CN111766315A - Method for distinguishing deteriorated rice seeds based on gluconic acid content change ratio - Google Patents
Method for distinguishing deteriorated rice seeds based on gluconic acid content change ratio Download PDFInfo
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Abstract
The invention belongs to the field of rice seeds and discloses a method for distinguishing deteriorated rice seeds based on a gluconic acid content variation ratio. The method comprises the following steps: extracting gluconic acid from rice seeds, and quantitatively determining the content of the gluconic acid in the rice seeds; determining the gluconic acid content in the new rice seeds by adopting the method as a reference activity index of the rice seeds, and determining the gluconic acid content in the rice seeds of which the seed activities need to be judged; calculating the ratio K of the gluconic acid content in the rice seeds of which the seed vigor needs to be judged to the standard vigor index of the rice seeds; calculating the ratio of the gluconic acid content of the rice seeds after natural aging for 2 years to the benchmark vitality index of the rice seeds, and recording the ratio as K1; and judging whether the stored rice seeds belong to deteriorated seeds or not according to whether the K value is more than or equal to K1, and judging that the stored rice seeds belong to deteriorated seeds when the K value is more than or equal to K1.
Description
Technical Field
The invention belongs to the field of rice seeds, and particularly relates to a method for distinguishing deteriorated rice seeds based on a gluconic acid content variation ratio.
Background
Rice seeds are important agricultural production data. With the change of the sowing mode and the frequent occurrence of extreme weather, the requirement of agricultural production on the sowing quality of the seeds is increased year by year. Due to the unpredictability of seed production, particularly hybrid rice seeds, it is often necessary to store the seeds. The seeds inevitably undergo an irreversible change process of activity reduction, namely, deterioration or aging of the seeds during the storage process, so that the germination rate, emergence rate and production potential are seriously reduced, and the production benefit of the rice is serious. In the practical production application in the south, the most common situation is that rice seeds are subjected to high temperature and high humidity conditions in the process of sale and transportation, so that the seeds are rapidly deteriorated, the vitality is rapidly reduced in a short time, and the commodity performance is lost, the deteriorated seeds are not stored for a long time and are relatively normal in appearance, and therefore, the rapid and effective judgment of the deterioration degree of the seeds is very important.
For a long time, the deterioration of seeds is mainly determined by measuring the germination rate and the seedling rate, but the measurement of the germination rate of the seeds is time-consuming and labor-consuming, and factors influencing data change are more, such as temperature, water absorption of the seeds and the like during measurement. Therefore, it is particularly important to find a fast discrimination method which is not affected by human factors.
Accumulation of storage material is the basis for the formation of seed vigor and is also an important cause of reduced seed vigor after storage. The harvested seeds are stored for a long time, substances stored in the seeds are converted into related metabolites, and the accumulated amount of certain metabolites is related to the substances of the seeds. Therefore, the degree of deterioration of the rice seeds can be determined by directionally measuring the content of the target metabolites in the seeds. In fact, a few patents have been published that use the metabolite content of seeds to identify seed attributes such as seed classification, seed vigor, etc. The patent "method for identifying japonica or indica rice varieties by using the content of metabolites of seeds" (application publication No. CN 103512973A) discloses that japonica or indica rice varieties are identified according to the ratio of the weight percentage content of asparagine to alanine in rice seeds. A method for determining activity of semen glycines based on nuclear magnetic resonance technology (application publication No. CN 106680307A) discloses determining activity of semen glycines by absolute content of metabolite in semen glycines extract, wherein the metabolite can be one or more of arginine, tryptophan, hypoxanthine, isopropanol, propylene glycol, phosphorylcholine, and dimethyl urate in semen glycines.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a method for judging deteriorated rice seeds based on a gluconic acid content change ratio; the inventor finds that a metabolite Gluconic acid (Gluconic acid) in the rice seeds is closely related to the aging degree of the rice seeds in the research process, and the deteriorated rice seeds can be distinguished by utilizing the change ratio of the Gluconic acid content.
The purpose of the invention is realized by the following technical scheme:
a method for distinguishing deteriorated seeds of rice based on a gluconic acid content change ratio comprises the following steps:
(1) extracting gluconic acid from rice seeds;
(2) quantitatively determining the content of gluconic acid in rice seeds, including absolute content or relative content;
(3) judging deteriorated seeds by using the gluconic acid content ratio of the seeds before and after storage;
taking the gluconic acid content of the new seeds before storage as a benchmark vitality index of the rice seeds, and marking as A0; determining the content of gluconic acid in the rice seeds after storage of the deteriorated seeds to be distinguished, and marking the content of the gluconic acid as A; measuring the content of gluconic acid in the rice seeds aged for 2 years, and recording the content as A1; calculating the ratio of the gluconic acid content A in the rice seeds to be distinguished after the storage of the deteriorated seeds and the benchmark vitality index A0 of the rice seeds, and recording the ratio as K; calculating the ratio of the gluconic acid content A1 in the rice seeds after natural aging for 2 years to the standard activity index A0 of the rice seeds, and marking as K1; and judging whether the stored rice seeds belong to deteriorated seeds or not according to whether the K value is more than or equal to K1, and judging that the stored rice seeds belong to deteriorated old seeds when the K value is more than or equal to K1.
The method for extracting the gluconic acid from the rice seeds in the step (1) specifically comprises the following steps: taking rice seeds or single rice embryo or endosperm, placing the rice seeds or the single rice embryo or endosperm in a tissue grinder precooled by liquid nitrogen, grinding the rice seeds or the single rice embryo or endosperm into powder, accurately weighing 50mg of the powder, placing the powder in a 2ml Eppendorf centrifuge tube, adding 800 mul of extracting solution of methanol, chloroform and water in a volume ratio of 5:2:2 into each tube, and adding 10 mul of ribitol as an internal standard; during extraction, the centrifugal tube is shaken up and down for 1min, vortexed for 5min, then centrifuged for 10min at 11000rpm and 4 ℃, 200 mu L of supernatant is transferred to a new 1.5mL centrifugal tube and placed in a vacuum concentration dryer for drying; adding 50 mu L of newly prepared methoxyamine hydrochloride-pyridine solution with the concentration of 20mg/mL into the dried gluconic acid sample, fully and uniformly mixing, and reacting for 2h at 37 ℃ and 200 rpm; adding 70 μ L N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA), mixing, and shaking at 37 deg.C and 200rpm for 30 min; after the reaction is finished, the gluconic acid derivatization sample is immediately sucked and transferred into a sample injection bottle provided with a 250 mu L inner insertion tube, the sample injection bottle is placed in an automatic sample injection disc, and the GC-MS analysis of the subsequent steps is carried out.
The method for quantitatively determining the relative content in the step (2) comprises the following steps: and (2) carrying out GC-MS analysis on the gluconic acid extracted in the step (1), preprocessing GC-MS original data, obtaining peak area information of the gluconic acid in different samples, and carrying out normalization processing on the peak area quantitative information to obtain the final relative content of the metabolite gluconic acid.
The specific conditions for the GC-MS analysis were as follows: the temperature of a sample inlet is 270 ℃; the carrier gas is high-purity helium with the purity of more than 99.999 percent and the flow rate of 1 mL/min; split-flow sample injection with a split-flow ratio of 50: 1; the temperature rising procedure is as follows: keeping at 85 deg.C for 5min, increasing to 205 deg.C at 8 deg.C/min for 5min, and increasing to 300 deg.C at 8 deg.C/min for 5 min; the sample volume is 1 mu L; the ion source EI of the mass spectrum has the ion source temperature of 230 ℃, the quadrupole rod temperature of 150 ℃, the electron energy of 70eV, the transmission line temperature of 280 ℃, the electron multiplier voltage of 1400V, the mass scanning range of 60-1000amu and the solvent delay of 5.00 min;
the preprocessing of the GC-MS raw data is to adopt AglientChemation software to preprocess the GC-MS raw data to obtain peak area information of gluconic acid in different samples, and to normalize the peak area of the gluconic acid by taking the peak area of an isotope internal standard as a reference to obtain the final relative content of the metabolite gluconic acid.
The method for quantitatively determining the absolute content in the step (2) comprises the following steps: and (2) carrying out GC-MS analysis on the gluconic acid extracted in the step (1), and calculating the absolute content (unit microgram/g) of the gluconic acid in the seeds by using a standard curve made of a gluconic acid standard substance.
The specific conditions for the GC-MS analysis were as follows: the gas chromatograph-mass spectrometer is a gas chromatograph-mass spectrometer (model is GCMS-7890A-5975C) of Agilent company; a DB-35UI MS capillary column (30m x 0.25mm x 0.25 μm) is adopted; the temperature of a sample inlet is 270 ℃; the carrier gas is high-purity helium with the purity of more than 99.999 percent and the flow rate of 1 mL/min; split-flow sample injection with a split-flow ratio of 50: 1; the temperature rising procedure is as follows: maintaining at 90 deg.C for 4min, increasing to 205 deg.C at 8 deg.C/min for 2min, and increasing to 310 deg.C at 15 deg.C/min for 5 min; the sample size was 1. mu.L. The mass spectrometry comprises the following steps of (1) mass spectrometry ion source EI, ion source temperature 230 ℃, quadrupole rod temperature 150 ℃, electron energy 70eV, transmission line temperature 300 ℃, mass scanning range 85-700amu, MS analysis by adopting SIM mode, and solvent delay 5.00 min;
the standard curve is prepared according to the following method: precisely weighing 1mg of gluconic acid standard substance, putting the gluconic acid standard substance into a 1.5ml sample bottle, adding methanol water for dissolving, and shaking up to prepare standard solution containing 0.1mg of gluconic acid in each 1 ml; firstly, diluting a standard solution of 0.1mg/ml (100ppm) to prepare standard solutions of 40ppm, 20ppm, 10ppm, 5ppm, 2ppm, 1ppm and 0.2ppm in series concentration; then respectively taking 50 mul of series concentration standard substance solution, and placing the standard substance solution in a vacuum concentration drying instrument for drying; adding 50 mu L of 20mg/mL methoxyamine hydrochloride solution into the dried standard substance, fully and uniformly mixing, and reacting for 2 hours at 37 ℃ and 200 rpm; adding 70 mu L N-methyl-N- (trimethylsilyl) trifluoroacetamide, mixing uniformly, and shaking at 37 ℃ and 200rpm for 30 min; after the reaction is finished, immediately absorbing the derivatization sample and transferring the derivatization sample to a sample injection bottle for GC-MS analysis; carrying out peak extraction on the collected mass spectrum data through MassHunter software to obtain peak area information of the gluconic acid in different samples; respectively drawing standard curves by taking the peak area as a vertical coordinate and the concentration as a horizontal coordinate;
the method comprises the steps of calculating the absolute content of gluconic acid in seeds, specifically, carrying out peak extraction on mass spectrum data acquired by GC-MS analysis through MassHunter software to obtain peak area information of the gluconic acid in different samples; and converting the absolute content of the gluconic acid according to the peak area, the standard curve and the weight of the sample.
The equation of the standard curve is that Y is 107937.485394X-17585.687560, R20.9722, Y is peak area and X is concentration.
The principle of the invention is as follows:
during the process of seed vitality reduction in the seed storage stage, the storage substance content in the seeds is generally reduced, and the storage protein and the like are degraded, so that the seeds are aged and deteriorated, the germination is delayed or the germination rate is reduced, and the seed vitality is obviously reduced. The research finds that the content of the gluconic acid in the seeds is continuously accumulated within a certain time range in the process of reducing the vitality of the seeds, and the accumulation of the content of the gluconic acid has a certain correlation with the aging degree of the seeds. Therefore, the aging deterioration degree of the seeds can be distinguished by directionally measuring the content of the target metabolite gluconic acid in the seeds.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the method overcomes the defects of time and labor consumption in judging the seed vitality based on the germination rate, overcomes the defects of extremely high cost and high cost of other metabolite methods and instruments, can make the judgment of the seed deterioration degree simpler and quicker by using the content of the gluconic acid in the seeds as an index for measuring the seed deterioration, improves the judgment efficiency and saves the seeds.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
The following examples use the following raw materials and methods:
1. all rice seeds were provided by jin rice cultivars ltd, guangdong province during the experiment.
2. The natural storage and aging method of the seeds comprises the following steps: the harvested rice seeds are respectively packed in mesh bags and stored indoors under natural conditions.
3. The artificial seed aging method comprises the following steps: three sealed dryers were prepared before aging, one of which was charged with supersaturated MgCl2The solution was left at 20 ℃ and the remaining two were each easily added with supersaturated KCl and left at 15 ℃ and 40 ℃ respectively. The harvested rice seeds are respectively packaged by a mesh bag, pre-treated in a drier with the temperature of 15 ℃ and the relative humidity (R.H) of 85 percent for 3 days, then respectively transferred to a balanced drier for treating for 8 days at the temperature of 43 ℃ and the relative humidity (R.H), then respectively dried for 3 days at the temperature of 25 ℃ and the relative humidity of 32 percent, and hermetically stored at-20 ℃.
4. The rice seed germination rate measuring method is carried out according to the rice seed germination rate measuring method (GB/T3543).
5. The calculation formula of the seed germination rate is as follows: the seed germination percentage is the number of germinated seeds/total seeds × 100%.
6. The method for extracting the gluconic acid from the rice seeds comprises the following steps:
taking rice seeds or single rice embryo or endosperm, placing the rice seeds or the single rice embryo or endosperm in a tissue grinder precooled by liquid nitrogen, grinding the rice seeds or the single rice embryo or endosperm into powder, accurately weighing 50mg of the powder, placing the powder in a 2ml Eppendorf centrifuge tube, adding 800 mul of extracting solution of methanol, chloroform and water in a volume ratio of 5:2:2 into each tube, and adding 10 mul of ribitol as an internal standard; during extraction, the centrifugal tube is shaken up and down for 1min, vortexed for 5min, then centrifuged for 10min at 11000rpm and 4 ℃, 200 mu L of supernatant is transferred to a new 1.5mL centrifugal tube and placed in a vacuum concentration dryer for drying; adding 50 mu L of newly prepared methoxyamine hydrochloride-pyridine solution with the concentration of 20mg/mL into the dried gluconic acid sample, fully and uniformly mixing, and reacting for 2h at 37 ℃ and 200 rpm; adding 70 μ L N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA), mixing, and shaking at 37 deg.C and 200rpm for 30 min; after the reaction is finished, immediately sucking a gluconic acid derivatization sample, transferring the gluconic acid derivatization sample into a sample injection bottle provided with a 250 mu L inner insertion tube, placing the sample injection bottle in an automatic sample injection disc, and performing GC-MS analysis of the subsequent steps;
7. and (3) determining the relative content of the gluconic acid in the rice seed sample:
the method for extracting the gluconic acid from the rice seeds is as before, and then the chromatographic-mass spectrometric analysis of metabolic fingerprint acquisition, data processing and the calculation of the relative content of the gluconic acid are carried out. Wherein the GC-MS analysis conditions are as follows: the gas chromatograph-mass spectrometer is a gas chromatograph-mass spectrometer of GCMS-7890A-5975C of Agilent company; using DB-35UI MS capillary column (30m x 0.25mmx 0.25 μm); the temperature of a sample inlet is 270 ℃; the carrier gas is high-purity helium with the purity of more than 99.999 percent and the flow rate of 1 mL/min; split-flow sample injection with a split-flow ratio of 50: 1; the temperature rising procedure is as follows: keeping at 85 deg.C for 5min, increasing to 205 deg.C at 8 deg.C/min for 5min, and increasing to 300 deg.C at 8 deg.C/min for 5 min; the sample volume is 1 mu L; the ion source EI of the mass spectrum has the ion source temperature of 230 ℃, the quadrupole rod temperature of 150 ℃, the electron energy of 70eV, the transmission line temperature of 280 ℃, the electron multiplier voltage of 1400V, the mass scanning range of 60-1000amu and the solvent delay of 5.00 min.
And then preprocessing GC-MS original data by adopting AglientChemsstion software, finally processing image information into data information for further processing and analysis, obtaining peak area information of gluconic acid in different samples, and normalizing the peak area quantitative information to obtain the final relative content of the metabolite gluconic acid.
8. Quantitative analysis of absolute content of gluconic acid in rice seed samples:
the gluconic acid extracted by the above method was subjected to GC-MS analysis, and the absolute content (in μ g/g) of gluconic acid in the seeds was calculated using a standard curve prepared from a gluconic acid standard.
The extraction of the gluconic acid in the seed sample is the same as the previous extraction. But the GC-MS analysis conditions were slightly different from the relative quantitation: the gas chromatograph-mass spectrometer is a gas chromatograph-mass spectrometer (model number is GCMS-7890A-5975C) of Agilent. A DB-35UI MS capillary column (30m x 0.25mm x 0.25 μm) is adopted; the temperature of a sample inlet is 270 ℃; the carrier gas is high-purity helium with the purity of more than 99.999 percent and the flow rate of 1 mL/min; split-flow sample injection with a split-flow ratio of 50: 1; the temperature rising procedure is as follows: maintaining at 90 deg.C for 4min, increasing to 205 deg.C at 8 deg.C/min for 2min, and increasing to 310 deg.C at 15 deg.C/min for 5 min; the sample size was 1. mu.L. The ion source EI of the mass spectrum has the ion source temperature of 230 ℃, the quadrupole rod temperature of 150 ℃, the electron energy of 70eV, the transmission line temperature of 300 ℃, the mass scanning range of 85-700amu, MS analysis is carried out by adopting an SIM mode, and the solvent delay is 5.00 min.
Preparing a gluconic acid standard curve: precisely weighing 1mg of gluconic acid standard substance, placing the gluconic acid standard substance into a 1.5ml sample bottle, adding methanol water for dissolving, and shaking up to prepare standard solution containing 0.1mg of gluconic acid in each 1 ml. First, 0.1mg/ml (100ppm) of a standard solution was diluted to prepare a series of standard solutions of 40ppm, 20ppm, 10ppm, 5ppm, 2ppm, 1ppm, and 0.2ppm concentrations. Then 50ul of standard solutions with different concentrations were taken and dried in a vacuum concentration dryer. Adding 50 mu L of 20mg/mL methoxyamine hydrochloride solution into the dried standard substance, fully and uniformly mixing, and reacting for 2 hours at 37 ℃ and 200 rpm; adding 70 mu L N-methyl-N- (trimethylsilyl) trifluoroacetamide, mixing uniformly, and shaking at 37 ℃ and 200rpm for 30 min; and after the reaction is finished, immediately sucking the derivatization sample and transferring the derivatization sample to a sample injection bottle for GC-MS analysis. And performing peak extraction on the collected mass spectrum data through MassHunter software to obtain peak area information of the gluconic acid in different samples. Respectively drawing standard curves (Y is 107937.485394X-17585.687560, R is20.9722, Y is peak area and X is concentration).
The method for calculating the absolute content of the gluconic acid in the seeds specifically comprises the step of carrying out peak extraction on mass spectrum data acquired by GC-MS analysis through MassHunter software to obtain peak area information of the gluconic acid in different samples. Converting the absolute content (mu g/g) of the gluconic acid according to the peak area, the standard curve and the weight of the sample, specifically, obtaining the concentration of the gluconic acid in the solution according to the peak area and the standard curve, and then calculating to obtain the content of the gluconic acid in the sample by using the following formula: gluconic acid content (μ g/g) ═ concentration of gluconic acid in solution x volume of extraction solution x dilution times divided by weight of sample.
9. Method for judging vitality of seeds by using change of gluconic acid content before and after aging
The gluconic acid content (absolute content or relative content) of the new seeds before aging is taken as a benchmark vitality index of the rice seeds and is marked as A0; determining the content (absolute content or relative content) of gluconic acid in the rice seeds of which the seed aging degree needs to be judged, and marking the content as A; measuring the content of gluconic acid in the rice seeds aged for 2 years, and recording the content as A1; calculating the ratio of the gluconic acid content A in the rice seeds of which the seed aging degree needs to be judged to the reference activity index A0 of the rice seeds, and recording the ratio as K; calculating the ratio of the gluconic acid content A1 in the rice seeds after natural aging for 2 years to the standard activity index A0 of the rice seeds, and marking as K1; and judging whether the stored rice seeds belong to deteriorated seeds or not according to whether the K value is more than or equal to K1, and judging that the stored rice seeds belong to deteriorated seeds when the K value is more than or equal to K1.
Example 1:
hybrid rice combinations such as QY3618, B3Y998, IIY998 and QY998 are selected as experimental materials, and harvested seeds are subjected to natural aging treatment for 2 years.
Then taking a certain amount of the rice seeds, and determining the germination rate of the rice seeds according to a rice seed germination rate determination method (GB/T3543); meanwhile, the extraction and quantitative determination of the seed metabolite gluconic acid are carried out according to the method for extracting the gluconic acid and quantitatively determining the relative content, the ratio of the gluconic acid in the rice seeds before and after storage is calculated and is recorded as K1 (table 1), wherein the content of the seed metabolite gluconic acid refers to the average content of the gluconic acid in the whole seeds, namely the content of the mixture of the embryo and the endosperm in the seeds.
As can be seen from Table 1, after all the hybrid rice seeds tested are naturally aged for 2 years, the germination rates are reduced, and the reduction degrees of the germination rates of different combinations are not completely the same, but the germination rates of the seeds in the experiment are all lower than 80%. According to the regulations in the 1 st part cereal of grain crop seeds in China, the seeds with the germination rate of the hybrid seeds of less than 80 percent are unqualified seeds. The germination rate of the seeds is also an important index for representing the vitality of the seeds, and the seeds with unqualified germination rate are naturally low-vitality seeds.
It can also be seen from table 1 that, as the germination rate of naturally aged seeds decreases, the gluconic acid content in all the seeds after storage increases, i.e., the gluconic acid ratio K1 before and after storage is greater than 1. The conclusion that the invention utilizes the gluconic acid content ratio as the identification index of the deteriorated seed is correct can be shown.
TABLE 1 change in the gluconic acid ratio K1 of naturally aged 2-year seeds and germination percentage of the seeds
| Variety/combination | Initial germination percentage (%) | Germination percentage after aging (%) | K1 value |
| QY3618 | 96.50 | 50.16 | 3.91047 |
| B3Y998 | 95.43 | 67.00 | 7.34477 |
| IIY998 | 96.58 | 44.82 | 11.7099 |
| QY998 | 96.42 | 67.43 | 6.4830 |
Remarking: k1 represents the ratio of the relative content of gluconic acid in seeds stored naturally for 2 years to the relative content of gluconic acid at 0 day.
The QY3618 hybrid rice combination after being stored for a certain time is randomly selected, the gluconic acid as a metabolite of the seed is extracted and quantitatively measured according to the method for extracting the gluconic acid and quantitatively measuring the relative content, the ratio K of the gluconic acid in the rice seeds before and after storage is calculated to be 5.63756, the value is larger than the corresponding K1 value 3.91047 in the table 1, the rice seeds are judged to belong to the deteriorated seeds, and meanwhile, the germination rate of the rice seeds is measured to be 37.21% according to the method for measuring the germination rate of the rice seeds (GB/T3543), so that the rice seeds can be confirmed to belong to the deteriorated seeds, and the method is proved to be correct.
Randomly selecting a B3Y998 hybrid rice combination which is naturally stored for a certain time, extracting and quantitatively measuring a metabolite gluconic acid of the seeds according to the method for extracting the gluconic acid and quantitatively measuring the relative content, calculating that the ratio K of the gluconic acid in the rice seeds before and after storage is 8.73556, the value is larger than the corresponding K1 value 7.34477 in the table 1, judging the rice seeds to belong to the deteriorated seeds, and simultaneously measuring the germination rate of the rice seeds to be 57.35% according to a method for measuring the germination rate of the rice seeds (GB/T3543), so that the rice seeds can be confirmed to belong to the deteriorated seeds, thereby proving that the method is correct.
Randomly selecting IIY998 hybrid rice combination after natural storage for a certain time, extracting and quantitatively measuring the metabolite gluconic acid of the seeds according to the method for extracting the gluconic acid and quantitatively measuring the relative content, calculating that the ratio K of the gluconic acid in the rice seeds before and after storage is 9.88965, the value is less than the corresponding K1 value 11.7099 in the table 1, judging the rice seeds not to belong to the deteriorated seeds, and simultaneously measuring the germination rate of the rice seeds to be 81.22% according to the method for measuring the germination rate of the rice seeds (GB/T3543), thus confirming that the rice seeds do not belong to the deteriorated seeds and proving that the method of the invention is correct.
The QY998 hybrid rice combination after being stored for a certain time is randomly selected, the extraction and quantitative determination of the gluconic acid as the metabolite of the seeds are carried out according to the method for extracting the gluconic acid and quantitatively determining the relative content, the ratio K of the gluconic acid in the rice seeds before and after storage is calculated to be 4.73256, the value is smaller than the corresponding K1 value 6.4830 in the table 1, the rice seeds are judged not to be the deteriorated seeds, meanwhile, the germination rate of the rice seeds is determined to be 85.31% according to the method for determining the germination rate of the rice seeds (GB/T3543), and the rice seeds can be confirmed not to be the deteriorated seeds, and the method is proved to be correct.
Example 2:
368 series hybrid rice combination is selected as an experimental material, and the harvested seeds are subjected to natural aging treatment for 2 years.
Then taking a certain amount of the rice seeds, and determining the germination rate of the rice seeds according to a rice seed germination rate determination method (GB/T3543); meanwhile, the method for extracting and quantitatively measuring the gluconic acid as the metabolite of the seeds is used for extracting and quantitatively measuring the gluconic acid, and the ratio of the gluconic acid in the rice seeds before and after storage is calculated and is marked as K1 (table 2).
As can be seen from Table 2, the germination rates of the 368 series hybrid rice seeds tested all decreased after natural aging for 2 years, and although the germination rates of the different combinations were not completely the same, the germination rates of the seeds in the experiment were all lower than 80%, which are not qualified seeds. It can also be seen from table 2 that, as the germination rate of naturally aged seeds decreases, the gluconic acid content in all the seeds after storage increases, confirming that the judgment of the invention using the gluconic acid content ratio as the identification index of the deteriorated seeds is correct.
TABLE 2 change of gluconic acid ratio K1 of naturally aged 2 years of hybrid rice 368 series seeds and germination rate of seeds
| Variety/combination | Initial germination percentage (%) | Germination percentage after aging (%) | Value of K |
| QY368 | 95.67 | 58.44 | 14.3463 |
| B3Y368 | 97.31 | 73.72 | 12.5696 |
| IIY368 | 96.12 | 53.37 | 10.4756 |
| TY368 | 95.75 | 75.76 | 14.3481 |
Randomly selecting a QY368 hybrid rice combination which is naturally stored for a certain time, extracting and quantitatively measuring a metabolite gluconic acid of the seeds according to the method for extracting the gluconic acid and quantitatively measuring the relative content, calculating that the ratio K of the gluconic acid in the rice seeds before and after storage is 12.33552, the value is smaller than the corresponding K1 value 14.3463 in the table 1, judging that the rice seeds do not belong to the deteriorated seeds, and simultaneously measuring that the germination rate of the rice seeds is 82.23% according to a method for measuring the germination rate of the rice seeds (GB/T3543), so that the rice seeds do not belong to the deteriorated seeds can be confirmed, and the method is proved to be correct.
Randomly selecting B3Y368 hybrid rice combination after natural storage for a certain time, extracting and quantitatively measuring the metabolite gluconic acid of the seeds according to the method for extracting the gluconic acid and quantitatively measuring the relative content, calculating that the ratio K of the gluconic acid in the rice seeds before and after storage is 14.7255, the value is larger than the corresponding K1 value 12.5696 in the table 1, judging the rice seeds to belong to the deteriorated seeds, and simultaneously measuring the germination rate of the rice seeds to be 65.44% according to the method for measuring the germination rate of the rice seeds (GB/T3543), so that the rice seeds can be confirmed to belong to the deteriorated seeds, and the method is proved to be correct.
Randomly selecting IIY368 hybrid rice combination after natural storage for a certain time, extracting and quantitatively measuring the metabolite gluconic acid of the seeds according to the method for extracting the gluconic acid and quantitatively measuring the relative content, calculating that the ratio K of the gluconic acid in the rice seeds before and after storage is 8.7876, the value is less than the corresponding K1 value 10.4756 in the table 1, judging the rice seeds not to be the deteriorated seeds, and simultaneously measuring the germination rate of the rice seeds to be 80.97% according to the method for measuring the germination rate of the rice seeds (GB/T3543), so that the rice seeds not to be the deteriorated seeds can be confirmed, and the method is proved to be correct.
The method is characterized in that TY368 hybrid rice combination after natural storage for a certain time is randomly selected, the extraction and quantitative determination of the gluconic acid as the metabolite of the seeds are carried out according to the methods for extracting the gluconic acid and quantitatively determining the relative content, the ratio K of the gluconic acid in the rice seeds before and after storage is calculated to be 17.2556, the value is larger than the corresponding K1 value 14.3481 in the table 1, the rice seeds are judged to belong to the deteriorated seeds, and the germination rate of the rice seeds is determined to be 65.25% according to the method for determining the germination rate of the rice seeds (GB/T3543), so that the rice seeds can be confirmed to belong to the deteriorated seeds.
Example 3:
taking Guangyou 8 you 165, Guangyou 8B and Guangyui 308 with natural aging treatment time of 2 years and Nanyouzhan with natural aging treatment time of 1.5 years, respectively carrying out extraction and quantitative determination on a seed metabolite gluconic acid according to the extraction and quantitative determination method of the relative content of the gluconic acid, and calculating the ratio of the gluconic acid in the rice seeds before and after storage, which is recorded as K1: the K1 value of Guang 8 you 165 is 3.156215678, the K1 value of Guang 8B is 3.787893221, the K1 value of Guanghui 308 is 2.255632878, and the K1 value of Nanyou is 3.117867776.
In the natural storage of rice seeds, 4 varieties of old seeds of Guangyou 165, Guangyou 8B, Guanghui 308, Nanyou account and the like after natural storage for a certain time and 4 kinds of new seeds (not stored) which are the same as the old seeds are randomly selected as materials, and then a certain number of rice seeds are taken to carry out experiments.
The first step is as follows: the absolute content of gluconic acid in the seeds was determined and the K value was calculated (table 3). And (3) determining the absolute content of the seed metabolite gluconic acid according to the method for extracting and quantifying the gluconic acid, and calculating the ratio of the gluconic acid in the rice seeds before and after storage. The specific data are shown in Table 3.
The second step is that: and judging the seed vitality according to the K value. In table 3, K values of 3 varieties are respectively greater than their corresponding K1 values, and we distinguish that the above 3 combined seeds belong to deteriorated seeds. And the K value of the Nanyou account variety is smaller than the corresponding K1 value, and the Nanyou account variety is judged to be qualified seed.
The third step: the germination rate of the rice seeds is determined according to a rice seed germination rate determination method (GB/T3543), and specific test data are shown in Table 4. Table 4 germination rate results show that all of the 3 randomly selected naturally stored combinations belong to deteriorated seeds with high aging degree, the germination rate is lower than 30%, the seeds basically have no vigor, and the storage time is actually more than 2 years. But the Nanyou accounts for 81.33% of the germination rate, belongs to unaged seeds and can be continuously used.
This example illustrates that the ratio of the change in the content of gluconic acid can be used to discriminate the inferior or old rice seeds according to the present invention.
TABLE 3 ratio K for verifying gluconic acid content in the experimental materials
Table 4 verifies the germination percentage in the experiment
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. A method for distinguishing deteriorated seeds of rice based on a gluconic acid content change ratio is characterized by comprising the following steps:
(1) extracting gluconic acid from rice seeds;
(2) quantitatively determining the content of gluconic acid in rice seeds, including absolute content or relative content;
(3) judging deteriorated seeds by using the gluconic acid content ratio of the seeds before and after storage;
taking the gluconic acid content of the new seeds before storage as a benchmark vitality index of the rice seeds, and marking as A0; determining the content of gluconic acid in the rice seeds after storage of the deteriorated seeds to be distinguished, and marking the content of the gluconic acid as A; measuring the content of gluconic acid in the rice seeds aged for 2 years, and recording the content as A1; calculating the ratio of the gluconic acid content A in the rice seeds to be distinguished after the storage of the deteriorated seeds and the benchmark vitality index A0 of the rice seeds, and recording the ratio as K; calculating the ratio of the gluconic acid content A1 in the rice seeds after natural aging for 2 years to the standard activity index A0 of the rice seeds, and marking as K1; and judging whether the stored rice seeds belong to deteriorated seeds or not according to whether the K value is more than or equal to K1, and judging that the stored rice seeds belong to deteriorated seeds when the K value is more than or equal to K1.
2. The method for distinguishing deteriorated seeds of rice based on the gluconic acid content variation ratio as claimed in claim 1, wherein: the method for extracting the gluconic acid from the rice seeds in the step (1) specifically comprises the following steps: taking rice seeds or single rice embryo or endosperm, placing the rice seeds or the single rice embryo or endosperm in a tissue grinder precooled by liquid nitrogen, grinding the rice seeds or the single rice embryo or endosperm into powder, accurately weighing 50mg of the powder, placing the powder in a 2ml Eppendorf centrifuge tube, adding 800 mul of extracting solution of methanol, chloroform and water in a volume ratio of 5:2:2 into each tube, and adding 10 mul of ribitol as an internal standard; during extraction, the centrifugal tube is shaken up and down for 1min, vortexed for 5min, then centrifuged for 10min at 11000rpm and 4 ℃, 200 mu L of supernatant is transferred to a new 1.5mL centrifugal tube and placed in a vacuum concentration dryer for drying; adding 50 mu L of newly prepared methoxyamine hydrochloride-pyridine solution with the concentration of 20mg/mL into the dried gluconic acid sample, fully and uniformly mixing, and reacting for 2h at 37 ℃ and 200 rpm; adding 70 mu L N-methyl-N- (trimethylsilyl) trifluoroacetamide, mixing, and shaking at 37 ℃ and 200rpm for 30 min; after the reaction is finished, the gluconic acid derivatization sample is immediately sucked and transferred into a sample injection bottle provided with a 250 mu L inner insertion tube, the sample injection bottle is placed in an automatic sample injection disc, and the GC-MS analysis of the subsequent steps is carried out.
3. The method for distinguishing deteriorated seeds of rice based on the gluconic acid content variation ratio as claimed in claim 1, wherein: the method for quantitatively determining the relative content in the step (2) comprises the following steps: and (2) carrying out GC-MS analysis on the gluconic acid extracted in the step (1), preprocessing GC-MS original data, obtaining peak area information of the gluconic acid in different samples, and carrying out normalization processing on the peak area quantitative information to obtain the final relative content of the metabolite gluconic acid.
4. The method for distinguishing deteriorated seeds of rice based on the gluconic acid content variation ratio as claimed in claim 3, wherein: the specific conditions for the GC-MS analysis were as follows: the temperature of a sample inlet is 270 ℃; the carrier gas is high-purity helium with the purity of more than 99.999 percent and the flow rate of 1 mL/min; split-flow sample injection with a split-flow ratio of 50: 1; the temperature rising procedure is as follows: keeping at 85 deg.C for 5min, increasing to 205 deg.C at 8 deg.C/min for 5min, and increasing to 300 deg.C at 8 deg.C/min for 5 min; the sample volume is 1 mu L; the ion source EI of the mass spectrum has the ion source temperature of 230 ℃, the quadrupole rod temperature of 150 ℃, the electron energy of 70eV, the transmission line temperature of 280 ℃, the electron multiplier voltage of 1400V, the mass scanning range of 60-1000amu and the solvent delay of 5.00 min;
the preprocessing of the GC-MS raw data is to adopt AglientChemation software to preprocess the GC-MS raw data to obtain peak area information of gluconic acid in different samples, and to normalize the peak area of the gluconic acid by taking the peak area of an isotope internal standard as a reference to obtain the final relative content of the metabolite gluconic acid.
5. The method for distinguishing deteriorated seeds of rice based on the gluconic acid content variation ratio as claimed in claim 1, wherein: the method for quantitatively determining the absolute content in the step (2) comprises the following steps: and (2) carrying out GC-MS analysis on the gluconic acid extracted in the step (1), and calculating the absolute content of the gluconic acid in the seeds by using a standard curve made of a gluconic acid standard substance, wherein the unit of the absolute content is mu g/g.
6. The method for distinguishing deteriorated seeds of rice based on the gluconic acid content variation ratio as claimed in claim 5, wherein: the specific conditions for the GC-MS analysis were as follows: the gas chromatograph-mass spectrometer is a gas chromatograph-mass spectrometer of Agilent company; a DB-35UI MS capillary column is adopted, and the size is 30m x 0.25mm x 0.25 mu m; the temperature of a sample inlet is 270 ℃; the carrier gas is high-purity helium with the purity of more than 99.999 percent and the flow rate of 1 mL/min; split-flow sample injection with a split-flow ratio of 50: 1; the temperature rising procedure is as follows: maintaining at 90 deg.C for 4min, increasing to 205 deg.C at 8 deg.C/min for 2min, and increasing to 310 deg.C at 15 deg.C/min for 5 min; the sample size was 1. mu.L. The mass spectrometry comprises the following steps of (1) mass spectrometry ion source EI, ion source temperature 230 ℃, quadrupole rod temperature 150 ℃, electron energy 70eV, transmission line temperature 300 ℃, mass scanning range 85-700amu, MS analysis by adopting SIM mode, and solvent delay 5.00 min;
the standard curve is prepared according to the following method: precisely weighing 1mg of gluconic acid standard substance, putting the gluconic acid standard substance into a 1.5ml sample bottle, adding methanol water for dissolving, and shaking up to prepare standard solution containing 0.1mg of gluconic acid in each 1 ml; firstly, diluting 0.1mg/ml standard substance solution to prepare standard substance solutions with series concentrations of 40ppm, 20ppm, 10ppm, 5ppm, 2ppm, 1ppm and 0.2 ppm; then respectively taking 50 mul of series concentration standard substance solution, and placing the standard substance solution in a vacuum concentration drying instrument for drying; adding 50 mu L of 20mg/mL methoxyamine hydrochloride solution into the dried standard substance, fully and uniformly mixing, and reacting for 2 hours at 37 ℃ and 200 rpm; adding 70 μ LN-methyl-N- (trimethylsilyl) trifluoroacetamide, mixing, and shaking at 37 deg.C and 200rpm for 30 min; after the reaction is finished, immediately absorbing the derivatization sample and transferring the derivatization sample to a sample injection bottle for GC-MS analysis; carrying out peak extraction on the collected mass spectrum data through MassHunter software to obtain peak area information of the gluconic acid in different samples; respectively drawing standard curves by taking the peak area as a vertical coordinate and the concentration as a horizontal coordinate;
the method comprises the steps of calculating the absolute content of gluconic acid in seeds, specifically, carrying out peak extraction on mass spectrum data acquired by GC-MS analysis through MassHunter software to obtain peak area information of the gluconic acid in different samples; and converting the absolute content of the gluconic acid according to the peak area, the standard curve and the weight of the sample.
7. The method for distinguishing deteriorated seeds of rice based on the gluconic acid content variation ratio as claimed in claim 6, wherein: the equation of the standard curve is that Y is 107937.485394X-17585.687560, R20.9722, Y is peak area and X is concentration.
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