CN103529139B - Method for analyzing metabolism difference of transgenic rice by using natural variation - Google Patents
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Abstract
The invention discloses a method for analyzing a metabolism difference of transgenic rice by using natural variation. The method comprises the following steps: adopting an ultra-high performance liquid chromatography-mass spectrometry and a gas chromatography-mass spectrometry to analyze extractives of rice seeds with natural varieties to obtain a rice metabolism outline spectrum so as to obtain a concentration change range of all metabolins in the rice with different natural varieties, namely obtaining a natural variation range of the metabolins in the rice with the natural varieties; analyzing the extractives of the transgenic rice seeds under the same testing condition; comparing an analyzing result with the natural variation range; if the analyzing result exceeds the natural variation range, showing that the variation of the metabolins is not predicated; and if the analyzing result is in the natural variation range, showing that the transgenic rice and the natural seeds are equal substantially on a seed metabolome level. The analyzing method has the advantages of high sensitivity and high throughput; the natural variation range of the metabolins is considered so that the method can provide data for deeply researching the safety of the transgenic rice.
Description
Technical field
The present invention relates to analytical chemistry field, be specifically related to a kind of method that transgenic paddy rice Difference of Metabolism is analyzed in natural variation.
Background technology
Genetically modified crops refer to the crop after utilizing technique for gene engineering genes of interest to be forwarded to the improvement formed in acceptor gene group.Genetically modified crops can improve the output of crop, and oil recovery enhancement and nutritive value, strengthen the resistances such as pest-resistant disease-resistant drought resisting.Up to the present the whole world has had 196 transgenic events to be commercially used, and comprises transgenosis cattle and sheep, transgenosis fishes and shrimps, transgenic crops, transgenic vegetable and fruit.But the worry of the public to safety of transgenic crops never stopped, be the worry of the secure context to edible enetically modified food on the one hand, worry that whether extrinsic protein is toxic to people, in enetically modified food, whether amount of allergenic protein present can increase.Be on the impact of genetically modified crops on ecologic environment on the other hand, worry that resistant gene can be diffused in other crops, particularly its wild affinity species in environment, thus produce superweed.True for consumption, when genetically modified crops and derivant thereof are used to food or feed, its security is controversial.Researchist is being that effort is done in transgenosis safe evaluation always, the Organization for Economic Cooperation and Development (Organization for Economic Cooperation and Development, OECD) first proposed " substantial equivalence " principle in 2006 for evaluating the security of genetically modified crops, this principle is united state grain and agricultural organization (Food and Agriculture Organization of the United Nations) and the World Health Organization (WHO) (World Health Organization) institute's refinement again.The concept of substantial equivalence is that so it just can be treated safely as common food if a kind of food or composition of food are actually identical with the food existed or composition of food, and namely it can be seen as and have identical security with conventional food.The content of its bag expansion three levels: chemical composition is equal to, eating effect is equal to, and environmental impact is equal to.
In recent years, the change of the group level of genetically modified crops was all paid close attention in much research.These group methods are considered to the non-target calibration method with high flux, high precision and stronger reliability, comprise cDNA microarray, Microrna fingerprint, Leaf proteins, metabolism group and toxicity profile.The security very general and success of application metabolism group method evaluation genetically modified crops.Metabolism group research is before all the metabolism group of genetically modified crops and the metabolism group of its acceptor are compared substantially, this method ignores interracial natural variation, likely cause misreading metabolism group data, namely we can not only according to the metabolism group of genetically modified crops with the metabolism group of its acceptor is variant just thinks these genetically modified crops whether safety.So we are in the urgent need to bringing the natural variation of crop varieties into safety of transgenic crops appraisement system.
Summary of the invention
The object of the invention is to the deficiency overcoming the existence of above-mentioned prior art, provide a kind of natural variation by crop varieties to bring the method analyzing transgenic paddy rice Difference of Metabolism with natural variation of safety of transgenic crops appraisement system into.The present invention utilizes and carries out metabolic profiling analysis based on Ultra Performance Liquid Chromatography-mass spectrometry and gas chromatography-mass spectrum contact system to the seed producing upper conventional parental rice, analyze and obtain the variation range of various metabolin in different rice varieties, and this natural variation is used for study the Difference of Metabolism of transgenosis and non-transgenic rice paddy seed.The method has high sensitivity, high-throughout advantage.Owing to considering the natural variation scope of metabolin, the method can provide basis for the security furtheing investigate transgenic paddy rice.
The object of the invention is to be achieved through the following technical solutions:
The present invention relates to a kind of method that transgenic paddy rice Difference of Metabolism is analyzed in natural variation, described method comprises the steps:
A, employing Ultra Performance Liquid Chromatography-mass spectrometry and Gas chromatographyMass spectrometry carry out analysis to natural kind rice paddy seed extract and obtain paddy rice metabolic profile spectrum, and then obtain the concentration change scope of various metabolin in difference nature kind paddy rice, be the natural variation scope of metabolin in natural kind paddy rice;
B, under the test condition identical with steps A, adopt Ultra Performance Liquid Chromatography-mass spectrometry and Gas chromatographyMass spectrometry to analyze transgenic paddy rice seed extract, the natural variation scope in analysis result and steps A compares; Metabolite concentration change exceedes natural variation scope, illustrates that the change of this metabolin is unexpected, and metabolite concentration changes within the scope of natural variation, illustrates that this transgenic paddy rice and natural kind are in fact equivalent in seed metabolism group level.
Preferably, described natural kind rice paddy seed comprises the acceptor rice paddy seed of described transgenic paddy rice seed; Described transgenic paddy rice is with non-transgenic paddy rice for acceptor carries out gene insertion, also selects the transgenic paddy rice with lysine-rich protein characteristic through selfing.
Preferably, described natural kind rice paddy seed is the core key variety comprised in rice in China database.
Preferably, described analysis rice paddy seed extract specifically comprises the steps:
A, water intaking rice sample, grind into powder in liquid nitrogen respectively;
Ultrapure water is added in b, described sample powder, concussion suspends, extract is added in order to the protein in deposit sample in suspending liquid, after centrifugal, three portions of supernatants got by often kind of sample, dry up rear freezingly to drain under nitrogen, form three parts of testing samples, carry out LC (+) respectively, LC (-), and GC-MS analyzes;
C, by LC (+), LC (-) in step b, and GC-MS analyzes the concentration that each kind of obtaining often plants metabolin and is converted into criterion score z-score; Rice paddy seed metabolite concentration (C) is taken the logarithm (that is: log2C), corresponding z-score=(Log2C-mean)/SD, wherein, C is the average that all kinds (comprising nature kind, transformed variety and acceptor thereof) often kind of metabolin three biology repeat concentration, mean is often kind of metabolite concentration average in all natural kinds, and SD is the standard deviation of often kind of metabolin in nature kind.
Preferably, described analysis rice paddy seed extract also comprises: adopt and carry out LC (+) respectively, LC (-) with reference to test set, and GC-MS analyzes, for the stability of testing and analysis system; Described with reference to test set be selected from testing mixture that three kinds of testing samples in described step b are mixed to form, the plasma sample of people, water one or more.
Preferably, in described step b, testing sample is used for being specially when LC (+) analyzes: add LC (+) dilution in testing sample after, adopt Ultra Performance Liquid Chromatography-MS (UHPLC-MS) to measure metabolin, and use UHPLC-MS (+) pattern to analyze.
Preferably, in described step b, testing sample is used for being specially when LC (-) analyzes: add LC (-) dilution in testing sample after, adopt Ultra Performance Liquid Chromatography-MS (UHPLC-MS) to measure metabolin, and use UHPLC-MS (-) pattern to analyze.
Preferably, in described step b, testing sample is used for being specially when GC-MS analyzes: in testing sample, add diluting solvent and derivatization reagent carries out derivative reaction, put into specimen chamber on GC/MS and detect after centrifugal.
Preferably, described analysis rice paddy seed extract also comprises: change into z-plot figure by the z-score value that step c obtains by biometric analysis software-R software.
Compared with prior art, the present invention has following beneficial effect:
1, method of the present invention has simple, quick, reproducible, the advantage such as high sensitivity, high flux, is applicable to the batch quantity analysis of actual sample.
2, the paddy rice metabolic profiling analysis method that the present invention sets up can be reduced in the error caused in experimentation, and method validation result is good.
3, by quality control (QC), achieve the monitoring to retention time and corresponding signal in instrument operational process, ensure that the reliability of data; And be selected from testing mixture that three kinds of testing samples in described step b are mixed to form, the plasma sample of people, the reference test set of water the quality control for metabolite determination of selecting provide important guarantee.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the Z-score figure of 121 kinds of metabolins in high lysine transgenic paddy, acceptor paddy rice and other 103 natural kind rice paddy seeds, and horizontal ordinate represents 121 kinds of metabolins, and ordinate represents Z-score value.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make certain adjustments and improvements.These all belong to protection scope of the present invention.
embodiment
The present embodiment relates to a kind of method that transgenic paddy rice Difference of Metabolism is analyzed in natural variation; Specifically comprise the steps:
1, paddy rice sample collection.Transgenic paddy rice sample is high-lysine paddy rice, and the promoter that transgenosis builds is the promoter of paddy rice hydroxyproline synzyme, and code area is the lysine-rich protein of goa bean.High-lysine paddy rice transports No. 9, round-grained rice for acceptor with force, through the elite plant strain that continuous multi-generation selfing and field are selected.Nature kind comprises 49 kinds of conventional Indica Rice Cultivars and 55 kinds of conventional japonica rice kinds (table 1).Transgenic paddy rice and acceptor kind (military fortune No. 9, round-grained rice) and other natural kinds all in 2011 in Shanghai plantation sowing.-80C is placed on after paddy rice sample collection.
Get transgenic paddy rice, acceptor paddy rice and other 103 natural kind body rice paddy seeds, grind into powder in liquid nitrogen respectively.The ground powder of each sample weighing 40mg is for extracting metabolin.
2, metabolin extracts: the sample taken out by-80 DEG C of refrigerators, places on ice.The ultrapure water of appropriate volume is added in the paddy rice powder weighed, concussion suspends, draw in suspending liquid 100 μ l to 96 deep-well plates, add proper amount of methanol extract (comprising 4 kinds of various criterion samples) for the protein in deposit sample, use Geno/Grinder2000 (GlenMillsINC., Clifton, NJ) 680r/min vibration 2min, vibration terminates the centrifugal 8min of rear 2800r/min, draw three parts of supernatants and be used for LC (+), LC (-), and GC-MS.Three increment product nitrogen dry up instrument and dry up, and then put into freezing machine of draining and drain 24h.Take out sample from freezing draining machine, add LC (+) and LC (-) dilution respectively; The GC sample of derivatization is needed to carry out derivatization operation in glove box.Ready sample is put into corresponding test system respectively and is carried out loading mensuration.
3, in detection sample, whether the potpourri (CMTRX) simultaneously also having prepared sample carrys out every instrument index in test experience process as independent testing sample and stablizes; The plasma sample of people is equally also as a reference standard of monitoring system stability; In addition, water is then as blank.The quality control for metabolite determination of selecting of these standard substances provides important guarantee.
4, derivative: to need to carry out derivatization treatment to the sample carrying out gas chromatographic analysis.By derivative reaction, the sample that should not gasify can be transformed into derivative products, thus they are become than being easier to gasification under gas phase condition.Derivatization reagent (BSTFA, silylating reagent) is added containing freezing intubate of taking out the gas phase sample bottle of thyraden.Whole application of sample process is carried out in the glove box being full of nitrogen, carries out application of sample by automatic sampler according to the program set.The sealing of jaw sample bottle is given rapidly after application of sample completes.60 DEG C reaction one hour after centrifugal complete preparation of samples and the sample prepared put into specimen chamber on GC/MS detect.
5, Ultra Performance Liquid Chromatography-MS (UHPLC-MS) measures metabolin: the sample WatersAcquity UPLC (Waters of preparation, Millford, MA) be separated, and using UHPLC-MS (+), UHPLC-MS (-) two patterns are analyzed.Positive ion mode mobile phase is: (A1)=0.1% aqueous formic acid, (B1)=0.1% formic acid methanol solution is (in 4 minutes: 0%B progressively rises to 70%B, 98%B is adjusted to by 70% in 0.5 minute, 98%B0.9 minute, negative ion mode mobile phase (A2) 6.5mM ammonium bicarbonate aqueous solution, pH8 and (concentration gradient is the same) in (B2) 6.5mM ammonium bicarbonate methanol aqueous solution, flow velocity is 350uL/min, applied sample amount 5ul, with LTQ (linear trapquadrupole) MS (mass spectrometer) (ThermoFisher Corporation) and Electrospray ionization (electrospray ionization, ESI).Chromatographic column (2.1mm100mm Waters BEH C181.7 μm of thickness), column temperature 40 DEG C, sample chamber temperature 12 DEG C, capillary exit temperature 350 DEG C, the flow velocity of blanket gas is 40, and assist gas flow velocity is 5.Mass Spectrometry Conditions: kation injection electric is 4.5KV, negative ion injection electric is 3.75kv; Mass scan range m/z99-1000; Sweep velocity is about 6scans/second; The ion filling time is 100-200ms.
6, GC-MS(gas chromatography-mass spectrography) (GC-MS) measures metabolin: the sample after derivative is detected by gaschromatographic mass spectrometry (Thermo-Finnigan Trace DSQ rapid scanning list level Four bar mass spectrum), the pillar used is 20m*O.18mm, containing the benzene of 5%, scanning temperature rose to 300 DEG C from from 40 DEG C within 16 minutes, carrier gas is nitrogen, scanning of the mass spectrum scope is 50-750amu, and each experiment will carry out calibration and the adjustment of instrument.
7, metabolin retrieval: rice paddy seed metabolic product is analyzed by GC/MS, LC/MS/MS full scan mode.The data obtained, are carried out pre-treatment and carry out retrieval to metabolin standard database subsequently producing to data by the independently developed a business data process software of Metabolon company of the U.S..In this experiment, 121 kinds of metabolins detected altogether, comprise amino acid, carbohydrates, lipid, coenzyme prothetic group and ucleotides compound (table 2).
8, transgenic paddy rice and natural kind rice seed are often planted metabolite concentration and be converted into z-score, be specially: the rice paddy seed metabolite concentration C of transgenic paddy rice and natural kind is got log
2, often kind of metabolin is just converted into standardized normal distribution in the distribution of different cultivars like this; Then criterion score and Z-score that each kind often plants metabolin is calculated, the computing formula of Z-score is: (X-mean)/SD, wherein X is the average that all kinds (comprising nature kind, transformed variety and acceptor thereof) often kind of metabolin three biology repeat concentration, i.e. log
2c, mean are the average of often kind of metabolite concentration in all natural kinds, and SD is the standard deviation of often kind of metabolin in nature kind.In a standardized normal distribution, average adds and subtracts the data that (mean ± 3SD) within the scope of 3 times of standard deviations contains this distribution 99.5%.If the Z-score of the metabolin in transgenic paddy rice seed is greater than-3 and be less than 3 (i.e. its-3≤Z-score≤3), then in transgenic paddy rice seed the concentration of metabolin within the scope of natural variation.From the results shown in Table 2, the Z-score value of these 121 kinds of metabolins turned in base rice paddy seed is all greater than-3 and is less than 3, so this transgenic paddy rice and natural kind are substantial equivalences in seed metabolism group.
9, utilize R software that the Z-score of 121 kinds of metabolins of transgenic paddy rice and 104 kinds of natural kinds is made Z-plot (as shown in Figure 1), grey point wherein represents the Z-score of 103 natural kind rice paddy seeds, 121 kinds of metabolins, black triangle represents the Z-score of transgenic paddy rice seed 121 kinds of metabolins, black point represents the Z-score of acceptor rice paddy seed 121 kinds of metabolins, in figure, two dotted lines refer to the position that Z-score is ± 3, and namely the metabolite concentration of natural kind 99.7% all drops between these two dotted lines.Can clearly be found out by Fig. 1, in two dotted lines that the point (i.e. black triangle) of transformed variety metabolin all falls in the drawings, represent the Z-score of transgenic paddy rice seed metabolin all drop on ± 3 scopes in, illustrate that the metabolism group of this transgenic paddy rice seed and the metabolism group of other natural kind rice paddy seed are substantial equivalences.
The background of the rice varieties used in table 1 the present embodiment and each kind
| Kind | Background | Kind | Background |
| Transgenic paddy rice | Japonica rice | Osmanthus is towards No. 2 | Long-grained nonglutinous rice |
| Military fortune No. 9, round-grained rice (acceptor) | Japonica rice | West Lake jewel | Long-grained nonglutinous rice |
| Elegant water 11 | Japonica rice | Survey 64-7 | Long-grained nonglutinous rice |
| Elegant water 04 | Japonica rice | Milyang 46 | Long-grained nonglutinous rice |
| Excellent No. 1 of river in Zhejiang Province | Japonica rice | Zhejiang spoke 802 | Long-grained nonglutinous rice |
| Gold is fine | Japonica rice | Nanfeng County is glutinous | Long-grained nonglutinous rice |
| Spring river 025 | Japonica rice | Raise glutinous No. 4 of spoke | Long-grained nonglutinous rice |
| Elegant water 09 | Japonica rice | IR24 | Long-grained nonglutinous rice |
| Elegant water 223 | Japonica rice | Good morning 935 | Long-grained nonglutinous rice |
| Elegant water 24 | Japonica rice | Close positive 23 | Long-grained nonglutinous rice |
| Samsara 422 | Japonica rice | 9311 | Long-grained nonglutinous rice |
| River in Zhejiang Province round-grained rice 44 | Japonica rice | Low pin crow point | Long-grained nonglutinous rice |
| Military fortune No. 7, round-grained rice | Japonica rice | Long filament accounts for | Long-grained nonglutinous rice |
| 02428 | Japonica rice | Special blue or green | Long-grained nonglutinous rice |
| Elegant water 110 | Japonica rice | Square is short | Long-grained nonglutinous rice |
| Taihu Lake is glutinous | Japonica rice | Land wealth number | Long-grained nonglutinous rice |
| Guanling perfume (or spice) is glutinous | Japonica rice | Triumph Xian | Long-grained nonglutinous rice |
| Town rice No. 2 | Japonica rice | Short pin Nan Te | Long-grained nonglutinous rice |
| Jiang Zhouxiang is glutinous | Japonica rice | Bright extensive 86 | Long-grained nonglutinous rice |
| Jiahua-1 | Japonica rice | No. 11, Cymbidium nanulum | Long-grained nonglutinous rice |
| Land-reclaimable 58 | Japonica rice | Special number of south | Long-grained nonglutinous rice |
| Elegant water 63 | Japonica rice | Guanglu ai 4 | Long-grained nonglutinous rice |
| No. 12, peaceful round-grained rice | Japonica rice | Short son accounts for | Long-grained nonglutinous rice |
| No. 24, peaceful round-grained rice | Japonica rice | Chicken is to human relations | Long-grained nonglutinous rice |
| The Liao Dynasty's No. 9, round-grained rice | Japonica rice | Richly shortly account for No. 1 | Long-grained nonglutinous rice |
| Shen Nong 962 | Japonica rice | Hua Xinzhan | Long-grained nonglutinous rice |
| Stone is hunted white hair | Japonica rice | BG90-2 | Long-grained nonglutinous rice |
| Sweet osmanthus is yellow | Japonica rice | Square is short by 3784 | Long-grained nonglutinous rice |
| Land-reclaimable 57 | Japonica rice | No. 1 is being come in platform | Long-grained nonglutinous rice |
| The capital of a country rising sun | Japonica rice | Raise rice No. 2 | Long-grained nonglutinous rice |
| Southwest 175 | Japonica rice | Huang Huazhan | Long-grained nonglutinous rice |
| Japan is fine | Japonica rice | IR8 | Long-grained nonglutinous rice |
| Bali draws | Japonica rice | IR26 | Long-grained nonglutinous rice |
| Wuyunjing No.8 | Japonica rice | Green grass or young crops two is short | Long-grained nonglutinous rice |
| Raise round-grained rice 4038 | Japonica rice | Ye Qinglun | Long-grained nonglutinous rice |
| Precious agriculture 203 | Japonica rice | Blue or green rich short | Long-grained nonglutinous rice |
| Precious agriculture 219 | Japonica rice | Square 13 | Long-grained nonglutinous rice |
| Shen excellent 254 | Japonica rice | Bright extensive 63 | Long-grained nonglutinous rice |
| Spend excellent 14 | Japonica rice | CDR22 | Long-grained nonglutinous rice |
| The fragrant round-grained rice in Soviet Union Shanghai | Japonica rice | Become extensive 448 | Long-grained nonglutinous rice |
| Elegant water 134 | Japonica rice | Guangdong perfume (or spice) accounts for | Long-grained nonglutinous rice |
| Autumn excellent Jin Feng | Japonica rice | Rich Australia accounts for | Long-grained nonglutinous rice |
| Shennong-265 | Japonica rice | Hunan No. 5, Xian in evening | Long-grained nonglutinous rice |
| Autumn fields little Ding | Japonica rice | Spy three is short No. 2 | Long-grained nonglutinous rice |
| Fragrant round-grained rice 111 | Japonica rice | No. 1, polyphyly | Long-grained nonglutinous rice |
| More light | Japonica rice | Praise and educate 293 | Long-grained nonglutinous rice |
| No. 23, Hejiang | Japonica rice | Zhejiang 733 | Long-grained nonglutinous rice |
| South round-grained rice 11 | Japonica rice | Zhejiang 852 | Long-grained nonglutinous rice |
| Town rice 88 | Japonica rice | IR661-1 | Long-grained nonglutinous rice |
| Two rich No. 1 | Japonica rice | Hunan No. 13, Xian in evening | Long-grained nonglutinous rice |
| Xu rice No. 3 | Japonica rice | ||
| No. 3, Shanghai drought | Japonica rice | ||
| Zhong Han 3 | Japonica rice | ||
| Lemont | Japonica rice | ||
| IAPAR9 | Japonica rice |
The 121 kinds of metabolins measured in table 2 transgenic paddy rice seed and the Z-score value of often kind of metabolin
In sum, the present invention utilizes Ultra Performance Liquid Chromatography-mass spectrometry and Gas chromatographyMass spectrometry to set up rice paddy seed metabolic profiling analysis method, evaluates transgenic paddy rice and not genetically modified natural kind paddy rice Difference of Metabolism in conjunction with data analysis.The paddy rice metabolic profiling analysis method that the present invention sets up adds interior mark in preprocessing process, and be farthest reduced in the error caused in experimentation, method validation result is good.In addition, achieve the monitoring to retention time and corresponding signal in instrument operational process by quality control (QC), ensure that the reliability of data.The change of transgenic paddy rice seed metabolite concentration compares with natural variation scope by the present invention, can illustrate whether the change of transgenic paddy rice metabolism group likely causes the unexpected effect of paddy rice better, scientific basis can be provided for the non-premixed flame of genetically modified plants and safety evaluatio.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (9)
1. analyze a method for transgenic paddy rice Difference of Metabolism with natural variation, it is characterized in that, described method comprises the steps:
A, employing Ultra Performance Liquid Chromatography-mass spectrometry and Gas chromatographyMass spectrometry carry out analysis to natural kind rice paddy seed extract and obtain paddy rice metabolic profile spectrum, and then obtain the concentration change scope of various metabolin in difference nature kind paddy rice, be the natural variation scope of metabolin in natural kind paddy rice;
B, under the test condition identical with steps A, adopt Ultra Performance Liquid Chromatography-mass spectrometry and Gas chromatographyMass spectrometry to analyze transgenic paddy rice seed extract, the natural variation scope in analysis result and steps A compares; Metabolite concentration change exceedes natural variation scope, illustrates that the change of this metabolin is unexpected, and metabolite concentration changes within the scope of natural variation, illustrates that this transgenic paddy rice and natural kind are in fact equivalent in seed metabolism group level.
2. the method for transgenic paddy rice Difference of Metabolism is analyzed in natural variation according to claim 1, and it is characterized in that, described natural kind rice paddy seed comprises the acceptor rice paddy seed of described transgenic paddy rice seed; Described transgenic paddy rice is with non-transgenic paddy rice for acceptor carries out gene insertion, also selects the transgenic paddy rice with lysine-rich protein characteristic through selfing.
3. the method for transgenic paddy rice Difference of Metabolism is analyzed in natural variation according to claim 2, and it is characterized in that, described natural kind rice paddy seed is the core key variety comprised in rice in China database.
4. the method analyzing transgenic paddy rice Difference of Metabolism with natural variation according to claim 1,2 or 3, it is characterized in that, described analysis rice paddy seed extract specifically comprises the steps:
A, water intaking rice sample, grind into powder in liquid nitrogen respectively;
Ultrapure water is added in b, described sample powder, concussion suspends, extract is added in order to the protein in deposit sample in suspending liquid, after centrifugal, three portions of supernatants got by often kind of sample, dry up rear freezingly to drain under nitrogen, form three parts of testing samples, carry out LC (+) respectively, LC (-), and GC-MS analyzes;
C, by LC (+), LC (-) in step b, and GC-MS analyzes the concentration that each kind of obtaining often plants metabolin and is converted into criterion score z-score; Rice paddy seed metabolite concentration C is got log
2, corresponding z-score=(Log
2c-mean)/SD, wherein, log
2c is the average that all kind rice paddy seeds often plant that metabolin three biology repeat concentration, and mean is often kind of metabolite concentration average in all natural kinds, and SD is the standard deviation of often kind of metabolin in nature kind.
5. the method for transgenic paddy rice Difference of Metabolism is analyzed in natural variation according to claim 4, it is characterized in that, described analysis rice paddy seed extract also comprises: adopt and carry out LC (+) respectively with reference to test set, LC (-), analyze, for the stability of testing and analysis system with GC-MS; Described with reference to test set be selected from testing mixture that three kinds of testing samples in described step b are mixed to form, the plasma sample of people, water one or more.
6. the method for transgenic paddy rice Difference of Metabolism is analyzed in natural variation according to claim 4, it is characterized in that, in described step b, testing sample is used for being specially when LC (+) analyzes: add containing after LC (+) dilution in testing sample, adopt Ultra Performance Liquid Chromatography-MS to measure metabolin, and use UHPLC-MS (+) pattern to analyze.
7. the method for transgenic paddy rice Difference of Metabolism is analyzed in natural variation according to claim 4, it is characterized in that, in described step b, testing sample is used for being specially when LC (-) analyzes: add LC (-) dilution in testing sample after, adopt Ultra Performance Liquid Chromatography-MS to measure metabolin, and use UHPLC-MS (-) pattern to analyze.
8. the method for transgenic paddy rice Difference of Metabolism is analyzed in natural variation according to claim 4, it is characterized in that, in described step b, testing sample is used for being specially when GC-MS analyzes: in testing sample, add diluting solvent and derivatization reagent carries out derivative reaction, put into specimen chamber on GC/MS and detect after centrifugal.
9. the method for transgenic paddy rice Difference of Metabolism is analyzed in natural variation according to claim 4, and it is characterized in that, described analysis rice paddy seed extract also comprises: the z-score value obtained by step c with R software changes into z-plot figure.
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