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Method for extracting sucrose synthase and sucrose phosphate synthase of pear fruit and activity determination method thereof

A phosphate synthase and sucrose synthase technology, applied in the field of plant physiology, can solve problems such as unscientific treatment methods, low efficiency, and inability to measure activity, and achieve the effects of improving fruit quality, effective activity, and being beneficial to accuracy.

Inactive Publication Date: 2012-10-24
NANJING AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0003] At present, there are reports on the methods of measuring the activity of sucrose synthase and sucrose phosphate synthase in pear fruit at home and abroad. However, due to the unscientific treatment method and the limitation of application range, the measurement of invertase activity is inaccurate or inefficient. The activity of the corresponding enzyme cannot even be determined according to the published method steps

Method used

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  • Method for extracting sucrose synthase and sucrose phosphate synthase of pear fruit and activity determination method thereof
  • Method for extracting sucrose synthase and sucrose phosphate synthase of pear fruit and activity determination method thereof
  • Method for extracting sucrose synthase and sucrose phosphate synthase of pear fruit and activity determination method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] (1) Experimental method

[0028] 1. The test material is Yali (Pyrus bretschneideri Rehd'yali').

[0029] 2. Sample collection: Take the fruit 105 days after the flowering of Yali pear, and randomly select 5 disease-free fruits according to the top, east, south, west, and north directions of the canopy when sampling, put them in an ice box, and bring them back to the experiment room processing.

[0030] 3. Sample treatment: clean the collected fruit, peel and remove the core, mix the pulp evenly and then sample by quartering. After quick freezing in liquid nitrogen, place it in a -70°C refrigerator for sucrose synthase and sucrose Determination of Phosphate Synthase Activity.

[0031] 4. Extraction of sucrose synthase and sucrose synthase enzyme:

[0032] The extraction of the enzyme solution was carried out at 0-4°C. Accurately weigh 1 g of pear pulp, put it into a pre-cooled mortar, grind in an ice bath, add 5 mL of extraction buffer (200 mmol L -1 Hepes-NaOH, 5m...

Embodiment 2

[0042] 1. Establishment of standard curve for sucrose synthase (decomposition direction):

[0043] 1) Dry the analytically pure fructose at 80°C to constant weight, accurately weigh 1g of fructose, dissolve it in water, and set the volume to 100mL to form a standard solution of fructose.

[0044] 2) Take 7 test tubes with a 25mL scale, number them, and add the solution accurately according to the amount shown in Table 2.

[0045] Table 2 makes the reagent quantity of standard curve

[0046]

[0047] 3) Shake each test tube evenly, heat in a boiling water bath for 5 minutes, cool to room temperature, and measure OD at a wavelength of 540nm 540 value. Taking the absorbance as the ordinate and the milligrams of fructose as the abscissa, the regression equation (y=2.3013x-0.1841) was obtained.

[0048] Calculation of enzyme activity:

[0049] Enzyme activity (μmol h -1 g -1 FW)=[Fructose content calculated according to the standard curve (mg) / 180.17 (molar mass)×volume of...

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Abstract

The invention belongs to the field of plant physiology, and discloses a method for extracting sucrose synthase and sucrose phosphate synthase of pear fruits and an activity determination method thereof. Small molecules such as sugar, ions and the like can be usually removed from a primary extraction enzyme solution through adding a salting-out step and a dialysis step in the extracting process of the sucrose synthase and the sucrose phosphate synthase, so that activities of the sucrose synthase and the sucrose phosphate synthase in the fruits can be accurately detected. The method provides physiological basis for disclosing accumulation differences of the sugar in the fruits, and further provides theoretical basis for improving the quality of fruit sweetness and flavor.

Description

technical field [0001] The invention belongs to the field of plant physiology, and relates to a method for extracting sucrose synthase and sucrose phosphate synthase from pear fruit and an activity assay method thereof. Background technique [0002] Soluble sugar in pear fruit is a key factor that directly affects fruit quality and plays an important role in the formation of fruit quality and flavor, so it has become one of the important standards to measure the quality of fruit. The main sugars in pear fruit are sucrose, glucose, fructose, sorbitol and so on. The accumulation of sugar in fruit is closely related to the sugar metabolizing enzymes in the fruit, especially sucrose synthase and sucrose phosphate synthase among the sucrose metabolizing enzymes. In the pool tissue of sucrose accumulation, sucrose phosphate synthase plays an important role in the accumulation of sucrose. The increase of sucrose phosphate synthase activity in fruits such as pears, peaches, oranges...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12Q1/48
Inventor 吴俊杨志军张绍铃齐开杰陶书田吴巨友
Owner NANJING AGRICULTURAL UNIVERSITY
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