CN105651888A - Method for analyzing influence of environmental factors on contribution mechanism of amino-acid nutrition - Google Patents
Method for analyzing influence of environmental factors on contribution mechanism of amino-acid nutrition Download PDFInfo
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Abstract
The invention provides a method for analyzing the influence of environmental factors on the contribution mechanism of amino-acid nutrition. The method comprises the following steps: adopting combination of sterile culture and an isotopic tracing technology to study the influence of the environmental factor on the plant growth and amino-acid absorption and determine long-term effect of the environmental factor; then adopting a method for absorbing and adding a metabolic inhibitor in a short term to study the influence of the environmental factor on the absorption of a root system to the amino acid in the short term and the absorption mode; and finally, adopting a combined detection technology with combination of isotopic tracing and gas chromatography-isotopic mass spectrometry to determine the speed-limiting step of using the environmental factors to limit the glycine metabolism. The method provided by the invention has the advantages that the design is reasonable and the operation is simple; the precision and the credibility of the combined detection technology with the isotopic tracing technology and gas chromatography-isotopic mass spectrometry are higher, and the influence of the environmental factors on the contribution mechanism of the amino-acid nutrition can be effectively analyzed.
Description
Technical field
The invention belongs to soil science field, relate to Plant Nutrition and plant physiology science study method, particularly relate to a kind of method that analysis environments factor affects amino acid nutrient contribution mechanism.
Background technology
Since 1840 Li Bixi (Liebig) founded " mineral nutrition of plant theory " rise, most research all for inorganic nitrogen launch, and organotrophic research and relatively backward. The research of last decade shows, most plants possesses absorption aminoacid, polypeptide, nucleic acid, the even ability of macro-molecular protein. Although plant can absorb organic nitrogen has become as common recognition, but under natural environment, soil amino acid turnover is rapid, and therefore under natural environment, amino acid whose nutrition is contributed still without final conclusion. The model that Lipson etc. absorb according to plant-microorganism competition, calculates the aminoacid nutrition contribution rate to acrophyta and is not less than 50%. But, in subtropical zone, according to13C,15N Double Labelling Technique research show that its nutrition contribution rate is lower than 1%. From the difference of different region amino acid nutrition contributions it can be seen that amino acid nutrient contribution is had important effect by environmental condition.
Envirment factor includes temperature, intensity of illumination, gas concentration lwevel, soil nitrogen composition and content, soil aciditiy, Soil structure, soil pollutant composition and concentration etc., and amino acid nutrient contribution can be produced large effect by these factors. These factors affect amino acid whose biological effectiveness in soil incessantly, also can root system of plant be absorbed, root system produces large effect to the transhipment of overground part, root system and the amino acid whose metabolism of overground part etc. But, research and research method about this aspect lack relatively, the method lacking system determination influences effect accurately, especially comprehensively analyze the environmental factors impact on amino acid nutrient metabolism from plant nutrient and plant physiology angle.
Summary of the invention
Present invention seek to address that the problem that Amino Acid Absorption metabolic effect method is lacked by research environment factor, a kind of method that analysis environments factor affects amino acid nutrient contribution mechanism is provided, being a kind of employing isotope tracer technique and gas chromatogram-isotope mass spectrometry multiple techniques, analysis environments factor affects the method for amino acid nutrient contribution mechanism.
The inventive method is realized by following steps:
1. the envirment factor impact on aminoacid Long-term absorption
Adopt sterile nutrient solution cultural method: with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, nutritional solution is denitrogenated other constituent content outer with reference to Huo Gelan nutrient solution prescription, after 50ml centrifuge tube cultivates plant 15-25d, the plant of growth selection state consistency, is replaced by 3mM5-55%15N-glycine is nitrogenous source, and in nutritional solution, other constituent content is with reference to Huo Gelan nutrient solution prescription, arranges corresponding environmental condition, such as intensity of illumination, nutritional solution pH, CO2Concentration etc., every 2-3d changes one time of nutrition liquid, destructive sampling after cultivating 6-25d, root system of plant is separated with overground part, root system cleans three times in clear water, is then positioned in ultrasonic washing unit and cleans 1 minute, adopts 0.5M calcium chloride solution to clean three times, finally use purified rinse water three times, be adsorbed on root system to remove15N, after adopting lyophilization or oven for drying, root system is put in together with overground part in ball milling instrument and pulverizes, adopt the plant nitrogen content and glycine absorbtivity that process when mass spectrometric analysis for isotope varying environment, can plant growing and the long-term of Amino Acid Absorption be affected analysis environments factor according to this step;
2. the envirment factor impact on the absorption of aminoacid short-term and absorption pattern
Adopt the cultural method as described in step 1, with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, nutritional solution is denitrogenated other constituent content outer with reference to Huo Gelan nutrient solution prescription, after 50ml centrifuge tube cultivates plant 15-25d, the plant of growth selection state consistency, adopt after sterile purified water repeatedly rinses root system and centrifuge tube, be positioned in clear water by plant 8-10h, afterwards its root system is positioned over 50 ��m of olL-1In CCCP solution 1 hour so that it is root system inactivates, and then to compare without the CCCP seedling processed, is positioned over plant containing 3mM98.10at.%15In the nutritional solution of N-glycine, denitrogenating other constituent content outer with reference to Huo Gelan nutrient solution prescription in nutritional solution, the short-term carrying out labeled amino acid absorbs and absorption pattern test, arranges corresponding environmental condition, such as intensity of illumination, nutritional solution pH, CO2Concentration etc., after cultivating 2-6h, destructive sampling, separates root system of plant with overground part, root system is put in clear water and cleans three times, be then positioned in ultrasonic washing unit and clean 1 minute, adopts 0.5M calcium chloride solution to clean three times, finally use purified rinse water three times, be adsorbed on root system to remove15N, after adopting lyophilization or oven for drying, root system is put in together with overground part in ball milling instrument and pulverizes, adopt the plant nitrogen content and glycine absorbtivity that process when mass spectrometric analysis for isotope varying environment, result according to step 1, may determine that the long term effect that glycine is absorbed by envirment factor, namely on the impact absorbed with follow-up metabolism, and the short-term according to step 2 absorbs result, can the impact on Root Absorption and absorption pattern of the analysis environments factor, and then speculate affect glycine nutrition contribute rate-limiting step.
3. envirment factor is on the aminoacid impact at plant intracellular metabolite
Adopt the sterile nutrient solution cultural method described in step 1, with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, nutritional solution is denitrogenated other constituent content outer with reference to Huo Gelan nutrient solution prescription, after 50ml centrifuge tube cultivates plant 15-25d, the plant of growth selection state consistency, adopt after sterile purified water repeatedly rinses root system and centrifuge tube, be positioned in clear water by plant 8-24h, be replaced by 3mM50-99.8% afterwards15N-glycine is nitrogenous source, nutritional solution is denitrogenated other constituent content outer with reference to Huo Gelan nutrient solution prescription, corresponding environmental condition is set, such as intensity of illumination, nutritional solution pH, CO2Concentration etc., after cultivating 10-24h, destructiveness is sampled, and is cleaned three times by root system of plant in clear water, is then positioned in ultrasonic washing unit and cleans 1 minute, adopts 0.5M calcium chloride solution to clean three times, finally uses purified rinse water three times, is adsorbed on root system with removal15N, after adopting lyophilization or oven for drying, is put in root system in ball milling instrument together with plant shoot and pulverizes;Weigh the 20mg plant sample pulverized after drying, add 4ml80% ethanol to be positioned in oscillator and extract 1h with 10-30r/min concussion, extracting solution is at the centrifugal 15min of 3500-4500g/min, retain supernatant, residue 1ml80% ethanol extracts 1h again, 10000-12000g is centrifuged 5-15min, the supernatant mixing of two times centrifugal, adopts N2Drying up or low-temp low-pressure Rotary Evaporators is evaporated at 25-35 DEG C, again dissolve with 2ml0.1mol/LHCl, centrifugal 10-15min under 10000-15000r/min rotating speed, it is 2cm that supernatant is poured into volume3Cation exchange column in (Dowex50WX8-200), this cation exchange column adopt 20ml ultra-pure water clean, aminoacid therein adopt 20ml4mol/LNH4OH eluting, eluting liquid nitrogen blows over night, removes NH therein3, postlyophilization, the aminoacid in remaining extract is just changed into corresponding fert-butyidimethylsilyl derivant, afterwards in extract amino acid concentration and15N abundance adopts gas chromatogram-isotope mass spectrometry combined instrument to carry out detecting (GasChromatographyMassSpectrometry).
Adopt the step 1 can the impact on plant Long-term absorption of the research environment factor, and the main research environment factor impact on root system of plant absorbability of step 2, the main research environment factor of step 3 is on the impact of amino acid metabolism in plant, in conjunction with three aspect results, just plant absorption can be utilized overall merit environmental factors amino acid whose impact, and explain concrete metabolism rate-limiting step.
The present invention is by adopting aseptic culture in conjunction with isotope tracer technique, the impact on plant growing and Amino Acid Absorption of the research environment factor, determine the long term effect of environmental factors, adopt the method that short-term absorbs and adds metabolic poison afterwards, root system short-term is absorbed the impact of aminoacid and absorption pattern by research environment factor, finally adopt isotopic tracing in conjunction with gas chromatogram-isotope mass spectrometry coupling detection technique, it is determined that the rate-limiting step of environmental factors restriction Glycine Metabolism. The method is simple to operate, adopt isotope tracer technique and gas chromatogram-isotope mass spectrometry coupling detection technique precision and credibility higher, effectively analysis environments factor can affect the mechanism of amino acid nutrient contribution, solve the problem that Amino Acid Absorption metabolic effect method is lacked by research environment factor.
Accompanying drawing explanation
Fig. 1 is the intensity of illumination impact on glycine Long-term absorption.
Fig. 2 is the impact that glycine short-term is absorbed by intensity of illumination.
Fig. 3 be intensity of illumination to Plantula Brassicae chinensis overground part containing15The amino acid whose impact of N.
Fig. 4 be intensity of illumination to Plantula Brassicae chinensis root system containing15The amino acid whose impact of N.
Fig. 5 is the impact on glycine Long-term absorption of the nutritional solution acidity.
Fig. 6 is the impact that glycine short-term is absorbed by nutritional solution acidity.
Fig. 7 be nutritional solution acidity to Plantula Brassicae chinensis overground part containing15The amino acid whose impact of N.
Fig. 8 be nutritional solution acidity to Plantula Brassicae chinensis root system containing15The amino acid whose impact of N.
Detailed description of the invention
The present invention is in conjunction with the drawings and specific embodiments, and the present invention is described in detail.
Embodiment 1 one kinds analyzes the method that intensity of illumination affects amino acid nutrient contribution mechanism
Operating procedure is as follows:
Step one: the intensity of illumination impact (for Plantula Brassicae chinensis) on aminoacid Long-term absorption
Adopt sterile nutrient solution cultural method, with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, nutritional solution is denitrogenated other constituent content outer with reference to Huo Gelan nutrient solution prescription, after 50ml centrifuge tube cultivates Plantula Brassicae chinensis 25d, the Plantula Brassicae chinensis of growth selection state consistency, is replaced by 3mM50.16%15N-glycine is nitrogenous source, and in nutritional solution, other constituent content is with reference to Huo Gelan nutrient solution prescription, and arranging illumination condition is 90,360,540 ��m of olm-2s-1(early-stage Study shows 360 ��m of olm-2s-1Growth of Cabbage it is best suitable under intensity, and 90 and 540 ��m of olm-2s-1Then distinguish too low and too high under intensity), every 2d changes one time of nutrition liquid, destructive sampling after cultivating 10d, Plantula Brassicae chinensis root system is separated with overground part, root system cleans three times in clear water, is then positioned in ultrasonic washing unit and cleans 1 minute, adopts 0.5M calcium chloride solution to clean three times, finally use purified rinse water three times, be adsorbed on root system to remove15N, after adopting lyophilization or oven for drying, is put in root system in ball milling instrument together with Plantula Brassicae chinensis overground part and pulverizes, and adopts the Plantula Brassicae chinensis nitrogen content and glycine absorbtivity that process when mass spectrometric analysis for isotope varying environment.Can the long-term impact on Growth of Cabbage and Amino Acid Absorption of the analysis environments factor according to this step.
Step 2: the envirment factor impact on the absorption of aminoacid short-term and absorption pattern
Adopt the employing sterile nutrient solution cultural method as described in step one, with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, nutritional solution is denitrogenated other constituent content outer with reference to Huo Gelan nutrient solution prescription, after 50ml centrifuge tube cultivates Plantula Brassicae chinensis 25d, the Plantula Brassicae chinensis of growth selection state consistency, adopt after sterile purified water repeatedly rinses root system and centrifuge tube, be positioned in clear water by Plantula Brassicae chinensis 10h, afterwards its root system is positioned over 50 ��m of olL-11h in CCCP solution so that it is root system inactivates, and then to compare without the CCCP seedling processed, is positioned over plant containing 3mM98.10at.%15In the nutritional solution of N-glycine, denitrogenating other constituent content outer with reference to Huo Gelan nutrient solution prescription in nutritional solution, the short-term carrying out labeled amino acid absorbs and absorption pattern test, and arranging illumination condition is 90,360,540 ��m of olm-2s-1, after cultivating 4h, destructive sampling, separates Plantula Brassicae chinensis root system with overground part, root system is put in clear water and cleans three times, be then positioned in ultrasonic washing unit and clean 1 minute, adopts 0.5M calcium chloride solution to clean three times, finally use purified rinse water three times, be adsorbed on root system to remove15N, after adopting lyophilization or oven for drying, root system is put in together with Plantula Brassicae chinensis overground part in ball milling instrument and pulverizes, adopt the Plantula Brassicae chinensis nitrogen content and glycine absorbtivity that process when mass spectrometric analysis for isotope varying environment, result according to step one, may determine that the long term effect that glycine is absorbed by envirment factor, namely on the impact absorbed with follow-up metabolism, and the short-term according to step 2 absorbs result, can the impact on Root Absorption and absorption pattern of the analysis environments factor, and then speculate affect glycine nutrition contribute rate-limiting step.
Step 3: envirment factor is on the aminoacid impact at plant intracellular metabolite
Adopt the sterile nutrient solution cultural method as described in step one, with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, nutritional solution is denitrogenated other constituent content outer with reference to Huo Gelan nutrient solution prescription, after 50ml centrifuge tube cultivates Plantula Brassicae chinensis 25d, the Plantula Brassicae chinensis of growth selection state consistency, adopt after sterile purified water repeatedly rinses root system and centrifuge tube, be positioned in clear water by Plantula Brassicae chinensis 10h, be replaced by 3mM99.81% afterwards15N-glycine is nitrogenous source, and in nutritional solution, other constituent content is with reference to Huo Gelan nutrient solution prescription, and arranging illumination condition is 90,360,540 ��m of olm-2s-1, after cultivating 12h, destructiveness is sampled, and is cleaned three times by Plantula Brassicae chinensis root system in clear water, is then positioned in ultrasonic washing unit and cleans 1 minute, adopts 0.5M calcium chloride solution to clean three times, finally uses purified rinse water three times, is adsorbed on root system with removal15N, after adopting freeze-drying or oven for drying, is put in root system in ball milling instrument together with Plantula Brassicae chinensis overground part and pulverizes; Weigh the 20mg plant sample pulverized after drying, add 4ml80% ethanol to be positioned in oscillator and extract 1h with 10r/min concussion, extracting solution, at the centrifugal 15min of 3500g/min, retains supernatant, and residue 1ml80% ethanol extracts 1h again, 12000g is centrifuged 10min, the supernatant mixing of two times centrifugal, adopts low-temp low-pressure Rotary Evaporators to be evaporated at 25 DEG C, again dissolves with 2ml0.1mol/LHCl, centrifugal 10min under 15000r/min rotating speed, it is 2cm that supernatant is poured into volume3Cation exchange column in (Dowex50WX8-200), this cation exchange column adopt 20ml ultra-pure water clean, aminoacid therein adopt 20ml4mol/LNH4OH eluting, eluting liquid nitrogen blows over night, removes NH therein3, postlyophilization, the aminoacid in remaining extract is just changed into corresponding fert-butyidimethylsilyl derivant, afterwards in extract amino acid concentration and15N abundance adopts gas chromatogram-isotope mass spectrometry combined instrument (GC-MS) to detect, result according to step one and step 2, the rate-limiting step affecting amino acid whose biological effectiveness can be speculated, and step 3 can verify that the rate-limiting step of step 2, and disclose the concrete rate-limiting step of amino acid metabolism further.
Adopt the step 1 can the impact on plant Long-term absorption of the research environment factor, and the impact that the main research environment factor of step 2 is on root system of plant absorbability, the main research environment factor of step 3 is on the impact of amino acid metabolism in plant, in conjunction with three aspect results, just plant absorption can be utilized overall merit environmental factors amino acid whose impact, and explain concrete metabolism rate-limiting step.
It will be seen from figure 1 that Plantula Brassicae chinensis root system and overground part glycine are absorbed by intensity of illumination has significant impact. At Low light intensity (90 ��m of olm-2s-1) and too high intensity of illumination (540 ��m of olm-2s-1) under, glycine absorbs and is substantially less than optimal light intensity (360 ��m of olm-2s-1), it was shown that too high or under high Low light intensity, be all not suitable for the absorption of glycine.
As can be seen from Figure 2, under too high intensity of illumination, the absorbtivity of glycine is suitable with under optimal light intensity, and the amount of Passive intake (add CCCP limit active absorption, add the CCCP Root Absorption amount processed and be Passive intake amount, be not added with the CCCP absorbtivity processed deduct add the CCCP absorbtivity processed and be active absorption amount) be significantly higher than under optimal light intensity, and it is slight to be substantially less than the suitableeest illumination under Low light intensity. From Long-term absorption, too high intensity of illumination glycine absorbtivity is substantially less than optimal light intensity, and does not have this kind of phenomenon in short-term absorbs. Long-term nutrition contribution is the comprehensive function of glycine absorption, transhipment, metabolism, time is only the short-term of 4h and absorbs, it is mainly used to analyze the absorption characteristic of root system, therefore conclude that the metabolism after being mainly Root Absorption glycine under high intensity of illumination limits the nutrition contribution of glycine.
From figure 3, it can be seen that glycine, serine, leucine, lysine, arginine content are than significantly higher under too high intensity of illumination under optimal light intensity. And aspartic acid, proline, alanine, ammonium nitrogen content are above optimal light intensity under too high intensity of illumination. This shows, in blade, under too high intensity of illumination, the integral dose of glycine is higher, and glycine is not the metabolic step of restriction glycine nutrition to the conversion of serine, and the secondary metabolism of the ammonia that Glycine Metabolism produces is the important step of restriction glycine nutrition contribution.
Fig. 4 shows, under too high light intensity, proline, ammonium nitrogen, cysteine content are significantly higher than under optimal light intensity, and glycine, tyrosine content are substantially less than under optimal light intensity. It can thus be seen that in root system, under too high intensity of illumination, glycine is not the metabolic step of restriction glycine nutrition to the conversion of serine, the secondary metabolism of the ammonia that Glycine Metabolism produces is the important step of restriction glycine nutrition contribution.
In sum, under low-light conditions, Root Absorption is the rate-limiting step of restriction glycine nutrition contribution, under high intensity of illumination, has a Glycine Metabolism and the secondary metabolism of ammonium nitrogen that comes is the rate-limiting step of restriction glycine nutrition contribution.
Embodiment 2 one kinds analyzes the method for nutritional solution Effect of Acidity On Absorption amino acid nutrient contribution mechanism, and its step is as follows:
Step one: the nutritional solution acidity impact (for Plantula Brassicae chinensis) on aminoacid Long-term absorption
Adopt sterile nutrient solution cultural method, with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, after cultivating Plantula Brassicae chinensis 25d in 50ml centrifuge tube, the Plantula Brassicae chinensis of growth selection state consistency, it is replaced by 3mM50.16%15N-glycine is nitrogenous source, in nutritional solution, other constituent content is with reference to Huo Gelan nutrient solution prescription, arranging nutritional solution acidity condition is pH=4.0, 6.0, 8.0 (early-stage Study show pH=6.0 be best suitable for Plantula Brassicae chinensis absorb glycine, pH=4.0 and pH=8.0 then distinguishes too low and too high), every 2d changes one time of nutrition liquid, destructive sampling after cultivating 7d, Plantula Brassicae chinensis root system is separated with overground part, root system cleans three times in clear water, then it is positioned in ultrasonic washing unit and cleans 1 minute, 0.5M calcium chloride solution is adopted to clean three times, finally use purified rinse water three times, it is adsorbed on root system to remove15N, after adopting lyophilization or oven for drying, is put in root system in ball milling instrument together with Plantula Brassicae chinensis overground part and pulverizes, and adopts mass spectrometric analysis for isotope respectively to process Plantula Brassicae chinensis nitrogen content and glycine absorbtivity.The long-term impact on Growth of Cabbage and Amino Acid Absorption of the nutritional solution acidity can be analyzed according to this step.
Step 2: the nutritional solution acidity impact on the absorption of aminoacid short-term and absorption pattern
Adopt the employing sterile nutrient solution cultural method as described in step one, with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, after 50ml centrifuge tube cultivates Plantula Brassicae chinensis 25d, the Plantula Brassicae chinensis of growth selection state consistency, after adopting sterile purified water repeatedly to rinse root system and centrifuge tube, it is positioned in clear water by Plantula Brassicae chinensis 10h, afterwards its root system is positioned over 50 ��m of olL-11h in CCCP solution so that it is root system inactivates, and then to compare without the CCCP seedling processed, is positioned over plant containing 3mM98.10at.%15In the nutritional solution of N-glycine, the short-term carrying out labeled amino acid absorbs and absorption pattern test, in nutritional solution, other constituent content is with reference to Huo Gelan nutrient solution prescription, arranging nutritional solution acidity condition is pH=4.0,6.0,8.0, destructive sampling after cultivating 4h, Plantula Brassicae chinensis root system is separated with overground part, root system is put in clear water and cleans three times, then it is positioned in ultrasonic washing unit and cleans 1 minute, 0.5M calcium chloride solution is adopted to clean three times, finally use purified rinse water three times, be adsorbed on root system to remove15N, after adopting lyophilization or oven for drying, is put in root system in ball milling instrument together with Plantula Brassicae chinensis overground part and pulverizes, and adopts mass spectrometric analysis for isotope respectively to process Plantula Brassicae chinensis nitrogen content and glycine absorbtivity. Result according to step one, may determine that the long term effect that glycine is absorbed by nutritional solution acidity, namely on the impact absorbed with follow-up metabolism, and the short-term according to step 2 absorbs result, the impact on Root Absorption and absorption pattern of the nutritional solution acidity can be analyzed, and then speculate the rate-limiting step affecting glycine nutrition contribution.
Step 3: nutritional solution acidity is on the aminoacid impact at plant intracellular metabolite
Adopt the sterile nutrient solution cultural method as described in step one, with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, after 50ml centrifuge tube cultivates Plantula Brassicae chinensis 25d, the Plantula Brassicae chinensis of growth selection state consistency, after adopting sterile purified water repeatedly to rinse root system and centrifuge tube, it is positioned in clear water by Plantula Brassicae chinensis 10h, is replaced by 3mM99.81% afterwards15N-glycine is nitrogenous source, in nutritional solution, other constituent content is with reference to Huo Gelan nutrient solution prescription, arranging nutritional solution acidity condition is pH=4.0,6.0,8.0, destructive sampling after cultivating 12h, Plantula Brassicae chinensis root system is cleaned three times in clear water, is then positioned in ultrasonic washing unit and cleans 1 minute, adopt 0.5M calcium chloride solution to clean three times, finally use purified rinse water three times, be adsorbed on root system to remove15N, after adopting freeze-drying or oven for drying, is put in root system in ball milling instrument together with Plantula Brassicae chinensis overground part and pulverizes; Weigh the 20mg plant sample pulverized after drying, add 4ml80% ethanol to be positioned in oscillator and extract 1h with 30r/min concussion, extracting solution, at the centrifugal 15min of 4500g/min, retains supernatant, and residue 1ml80% ethanol extracts 1h again, 15000g is centrifuged 10min, the supernatant mixing of two times centrifugal, adopts low-temp low-pressure Rotary Evaporators to be evaporated at 25 DEG C, again dissolves with 2ml0.1mol/LHCl, centrifugal 10min under 15000r/min rotating speed, it is 2cm that supernatant is poured into volume3Cation exchange column in (Dowex50WX8-200intheH+Form), this cation exchange column adopts 20ml ultra-pure water to clean, and aminoacid therein adopts 20ml4mol/LNH4OH eluting, eluting liquid nitrogen blows over night, removes NH therein3, postlyophilization, the aminoacid in remaining extract is just changed into corresponding fert-butyidimethylsilyl derivant, afterwards in extract amino acid concentration and15N abundance adopts gas chromatogram-isotope mass spectrometry combined instrument (GC-MS) to detect.According to step one and step 2 as a result, it is possible to speculate the rate-limiting step affecting amino acid whose biological effectiveness, and step 3 can verify that the rate-limiting step of step 2, and discloses the concrete rate-limiting step of amino acid metabolism further.
Adopt step one can study the impact on plant Long-term absorption of the nutritional solution acidity, and step 2 mainly studies the impact on root system of plant absorbability of the nutritional solution acidity, step 3 mainly studies nutritional solution acidity to the impact of amino acid metabolism in plant, in conjunction with three aspect results, just plant absorption can be utilized overall merit nutritional solution acidity amino acid whose impact, and explain concrete metabolism rate-limiting step.
Fig. 5 shows, with glycine for nitrogenous source, it is that pH=7.0, pH=4.0 seriously limit the absorption of glycine and the growth of Plantula Brassicae chinensis that optimum Plantula Brassicae chinensis absorbs the nutritional solution acidity of glycine, and pH=8.0 also limits the absorption of glycine to a certain extent.
From fig. 6, it can be seen that glycine is absorbed nutritional solution acidity and absorption pattern has significant impact. PH=4 nutritional solution processes, and Plantula Brassicae chinensis Passive intake amount is significantly higher than other two kinds of acidity and processes, and integral dose is suitable with pH=7. And process at pH=8 nutritional solution, Plantula Brassicae chinensis active absorption and Passive intake amount are all substantially less than pH=7 process, it was shown that when pH=8, Root Absorption limits the nutrition contribution of Plantula Brassicae chinensis glycine, and when pH=4, glycine absorbs is not limiting factor.
From figure 7 it can be seen that between each process except ammonium nitrogen and integral dose, all the other each seed amino acids all do not show significant difference, it was shown that nutritional solution acidity is less on the impact of overground part.
As can be seen from Figure 8, the impact of root system amino acid content is greater than overground part by nutritional solution acidity. When pH=4, Glycine Levels is significantly higher than pH=7 process, it was shown that glycine to the conversion of serine be restriction pH=4 when amino acid nutrient contribution restriction factor. Although it should be noted that the nutrition of glycine contribution is the highest when pH=7, but its root system presents higher ammonia nitrogen content, it was shown that the accumulation of ammonium nitrogen is the key factor limiting amino acid nutrient. Research shows, gets rid of the interference of environmental factors, and restriction plant absorption utilizes the assimilation that amino acid whose subject matter is then the ammonium nitrogen of this raw metabolism generation.
Claims (3)
1. the method that an analysis environments factor affects amino acid nutrient contribution mechanism, it is characterised in that realized by following steps:
(1) envirment factor impact on aminoacid Long-term absorption
Adopt sterile nutrient solution cultural method: with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, nutritional solution is denitrogenated other constituent content outer with reference to Huo Gelan nutrient solution prescription, after 50ml centrifuge tube cultivates plant 15-25d, the plant of growth selection state consistency, is replaced by 3mM5-55%15N-glycine is nitrogenous source, and in nutritional solution, other constituent content is with reference to Huo Gelan nutrient solution prescription, arranges intensity of illumination, nutritional solution pH, CO2The corresponding environmental condition of concentration, every 2-3d changes one time of nutrition liquid, and after cultivating 6-25d, destructive sampling, separates root system of plant with overground part, removes and is adsorbed on root system15N, clean root system, then it is positioned in ultrasonic washing unit and cleans, clean with 0.5M calcium chloride solution again, finally with purified rinse water, after drying, it is put in ball milling instrument together with overground part by root system to pulverize, adopt the plant nitrogen content and glycine absorbtivity that process when mass spectrometric analysis for isotope varying environment, the long-term impact on plant growing and Amino Acid Absorption of the analysis environments factor;
(2) envirment factor impact on the absorption of aminoacid short-term and absorption pattern
Adopt the sterile nutrient solution cultural method described in step (1), with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, nutritional solution is denitrogenated other constituent content outer with reference to Huo Gelan nutrient solution prescription, after 50ml centrifuge tube cultivates plant 15-25d, the plant of growth selection state consistency, after adopting sterile purified water to rinse root system and centrifuge tube, it is positioned in clear water by plant 8-10h, afterwards its root system is positioned over 50 ��m of olL-1In CCCP solution 1 hour so that it is root system inactivates, and then to compare without the CCCP seedling processed, is positioned over plant containing 3mM98.10at.%15In the nutritional solution of N-glycine, denitrogenating other constituent content outer with reference to Huo Gelan nutrient solution prescription in nutritional solution, the short-term carrying out labeled amino acid absorbs and absorption pattern test, arranges intensity of illumination, nutritional solution pH, CO2The corresponding environmental condition of concentration, after cultivating 2-6h, destructive sampling, separates root system of plant with overground part, root system is put in clear water and cleans, be then positioned in ultrasonic washing unit and clean, then cleans with 0.5M calcium chloride solution, finally with purified rinse water, it is adsorbed on root system to remove15N, after drying, root system is put in together with overground part in ball milling instrument and pulverizes, adopt the plant nitrogen content and glycine absorbtivity that process when mass spectrometric analysis for isotope varying environment, the impact on Root Absorption and absorption pattern of the analysis environments factor, and then the rate-limiting step of analyzing influence glycine nutrition contribution;
(3) envirment factor is on the aminoacid impact at plant intracellular metabolite
Adopt the sterile nutrient solution cultural method described in step (1), with 2mM sodium nitrate+0.5mM ammonium sulfate+0.5mM glycine for nitrogenous source, nutritional solution is denitrogenated other constituent content outer with reference to Huo Gelan nutrient solution prescription, after 50ml centrifuge tube cultivates plant 15-25d, the plant of growth selection state consistency, after adopting sterile purified water to rinse root system and centrifuge tube, it is positioned in clear water by plant 8-24h, is replaced by 3mM50-99.8% afterwards15N-glycine is nitrogenous source, and in nutritional solution, other constituent content is with reference to Huo Gelan nutrient solution prescription, arranges corresponding environmental condition, such as intensity of illumination, nutritional solution pH, CO2Concentration etc., after cultivating 10-24h, destructive sampling, cleans root system of plant in clear water, is then positioned in ultrasonic washing unit and cleans, then cleans with 0.5M calcium chloride solution, finally with purified rinse water, is adsorbed on root system to remove15N, after drying, is put in root system in ball milling instrument together with plant shoot and pulverizes; Weigh the 20mg plant sample pulverized after drying, add 4ml80% ethanol to be positioned in oscillator and extract 1h with 10-30r/min concussion, extracting solution is at the centrifugal 15min of 3500-4500g/min, retain supernatant, residue 1ml80% ethanol extracts 1h again, 10000-12000g is centrifuged 5-15min, the supernatant mixing of two times centrifugal, adopts N2Drying up or low-temp low-pressure Rotary Evaporators is evaporated at 25-35 DEG C, again dissolve with 2ml0.1mol/LHCl, centrifugal 10-15min under 10000-15000r/min rotating speed, it is 2cm that supernatant is poured into volume3Cation exchange column in, this cation exchange column adopt 20ml ultra-pure water clean, aminoacid therein adopt 20ml4mol/LNH4OH eluting, eluting liquid nitrogen blows over night, removes NH therein3, postlyophilization, the aminoacid in remaining extract is just changed into corresponding fert-butyidimethylsilyl derivant, afterwards in extract amino acid concentration and15N abundance adopts gas chromatogram-isotope mass spectrometry combined instrument to be measured.
2. a kind of method measuring amino acid metabolism rate-limiting step according to claim 1, it is characterised in that dry described in step (1)-(3), selects lyophilization or oven for drying.
3. a kind of method measuring amino acid metabolism rate-limiting step according to claim 1, it is characterized in that, in conjunction with the result of (1)-(3), plant absorption is utilized amino acid whose impact by overall merit environmental factors, measures amino acid metabolism rate-limiting step.
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