Embodiment
Below by drawings and Examples, the present invention is described in further detail, but protection domain of the present invention is not limited to described content.In embodiment, method if no special instructions, carry out routinely, the reagent that uses if no special instructions reagent to be conventional commercial reagent or to prepare according to a conventional method by operation.
Embodiment 1: the MgCl that promotes India canna nitrate nitrogen absorption and assimilation
2the application of promoter, comprises the steps:
(1) experiment material is India canna seedling; Select the India canna seedling of plant height 30-40cm in wetland soil to dig out, be placed on water planting in running water, change every three days water one time, cultivate about 30 days extremely new roots and grow up to for this experiment;
(2) preparation amount of substance concentration is the MgCl of 150 μ mol/L
2solution;
(3) India canna in (1) is placed in to above-mentioned steps (2) MgCl
2in solution, India Canna Rhizome is carried out to 8h pretreatment; Then the plant of processing is transferred to volume and be in the agricultural wastewater that 3L contains different nitrates; Take without the pretreated India canna of superchlorination magnesium is contrast (CK) simultaneously, is also placed in volume and is in the agricultural wastewater that 3L contains different nitrates; Measure the impact of magnesium chloride on nitrate nitrogen efficiency and relative physiologic index in India canna absorption agricultural wastewater.
Embodiment 2: the India canna checking MgCl after adopting embodiment 1 to process
2promoter absorbs the impact of nitrate speed on India canna, comprise the steps:
At tri-time points of 2h, 12h, 22h of processing, get respectively agricultural wastewater water sample 5 mL of experimental group and the processing of control group India canna, add 2 aluminium hydroxide suspension, standing flocculation, centrifugal rear supernatant is measured for nitrate, it during mensuration, with optical length, is the quartz colorimetric utensil of 10 mm, take fresh deionized water as reference, at 220 nm and 275 nm wavelength places, detect respectively, the corrected value of nitrate nitrogen absorbance is that OD220 value deducts the OD275 value of 2 times, checks in nitrate from calibration curve.3 repetitions are done in each processing, get the mean value of measurement result; By the method, verify MgCl
2activator is used the impact that India canna is absorbed to nitrate speed.
Fig. 1 demonstration, A figure is without MgCl
2process the absorption result of India Canna Rhizome to nitrate, B figure is through MgCl
2the absorption result of India Canna Rhizome to nitrate after processing.MgCl
2the result that affects that processing absorbs nitrate speed to India canna shows, adopts MgCl of the present invention
2inorganic agent is processed after India canna, and India canna seedling is significantly higher than and does not pass through MgCl the absorption rate of nitrate
2the control group of processing.
Embodiment 3: the India canna checking MgCl after adopting embodiment 1 to process
2promoter is to India canna plasma membrane H
+the impact of-ATPase activity, comprises the steps:
After A, the wastewater treatment of variable concentrations nitrate nitrogen, the root, 0.5 g of leaf texture that get India canna under different disposal, use liquid nitrogen Rapid-Freezing Method, adds Extraction of plasma membrane liquid (the 0.25 mol/L sorbierite of 1 mL after liquid nitrogen grinding is powder, 1 mmol/L EDTA, 5 mmol/L MgSO
4, 10 mmol/L Tris-HCl pH7.4); Homogenate is with 9000 g, and 4 ℃ of centrifugal 20min remove precipitation, and supernatant is through 30000g, 4 ℃ of centrifugal 1h; Collecting precipitation is also suspended in 0.5mL said extracted liquid it, and gained is cytoplasma membrane albumen;
B, by Brodford method, measure plasmalemma protein concentration: in the distilled water of 800 μ L, add the plasmalemma protein of 5 μ L to mix, then the commercial Brodford solution that adds 200 μ L, under OD595 wavelength, detect protein concentration, and calculate volume corresponding to 500 μ g plasmalemma protein;
C, H
+in the reaction system that is determined at 0.5 mL of-ATPase activity, carry out; Reaction system comprises 50 mmol/L BTP/MES, 5 mmol/L MgSO
4, 50 mmol/L KCl, 0.02% Brij-35 (w/v), 50 mmol/L KNO
3, 1 mmol/L(NH
4)
2moO
4, 1 mmol/L NaN
3, 4 mmol/L ATP-Na
2, start reaction after adding the plasmalemma protein extract of 500 μ g;
Reactant mixture is placed in after 30 ℃ of water-bath 30min, adds reaction terminating liquid 1 ml (2 % H
2sO
4(v/v), 5 % SDS (w/v) and 0.7 % (NH
4)
2moO
4(w/v)), add immediately 50 μ L 10% Vc (w/v) and under room temperature, place 20 min, measuring wavelength is the light absorption value at 700 nm places.
D, according to Phos calibration curve, calculate the release content of Phos, the plasma membrane H of 1 unit
+-ATPase activity is defined as: under the reaction condition of 30 ℃, in 1 minute, every milligram of albumen catalysis ATP decomposes the micromole's number that discharges inorganic phosphate.
Fig. 2 demonstration, A figure is MgCl
2plasma membrane H in India Canna Rhizome before and after processing
+-ATPase activity change, wherein without MgCl
2the India canna of processing is control group, represents, through MgCl with CK
2the India canna of processing is experimental group; B figure is MgCl
2plasma membrane H in India canna leaf before and after processing
+-ATPase activity change, wherein without MgCl
2the India canna of processing is control group, represents, through MgCl with CK
2the India canna of processing is experimental group.MgCl
2processing is to India canna plasma membrane H
+the result that affects of-ATPase activity shows, adopts MgCl of the present invention
2inorganic agent is processed after India canna, plasma membrane H in India canna shoot root and leaf
+-ATPase activity is significantly higher than control group CK.
Embodiment 4: the India canna checking MgCl after adopting embodiment 1 to process
2promoter is to H in India Canna Rhizome
+the impact of-pump activity, comprises the steps:
After A, the wastewater treatment of variable concentrations nitrate nitrogen, get root 0.5 g of India canna under different disposal, use liquid nitrogen Rapid-Freezing Method, after liquid nitrogen grinding is powder, add Extraction of plasma membrane liquid (0.25 mol/L sorbierite, 1 mmol/L EDTA, the 5 mmol/L MgSO of 1 mL
4, 10 mmol/L Tris-HCl pH7.4); Homogenate is with 9000g, and 4 ℃ of centrifugal 20 min remove precipitation, and supernatant is through 30000g, 4 ℃ of centrifugal 1h; Collecting precipitation is also suspended in 0.5ml said extracted liquid it, and gained is cytoplasma membrane albumen;
B, use Brodford method are measured plasmalemma protein concentration.In the distilled water of 800 μ L, add the plasmalemma protein extract of 5 μ L to mix, then add the commercial Brodford solution of 200 μ L, under OD595 wavelength, detect protein concentration, and calculate volume corresponding to 100 μ g plasmalemma protein;
In C, reaction system 1.5 ml, contain 5 mmol/L BTP/MES (pH 6.0), 12 μ mol/L AO, 300 mmol/L KCl, 250 mmol/L sucrose, 0.5 mmol/L EGTA(is used BTP to adjust pH to 6.0), 1 mmol/L NaN
3, 1 mmol/L Na
2moO
4, 50 mmol/L KNO
3, 0.05% Brij-35 (w/v) and 100 μ g plasmalemma proteins; Add abstergent Brij-35 and make the upset of original position film, reaction mixture is at room temperature placed after 20 min, adds 5 mmol/L ATP/BTP (pH 6.0) to start reaction;
D, proton are to carry out in the method for 492 nm light absorption value cancellation according to measuring acridine orange (AO) from outside pumping in film; By the method, verify MgCl
2activator is used H in India Canna Rhizome
+the impact of-pump activity.
Fig. 3 demonstration, A figure is not for passing through MgCl
2the control group of processing, B figure is through MgCl
2the experimental group of processing.Through MgCl
2processing is to India canna root H
+the result that affects of-pump activity shows, adopts MgCl of the present invention
2inorganic agent is processed after India canna, India canna root H
+-pump activity is significantly higher than control group CK.
Embodiment 5: the India canna checking MgCl after adopting embodiment 1 to process
2the impact of promoter on India canna nitrate reductase activity, comprises the steps:
After A, the wastewater treatment of variable concentrations nitrate nitrogen, get root, the 0.5g of leaf texture of India canna under different disposal, use liquid nitrogen Rapid-Freezing Method, after liquid nitrogen grinding is powder, add 1.5mL extract (25 mmol/L phosphate buffer pH7.4,1 mmol/L EDTA, 10 mmol/L cysteines) grind homogenate, be transferred in 2 mL EP pipes 4000 rpm, 4 ℃, centrifugal 15 min, remove precipitation supernatant and are zyme extract;
B, get 0.2 mL zyme extract in 2 mL EP pipes, add 0.5 mL 0.1 mmol/L KNO
3phosphate buffer and 0.3 mL NADH solution, mix, 25 ℃ of water-bath 30 min, after finishing, water-bath adds 0.5 mL sulfanilamide (SN) cessation reaction, add again 0.5 mL alpha-naphthylamine, mix, after colour developing 15 min, with spectrophotometer, in 520 nm places, carry out colorimetric estimation, write down OD value, using and do not add NADH(and add 0.2mL water) as blank;
C, according to calibration curve, calculate reactant liquor Central Asia nitrate nitrogen content, the nitrite nitrogen micrograms producing with every gram of fresh weight per hour represents nitrate reductase activity;
Fig. 4 demonstration, A figure is MgCl
2before and after processing, in India Canna Rhizome, nitrate reductase activity changes, wherein without MgCl
2the India canna of processing is control group, represents, through MgCl with CK
2the India canna of processing is experimental group; B figure is MgCl
2before and after processing, in India canna leaf, nitrate reductase activity changes, wherein without MgCl
2the India canna of processing is control group, represents, through MgCl with CK
2the India canna of processing is experimental group.MgCl
2processing shows the result that affects of India canna nitrate reductase activity, adopts MgCl of the present invention
2inorganic agent is processed after India canna, and in India Canna Rhizome, leaf, the activity of nitrate reductase is significantly higher than control group CK.
Embodiment 6: the India canna checking MgCl after adopting embodiment 1 to process
2the impact of promoter on India canna activity of glutamine synthetase, comprises the steps:
After A, the wastewater treatment of variable concentrations nitrate nitrogen, the root, the 0.5g of leaf texture that get India canna under different disposal, use liquid nitrogen Rapid-Freezing Method, adds 1.5 mL extracts to grind homogenate after liquid nitrogen grinding is powder, transfer them to 4500 rpm/min in centrifuge tube, 4 ℃ of centrifugal 10 min, abandon precipitation, get supernatant centrifugal 10 min of 4500 rpm again, be precipitated as chloroplast part, get centrifugal 20 min of supernatant 12000rpm at 4 ℃, be precipitated as mitochondria part, supernatant is crude enzyme liquid;
B, by Brodford method, measure thick liquid protein concentration, in the distilled water of 800 μ L, add the GS albumen crude enzyme liquid of 5 μ L to mix, then the commercial Brodford solution that adds 200 μ L detects protein concentration under OD595 wavelength, and calculates 0.7 mL crude enzyme liquid soluble protein content;
C, get the thick zyme extract of 0.7 mL and 0.7 mL ATP (40 mmol/L) solution adds (80 mmol/L hydroxylamine hydrochlorides, 80 mmol/L MgSO in 1.6 mL reactant liquor B
4, 20 mmol/L sodium glutamates, 20 mmol/L cysteines, 2 mmol/L EGTA, 0.1 mol/L Tris-HCl pH7.4), put upside down and mix, 37 ℃ of standing 30 min, add 1ml developer (0.2 mol/L TCA, 0.37 mol/L FeCl
3with 0.6 mol/L HCl mixed liquor), put upside down and mix, centrifugal 10 min of 5000 rpm, get supernatant and read absorbance at 540nm place.With not overstriking zyme extract, add 0.7mL reactant liquor A(80 mmol/L MgSO
4, 20 mmol/L sodium glutamates, 20 mmol/L cysteines, 2 mmol/L EGTA, 0.1 mol/L Tris-HCl pH7.4) and as blank;
D, GS vigor (A/mg proteinh)=OD540nm/(soluble protein content * 0.5) calculate enzyme and live; By the method, verify MgCl
2promoter is respectively organized the impact of activity of glutamine synthetase on India canna.
Fig. 5 demonstration, A figure is MgCl
2before and after processing, in India Canna Rhizome, activity of glutamine synthetase changes, wherein without MgCl
2the India canna of processing is control group, represents, through MgCl with CK
2the India canna of processing is experimental group; B figure is MgCl
2before and after processing, in India canna leaf, activity of glutamine synthetase changes, wherein without MgCl
2the India canna of processing is control group, represents, through MgCl with CK
2the India canna of processing is experimental group.MgCl
2processing shows the result that affects of India canna activity of glutamine synthetase, adopts MgCl of the present invention
2inorganic agent is processed after India canna, and in India Canna Rhizome and leaf, the activity of glutamine synthelase is significantly higher than control group CK.
Embodiment 7: the MgCl that promotes India canna nitrate nitrogen absorption and assimilation
2the application of promoter, comprises the steps:
(1) preparation amount of substance concentration is the MgCl of 130 μ mol/L
2solution;
(2) select the India canna seedling of plant height 30-40cm in wetland soil to dig out, be placed in running water and cultivate, change every three days water one time, cultivate about 30 days extremely new roots and grow up to for this experiment;
(3) India canna in (2) is placed in to above-mentioned steps (1) MgCl
2in solution, India Canna Rhizome is carried out to 6h pretreatment; Then the plant of processing is transferred to volume and be in the agricultural wastewater that 3L contains different nitrates; Take without the pretreated India canna of superchlorination magnesium is contrast (CK) simultaneously, is also placed in volume and is in the agricultural wastewater that 3L contains different nitrates; Measure MgCl
2promoter absorbs nitrate speed to India canna, and method is with embodiment 2.
Fig. 6 demonstration, A figure is not for passing through MgCl
2the control group of processing, B figure is through MgCl
2the experimental group of processing.MgCl
2the result that affects that processing absorbs nitrate speed to India canna shows, adopts MgCl of the present invention
2inorganic agent is processed after India canna, India canna seedling to the absorption rate of nitrate higher than control group CK.
Embodiment 8: the MgCl that promotes India canna nitrate nitrogen absorption and assimilation
2the application of promoter, comprises the steps:
(1) preparation amount of substance concentration is the MgCl of 180 μ mol/L
2solution;
(2) select the India canna seedling of plant height 30-40cm in wetland soil to dig out, be placed in running water and cultivate, change every three days water one time, cultivate about 30 days extremely new roots and grow up to for this experiment;
(3) India canna in (2) is placed in to above-mentioned steps (1) MgCl
2in solution, India Canna Rhizome is carried out to 7h pretreatment; Then the plant of processing is transferred to volume and be in the agricultural wastewater that 3L contains different nitrates; Take without the pretreated India canna of superchlorination magnesium is contrast (CK) simultaneously, is also placed in volume and is in the agricultural wastewater that 3L contains different nitrates; Measure MgCl
2promoter absorbs nitrate speed to India canna, and method is with embodiment 2.
Fig. 7 demonstration, A figure is not for passing through MgCl
2the control group of processing, B figure is through MgCl
2the experimental group of processing.MgCl
2the result that affects that processing absorbs nitrate speed to India canna shows, adopts MgCl of the present invention
2inorganic agent is processed after India canna, India canna seedling to the absorption rate of nitrate higher than control group CK.