CN102478563B - Method for studying metabolic difference of transgenic rice and non-transgenic rice - Google Patents
Method for studying metabolic difference of transgenic rice and non-transgenic rice Download PDFInfo
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Abstract
The invention discloses a method for studying metabolic difference of transgenic rice and non-transgenic rice. The method analyzes rice seed extract with liquid chromatography-mass spectrometry (LC-MS) technology to obtain metabolic profiling of rice, studies integral difference of metabolic profiling data between transgenic rice and non-transgenic rice by multivariate statistics method, and finds out compound with significant contribution to metabolic phenotype difference, to provide a basis for evaluation of unintended effect and safety of transgenic rice. The simple and rapid analysis method with good repeatability is suitable for batch analysis of practical samples.
Description
Technical field
The present invention relates to analytical chemistry field, is a kind of method that paddy rice metabolic profile spectrum based on metabonomic technology is studied transgenosis and non-transgenic paddy rice Difference of Metabolism.
Background technology
The fast development of transgenic technology, has caused the arguement of genetically modified plants to human health and eco-environmental impact.People are mainly reflected in following several respects to the worry of transgenic plant safety at present: the toxicity of (1) foreign gene how; (2) have or not anaphylaxis; (3) whether produce noxious material; (4) nutritive value is had or not to impact.In order to ensure the security of genetically modified plants, some international organizations such as many countries and FAO, WHO including the U.S., European Union, Japan and China have worked out a series of control measures for transgenic plant safety one after another.Wherein " principle of substantial equivalence (substantial equivalence) " is the generic principles that current internationally recognized transgenic foods safety is evaluated.It comprises the content of three levels: (1) chemical composition is equal to; (2) eating effect is equal to; (3) environmental impact is equal to.Can find out, one of key content to transgenic plant safety evaluation is the composition Research on differences of carrying out between genetically modified plants and non-transgenic plant.
Metabolites kinds in paddy rice is various, and existing activity and the necessary nascent metabolic product that grows of sustaining life also has that utilize that nascent metabolin generates and the secondary metabolite in close relations such as disease-resistant and degeneration-resistant; Existing inorganic ions also has organic compound; Existing hydrophilic compounds also has lipophilic compound.In addition, the content difference of various materials is also very greatly different, and this has just proposed very high requirement to analytical approach.And the realization that appears as above-mentioned target of metabonomic technology provides possibility.Metabolism group be variation by investigating (as by after certain specific genetic mutation or environmental change) its metabolic product after living things system irriate or disturbance or its over time, carry out a kind of technology of postgraduate's objects system metabolic pathway.Based on the method for profile analysis of metabolism group, can non-selectively detect genetically modified plants and change at the potential physiology of metabolite level, can detect unexpected effect, for the safety evaluatio of genetically modified plants provides basis.
The technology that pin metabolism group research at present adopts has: chromatogram, mass spectrum, nuclear magnetic resoance spectrum (NMR) etc.Nuclear magnetic resonance technique is without complicated sample pretreatment process, and without skewed popularity, but its sensitivity is too low.Combined gas chromatography mass spectrometry combines chromatogram and mass spectrographic advantage, there is high separation, high flux and highly sensitive feature, that metabolism group is studied indispensable separate analytical technique, and high performance liquid chromatography mass spectrometric hyphenated technique due to applied range, be not subject to sample thermal stability and volatile impact, become the main flow analysis means of metabolism group research.Transgenic paddy rice changes because the variation of gene structure can cause the physiology on some row metaboilic levels, adopt LC-MS-MS to find these to change, for the unexpected effect of transgenic paddy rice and safety evaluatio provide according to being the core content of this research.There is no so far and adopt liquid chromatography mass combined system to carry out the report of rice paddy seed metabolic profiling analysis.Because preprocessing process point multistep is carried out, in preparation of samples process, easily introduce error; If sample number is many, the time of mass spectrum operation is longer, and due to the attenuation effect of electric spray ion source itself, the mass spectrum response signal of sample can produce certain deviation.This all will cause experimental data to depart from actual value, affect the confidence level of net result.For above-mentioned situation, we add the measure such as interior mark and quality control (QC) sample correction experimental bias to guarantee the reliability of data in experimentation.
Summary of the invention
The object of the invention is to set up and a kind of carry out rice paddy seed metabolic profiling analysis based on high performance liquid chromatography mass spectrometry system, and the method that transgenosis and non-transgenic rice paddy seed Difference of Metabolism are studied.The method has high sensitivity, high-throughout advantage, can provide basis for the security of further investigation transgenic paddy rice according to the method.
For achieving the above object, the technical solution used in the present invention is as follows:
A kind of for studying the method for transgenosis and non-transgenic paddy rice Difference of Metabolism, adopt HPLC-MS technology to analyze and obtain rice paddy seed metabolic profile paddy rice powder extract, then distinguish transgenosis and non-transgenic paddy rice data by Multivariable Statistical Methods, find metabolic phenotype difference to have the compound of significant contribution.
Concrete steps are as follows,
1) by representative transgenic paddy rice seed sample and non-transgenic rice paddy seed sample, after grinding with mortar respectively, cross 60 mesh sieves, rice paddy seed powder can be stored in ultra low temperature freezer (80 DEG C), for subsequent use;
2) paddy rice sample pretreatment: take respectively a certain amount of rice paddy seed sample in glass tube, in every arm, add the extraction solvent (methanol/water containing interior mark lysoPC (19:0), v/v is 3/2), mediate, centrifugal after ultrasonic processing, get supernatant freeze-drying and be heavily dissolved in methanol/water (v/v 3/2) solution, mediate, after centrifugal, supernatant is moved in sample injection bottle.
3) paddy rice sample is carried out to liquid chromatography mass combination analysis successively;
Liquid-phase condition is: chromatographic column is Zorbax SB-Aq post (1.8 μ m, 3.0 × 100 mm), and mobile phase A is volumetric concentration 0.1% formic acid solution, and B is acetonitrile; Condition of gradient elution: 0～2min 95/5(A/B, V/V), 4 min 60/40 (A/B, V/V), 20 min 5/95 (A/B, V/V), 22.1min 0/100 acetonitrile continual rinsing chromatographic column 4 min, 28 min 95/5(A/B, V/V), start gradient balance 7 min subsequently; Flow velocity 0.30 mL/min, after post, efflux directly imports mass spectrometer system detection without shunting;
Mass spectrum condition is: electric spray ion source (ESI), adopts positive ion mode to detect; Use high-purity N 2 assistant spray ionization and desolventizings, 350 DEG C of dry gas temperature, dry gas flow velocity is 11 L/min; 350 DEG C of ion source temperatures, capillary voltage is 4000 V, solvation ion removes bunch voltage 175 V, Centroid type collection mass spectrometric data, mass scanning scope m/z 100～1000, obtains the total ion current figure of analyzed sample; Finally obtain the total ion current figure of paddy rice sample, it is rice paddy seed metabolic profile spectrum.
Using the potpourri of randomly drawing a sample or more than one sample in rice paddy seed sample as QC sample, as the quality control standard in Mass Spectrometer Method process.Paddy rice sample seed is carried out in liquid chromatography mass combination analysis process successively, first by QC sample sample introduction once, then after 4-8 sample of every analysis, again by QC sample sample introduction once, guarantee that by the repeatability that ensures QC sample total ion current figure in experimentation, liquid chromatography mass combined instrument has good stability.
4) adopt LC-MS data processing software to carry out peak extraction to the paddy rice metabolic profile spectrum of paddy rice sample, after peak match, form a data matrix.First by data normalization to total peak area, then the variable in data matrix is carried out to missing values processing, then by the variable deletion of RSD>25% in QC sample, next data are carried out utilizing offset minimum binary-discriminatory analysis (PLS-DA) method to carry out statistical study to data matrix after scale, the Metabolic Fingerprinting data of transgenic paddy rice and non-transgenic paddy rice are carried out to modeling.10 or 10 above detection ions according to the variable importance factor screening in model, classification being had significant contribution, the metabolin that these detection ion pairs are answered can be considered to be in the compound playing an important role in the non-transgenic paddy rice classification of transgenic paddy rice and correspondence thereof.
5) compound filtering out is carried out to t inspection, obtain the significant important compound of content difference.
6) important compound filtering out is carried out to Structural Identification.
The effect that the present invention has is: the storage of sample, pre-service adopt the autonomous standardized program of setting up, and avoid introducing personal error.Paddy rice sample pretreatment process is relatively simple, does not need derivatization, and the error of introducing is little, has increased the reliability of data.The research platform that adopts the metabolism group based on high separation fast liquid chromatography-quadrupole rod flight time mass spectrum (RRLC/QTOF MS) still can keep good degree of separation under higher linear velocity, has shortened analysis time, has improved analysis throughput.Meanwhile, the resolution of flight time mass spectrum is high, and sweep velocity is fast, once analyzes and can obtain the macrometabolic element information that quality precision is high, for the qualification of mark provides reliable foundation, has also saved qualification time.In sample preprocessing process, add interior mark, proofread and correct in preprocessing process and instrument operational process and respond the error that small drift brings.In sample sequence analytic process, insert quality control (QC) sample being mixed by all paddy rice samples, by the monitoring to QC sample total ion current figure, can detect the response of analytical sequence Instrumental and have or not drift, guaranteed the reliability of experimental data.
Analytical approach of the present invention is simple, fast, reproducible, is suitable for the batch quantity analysis of actual sample.
Brief description of the drawings
The contrast of the little molecule component of Fig. 1 paddy rice LC-MS metabolic profile.(A) total ion current of non-transgenic sample (TIC) figure; (B) TIC of transgene sample figure;
Fig. 2 PLS-DA model (A) score matrix.
for transgenic paddy rice, ■ is non-transgenic paddy rice, and each point represents a sample, and t represents that sample is in principal component projection value.(B) S-plot X-axis represents covariance, and it has represented the contribution degree of variable to classification in PLS-DA loading matrix.Y-axis has represented correlativity, and it represents the reliability of variable in classification.
Embodiment
embodiment 1
1, paddy rice sample collection
Transgenic paddy rice sample is Marker-free divalent insect-resistant strain material; Comprise 3 strains, be respectively N6080, N6049, N6130, is bright extensive 86 to transform as acceptor, the elite plant strain of selecting through continuous multi-generation selfing and field.Transgenic paddy rice and non-transgenic paddy rice bright extensive 86 is to be all in Hainan plantation, the sowing same period, sowing.After paddy rice sample collection, be placed on-80 DEG C of Refrigerator stores.
2, analytical approach
2.1 rice paddy seed sample preprocessings
Rice paddy seed sample takes out from-80 DEG C of refrigerators, waits for that its temperature returns to room temperature in exsiccator.By being placed in mortar and grinding after every kind of rice paddy seed shelling, cross 60 mesh sieves, obtain the powder of uniform particle diameter.The packing of paddy rice powder is to plastic tube, for subsequent use.
(every part 0.1500 g), is placed in respectively 5 mL glass tubes to take each 5 parts of N6080 or N6049.(every part 0.1500 g), is placed in respectively 5 mL glass tubes to take 4 parts of N6130.
(every part 0.1500 g), is placed in respectively 5 mL glass tubes to take bright extensive 86 5 parts of non-transgenic paddy rice.
As quality control (QC) sample, take every kind of (N6080, N6049, N6130, bright extensive 86) paddy rice 0. 5 g, mix, (every part 0.1500 is g), in 5 mL glass tubes then to take 4 parts, QC sample.
Take interior mark lysopc (19:0) 1.0 mg in 1.5 mL centrifuge tubes, add 1 mL methyl alcohol.Mediate, ultrasonic, make it to dissolve completely, obtaining concentration is the inner mark solution of 1 mg/mL.
Get 20 mL water, 30 mL methyl alcohol, ultrasonic mixing, configuration 50 mL 60% methyl alcohol (v/v) solution are as extracting solvent.To extracting the inner mark solution that adds 50 μ L 1 mg/mL in solvent, mix.
In each glass tube, add mark in 2 mL bands to extract solvent, 10 s that mediate, ultrasonic 40 minutes.Take out 1.2 mL and move in eppendorf pipe, in 4 DEG C, under 14000 rpm centrifugal 10 minutes.After centrifugal end, get 1.0 mL supernatants and be placed in freeze dryer freeze-drying.Before instrumental analysis, add 200 μ L water/methyl alcohol (v/v 2:3) solution to redissolve.30 s that mediate, in 4 DEG C, move to sample injection bottle by supernatant after centrifugal 10 minutes under 14000 rpm, for high performance liquid chromatography mass spectrophotometry.
2.2 high performance liquid chromatography mass spectrophotometry
Liquid-phase condition is: chromatographic column is Zorbax SB-Aq post (1.8 μ m, 3.0 × 100 mm), 60 DEG C of column temperatures, and sample size 2 μ L, mobile phase A is volumetric concentration 0.1% formic acid solution, B is acetonitrile; Condition of gradient elution: 0～2 min 95/5(A/B, V/V), 4 min 60/40 (A/B, V/V), 20 min 5/95 (A/B, V/V), 22.1 min 0/100 acetonitrile continual rinsing chromatographic column 4 min, 28 min 95/5(A/B, V/V), start gradient balance 7 min subsequently; Flow velocity 0.30 mL/min, after post, efflux directly imports mass spectrometer system detection without shunting;
Mass spectrum condition is: electric spray ion source (ESI), adopts positive ion mode to detect; Use high-purity N 2 assistant spray ionization and desolventizings, 350 DEG C of dry gas temperature, dry gas flow velocity is 11 L/min, atomization air pressure 45 psig; 350 DEG C of ion source temperatures, capillary voltage is 4000 V, solvation ion removes bunch voltage 175 V, taper hole voltage 65 V, Centroid type collection mass spectrometric data, every 0.50 s gathers a secondary data; Mass scanning scope m/z 100～1000, obtains the total ion current figure of analyzed sample; Finally obtain the total ion current figure of rice paddy seed, it is rice paddy seed metabolic profile spectrum.
19 paddy rice samples are carried out in high performance liquid chromatography mass spectrometry analytic process successively, first by QC sample sample introduction once, then after 5 samples of every analysis, again by QC sample sample introduction once, by ensureing the reappearance of QC sample retention time and response signal, guarantee that in experimentation, liquid chromatography mass combined instrument has good stability.
According to above operation, obtain the profile (Fig. 1) of the little molecule metabolites of rice paddy seed.14 transgene samples and 5 non-transgenic contrast paddy rice samples are analyzed.Sample analysis is sequentially for transgenosis and control group hocket.In analytical sequence, insert QC sample, QC sample is the potpourri of transgenic paddy rice and non-transgenic paddy rice.Within analysis time, the repeatability of QC sample is good, does not occur obvious change of component, and drift does not appear in retention time, and known all paddy rice samples are also stable (tables 1) within whole analysis time.By interior mark and QC sample, can guarantee the reliability of data.
3 pattern-recognitions and mark screening
The raw data of the total ion current figure of obtained all rice paddy seeds is utilized to MFE(molecular feature extraction) software extraction compound information, in this step by retention time at 0 to 20 minute, m/z value is 100 to 1000, the peak reservation that peak height is greater than 1000, and calculate accurate compound molecular weight, generate MHD file.MHD file is imported to gene spring software and carry out obtaining parent mass peak table after peak match, in this step, the slope of mass deviation is made as 5.0ppm, and intercept is made as 2.0mDa, and other parameters are software default value.Parent mass peak table is normalized to total peak area and reduces systematic error, then missing values is processed.
Then utilize SIMCA-P 11.5 (Umetrics, Umea, Sweden) to carry out PLS-DA analysis in peak after treatment missing values table.T inspection adopts SPSS 13.0(SPSS Inc., Chicago, IL) carry out.
In order to investigate the metabolic profile difference of transgenosis and non-transgenic paddy rice, we carry out peak identification, peak match the measurement data of each sample.After peak identification coupling, form an EXCEL peak table being formed by retention time, mass number and peak area.First by data normalization to total peak area, then by data according in QC sample 80% exist variable retain principle, missing values is processed.Then delete the variable that in QC sample, relative standard deviation is greater than 25%, obtain the reliable peak of a data table.
In rice paddy seed metabolic profile, there is hundreds of component, and utilize the multivariable technique can be by Data Dimensionality Reduction, from a large amount of extracting data Useful Informations.PLS-DA is a kind of multivariable classification method based on partial least square method, and it has simulated the relation of data group matrix and classification subordinate matrix, the maximum deviation in decryption matrix between defined classification sample.Utilize PLS-DA can explain the maximum deviation between transgene sample and non-transgenic sample.
Square root by the each variable in peak table (LC/MS analyzes the mass spectra peak obtaining) divided by its standard deviation, carry out par scale, to a certain degree reducing the deviation causing because of response intensity in PLS-DA computing, also can make the effect of the metabolin molecule of low concentration be embodied.Transgene sample is given classification value 1, and non-transgenic paddy rice is given classification value 2, and all calculating parameters are software default value, obtains thus PLS-DA model.The related data of PLS-DA model is as follows: A=2, R
2x=0.513, R
2y=0.97, Q
2y=0.955.Wherein A is the number of principal components that model uses, R
2x, R
2y distinguishes representative model to former data matrix X, the explanation degree of Y information, Q
2y is the predictive ability of model.The bright model of above-mentioned data has good stability and predictive ability.
Model has 2 major components, projection according to each sample in major component, obtain score matrix figure (Fig. 2), therefrom known, 14 transgene samples and being projected on first principal component of 5 non-transgenic samples can well be separated, and show that the metabolic profile of transgenic paddy rice and non-transgenic paddy rice exists significantly difference.Projection according to each variable in major component, obtains the load moment system of battle formations, on this basis, can calculate its variable importance factor VIP(VIP directly obtained by PLS-DA according to the coordinate on load diagram of each variable):
(1)
In formula (1), A is model number of principal components, and K is variable number, w
ak for the weight of variable in PLS, SSY represents a the explainable SS of composition, and SS is the quadratic sum of a upper major component residual error, and a represents a composition.
S-plot has showed contribution degree and the reliability (Fig. 2 B) of variable to classification in patterned mode simultaneously, and X-axis represents covariance, and it has represented the contribution degree of variable to classification in PLS-DA loading matrix.The variable with low covariance may derive from analytical error and noise bad student's signal, and Y-axis has represented correlativity, and it represents the reliability of variable in classification.Variable is S-type in the drawings.Be distributed in the variable at S two ends, contribution degree and reliability are all higher, and they are metabolins that classification is played an important role.Because S-plot combines the information of covariance and correlativity, can reduce mark and choose the false-positive risk existing in process.
To be positioned at S-plot figure two ends and VIP(VIP>1) information extraction of the variable of maximum 10, and its content information is imported to SPSS carry out independent sample t inspection, the variable (p<0.05) of checking by t thinks that the metabolism of transgenosis and non-transgenic paddy rice has the compound (table 2) of significant difference.
Ion in his-and-hers watches 2 carries out Structural Identification, is respectively tryptophan, linolenic acid, phytosphingosine, 9,10,13-TriHOME, LPE (16:0), palmitic acid etc.They are compounds that transgenosis and non-transgenic differ greatly, and the unexpected effect and the safety evaluatio that can be transgenic paddy rice provide foundation.
The present invention utilizes high performance liquid chromatography mass spectrometric hyphenated technique to set up rice paddy seed metabolic profiling analysis method, finds the Difference of Metabolism of transgenic paddy rice and non-transgenic paddy rice in conjunction with multivariate data.Through ultrasonic auxiliary extraction technology, paddy rice component is carried out after pre-service, utilize LC-MS system to separate the little molecule metabolites detecting in rice paddy seed, obtain rice paddy seed metabolic profile information.By data be normalized, missing values sets up PLS-DA model after processing, and the metabolism of transgenic paddy rice and non-transgenic rice paddy seed is carried out to entirety differentiation.Meanwhile, go out classification is had to the compound of significant contribution in conjunction with load factor and S-plot figure examination, and it is carried out to t inspection.The paddy rice metabolic profiling analysis method that the present invention sets up adds interior mark in preprocessing process, is farthest reduced in the error causing in experimentation, and method validation result is good.In addition, realize the monitoring to retention time and corresponding signal in instrument operational process by quality control (QC), guaranteed the reliability of data.The present invention filters out multiple compounds that classification had to significant contribution, can provide foundation for unexpected effect and the safety evaluatio of genetically modified plants.
Claims (2)
1. for studying a method for transgenosis and non-transgenic paddy rice Difference of Metabolism, it is characterized in that: adopt LC-MS-MS to analyze and obtain paddy rice metabolic profile spectrum rice paddy seed extract; Then by the overall difference of the metabolic profile data of Multivariable Statistical Methods research transgenic paddy rice and non-transgenic paddy rice, find metabolic phenotype difference to have the compound of significant contribution, for unexpected effect and the safety evaluatio of transgenic paddy rice provide foundation;
Metabolic profile is composed the concrete steps of obtaining,
1) get transgenic paddy rice seed sample and non-transgenic rice paddy seed sample, grind into powder sieving respectively, obtains the paddy rice sample of two class uniform particle diameter;
2) paddy rice sample pretreatment: take respectively two class rice paddy seed samples in glass tube, in every test tube, add the extraction solvent methanol/water containing interior mark 19 acyls-lysophosphatidyl choline, wherein the volume ratio of methanol/water is 3/2, vortex, centrifugal after ultrasonic processing, get in the solution of volume ratio=3/2 that supernatant freeze-drying is heavily dissolved in methanol/water vortex, after centrifugal, supernatant is moved in sample injection bottle for liquid chromatography mass combination analysis;
3) paddy rice sample is carried out to liquid chromatography mass combination analysis successively;
Liquid-phase condition is: chromatographic column is that Zorbax SB-Aq post is 1.8 μ m, 3.0 × 100mm, and mobile phase A is volumetric concentration 0.1% formic acid solution, B is acetonitrile; Flow velocity 0.30mL/min, after post, efflux directly imports mass spectrometer system detection without shunting;
Mass spectrum condition is: electric spray ion source ESI, adopts positive ion mode to detect; Use high-purity N 2 assistant spray ionization and desolventizings, 350 DEG C of dry gas temperature, dry gas flow velocity is 11L/min; 350 DEG C of ion source temperatures, capillary voltage is 4000V, solvation ion removes a bunch voltage 175V, Centroid type collection mass spectrometric data; Mass scanning scope m/z100～1000, obtain the total ion current figure of analyzed sample; Finally obtain the total ion current figure of rice paddy seed sample, it is rice paddy seed metabolic profile spectrum;
Using the potpourri of randomly drawing a sample or more than one sample in rice paddy seed sample as QC sample, as the quality control standard in Mass Spectrometer Method process; Paddy rice sample is carried out in liquid chromatography mass combination analysis process successively, first by QC sample sample introduction once, then after 4-8 sample of every analysis, again by QC sample sample introduction once, guarantee that by the repeatability that ensures QC sample total ion current figure in experimentation, liquid chromatography mass combined instrument is without larger system deviation, data are reliable;
Adopt LC-MS data processing software to carry out peak extraction to the metabolic profile spectrum of rice paddy seed sample, after peak match, form an EXCEL data matrix; Variable in data matrix is carried out to missing values processing, then by the variable deletion that RSD is greater than 25% in QC sample, next utilize offset minimum binary-discriminatory analysis (PLS-DA) method to carry out holistic approach to data matrix, the Metabolic Fingerprinting data of transgenic paddy rice and non-transgenic paddy rice are carried out to modeling; 10 or 10 above detection ions according to the variable importance factor screening in model, classification being had significant contribution, the metabolin that these detection ion pairs are answered can be considered to be in the compound playing an important role in the non-transgenic paddy rice classification of transgenic paddy rice and correspondence thereof;
The compound filtering out is carried out to t inspection; Then important compound is carried out to Structural Identification.
2. method according to claim 1, is characterized in that:
Described transgenic paddy rice is to carry out gene insertion taking described non-transgenic paddy rice as acceptor, the transgenic paddy rice of also selecting the sample with insect resistace to obtain through selfing;
By what find out, metabolic phenotype difference is had to the compound of significant contribution, further know by gene and insert the impact on acceptor.
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