemical messenger that initiates α-amylase synthesis is gibberellin. The embryo of germinating barley seeds produces gibberellins that diffuse into the aleurone layer of the endosperm, stimulating the synthesis of α-amylase in the aleurone cells. The synthesized amylase enters the endosperm and hydrolyzes the starch stored in the endosperm into reducing sugars. Therefore, without the activity of embryonic release of gibberellins, α-amylase cannot be synthesized. The addition of gibberellins can replace the release of embryos and induce the synthesis of α-amylase. This extremely specific reaction was used as a bioassay for gibberellins. Within a certain range, the amount of reducing sugar produced by degerminated imbibed barley kernels is proportional to the logarithm of the concentration of added gibberellin. Based on the principle that starch can develop blue color with I2-KI, but reducing sugar, the product of starch decomposition, cannot develop color with I2-KI, the activity of α-amylase can be analyzed qualitatively and quantitatively.

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(1) Experimental materials

Barley (or wheat) seeds .

(2) Reagents

1.1% sodium hypochlorite solution.

2. 0.1% starch solution: Take 1g starch and 8.16g

KH2PO4, and prepare a 1000mL solution with distilled water.

3. 2×10-5mol/L gibberellin solution: weigh 6.8mg of GA3

into a beaker, add a small amount of 95% alcohol to dissolve it, and transfer it to 1000mL capacity In the bottle, add water to bring to volume. Then dilute it into three concentrations of gibberellin solutions: 2×10-6mol/L, 2×10-7mol/L and 2×10-8mol/L.

4. 10-3mol/L acetic acid buffer (pH4.8): take 2mL

0.2mol/L acetic acid (11.55mL glacial acetic acid diluted to 1000mL), 3mL< /p>

0.2

mol/L sodium acetate (16.4g anhydrous sodium acetate to make 1000mL) and 1g streptomycin, dilute to 000mL, each mL buffer contains streptomycin 1 mg.

5. I2-KI solution: Dissolve 0.6gKI and 0.06gI2 in 1000mL0.05mol/L HCl.

(3)

Instruments and equipment

Spectrophotometer, constant temperature oscillator, water bath, 1 2mL pipette, 2 50mL beakers, test tubes 6 bottles, 6 vials of penicillin, 1 pair of tweezers, razor blade.

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Sampling

Select the ones that are consistent in size and intact 50 barley or wheat seeds, cut each seed transversely into embryonic and non-embryonic half with a razor blade, and divide into 2 beakers.

Surface disinfection

Add 1% sodium hypochlorite solution to 2 beakers to the extent that the seeds are submerged. Disinfection

After 15 minutes, rinse 3 times with sterile water and set aside.

Treatment concentration

After numbering the 6 penicillin vials, add the solution and materials according to Table 40-1. After the solution is mixed, the final concentrations of gibberellins in vials 1 to 6 are: 0, 0, 2×10-5, 2×10-6, 2×10-7 and 2×10-8mol/L respectively. Place the penicillin vial in a constant-temperature oscillator and incubate with shaking at 25°C for 24 hours.

Table

40-1

Treatment concentration and method

Penicillin vial number

Gibberellin solution

Acetate buffer (mL)

Experimental materials

Concentration (mol/L)

10 embryoless half grains

10 embryonic halves

2×10-5

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