CN105445209A - Method for screening out pesticide or safener for tobacco and application - Google Patents
Method for screening out pesticide or safener for tobacco and application Download PDFInfo
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- CN105445209A CN105445209A CN201511025441.8A CN201511025441A CN105445209A CN 105445209 A CN105445209 A CN 105445209A CN 201511025441 A CN201511025441 A CN 201511025441A CN 105445209 A CN105445209 A CN 105445209A
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- tobacco
- gsts
- agricultural chemicals
- safener
- screening
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/32—Ingredients for reducing the noxious effect of the active substances to organisms other than pests, e.g. toxicity reducing compositions, self-destructing compositions
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- General Health & Medical Sciences (AREA)
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- Spectroscopy & Molecular Physics (AREA)
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- Pest Control & Pesticides (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
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- Toxicology (AREA)
- Agronomy & Crop Science (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for screening out a pesticide or a safener for tobacco. Different to-be-screened pesticides or safeners for the tobacco are sprayed to the tobacco, leaves and roots are taken as samples, meanwhile, leaves and roots of the tobacco which is not subjected to pesticide spraying are taken as controls, the method is characterized in that whole protein extraction solutions of the samples and the controls are extracted, GSH (glutathione) and CDNB (1-chloro-2,4-dinitrobenzene) are taken as reaction substrates, light absorption values of all whole protein extraction solutions under 340 nm are detected, GS-CDNB coupled product quantities are calculated, then the activity of GSTs (glutathione s-transferases) is determined, and the pesticide or the pesticide safener capable of increasing the activity of the GSTs is screened out. The method has the characteristics of simplicity in operation, rapidness in analysis and high flux, requirements for instruments are not high, rapid screening of the pesticide for the tobacco can be realized, and the method has certain application value and economic value in screening of the pesticide for the tobacco.
Description
Technical field
The invention belongs to biological technical field, be specifically related to detoxification metabolic pathway key enzyme---glutathione transferase and the application in cigarette chemistry security agent screening thereof.
Background technology
Weeds and various pathogen infection are the formidable enemies of agricultural production, and therefore the use of the chemical agent such as various herbicide, germifuge is very general, and these medicaments also result in Pesticide Residue higher in environmental pollution and crops while weeding sterilization.Entering in the pesticide metabolism done in object, glutathione transferase (GSTs) is a wherein more crucial class solution toxalbumin.The major function of GSTs is by catalytic reduction type glutathione and combination that is endogenous or external source noxious material, forms less toxicity and comparatively highly-water-soluble bond, and can be secreted into apoplast or separating further, reach and fall hypotoxic object.
In recent years in order to reduce agricultural chemicals do the accumulation of the toxicity in object and improve crop on the resistance of agricultural chemicals, reduce agricultural chemicals to the impact of plant growth, the research and development of chemistry security agent and use more and more.Chemistry security agent refers to the expression that can improve enzyme (as GSTs) in detoxification metabolic pathway, thus improves crop to a class agricultural chemicals of the tolerance of herbicide etc.Some crop tolerance agricultural chemicals doing the expression that also can improve enzyme in crop detoxification metabolic pathway in object, thus reduce the toxicity of agricultural chemicals and the impact on plant growth thereof.It is generally acknowledged that plant GSTs activity is an important indicator of the agricultural chemicals abilities such as plant resistant herbicide, therefore, by analyzing the change of crop GSTs activity after applying pesticides, screen the agricultural chemicals by GSTs degraded detoxification and safener, contribute to guiding agricultural production, increase the security of crop.
Summary of the invention
The invention provides a kind of method of screening cigarette agricultural chemicals or safener, is realized by the impact of monitoring agricultural chemicals on tobacco GSTs activity change.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of method and application of screening cigarette agricultural chemicals or safener, tobacco is sprayed with difference cigarette agricultural chemicals to be screened or safener, get blade and root as sample, simultaneously with the tobacco leaf of non-dispenser and root for control sample, extract the holoprotein extract of sample and control sample, with GSH and CDNB for reaction substrate, detect the light absorption value under each group of holoprotein extract 340nm, calculate GS-CDNB coupled product amount, and then determine that GSTs is active, the agricultural chemicals that screening can make GSTs activity raise or chemistry security agent.
Described GSTs refers to tobacco GSTs family, comprises all GSTs expressed in tobacco.
In described glutathione transfers enzyme activity analytic process, the substrate of use is GSH and CDNB.
Application during described GSTs activity analysis detects as agricultural chemicals detoxification metabolism.
Beneficial effect of the present invention: analyzed by the glutathione transfers enzyme activity in the holoprotein extract of the tobacco to different cigarette agricultural chemicals or safener process, agricultural chemicals or the safener of GSTs activity are raised in screening.The method have simple to operate, analyze quick, high-throughout feature, and not high to instrument requirements, the rapid screening to cigarette agricultural chemicals can be realized, in cigarette pesticide screening test, there is certain using value and economic worth.
Accompanying drawing explanation
Fig. 1 is the impact of different agricultural chemicals on GSTs activity in Tobacco Root;
Fig. 2 is the impact of different agricultural chemicals on GSTs activity in tobacco leaf.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further, these embodiments are only not used in for illustration of the present invention and limit the scope of the invention.The test method of unreceipted actual conditions in the following example, usually conveniently condition.
Tobacco is sprayed with difference cigarette agricultural chemicals to be screened or safener, get blade and root as sample, simultaneously with the tobacco leaf of non-dispenser and root for control sample, extract the holoprotein extract of sample and control sample, with GSH and CDNB for reaction substrate, detect the light absorption value under each group of holoprotein extract 340nm, calculate GS-CDNB coupled product amount, and then determine that GSTs is active, the agricultural chemicals that screening can make GSTs activity raise or chemistry security agent.
Concrete steps are as follows: preparation of samples: purchase Common Herbicides napropamide tobacco agriculture, paraquat and germifuge thiophanate methyl, Mancozeb from market; When in laboratory, earth culture cultivation tobacco (NicotianatobacumL.) grows to 6 leaf age, illustrate that the consumption of suggestion sprays respectively on the soil surface or tobacco leaf of plantation tobacco according to using, respectively before applying pesticides, use after 5 hours, 1 day, within 2 days, get root and third and fourth blade of tobacco seedling, get the tobacco control sample of non-applying pesticides simultaneously.
The preparation of holoprotein extract: use 100mM phosphate buffer (pH7.5) to add in tobacco sample according to the ratio of 3:1 (v/w), ice bath is ground to homogenate, centrifugal (14000rpm, 20min, 4 DEG C), supernatant is the holoprotein extract of Tobacco Root or blade, and utilize BCA kit (Thermo, pierce) to measure the protein concentration of holoprotein extract.
The detection of light absorption value: use ELISA Plate as cuvette, add 100 μ l holoprotein extracts, the above-mentioned 100mM phosphate buffer of 97.8 μ l, 2 μ lGSH (200mM), 0.2 μ lCDNB (1M) successively, blank is the above-mentioned 100mM phosphate buffer of 197.8 μ l, 2 μ lGSH (200mM), 0.2 μ lCDNB (1M), each sample arranges 3 repetitions, puts into the microplate reader continuously absorbance value change of 5min under monitoring 340nm.
Active calculating: when using reaction beginning and react 2min, the reading at 340nm place calculates Δ A340/min, according to formula
Activity=[Δ A340/min (sample)-Δ A340/min (blank)] × 0.2ml/5.3 (mM-1cm-1)/0.1ml/Con (g/ml) × 1000
The GSTs calculating holoprotein crude extract is active, wherein 0.2ml is reaction cumulative volume, and 0.1ml is the addition of protein crude extract, and 5.3 is molar extinction coefficient, Con is the protein concentration of crude extract, and active unit is micromole's number of the CDNB coupled product of every gram of albumen generation per minute.
The reaction equation of GSH and CDNB coupling is:
C
6H
3(NO
2)
2Cl+GSH→C
6H
3(NO
2)
2SG+H++Cl-
According to the change speed of 340nm place absorbance value, interpretation of result: this reaction can be carried out fast under GSTs participates in, and CDNB-SG coupled product has characteristic absorption peak at 340nm place, therefore can differentiate that the activity of GSTs is strong and weak.
After different pesticide-treated, in Tobacco Root and blade holoprotein extract, the change of CDNB coupling activity is as depicted in figs. 1 and 2, napropamide and Mancozeb can significantly improve the activity of the GSTs in Tobacco Root or blade, illustrate that napropamide and the expression of Mancozeb to tobacco GSTs have inducing action, high GSTs activity is conducive to the metabolism detoxification of xenobiotic.Therefore according to the effect of GSTs in the metabolism of allos noxious material, in several agricultural chemicals that this experiment is screened, Herbicide DICAOAN and germifuge Mancozeb can priority application in tobacco agriculture actual production, in fact agricultural production practice also shows that napropamide and Mancozeb are safety, low toxicity, effective agricultural chemicals reagent for many years, and the application in tobacco agriculture widely.
Claims (4)
1. the method for a cigarette agricultural chemicals or safener screening, tobacco is sprayed with difference cigarette agricultural chemicals to be screened or safener, get blade and root as sample, simultaneously with the tobacco leaf of non-dispenser and root for control sample, it is characterized in that: the holoprotein extract extracting sample and control sample, with GSH and CDNB for reaction substrate, detect the light absorption value under each group of holoprotein extract 340nm, calculate GS-CDNB coupled product amount, and then determine that GSTs is active, the agricultural chemicals that screening can make GSTs activity raise or chemistry security agent.
2. the method for cigarette agricultural chemicals as claimed in claim 1 or safener screening, is characterized in that: described GSTs refers to tobacco GSTs family, comprises all GSTs expressed in tobacco.
3. the method for cigarette agricultural chemicals as claimed in claim 2 or safener screening, it is characterized in that: in described glutathione transfers enzyme activity analytic process, the substrate of use is GSH and CDNB.
4. one kind is screened the application of method in detecting as agricultural chemicals detoxification metabolism of cigarette agricultural chemicals or safener.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511025441.8A CN105445209A (en) | 2015-12-31 | 2015-12-31 | Method for screening out pesticide or safener for tobacco and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511025441.8A CN105445209A (en) | 2015-12-31 | 2015-12-31 | Method for screening out pesticide or safener for tobacco and application |
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| CN105445209A true CN105445209A (en) | 2016-03-30 |
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| CN201511025441.8A Pending CN105445209A (en) | 2015-12-31 | 2015-12-31 | Method for screening out pesticide or safener for tobacco and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114807081A (en) * | 2022-05-18 | 2022-07-29 | 云南中烟工业有限责任公司 | Gene related to tobacco glutathione transfer and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1680588A (en) * | 2004-03-25 | 2005-10-12 | 株式会社佐竹 | Method for analyzing residual agricultural chemical |
| CN101302208A (en) * | 2008-03-04 | 2008-11-12 | 刘庆山 | Compound inhibiting glutathion S-transferase activity, preparation and use thereof |
| CN101948817A (en) * | 2010-08-11 | 2011-01-19 | 中国矿业大学(北京) | Enzyme for degrading acephate pesticides, coding gene, expression vector, industrial bacterial strain and application thereof |
| CN103170089A (en) * | 2012-06-13 | 2013-06-26 | 中国矿业大学(北京) | Application of glutathione-S transferase (GST) protein to degradation of multiple pesticides |
-
2015
- 2015-12-31 CN CN201511025441.8A patent/CN105445209A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1680588A (en) * | 2004-03-25 | 2005-10-12 | 株式会社佐竹 | Method for analyzing residual agricultural chemical |
| CN101302208A (en) * | 2008-03-04 | 2008-11-12 | 刘庆山 | Compound inhibiting glutathion S-transferase activity, preparation and use thereof |
| CN101948817A (en) * | 2010-08-11 | 2011-01-19 | 中国矿业大学(北京) | Enzyme for degrading acephate pesticides, coding gene, expression vector, industrial bacterial strain and application thereof |
| CN103170089A (en) * | 2012-06-13 | 2013-06-26 | 中国矿业大学(北京) | Application of glutathione-S transferase (GST) protein to degradation of multiple pesticides |
Non-Patent Citations (4)
| Title |
|---|
| 胡利锋 等: "谷胱甘肽-Ｓ-转移酶对除草剂代谢作用的研究进展", 《农田杂草与防控》 * |
| 郭玉莲 等: "植物谷胱甘肽S- 转移酶(GSTs) 及除草剂解毒剂的诱导作用", 《东北农业大学学报》 * |
| 郭玉莲 等: "玉米谷胱甘肽转移酶(GSTs )特性及除草剂的诱导作用", 《玉米科学》 * |
| 陶波 等: "乙草胺对玉米中谷胱甘肽转移酶的诱导作用", 《东北农业大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114807081A (en) * | 2022-05-18 | 2022-07-29 | 云南中烟工业有限责任公司 | Gene related to tobacco glutathione transfer and application thereof |
| CN114807081B (en) * | 2022-05-18 | 2023-11-03 | 云南中烟工业有限责任公司 | Tobacco glutathione transfer related gene and application thereof |
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Application publication date: 20160330 |