CN109283160A - A kind of new method facilitating Sensitive Detection sheep's hay blade phosphorus content - Google Patents

A kind of new method facilitating Sensitive Detection sheep's hay blade phosphorus content Download PDF

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Publication number
CN109283160A
CN109283160A CN201710590084.2A CN201710590084A CN109283160A CN 109283160 A CN109283160 A CN 109283160A CN 201710590084 A CN201710590084 A CN 201710590084A CN 109283160 A CN109283160 A CN 109283160A
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sheep
hay
blade
phosphorus content
atpase activity
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CN109283160B (en
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黄芳
李玲玉
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Institute of Botany of CAS
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Institute of Botany of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention provides a kind of method of quickly detection sheep's hay phosphorus content, it include: 1) to carry out different known phosphorus concentrations to chinense seedlings to handle, carry out phosphorus detection and the atpase activity measurement of sheep's hay blade respectively to same blade, the atpase activity after the phosphorus content of sheep's hay blade and different phosphorus concentrations are handled after different phosphorus concentrations are handled in sheep's hay blade is respectively obtained, the corresponding relationship of sheep's hay blade phosphorus content and atpase activity is obtained；2) atpase activity measurement is carried out to phosphorus content sheep's hay blade to be measured and sheep's hay blade phosphorus content is obtained according to the corresponding relationship of above-mentioned sheep's hay blade phosphorus content and atpase activity.The present invention carries out the measurement of chlorophyll fluorescence parameters P515 using non-damaging, easy, sensitive chlorophyll fluorescence techniques to sheep's hay, analyze the variation of its atpase activity under different phosphate concentration conditions, it not only facilitates and further understands photosynthetic structures ATP enzyme to the response mechanism of different phosphorus concentrations and reference is provided, and to effective management and use sheep's hay resource, the sustainable development of its resource is promoted to have important value.

Description

A kind of new method facilitating Sensitive Detection sheep's hay blade phosphorus content
Technical field
The present invention relates to a kind of new methods for facilitating Sensitive Detection sheep's hay blade phosphorus content.
Background technique
Sheep's hay [Leymus chinensis (Trin.)] is the Dominant Species of Forage Grass and dominant species on northern China grassland, life Long situation directly determines the grassland productivity of this area.A great number of elements necessary to phosphorus is plant growth and develops, is not only to plant The constituent of the important substance such as object biomembrane, nucleic acid, protein, phosphatide, ATP, while participating in plant in many ways Physiology course.
It is mainly spectrophotometry that the method for plant phosphorus content is detected in currently available technology, and spectrophotometry generally has molybdenum Sour anti-, P-Mo blue and phosphorus vanadium molybdenum Huang spectrophotometry etc., but there are commonly P-Mo blues and phosphorus vanadium molybdenum Huang spectrophotometry.P-Mo blue The Sensitivity of Absorption Photometry is high, suitable for the measurement of microcomponent, but needs expensive spectrophotometer.Phosphorus vanadium molybdenum Huang point Light photometry is easy, quickly, easily grasps, but remolding sensitivity P-Mo blue is low, and accuracy is low, it is also desirable to expensive light splitting light Spend instrument.
Summary of the invention
The object of the present invention is to provide a kind of methods for facilitating Sensitive Detection sheep's hay blade phosphorus content.
The method of detection sheep's hay blade phosphorus content provided by the present invention, includes the following steps:
1) it carries out different known phosphorus concentrations to chinense seedlings to handle, the phosphorus for carrying out same blade respectively in sheep's hay blade contains The measurement of fixed and atpase activity is measured, is respectively obtained after different phosphorus concentrations are handled at the phosphorus content and different phosphorus concentrations of sheep's hay blade Atpase activity after reason in sheep's hay blade obtains sheep's hay blade phosphorus content pass corresponding with the atpase activity in sheep's hay blade System；
2) atpase activity measurement is carried out to phosphorus content sheep's hay blade to be measured, according to above-mentioned sheep's hay blade phosphorus content and sheep The corresponding relationship of atpase activity in blade of grass piece assesses sheep's hay blade phosphorus content.
In above method step (1), the different known phosphorus concentration concretely 250uM, 2.5uM and 0uM.
The measuring method of the phosphorus content of the sheep's hay blade is molybdenum blue method.
The measuring method of the atpase activity of the sheep's hay blade be chlorophyll fluorescence techniques, i.e., using it is non-damaging, easy, Sensitive chlorophyll fluorescence techniques carry out the measurement of P515 parameter to sheep's hay blade, obtain the atpase activity data of sheep's hay blade.
The corresponding relationship of atpase activity in sheep's hay blade phosphorus content and sheep's hay blade can use following functional relation tables Show: Y=0.2337X+15.421R2=0.9937, wherein Y represents atpase activity gH+(S-1)；X represents blade phosphorus content (with μ M is indicated).
In step (2), atpase activity measurement is carried out to phosphorus content sheep's hay blade to be measured using chlorophyll fluorescence techniques.
The concrete operations of the atpase activity measurement are as follows: first by after the abundant dark adaptation of sheep's hay 10 hours, pass through DUAL-PAM 100 irradiation activated light (325 μ Es-1m-2) 10min, close activated light after observe 0-500ms in P515 value variation.
The present invention carries out chlorophyll fluorescence parameters to sheep's hay using non-damaging, easy, sensitive chlorophyll fluorescence techniques The measurement of P515, analyzes the variation of its atpase activity under different phosphate concentration conditions, and establishes between content of inorganic phosphorus Linear relationship, to effective management Leymus Chinensis Steppe, promotes its sustainable development to have weight for assessing the phosphorus element situation of the economic plants It is worth.
Our new method is not only simple, quickly, high sensitivity, it is most important that this method is non-damaging to experimental material , without tedious steps such as extraction, reaction, colorimetrics.It only need to be living by measurement blade ATP enzyme undamaged after material dark processing Property, its phosphorus content is calculated using formula.
Detailed description of the invention
Fig. 1 is the growing state of sheep's hay after different phosphorus concentration processing in the embodiment of the present invention.
Fig. 2A is the variation of sheep's hay blade phosphorus content after the processing of different phosphorus concentrations.
Fig. 2 B is the variation of the atpase activity of sheep's hay blade after different phosphorus concentrations are handled.
Specific embodiment
The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments Reagent, biomaterial etc., are commercially available unless otherwise specified.
One, material
Vegetable material:
Sheep's hay [Leymus chinensis (Trin.)] seed is provided by grassland research institute, China Agriculture academy of sciences, seed It acquires from Xilin Hot, Inner Mongolia.
Two, method
1 material culture:
1) normal Hoagland culture solution: Ca (NO3)2·4H2O 236g/L, KNO3102g/L, MgSO4·7H2O98g/L, kH2PO427g/L, K2SO488g/L, CaCL2111g/L, NaH2PO424g/L, NaNO3170g/L, Na2SO421g/L, Fe-EDTA: (Na2EDTA 7.45g/L；FeSO4·7H2O 5.57g/L), microelement: H3BO32.86g/L MnSO41.015g/L CuSO4·5H2O 0.079g/L, ZnSO4·7H2O 0.22g/L, H2MoO40.09g/L；
2) Leymus chinensis seeds are sterilized at KMnO4 (0.7%, w/v) and are used twice of distilled water flushing after ten minutes, set wet filter paper On sprouted in illumination box.Condition of culture be 25 DEG C of daytime, 20 DEG C of night, 270 μ Es of intensity of illumination-1m-2, humidity is 50%, 14 hour/day of light application time.The seedling for sprouting 10 days or so is placed in the Hoagland solution of 1/4 intensity training of ventilating It supports, replaces a culture solution weekly, handled after 4 weeks for different phosphorus concentrations.
2 different phosphorus concentration processing
The chinense seedlings of above-mentioned culture are divided into control (250uM), 2.5uM and tri- groups of 0uM process experiment.In above-mentioned training It supports in case and carries out hydroponics.Control group is carried out using the above-mentioned complete culture solution (normal Hoagland culture solution) of Fresh Culture.The equimolar K of 2.5uM and 0uM group2SO4Instead of the KH in above-mentioned complete culture solution2PO4.Every group of processing sets three weights It is multiple.Third piece leaf after culture 20 days is measured for atpase activity, and using the phosphorus content of phosphorus molybdenum orchid method measurement third piece leaf.
3ATP enzyme assay
When carrying out atpase activity measurement, first by after the abundant dark adaptation of sheep's hay 10 hours, irradiated by DUAL-PAM 100 Activated light (325 μ Es-1m-2) 10min, close activated light after observe 0-500ms in P515 value variation.
Q circulation in the separation of charge of PSII and PSI reaction center, Cytb6/f complex can cause cross-film charge, and Proton outflow on ATP enzyme complex can restore this cross-film charge.The process can be arrived by P515 signal detection, and be used to reflect Thylakoid membrane and atpase activity.
Three, result
1, the growing state after the processing of sheep's hay difference phosphorus concentration
Fig. 1 is the growing state after the processing of sheep's hay difference phosphorus concentration.
As shown in Figure 1, by the processing of 20 days different phosphorus concentrations, the growth differences of sheep's hay are more apparent, i.e., with phosphorus concentration Reduction, reduced trend is presented in the growth of sheep's hay.
2, variation of the sheep's hay in different phosphorus concentrations treated blade phosphorus content and atpase activity
Fig. 2A is the variation of the phosphorus content of sheep's hay blade after different phosphorus concentrations are handled.Pass through the place of 20 days different phosphorus concentrations Reason, we have carried out the measurement of phosphorus content to sheep's hay third piece leaf.It was found that with the reduction of phosphorus concentration in culture solution, phosphorus in blade Concentration also decreases.
Fig. 2 B is the variation of sheep's hay blade atpase activity after the processing of different phosphorus concentrations.As can be seen that with the drop of phosphorus concentration Low, the activity of ATP enzyme also decreases.Illustrate phosphorus concentration and atpase activity is relevant.
The corresponding several groups of numerical value of phosphorus content and atpase activity is imported into SPSS EXEL software, scatter plot is inserted into, adds Add Trendline, and shows equation and R2Value.
The corresponding relationship of atpase activity in sheep's hay blade phosphorus content and sheep's hay blade is Y=0.2337X+15.421R2 =0.9937, wherein Y represents atpase activity gH+(S-1)；X represents blade phosphorus content (μM).

Claims (7)

1. a kind of method for detecting sheep's hay blade phosphorus content, includes the following steps:
1) it carries out different known phosphorus concentrations to chinense seedlings to handle, the phosphorus content for carrying out same blade respectively in sheep's hay blade is surveyed The measurement of fixed and atpase activity respectively obtains after the phosphorus content of sheep's hay blade and different phosphorus concentrations are handled after different phosphorus concentrations processing Atpase activity in sheep's hay blade obtains the corresponding relationship of the atpase activity in sheep's hay blade phosphorus content and sheep's hay blade；
2) atpase activity measurement is carried out to phosphorus content sheep's hay blade to be measured, according to above-mentioned sheep's hay blade phosphorus content and sheep's hay leaf The corresponding relationship of atpase activity in piece assesses sheep's hay blade phosphorus content.
2. according to the method described in claim 1, it is characterized by: in step (1), the different known phosphorus concentration is 250uM, 2.5uM and 0uM.
3. method according to claim 1 or 2, it is characterised in that: in step (1), the phosphorus content of the sheep's hay blade Measuring method is molybdenum blue method.
4. method according to any one of claim 1-3, it is characterised in that: in step (1), the sheep's hay blade The measuring method of atpase activity is chlorophyll fluorescence techniques, i.e., carries out chlorophyll fluorescence to sheep's hay using chlorophyll fluorescence techniques The measurement of parameter P515 obtains the atpase activity data of sheep's hay blade.
5. method according to any of claims 1-4, it is characterised in that: in step (1), sheep's hay blade phosphorus content with The corresponding relationship of atpase activity in sheep's hay blade is indicated with following functional relations: Y=0.2337X+15.421R2= 0.9937, wherein Y represents atpase activity gH+(S-1)；X represents blade phosphorus content (indicating with μM).
6. method according to any one of claims 1-5, it is characterised in that: in step (2), use chlorophyll fluorescence skill Art carries out atpase activity measurement to phosphorus content sheep's hay blade to be measured.
7. method according to claim 1 to 6, it is characterised in that: the operation of the atpase activity measurement are as follows: First by after the abundant dark adaptation of sheep's hay 10 hours, activated light 10min is irradiated by DUAL-PAM 100, observes 0- after closing activated light The variation of P515 value in 500ms.
CN201710590084.2A 2017-07-19 2017-07-19 Novel method for conveniently and sensitively detecting phosphorus content of leymus chinensis leaves Active CN109283160B (en)

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CN102598987A (en) * 2012-03-26 2012-07-25 天津师范大学 Method for using cerium to improve chlorophyll fluorescence dynamic of ryegrass in arid environments
CN103868894A (en) * 2013-06-03 2014-06-18 北京林业大学 Method for evaluating total content of nitrogen in coniferous tree seedling
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