CN103114126A - Method for distinguishing calcium absorption difference between transgenic rape protoplast and common rape protoplast by using 45Ca - Google Patents
Method for distinguishing calcium absorption difference between transgenic rape protoplast and common rape protoplast by using 45Ca Download PDFInfo
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- CN103114126A CN103114126A CN2013100317286A CN201310031728A CN103114126A CN 103114126 A CN103114126 A CN 103114126A CN 2013100317286 A CN2013100317286 A CN 2013100317286A CN 201310031728 A CN201310031728 A CN 201310031728A CN 103114126 A CN103114126 A CN 103114126A
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Abstract
The invention discloses a method for distinguishing calcium absorption difference between transgenic rape protoplast and common rape protoplast by using 45Ca. The method comprises the steps of: rape planting, protoplast extraction, protoplast activity identification, protoplast culture and protoplast calcium ion absorptive amount measurement. Experimental results prove that the protoplast with a heat resisting gene TR1 transferred improves Ca<2+> absorption of the rape at a low Ca<2+> concentration and can reduce amount of Ca<2+> that enters cells at a high Ca<2+> concentration. The method for distinguishing calcium absorption difference between transgenic rape protoplast and common rape protoplast provided by using 45Ca to lays a foundation for researching the cultivation of high-yield rape with better temperature regulation and control capability.
Description
Technical field
The present invention relates to the discriminating that transgene rape and ordinary cole protoplastis absorb calcium contents difference, particularly relate to use
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium contents difference.
Background technology
Isotopic tracer technique is to add the isotropic substance almost completely identical with object element character, and isotropic substance and object element move jointly, by isotropic substance realize following the trail of the objective element or be labeled a kind of method of operation or the variation of material.Isotropic substance can be radio isotope, can be also stable isotope.Radio isotope is not only highly sensitive as tracer agent, and measuring method is simple and easy to do, and has the energy accurate quantitative analysis, accurately locates and meet the characteristics such as physiological condition of institute's research object.Isotopic tracer technique is used extremely extensive in life science, can be used for investigating certain element Absorption And Metabolism process in life entity, can utilize isotopic tracer technique research photosynthesis, can utilize the function of isotopic labeling nucleic acids research biological gene etc.In addition, radioautograph can be caught substance transfer on tissue or cell levels dynamic, and radioimmunoassay is a kind of method of antigen antibody reaction that isotropic substance is introduced, and this method is sensitiveer than fluorescent antibody technique, also easier quantitatively.Isotopic tracer technique has now become the indispensable method of research biological phenomenon.
Calcium ion is intracellular second messenger's material, Ca
2+Be subject to special concern aspect the Heat Resistance of Plant Related Signaling Mechanisms.Studies show that, multiple environment-stress stimulates all may cause Ca in cell
2+Transfer transportation, illustrate that the various abiotic stress that calcium has participated in plant responds.Zhang Shenzong, government official learn the employing Ca such as bright
2+Pre-treatment has significantly improved the thermotolerance of its plant of studying, so in cell, whether the content of self calcium exists certain relation with plant heat resistance property, and whether thermophytes and common plant self institute's calcium ions is variant, is worth very much studying.
By transgenic method cultivate thermophytes and therewith the research of relevant heat-resisting mechanism be the research topic of frontier nature, all rarely have report both at home and abroad.TR1(
THermo
REsistance
1) be that of finding of the employing genetic engineering technique such as school of life and health sciences professor Yang Yi of Sichuan University can improve plant and the stable on heating gene of microorganism, this gene through the Chinese Academy of Sciences, academy of agricultural sciences etc. in the industry the authoritative sources expert group of evaluation committee that forms think, this is a kind of heat resistance gene that is positioned on film, under high temperature stress, compare with control group, the index such as Pollen Activity, setting percentage, single plant yield that changes the rice strain of this gene over to all is significantly increased.
Temperature is to affect one of ripe of paramount importance environmental factors of plant-growth.Along with Global Emissions of Greenhouse Gas increases, the Greenhouse effect aggravation, various extreme climates frequently occur, and high temperature has become the large problem of restriction crop growth.Rapeseed oil is one of main edible oil of China, and the high yield rape that temperature is had better ability of regulation and control is cultivated in research, and is most important to China's edible oil safety.Whether the rape that changes over to after heat-resisting gene by investigation has better ability of regulation and control than ordinary cole to calcium ion, study the resistance effect that heat-resisting gene plays in rape, there is the high yield rape of better ability of regulation and control very important for cultivating to temperature, but until before the present invention completed, the contriver had no the research report of relevant this respect.
Summary of the invention
The purpose of this invention is to provide a kind of utilization
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, and cultivating for research has the high yield rape of better ability of regulation and control to lay the foundation to temperature.
Utilization provided by the invention
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, mainly comprise the following steps:
(1) rape cultivation: with ordinary cole and transgenosis TR1 rapeseed cultivation in the Nutrition Soil that contains the MS substratum, in illumination 15-17 hour/day, cultivated 45-55 days under temperature 23-25 ℃ of condition, grow to true leaf 7-10 sheet, get respectively two kinds of rape leafs and extract protoplasma;
(2) protoplastis extracts: two kinds of rape leafs of above-mentioned cultivation are cut into the thread enzymolysis solution that is placed in respectively of 0.4-0.6mm, at 24-26 ℃ of enzymolysis 8-10h, cell suspending liquid after enzymolysis is through 100-120 order nylon net filter, gained filtrate is removed supernatant liquor through centrifugation, the gained precipitation adds WI protoplastis curing fluid capable, obtains protoplastis through centrifuge washing;
(3) protoplastis activity identification: the protoplastis of two kinds of rapes that step (2) is extracted is with luciferase FAD(Fluorescein Diacetate) dyeing observes, and the protoplastis of choosing microscopy cell survival rate＞90% absorbs the calcium ion experiment;
(4) protoplastis is cultivated: the protoplastis of the satisfactory two kinds of rapes of step (3) microscopy is placed in respectively contains
45Cultivate 11 ~ 13h in the WI protoplastis curing fluid capable of Ca tracer agent;
(5) protoplastis calcium ion absorbed dose is measured: will cultivate the protoplastis that obtains with the EDTA(ethylenediamine tetraacetic acid (EDTA) of concentration 90 ~ 110mmol/L through step (4)) washings washs and removes the calcium on protoplastis surface, gets 0.1mL protoplastis suspension through clearing up measurement
45The counting of Ca, get 0.1mL protoplastis suspension and be used for protoplastis number counting, with remaining protoplastis suspension centrifugation, get the 0.1mL supernatant liquor and be used for the supernatant liquor counting, the protoplastis that calculates respectively two kinds of rapes by following formula absorbs calcium ion amount q to differentiate the difference of two kinds of Rapeseed Protoplasts absorption calcium:
Wherein: T is the calcium ion actual count that the 0.1mL protoplastis absorbs, and the cpm of unit, H are 0.1mL supernatant liquor counting, and the cpm of unit, S are 0.1mL protoplasm somatocyte number, and unit is individual, and K is calcium ion actual amount and liquid flashing counting scale-up factor, the mol/cpm of unit.
In technique scheme of the present invention, described MS substratum is common substratum in the biological culture field, forms component and mainly comprises KNO
3, NH
4NO
3, KH
2PO
4, MgSO
47H
2O, CaCl
22H
2O, FeSO
47H
2O, Na
2EDTA etc.; Described WI protoplastis curing fluid capable is the common curing fluid capable in the biological culture field, forms component and mainly comprises MES damping fluid, KCl, N.F,USP MANNITOL etc.
In technique scheme of the present invention, described Nutrition Soil preferably contains the vermiculite of weight 25 ~ 35%.The Nutrition Soil of the described MS of containing substratum is preferably with the MS culture medium solution and mixes wet Nutrition Soil.
In technique scheme of the present invention, 0.1mL protoplastis suspension is cleared up measurement
45The counting of Ca preferably adds 0.1mL concentration 14-15mol/L nitric acid in sample, clear up 1 ~ 3h under 70 ~ 80 ℃, after clearing up, it is all transferred to 10mL liquid and dodges measurement count in liquid.The protoplastis suspension is cleared up measurement
45The counting of Ca, the counting of the protoplastis number of protoplastis suspension and the counting of supernatant liquor be available liquid flashing counting device measurement count all.
In technique scheme of the present invention, the rape leaf that is cut into the 0.4-0.6mm silk is placed in enzymolysis solution and carries out enzymolysis, preferably carries out enzymolysis under dark has the condition of vibration.
In technique scheme of the present invention, described protoplastis is observed with luciferase FAD dyeing, preferably by the protoplastis suspension is joined the FDA dyeing solution, mixing dyeing is after 1-2 minute, getting mixed solution is carried on slide and is placed in microscopically with cover plate, observe with the blue light irradiation, represent to have activity if green fluorescence appears in cell, otherwise non-activity.Described FDA dyeing solution can be by first being dissolved in acetone with FDA, and the acetone mixed solution that will be dissolved with again afterwards FDA joins in N.F,USP MANNITOL to be produced.
In technique scheme of the present invention, preferably the protoplastis of the satisfactory two kinds of rapes of microscopy is placed in respectively and contains
45Ca tracer agent and calcium ion concn are respectively in the nutrient solution of 0.1mmol/L, 0.3mmol/L, 1mmol/L, 3mmol/L, 50mmol/L, 100mmol/L and cultivate 11 ~ 13h, to turn out a plurality of measure sample.
In technique scheme of the present invention, described transgenosis TR1 rape is for changing respectively Z63-2, the Z644-21 of heat-resisting gene TR1 and the transgene rape of Z654-9 over to.
The present invention shows by experiment, and along with the increase of calcium ion concn, protoplastis increases gradually to the absorption of calcium.What is interesting is that working as calcium ion concn is 0.1mmol/L, during 0.3mmol/L, transgene rape has absorbed more calcium ion than ordinary cole protoplastis; When calcium ion concn was common 1 to 3mmol/L, the calcium ion that transgene rape and ordinary cole absorb did not almost have difference; And work as Ca
2+Concentration is 50mmol/L, and during 100mmol/L, the transgene rape protoplastis is than ordinary cole protoplastis absorption Ca still less
2+Experimental result confirms, changes the protoplastis of heat-resisting gene TR1 over to, low Ca
2+Improved rape during concentration to Ca
2+Absorption, and at high Ca
2+During concentration, can reduce Ca
2+Enter cell.
Description of drawings
Accompanying drawing 1 is the microscopic examination figure of logical Rapeseed Protoplast.
Accompanying drawing 2 is the microscopic examination figure that change the Rapeseed Protoplast of heat-resisting gene TR1 over to.
Embodiment
1, rape cultivation
The Nutrition Soil of vermiculite weight content 30% is mixed wet with the common MS culture medium solution of dilution after twice, plantation ordinary cole and change respectively Z63-2, Z644-21, Z654-9 transgene rape in heat-resisting gene TR1 over to, in illumination 15-17h/d, cultivate under temperature 23-25 ℃ of condition, incubation time 45-55 days, grow to true leaf 7-10 sheet, get the moderate blade of well-grown leaf age and extract protoplastis.
2, protoplastis extracts
the above-mentioned blade of choosing is cut into thread enzymolysis solution (the 1% cellulase cellulase R-10 that is placed in of 0.4-06mm, 0.1% polygalacturonase Mecerozyme R-IO, 0.4mol/L N.F,USP MANNITOL, 20mmol/L Repone K, the MES of 20mmol/L, 10mmol/L calcium chloride, 1% bovine serum albumin, push away filter sterilization) in, be placed in upward shaking culture enzymolysis 9h under 25 ℃ of dark conditions of low speed shaking table (54rpm), cell suspending liquid after enzymolysis is through 100-120 order nylon net filter, filtrate is through centrifugal (100g, 3min) supernatant liquor is removed in separation, add WI protoplastis curing fluid capable in precipitation, centrifuge washing three times, obtain protoplastis.
3, protoplastis activity identification
Protoplastis after washing is with luciferase FAD(Fluorescein Diacetate) dyeing observes that (5mgFDA is dissolved in 1mL acetone, lucifuge is stored in 4 ℃ of refrigerators, get during use in the N.F,USP MANNITOL that the 0.4mLFDA storage liquid joins 5mL0.65mol/L, obtain the FDA staining fluid, get 0.2mL protoplastis suspension during dyeing, add 0.1mL FDA staining fluid, gently mixing.After dyeing, after 1-2 minute, the mixed solution that takes a morsel is added on slide glass, with cover plate, be placed in microscopically, with the blue light irradiation, if green fluorescence appears in cell, expression has activity, otherwise non-activity), absorb the calcium ion experiment through the protoplastis that is used for of microscopy cell survival rate＞90%.
4, protoplastis is cultivated
The preparation calcium ion concn is respectively the WI curing fluid capable of 0.1mmol/L, 0.3mmol/L, 1mmol/L, 3mmol/L, 50mmol/L, 100mmol/L, and all adds
45Ca carries out spike.Here what deserves to be explained is, in order to keep homeo-osmosis, along with the corresponding minimizing of amount of N.F,USP MANNITOL in the raising WI curing fluid capable of calcium ion concn.During experiment, the WI curing fluid capable of each calcium ion concn is got 4 parts, every part of 1mL, add respectively the protoplastis of ordinary cole and Z63-2, Z644-21, three kinds of transgene rapes of Z654-9, then mix gently, be placed on the 54rpm shaking table and cultivate 12h under 25 ℃ of dark conditions.
5, protoplastis is measured the calcium ion absorbed dose
Cultivate the protoplastis that takes a morsel later and again carry out the activity investigation, after active the investigation, at first centrifugal (100g on low speed centrifuge, 3min) remove supernatant liquor, precipitation adds the EDTA(ethylenediamine tetraacetic acid (EDTA) again) washings (being dissolved with the WI curing fluid capable of the EDTA sodium of 100mmol/L), make protoplastis resuspended, recentrifuge removes supernatant liquor, three times repeatedly.Go for the last time to add the common WI nutrient solution of 1.5mL after supernatant liquor.Then allow protoplastis be uniformly dispersed in WI liquid, with the micro-taken amount device pipettor of 0.1mL, the protoplastis suspension of respectively getting 4 minutes (every part of 0.1mL) from each group is stored in respectively in 4 EP pipes, is used for clearing up and measuring
45The counting of Ca.Getting respectively 0.1mL in the suspension of remainder joins in 4 EP pipes for protoplastis number counting.Then with remaining centrifugal and get the 0.1mL supernatant liquor and preserve, use liquid scintillation counter measurement, result is as the counting of supernatant liquor.
Protoplastis number counting is counted with blood counting chamber, and the protoplastis suspension is dripped one side in the count block in right amount, allows suspension automatically be full of space between cover glass and tally, then counts at microscopically.At first four large lattice total cellular score in the microscopically computing board, line ball cell are only counted left side and top.Then by formula calculate: the large lattice cell count sum of cell count S=four/4 * 1000 in 0.1mL, each group is all done counting 4 times, then average and finally count as the protoplastis of this group.
Liquid flashing counting is measured: sample is placed under 80 ℃, allows moisture evaporate approximately half, then add the 0.1mL concentrated nitric acid, clear up 2h under 80 ℃.Sample is all after dissolving, it all transferred to fill 10mL liquid and dodge in liquid, and vibration is fully after dissolving, with liquid flashing counting device measurement count.
6, protoplastis absorbs the calculating of calcium ion amount
Protoplastis absorbs calcium ion amount q and records isotropic substance by the liquid flashing counting device
45The activity of Ca is calculated, and calculation formula is as follows:
Wherein: T is the calcium ion actual count that the 0.1mL protoplasma absorbs, the cpm of unit;
H is 0.1mL supernatant liquor counting, the cpm of unit;
S is 0.1mL protoplasm somatocyte number, and unit is individual;
K is calcium ion actual amount and liquid flashing counting scale-up factor, the mol/cpm of unit.
7, the absorption of protoplastis to calcium ion
The protoplastis activity that the method that employing Sichuan University Life Science College is recommended is extracted is very high, surpasses 90%, and high like this activity is higher than the protoplastis of taking to filter a centrifugal floating method acquisition of domestic similar information report.After cultivating 12h, the activity of protoplastis still can arrive approximately 90%.
Ordinary cole after cultivating 12h under the different calcium ion concentration gradient and the Rapeseed Protoplast that changes heat-resisting gene TR1 over to see Table 1 to the absorbed dose of calcium ion.By as seen from Table 1, along with the increase of calcium ion concn, protoplastis increases gradually to the absorption of calcium.But the concentration of working as calcium ion is 0.1mmol/L, and during 0.3mmol/L, the transgene rape protoplastis absorbs more calcium ion than the primary mass-energy body of ordinary cole; When calcium ion concn was general value 1 to 3mmol/L, the calcium ion that transgene rape and ordinary cole absorb did not almost have difference; And work as Ca
2+Concentration is 50mmol/L, and during 100mmol/L, the protoplastis of transgene rape is than the protoplastis absorption Ca still less of ordinary cole
2+In general, Ca
2+Entering cell can have various ways, and the active transport that requires the expenditure of energy, the assistance that does not require the expenditure of energy transportation are wherein arranged, and reaches the free diffusing that occurs due to concentration difference.Ca in the residing environment of protoplastis
2+When concentration was very low, free diffusing can not occur, and just needed active transport, and this moment, the ability of active transport just determined that the amount of absorption calcium ion is big or small, and experiment shows, after changing heat-resisting gene over to, at lower concentration Ca
2+Nutrient solution in, the Rapeseed Protoplast that changes heat-resisting base over to more can effectively absorb calcium ion than the protoplastis of coventional type rape.And active transport and assist transportation all with cytolemma on Ca
2+Passage is closely related, and experimental result has confirmed that also heat-resisting TR1 gene has improved calcium channel and absorbed Ca
2+Ability.On the other hand, when in environment, calcium ion is excessive, because the too much calcium ion of free diffusing has entered in cell, the active transport of cell will can not carried out again this moment, but need to get rid of the too much Ca of tenuigenin
2+Damage avoiding, by seeing in table 1, although transgene rape has also accumulated very high calcium, lower than coventional type rape.This shows that rape changes over to has increased the ability that cytolemma is got rid of too much calcium ion after heat-resisting gene.
Table 1 transgene rape and ordinary cole protoplastis absorb calcium ion
Claims (10)
1. utilization
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, is characterized in that mainly comprising the following steps:
(1) rape cultivation: with ordinary cole and transgenosis TR1 rapeseed cultivation in the Nutrition Soil that contains the MS substratum, in illumination 15-17 hour/day, cultivated 45-55 days under temperature 23-25 ℃ of condition, grow to true leaf 7-10 sheet, get respectively two kinds of rape leafs and extract protoplasma;
(2) protoplastis extracts: two kinds of rape leafs of above-mentioned cultivation are cut into the thread enzymolysis solution that is placed in respectively of 0.4-0.6mm, at 24-26 ℃ of enzymolysis 8-10h, cell suspending liquid after enzymolysis is through 100-120 order nylon net filter, gained filtrate is removed supernatant liquor through centrifugation, the gained precipitation adds WI protoplastis curing fluid capable, obtains protoplastis through centrifuge washing;
(3) protoplastis activity identification: the protoplastis of two kinds of rapes that step (2) is extracted is observed with luciferase FAD dyeing, and the protoplastis of choosing microscopy cell survival rate＞90% absorbs the calcium ion experiment;
(4) protoplastis is cultivated: the protoplastis of the satisfactory two kinds of rapes of step (3) microscopy is placed in respectively contains
45Cultivate 11 ~ 13h in the WI protoplastis curing fluid capable of Ca tracer agent;
(5) protoplastis calcium ion absorbed dose is measured: will cultivate the protoplastis that obtains through step (4) and remove the calcium on protoplastis surface with the EDTA washings washing of concentration 90 ~ 110mmol/L, and get 0.1mL protoplastis suspension through clearing up measurement
45The counting of Ca, get 0.1mL protoplastis suspension and be used for protoplastis number counting, with remaining protoplastis suspension centrifugation, get the 0.1mL supernatant liquor and be used for the supernatant liquor counting, the protoplastis that calculates respectively two kinds of rapes by following formula absorbs calcium ion amount q to differentiate the difference of two kinds of Rapeseed Protoplasts absorption calcium:
Wherein: T is the calcium ion actual count that the 0.1mL protoplastis absorbs, and the cpm of unit, H are 0.1mL supernatant liquor counting, and the cpm of unit, S are 0.1mL protoplasm somatocyte number, and unit is individual, and K is calcium ion actual amount and liquid flashing counting scale-up factor, the mol/cpm of unit.
2. utilization according to claim 1
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, it is characterized in that, in described Nutrition Soil, the weight content of vermiculite is 25 ~ 35%.
3. utilization according to claim 2
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, it is characterized in that, the Nutrition Soil of the described MS of containing substratum is for to mix wet Nutrition Soil with the MS culture medium solution.
4. utilization according to claim 1
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, it is characterized in that, 0.1mL protoplastis suspension is cleared up measurement
45The counting of Ca is to add 0.1mL concentration 14-15mol/L nitric acid in sample, clears up 1 ~ 3h under 70 ~ 80 ℃, after clearing up, it is all transferred to 10mL liquid and dodges measurement count in liquid.
5. utilization according to claim 4
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, it is characterized in that, the protoplastis suspension is cleared up measurement
45The counting of Ca, the counting of the protoplastis number of protoplastis suspension and the counting of supernatant liquor are all used liquid flashing counting device measurement count.
6. utilization according to claim 1
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, it is characterized in that, the rape leaf that is cut into the 0.4-0.6mm silk is placed in the enzymolysis solution enzymolysis to carry out under dark oscillating condition.
7. described utilization of one of according to claim 1 to 6
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, it is characterized in that, described protoplastis is observed with luciferase FAD dyeing, by the protoplastis suspension is joined the FDA dyeing solution, after mixing dyeing 1-2 minute, get mixed solution and be carried on slide and be placed in microscopically with cover plate, observe with the blue light irradiation, if cell green fluorescence occurs and represents to have activity, otherwise non-activity.
8. utilization according to claim 7
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, it is characterized in that, producing of FDA dyeing solution is first FDA to be dissolved in acetone, and the acetone mixed solution that will be dissolved with again afterwards FDA joins in N.F,USP MANNITOL to be produced.
9. described utilization of one of according to claim 1 to 6
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, it is characterized in that, the protoplastis of the satisfactory two kinds of rapes of microscopy is placed in respectively and contains
45Ca tracer agent calcium ion concn is respectively in the nutrient solution of 0.1mmol/L, 0.3mmol/L, 1mmol/L, 3mmol/L, 50mmol/L, 100mmol/L and cultivates 11 ~ 13h.
10. described utilization of one of according to claim 1 to 6
45Ca differentiates that transgene rape and ordinary cole protoplastis absorb the method for calcium difference, it is characterized in that, described transgenosis TR1 rape is for changing respectively Z63-2, the Z644-21 of heat-resisting gene TR1 and the transgene rape of Z654-9 over to.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116143082A (en) * | 2023-04-21 | 2023-05-23 | 中国中医科学院中药研究所 | Isotope-labeled selenium nanoparticle and application thereof |
-
2013
- 2013-01-28 CN CN2013100317286A patent/CN103114126A/en active Pending
Non-Patent Citations (3)
| Title |
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| 李廷强等: "锌在超积累植物东南景天叶片细胞水平的吸收积累特征", 《植物营养与肥料学报》 * |
| 许发伦等: "45Ca示踪技术对转基因油菜耐热机理的研究", 《中国核科学技术进展报告》 * |
| 谭伟等: "Ca2+提高烟草叶片光合作用对高温的耐热性", 《中国植物生理学会第十次会员代表大会暨全国学术年会》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116143082A (en) * | 2023-04-21 | 2023-05-23 | 中国中医科学院中药研究所 | Isotope-labeled selenium nanoparticle and application thereof |
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Application publication date: 20130522 |