CN103257235A - Method of evaluating security of transgenic plants - Google Patents
Method of evaluating security of transgenic plants Download PDFInfo
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- CN103257235A CN103257235A CN201210035564XA CN201210035564A CN103257235A CN 103257235 A CN103257235 A CN 103257235A CN 201210035564X A CN201210035564X A CN 201210035564XA CN 201210035564 A CN201210035564 A CN 201210035564A CN 103257235 A CN103257235 A CN 103257235A
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Abstract
The invention discloses a method of evaluating security of transgenic plants. The method comprises: (1) extracting total proteins of to-be-detected samples, performing 2D-DIGE electrophoresis and fluorescence image analysis, thereby obtaining proteome maps of the to-be-detected samples, wherein the to-be-detected samples comprises samples 1, 2, 3 and 4, the sample 1 is a to-be-detected transgenic plant, the sample 2 is a parent plant of the to-be-detected transgenic plant, the sample 3 and the sample 2 are different plants of a same cultivated species, and the sample 4 and the sample 2 are different cultivated species; (2) selecting proteins appearing in more than 70 % of proteome maps for principal component analysis; and (3) determining according to results of the principal component analysis. The method provided by the invention has practical application values for systematic assessment of molecular characteristics and biological safety of genetically modified crops.
Description
Technical field
The present invention relates to a kind of method of estimating transgenic plant safety.
Background technology
Paddy rice is one of most important cereal crops in the world, is the first cereal crops of China.Along with deeply perfect with transgenic technology of molecule genetics research, transgenic technology has become the most important technological means of crop economical character such as improveing paddy rice.But, the crops that transgenosis is modified still there is much controversy in application in agriculture, the focus of dispute is whether transgenosis can bring unexpected effect, works the mischief to human health, and it is particularly important in this case transgenic crop to be carried out safety evaluation.
The means of assessment genetically modified crops security mainly are the analyses that target is decided composition at present, have certain limitation, only can detect limited amount molecule.And at present anxious problem to be solved be objective, comprehensive assessment transgenosis to influence and the influence degree of crop characterization of molecules stability, this will be related to the industrialization of genetically modified crops and society to the concern of genetically modified crops security.
Proteomic techniques identifies that scope is wide, identify the flux height, receives increasing concern in genetically modified crops safety evaluation field.Dielectrophoresis is technology the most frequently used in the proteomics research field in conjunction with the mass spectrum authenticate technology, and the 2D-DIGE isolation technics that development in recent years is got up has solved common dielectrophoresis repeatability, quantitative poor accuracy and the big problem of workload especially, becomes the important technical of Comparative Proteomics.
There have been many work to utilize proteomic techniques to study the protein stack features of genetically modified crops.For example, utilize the 2-DE protein technique to analyze the contrast of antiviral transgene tomato and non-transgenic, but the antiviral proterties that the result shows introducing genetic stability in three generations at least, and the economical character of crop is without any change.Equally, between genetically engineered soybean, potato and corn and the contrast of its non-transgenic, found " substantial equivalence ".Yet, in the paddy endosperm that changes human GM-CSF (hGM-CSF) over to, marked change (p＜0.05) has taken place in 103 protein, wherein downward modulation has taken place in endogenous storage protein and most sugars metabolism associated protein, and rise has taken place for 26S proteasome related protein and molecular chaperones.In addition, also observe the induced expression endoplasmic reticulum of hGM-CSF and coerce and activate ubiquitin/26S proteasome pathway, caused foreign protein product ubiquitinization.
For whether the objective evaluation transgenosis causes the bio-safety problem, need set up a kind of technical standard and proteomic techniques of energy real assessment genetically modified crops characterization of molecules.
Summary of the invention
The purpose of this invention is to provide a kind of method of estimating transgenic plant safety.
The method of evaluation transgenic plant safety provided by the invention comprises the steps:
(1) sample to be tested is extracted total protein, and carry out 2D-DIGE electrophoresis and fluoroscopic image analysis, obtain the proteomic map of each sample to be tested; Described sample to be tested is sample first, sample second, sample third and sample fourth; Described sample first is genetically modified plants to be measured; Described sample second is the mother plant of genetically modified plants to be measured; Described sample third is the different plants that are subordinate to same cultigen with sample second; Described sample fourth is the plant (having different natural variation hereditary capacities) that is subordinate to different cultigens with sample second; The quality such as total protein of described sample first, described sample second, described sample third and described sample fourth are mixed, as the interior mark of 2D-DIGE electrophoresis;
(2) be chosen at more than 70% the albumen that occurs in the protein profiling of (preferred more than 85%, more preferably 100%) and carry out principal component analysis (PCA);
(3) result according to described principal component analysis (PCA) judges as follows: if the otherness of described sample first and described sample second is lower than the otherness of described sample second and described sample third and is lower than the otherness of described sample second and described sample fourth, described sample first is the genetically modified plants of candidate's safety.
Described plant can be paddy rice.Described cultigen specifically refers to round-grained rice subspecies (japonica rice) and Xian subspecies (long-grained nonglutinous rice).
Described method also can comprise the steps: interior target with reference under, with the protein profiling of the protein profiling of described sample first and described sample second relatively, the albumen of choosing expression ratio and being more than 1.2 carries out the mass spectrum evaluation; Interior target with reference under, with the protein profiling of the protein profiling of described sample second and described sample third relatively, the albumen of choosing expression ratio and being more than 1.2 carries out the mass spectrum evaluation; Interior target with reference under, with the protein profiling of the protein profiling of described sample second and described sample fourth relatively, the albumen of choosing expression ratio and being more than 1.2 carries out the mass spectrum evaluation.
Described method also comprises the step of the albumen after the mass spectrum evaluation being carried out function classification and/or cluster analysis and/or principal component analysis (PCA) and/or expression pattern analysis.In the differential expression protein of sample first and sample second, if there are not related proteins such as toxin, anaphylactogen, can judge further that then the sample first is candidate's genetically modified plants.
In described step (1), the total protein of described sample to be tested can adopt different fluorescein-labelled.Described fluorescein can be at least a among cy3 and the cy5.In described step (1), described interior mark fluorescein cy2 mark.
Described sample first, described sample second and described sample third specifically can belong to the Xian subspecies, and described sample fourth can belong to the round-grained rice subspecies.Described sample first specifically can be transgenic paddy rice 2036-1a, and described sample second specifically can be bright extensive 86, and described sample third specifically can be bright extensive 63, and described sample fourth specifically can be spends No. 10 in the japonica rice.Described sample first specifically can be transgenic paddy rice Bar68-1, and described sample second specifically can be D68, and described sample third specifically can be bright extensive 63, and described sample fourth specifically can be spends No. 10 in the japonica rice.Described sample first specifically can be transgenic paddy rice 2036-1a and transgenic paddy rice Bar68-1 (two transgenosis samples), described sample second specifically can be bright extensive 86 and D68 (two transgenosis sample parents separately), described sample third specifically can be bright extensive 63, and described sample fourth specifically can be spends No. 10 in the japonica rice.
Described method comprises the steps: also to identify whether foreign gene has inserted and expression in genetically modified plants to be measured.
In the described 2D-DIGE electrophoresis, all add described interior standard specimen product on each gel.In the described 2D-DIGE electrophoresis, in the same gel, the total protein of different fluorescein-labelled each samples to be tested of usefulness and interior standard specimen are originally.
The present invention relates to the application of proteomic techniques in genetically modified crops protein component subcharacter detects.Particularly, the present invention relates to the technical standard of genetically modified crops characterization of molecules detection and the method that proteomic techniques detects genetically modified crops protein component subcharacter, especially two-way difference gel electrophoresis (2D-DIGE) connexus spectral technology detects the method for genetically modified crops protein component subcharacter.The present invention contrasts except the parent is set for genetically modified plants, also be provided with the parent and contrast the different material of genetic background (being used for analyzing the characterization of molecules difference that natural variation causes), and belong to the material (material of deriving as conventional genetic breeding of same cultigen with parent contrast, be used for the characterization of molecules difference that the analytic routines genetic breeding causes), utilize parallel, the repeatable technique system is the difference of above-mentioned material characterization of molecules relatively, consider transgenosis, conventional genetic breeding quiding gene, and natural variation causes factors such as characterization of molecules difference, compare the difference of protein group between different materials and the functional character of differential expression protein, determine the characterization of molecules of genetically modified crops, estimate the biological safety of genetically modified crops.Method of the present invention has actual using value for characterization of molecules and the biological safety of system evaluation genetically modified crops.
Description of drawings
Fig. 1 is the detection of expression electrophoretogram of foreign gene in the transgenic paddy rice genome; A figure is that the expression of bar gene in Bar68-1 identified; B figure is that cry1Ac gene and the expression of sck gene in 2036-1a are identified.
Fig. 2 is the DIGE fluorogram; The gel numbering is labeled in the lower right corner of every figure, and sample distribution sees Table 1.
Fig. 3 is the principal component scores figure of principal component analysis (PCA) (PCA-X) model.A figure is PCA t1/t2 shot chart; B figure is PCA t1/t3 shot chart.Wherein various samples mark with distinct symbols: Bar68-1 (▲), D68 (△), MH63 (*), 2036-1a (◆), MH86 (◇) and ZH10 (●).
Fig. 4 identifies the function classification of the differential protein that obtains for mass spectrum.The grey pillar is represented the differential protein quantity that all identify, and the black pillar is represented chromosomal foci number identical protein quantity, and numeral is expression quantity separately respectively; 1 to 9 represents classification, is followed successively by metabolic protein and synthesizes with target, defense reaction, the unknown, cell growth and division, PPDK, signal transduction, transcribes, transports.
Fig. 5 is the cluster analysis of differential protein.Sample from left to right represents Bar68-1, D68, MH63,2036-1a, MH86 and ZH10 respectively among the figure.
Fig. 6 is the expression pattern figure of 9 major function classes and function subclass differential protein.Sample from left to right represents Bar68-1, D68, MH63,2036-1a, MH86 and ZH10 respectively among the figure.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is conventional method.Used test material among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all arranges repeated experiments three times, results averaged.Be used for three kinds of fluoresceins (Cy2, Cy3 and Cy5) of labelled protein quality sample among the embodiment all available from GE company.
One, the selection of experiment material
Transgenic paddy rice 2036-1a: the transgenic paddy rice (offspring that proterties is stable) that transforms anti insect gene cry1Ac/sck; The public can obtain from Institute of Botany, Chinese Academy of Sciences; Mention the list of references of this vegetable material: Liu Yufang etc., 2007, Scientia Agricultura Sinica, 40:1181-1189.
Bright extensive 86 (MH86): parent's contrast (long-grained nonglutinous rice) of transgenic paddy rice 2036-1a; The public can obtain from Institute of Botany, Chinese Academy of Sciences; Mention the list of references of this vegetable material: Zhang Jianxin etc., 2000, Agricultural University Of Jiangxi's journal, 22:485-490.
Transgenic paddy rice Bar68-1: the transgenic paddy rice that transforms anti-herbicide gene bar; The public can obtain from Institute of Botany, Chinese Academy of Sciences; Mention the list of references of this vegetable material: Xiao Guoying etc., 2007, hybrid rice, 22:57-61.
D68: parent's contrast (long-grained nonglutinous rice) of transgenic paddy rice Bar68-1; The public can obtain from Institute of Botany, Chinese Academy of Sciences; Mention the list of references of this vegetable material: Xiao Guoying etc., 2007, hybrid rice, 22:57-61.
Bright extensive 63 (MH63): cultivate bright extensive 86 parent material (long-grained nonglutinous rice) by conventional breeding, be used for the characterization of molecules difference that the analytic routines genetic breeding causes; The public can obtain from Institute of Botany, Chinese Academy of Sciences; Mention the list of references of this vegetable material: thank to Huaan, 1998, Fujian agriculture journal, 13:1-6.
In spend (ZH10) No. 10: existing japonica rice variety is used for analyzing the characterization of molecules difference that natural variation causes; The Chinese Academy of Agricultural Sciences is on sale.
Two, detect insertion and the expression of foreign gene in transgenic paddy rice seed
Extensive 86 to throwing light on to transgenic paddy rice 2036-1a and its parent respectively, and transgenic paddy rice Bar68-1 and its parent contrast D68 and carry out following experiment:
1, detects the insertion of foreign gene in transgenic paddy rice seed
Utilizing the CTAB method to extract the genomic DNA of paddy rice mature seed, is that template is carried out the PCR evaluation with the genomic DNA.
The primer of identifying the cry1Ac gene is to following (target sequence is 1709bp):
F_cry1Ac (sequence 1): 5 '-CCCAAACATCAACGAATGCA-3 ';
R_cry1Ac (sequence 2): 5 '-CAAAGTAACCGAAATCGCTGG-3 '.
Detect the pcr amplification condition of cry1Ac gene: 94 ℃ of sex change 1 minute; 30 seconds, 55 ℃ annealing of 94 ℃ of sex change were extended 32 circulations 2 minutes for 30 seconds, 72 ℃; 72 ℃ were extended 10 minutes.
The primer of identifying the sck gene is to following (target sequence is 358bp):
F_sck：5′-CTCTTTTGTGCCTTCACCACC-3′；
R_sck：5′-CTTCATCCCTGGACTTGCAAG-3′。
Identify the pcr amplification condition of sck gene: 94 ℃ of sex change 30 seconds; Carry out: 30 seconds, 55 ℃ annealing of 94 ℃ of sex change were extended 32 circulations 1 minute for 30 seconds, 72 ℃; 72 ℃ were extended 10 minutes.
The primer of identifying the bar gene is to following (target sequence is 568bp):
F_bar：5′-TTGGATCCATGAGCCCAGAACGACGC-3′；
R_bar：5′-TAGAGCTCCTAAAATCTCGGTGACGGC-3′。
Identify the pcr amplification condition of bar gene: 94 ℃ of sex change 30 seconds; Carry out: 30 seconds, 55 ℃ annealing of 94 ℃ of sex change were extended 32 circulations 1 minute for 30 seconds, 72 ℃; 72 ℃ were extended 10 minutes.
The result shows: contain the bar gene in the genome of transgenic paddy rice Bar68-1, contain cry1Ac gene and sck gene in the genome of transgenic paddy rice 2036-1a, all do not contain corresponding gene in the genome of each parent's contrast.
2, detect the expression of foreign gene in transgenic paddy rice seed
Use RNAprep pure plant total RNA extraction reagent box (day root, China), according to the RNA of operation manual extraction paddy rice mature seed, utilize RT-PCR to detect expression of exogenous gene (adopting the TubA gene as the reference gene).Detect primer that foreign gene adopts to the equal same step 1 of pcr amplification condition.
Detect the primer of TubA gene to as follows:
Tub-F：5′-TCAGATGCCCAGTGACAGA-3′；
Tub-R：5′-TTGGTGATCTCGGCAACAGA-3′。
Identify the pcr amplification condition of TubA gene: 94 ℃ of sex change 30 seconds; 30 seconds, 55 ℃ annealing of 94 ℃ of sex change were extended 25 circulations 1 minute for 30 seconds, 72 ℃; 72 ℃ were extended 10 seconds.
The results are shown in Figure 1.The bar gene is expressed in transgenic paddy rice Bar68-1 seed, and transcriptional level is moderate.Cry1Ac gene and sck gene are all expressed in transgenic paddy rice 2036-1a seed and expression difference to some extent, and cry1Ac gene transcription level is than sck gene height.
Three, the 2D-DIGE of protein analyzes
Respectively to transgenic paddy rice 2036-1a, bright extensive 86, transgenic paddy rice Bar68-1, D68, bright extensive 63 and in spend and carry out following experiment No. 10:
1, the preparation of protein example
Grinding buffer solution: solvent is the Tris-HCl damping fluid (pH7.5) of 20mM, and solute and concentration thereof are as follows: 250mM sucrose, 10mM EGTA (ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA)), 10mM PMSF (phenylmethylsulfonyl fluoride).
Lysis buffer: solvent is water, and solute and concentration thereof are as follows: the quality percentage composition is 4% CHAPS (3-(3-cholamine propyl group) dimethylamino-1-propane sulfonic acid), 20mM Tris, 7M urea, 2M thiocarbamide.
(1) the ripe rice paddy seed of 400mg is ground in the mortar of 4 ℃ of precoolings, to grinding the 4ml grinding buffer solution that adds 4 ℃ of precoolings in the powder completely, fully mixing is poured in the 50ml centrifuge tube, centrifugal 15 minutes of 4 ℃, 15000rpm.
(2) supernatant of step (1) is transferred in the 10ml centrifuge tube, placed on ice.
(3) precipitation with step (1) is resuspended in the 2ml grinding buffer solution, and 4 ℃, 15, centrifugal 15 minutes of 000rpm gets in the centrifuge tube of supernatant adding step (2).
(4) with 4 ℃ of the centrifuge tubes of completing steps (3), 15, centrifugal 15 minutes of 000rpm gets supernatant.
(5) supernatant with step (4) is transferred in the 50ml centrifuge tube, adds the 50%TCA (trichloroacetic acid) of 1/4 volume, places 45-60 minute for 4 ℃ behind the mixing, and centrifugal 15 minutes of 4 ℃ then, 18000rpm are got precipitation.
(6) with the precipitation of 80% acetone cleaning step (5), add 4ml, repeated washing 3 times will precipitate dry at every turn.
(7) add lysis buffer to the precipitation of step (6), 28 ℃, 250rpm vibration 1.5 hours, during every ultrasonic 30 seconds of 30min (ultrasonic power is 100 watts).
(8) with the system room temperature 18 of step (7), centrifugal 20 minutes of 000rpm gets supernatant, regulates about pH to 8.5 with NaOH or HCl, utilizes Bradford standard measure protein concentration, and protein concentration is between 5 μ g-10 μ g.
Relate to 6 kinds of biomaterials in the experiment altogether, every kind of material carries out repeating for three times extracting, and obtains 18 parts of protein examples (solution form, every kind of protein example contains a kind of total protein) altogether.
2,2D-DIGE experimental design
18 parts of protein examples are mixed, the quality of various total proteins in total system equated, standard specimen this (internal standard) in being, mark is in order to eliminate the error that 2D-DIGE image comparison procedure occurs in using.18 parts of protein examples and interior standard specimen are originally carried out the 2D-DIGE analysis by the design of table 1, and this design can realize the comparison between all sample protein matter groups to be analyzed.In the table 1, numeral in the bracket repeats used sample number into spectrum (being followed successively by 1,2,3) at every turn, can hold two kinds of samples and a kind of interior standard specimen product (interior target exists can be increased the reliability of mating between glue and the glue and allow to produce statistics numerical value accurately between glue and glue) on the every DIGE glue simultaneously.
Table 12D-DIGE design of experiment
| Gel number | | Cy3 | Cy5 | |
| 1 | Internal standard | Bar68-1(1) | D68(1) | |
| 2 | Internal standard | 2036-1a(1) | MH86(1) | |
| 3 | Internal standard | MH63(1) | ZH10(1) | |
| 4 | Internal standard | Bar68-1(2) | 2036-1a(2) | |
| 5 | Internal standard | D68(2) | MH86(2) | |
| 6 | Internal standard | ZH10(2) | D68(3) | |
| 7 | Internal standard | MH86(3) | ZH10(3) | |
| 8 | Internal standard | MH63(2) | Bar68-1(3) | |
| 9 | Internal standard | 2036-1a(3) | MH63(3) |
3,2D-DIGE analyzes
(1) fluorochrome label sample
According to experimental design, (wavelength of transmitted light under the 532nm excitation wavelength is 580nm to use fluorescein Cy3 respectively, shown in green) and fluorescein Cy5 (wavelength of transmitted light under the 633nm excitation wavelength is 670nm, shown in red) each protein example of mark, with mark in fluorescein Cy2 (wavelength of transmitted light under the 488nm excitation wavelength is 520nm, the is shown as blueness) mark.Labeling method: in centrifuge tube, add 50 μ g protein examples, add the 400pmol fluorescein, mixing is also of short duration centrifugal, 4 ℃ of dark reactions 30 minutes, add the lysine mixing of 1 μ l 10mM and of short duration centrifugal then, place on ice dark to hatch 10 minutes with cessation reaction.
(2) go up sample
Two kinds of protein examples that are arranged in the preliminary experiment design on the same DIGE glue are mixed with the standard specimen product (in total system, two kinds of protein example amounts and interior mark sample size are 50 μ g), add isopyknic 2 * sample buffer (8M urea, 130mM DTT, 4g/100ml CHAPS, volumn concentration is 2% pharmalyte pH3-10), mixing and of short duration centrifugal being placed on 10 minutes on ice.Volume according to protein sample and 2 * sample buffer, calculate rehydration damping fluid (8M urea, 13mM DTT, 4g/100ml CHAPS, volumn concentration is 0.5% pharmalyte pH 3-10) volume, and add the rehydration damping fluid of respective volume in the 10ml centrifuge tube, make cumulative volume reach 500 μ l.In the 10ml centrifuge tube that the rehydration damping fluid is housed, add protein sample and the 2 * sample buffer of mixing before.20000 * rpm goes up sample under the room temperature after centrifugal 20 minutes.
(3) 2D-DIGE electrophoretic analysis
Select the adhesive tape (GE company) of pH4-7 for use, labeling dye may decompose under the light in order to prevent from being exposed to for a long time, so need lucifuge when electrophoresis.
Being used for first instrument to isoelectric focusing electrophoresis is the Ettan IPGphor isoelectric focusing system of GE company, uses 24cm solid phase pH gradient adhesive tape (GE company), is placed in the solid phase pH gradient adhesive tape groove (IPG strip holder).Electrophoretic procedures: 6h → 30V, 6h → 60V, 1h → 500V, 1h → 1000V, 9:40h → 8000V is 65KVh when always lying prostrate.After isoelectric focusing is finished, adhesive tape equilibrium liquid (50mM Tris-HCl pH8.8,6M urea, 30% glycerine, 2%SDS, 0.002% bromjophenol blue, 1%DTT) in balance 20 minutes; Adhesive tape is respectively through ddH then
2Put into the middle balance of equilibrium liquid (50mM Tris-HCl pH8.8,6M urea, 30% glycerine, 2%SDS, 0.002% bromjophenol blue, 2.5% iodoacetamide) after O and the washing of 1 * electrophoretic buffer 20 minutes, and utilized ddH
2O and 1 * electrophoretic buffer wash.
Adhesive tape after the balance is put on 12.5% polyacrylamide gel, with confining liquid (electrophoretic buffer that contains 0.5% agarose, 0.002% bromjophenol blue) sealing, in Ettan DALT Six electrophoresis system (GE company), carry out second to SDS-PAGE (second direction that albumen moves in the electrophoresis is vertical with first direction that albumen moves in the electrophoresis).Electrophoretic buffer consists of: 25mM Tris-base, 192mM glycocoll, 0.1%SDS, all the other are water.Deposition condition: current stabilization, power 2W carries out electrophoresis, after the bromjophenol blue indicator enters polyacrylamide gel fully, adds high-powerly, when indicator arrives the feather edge of gel, stops electrophoresis.
(4) image scanning
Utilize fluorescent scanning instrument (the Typhoon 9400 series Variable Mode Imager of GE company) that the DIGE glue behind the electrophoresis is scanned, obtain fluoroscopic image.Light with wavelength 488/520nm excites the image that obtains Cy2 on every glue as exciting light/emission light (absorption light).Light with wavelength 532/580nm excites the image that obtains Cy3 on every glue as exciting light/emission light (absorption light).Light with wavelength 633/670nm excites the image that obtains Cy5 on every glue as exciting light/emission light (absorption light).Adjust suitable exciting voltage, make the maximum signal level of each image all about (80000 ± 3000) and resultant signal value (23000000 ± 3000000), to reduce every glue because of the error that scans or mark brings.Finally obtained 9 opening and closing fluoroscopic image (see figure 2) also through overscanning, the fluoroscopic image of each merging can be decomposed into three the single fluoroscopic images corresponding with Cy2, Cy3 and Cy5, have 27 images, these images are used for comparison and the differential expression protein analysis between the protein group.
(5) initial analysis of different sample protein matter groups
Utilize V.6.5 software (GE company) of DeCyder, according to the Guide Book of software the image that obtains is analyzed.
At first in glue the difference analysis module (Differential in gel analysis carries out the common detection of protein site to protein example and interior standard specimen product in DIA).The initial value of protein site is made as 5000, detects automatically.Automatically after detection is finished, manually adjust filtration parameter the falseness point is filtered for the first time, detect the protein site about 2250 on final every glue.
(biological variation analysis carries out coupling and statistical study between the different gels in BVA) at the biomutation analysis module then.Owing to all comprise identical interior standard specimen product on every glue, therefore by this common reference, the protein site that is in any two samples on the different glue can mate and compare.Equally, in order to ensure the expression data of reliable protein site, only consider 1233 points that those all exist on greater than 21 images (7 glue * 3 image/glue).
(normalized expression amount i.e. this albumen is carried out the expression that obtains after the normalization at interior mark albumen to utilize the normalized expression amount of 1233 protein; This normalization by DeCyder V.6.5 software carry out automatically) carry out principal component analysis (PCA) (princ ipal component analysis), student t check and variance analysis (ANOVA).
Principal component analysis (PCA) can become several new variablees with original a plurality of variablees through linear transformation, and new variables can be contained the information of primal variable.SIMCA-P is used in principal component analysis (PCA), and (Umea Sweden) carries out for version 12.0, Umetrics, and parameter is set to acquiescence.Through after the principal component analysis (PCA): do not distinguish (Fig. 3) between two groups of transgenic paddy rices and its parent's contrast separately; D68 and other three kinds (MH63, MH86 and ZH10) can distinguish on first principal component; ZH10 and two kinds bright extensive (MH63 and MH86) can distinguish (Fig. 3 A) on Second principal component,, and two kinds bright extensive (MH63 and MH86) separate (Fig. 3 B) in the 3rd major component district.
Choosing t check and variance analysis all has the significance difference opposite sex (p＜0.01) and variation multiple and enters next step mass spectrum evaluation program greater than 1.2 times protein.Be specially: interior target with reference under, with the protein profiling of transgenic paddy rice 2036-1a and bright extensive 86 protein profiling relatively, the albumen of choosing average expression amount ratio and being more than 1.2 carries out the mass spectrum evaluation; Interior target with reference under, with bright extensive 86 protein profiling and bright extensive 63 protein profiling relatively, the albumen of choosing average expression amount ratio and being more than 1.2 carries out the mass spectrum evaluation; Interior target with reference under, with bright extensive 86 protein profiling with in spend No. 10 protein profiling relatively, the albumen of choosing average expression amount ratio and being 1.2 or more carries out the mass spectrum evaluation; Interior target with reference under, with the protein profiling of the protein profiling of transgenic paddy rice Bar68-1 and D68 relatively, the albumen of choosing average expression amount ratio and being more than 1.2 carries out the mass spectrum evaluation; Interior target with reference under, with the protein profiling of D68 and bright extensive 63 protein profiling relatively, the albumen of choosing average expression amount ratio and being more than 1.2 carries out the mass spectrum evaluation; Interior target with reference under, with the protein profiling of D68 with in spend No. 10 protein profiling relatively, the albumen of choosing average expression amount ratio and being 1.2 or more carries out the mass spectrum evaluation.Average relative expression quantity ratio by DeCyder V.6.5 software calculate automatically.
3, the mass spectrum of protein is identified
(1) in-gel digestion
Successfully be recovered to 264 protein spot on the gel behind the electrophoresis, be cut into about 1mm
3Granule, change in the centrifuge tube.Add about 50 μ l ammonium bicarbonate-acetonitrile solutions (50mM ammonium bicarbonate and pure acetonitrile equal-volume mix) and make gel about 30min that decolours.Remove liquid, earlier with the 50% acetonitrile 15min that dewaters, dewatering to micelle with 100% acetonitrile is white in color again.Remove acetonitrile, place the interior 5-10min of SpeedVac (Speed-Vac), make the micelle bone dry.The 25mM ammonium bicarbonate that contains 10mM DTT that adds the new preparation of 50 μ l, 56 ℃ of heating 60min go back raw sample.Cool to room temperature, abandoning supernatant adds the 25mM ammonium bicarbonate that 50 μ l contain 55mM iodacetyl ammonium, and room temperature is placed 45min in the darkroom, the sulfydryl on the alkylation cysteine residues.Supernatant discarded night, with the washing of 25mM ammonium bicarbonate once.Abandoning supernatant is earlier with 50% acetonitrile treatment 15min, again with the dehydration of 100% acetonitrile, to bleach fully (approximately 15min).Remove acetonitrile, place in the SpeedVac (Speed-Vac), 5-10min makes the micelle bone dry.Add about 10 μ l trypsin solutions and place 30min on ice, make micelle rehydration, remove excessive trypsin solution, add minimum (about 5 μ 1) 25mM ammonium bicarbonate and cover micelle, keep enzyme to cut and make micelle moistening.37 ℃ of placements are spent the night (about general 16h), are used for mass spectrophotometry after the desalination of gained solution.
(2) proteomic image analysis
The instrument that is used for identification of proteins is the Ultroflex II MALDI flight time/time of-flight mass spectrometer (MALDI-TOF/TOF MS) of Bruker company.
1. point sample: add 1 μ l protein example at MTP AnchorChip 400/384 (Bruker) target, dry;
2. put matrix: (solvent is water to add 0.1 μ l matrix; Solute and concentration thereof are as follows: volumn concentration is 50% TFA, 8mg/ml α-cyano-4-hydroxcinnamic acid), dry;
3. desalination: add 1 μ l, 0.1% trifluoroacetic acid at each protein site, siphon away behind the 1-2min;
4. put standard specimen: per 4 sample centers add the PeptideMixMonoAutoflex standard items, 6 peptide species that it contains and mass number difference thereof: Augiotensin II:1046.54180; Auguotensin I:1296.68478; Substance P:1347.73543; Bombesin:1619.82235; ACTA (1-17): 2093.08617; ACTA (18-39): 2465.19834.
5. Ultroflex II MALDI TOF/TOF mass spectrometer (Bruker Daltonics packs 384 targets into, Germany) in, by flexanalysis 3.3 software analysis obtain protein site finger-print (peptife mass fingerprint, PMF).Calibrated by autotomy peak 906.51Da and 2273.16Da of interior mark and trypsase.The degree of accuracy that mass spectrum is set is in 25ppm, and corresponding resolution surpasses 25000.
(3) MALDI-TOF MS data analysis
PMF input BioTools 3.0 interface (Bruker Daltonics) with mass spectrum obtains use Mascot software (www.matrixscience.com then in NCBInr (http://www.ncbi.nlm.nih.gov/) albumen database; Matrix Science, London UK) searches for.Parameter is as follows: species classification (differential protein between transgenosis and the contrast is searched at all species): paddy rice; Peptide mass number character: single electric charge and [M+H]+; Mass number error range: ± 100ppm; Mistake shearing site number: 1; Modify for fixing with carbamidomethyl (C), oxidation (M), pyro-glu (N-term Q) is variable modification.234 protein spots have obtained the mass spectrum result, wherein only contain a kind of protein on 218 protein sites, contain two kinds of protein on other 16 protein sites, obtain 250 kinds of protein thus altogether.In these 250 kinds of protein, chromosomal foci number identical protein quantity is 146.
4, the transgenic paddy rice characterization of molecules is analyzed
According to experiment purpose mass spectrum is identified that the protein of finishing randomly carries out function classification, cluster analysis, principal component analysis (PCA), expression pattern analysis etc.
At first will identify differential protein and be divided into 8 function classes, that is: metabolism, protein synthesize with target, defense reaction, cell growth and division, PPDK, signal transduction, transcribe, transport; (see figure 4) in " the unknown " class that do not have the protein of clear and definite note to be divided into.Most of differentially expressed proteins (78.8%) participate in following three kinds of biological processes: metabolism (31.2%), protein synthesize and target (25.2%) and defense reaction (22.4%).More particularly, 64.1% metabolic related protein participates in center carbon metabolism (glycolysis and tricarboxylic acid cycle) and starch metabolism, and 74.6% protein is synthetic to participate in protein folding and modification with the target related protein.These results have disclosed and have participated in center carbon metabolism and starch is synthetic, protein folding changes greatly in the process of the evolution of response nature, conventional breeding or transgenic event with the protein of modification, defense reaction.
In order to study differential protein in the expression trend of each sample room, utilize GeneCluster software that 218 differential protein spots that contain single albumen have been done cluster analysis.Cluster analysis result shows that differential protein is divided into the three major types type: c0c3 type, c1c4 type and c2c5 type (Fig. 5).Sample is followed successively by in proper order: Bar68-1, D68, MH63,2036-1a, MH86 and ZH10.Each is expressed in the type and has comprised antipodal two subclass of expression pattern.Wherein, 71 albumen that the c0c3 type comprises show as: 31 albumen expression height in long-grained nonglutinous rice D68 and MH63, expression low (c0) in long-grained nonglutinous rice MH86 and japonica rice ZH10; The expression of all the other 40 albumen is opposite (c3) just in time.78 albumen that the c1c4 type comprises show as: 42 albumen expression in two kinds of bright extensive strains is low, expression height (c1) in D68 and ZH10; The expression of all the other 36 albumen is opposite (c4) just in time.The third is expressed type c2c5 type and has comprised 69 albumen, and these albumen show as: 34 albumen expression height in ZH10, expression low (c2) in three kinds of long-grained nonglutinous rices; The expression of all the other 35 albumen is opposite (c5) just in time.But no matter in any expression type, the protein expression intensity of variation of two groups of transgenosiss and its parent contrast is all very little, less than the protein expression intensity of variation between Different Rice Varieties.This shows that protein expression level does not change substantially than its non-transgenic contrast in the transgenic paddy rice seed.
Carry out the principal component analysis (PCA) second time at 218 differential proteins, with the contribution of evaluation differential protein confrontation paddy rice sample room difference.First three major component respectively with above-mentioned three big cluster (c1c4, c2c5 and c0c3) height correlations.The bigger protein of first principal component contribution is mainly participated in defense reaction, disclosed aspect the reaction that between D68/Bar68-1 and all the other samples maximum difference is that sample stimulates to external world.Yet the protein that has big load at Second principal component, belongs to respectively that glycolysis, starch are synthetic, protein folding and modification and defense reaction class, hints these biology activities difference maximum between japonica rice and long-grained nonglutinous rice; The protein that has big load in the 3rd major component mainly belongs to the synthetic and defense reaction class of starch, illustrates that the difference between these two function classes can be distinguished two kinds of bright extensive kinds.
The distribution situation of assessment differential protein in difference in functionality class and function subclass therefrom selected to have 9 function classes of more differential protein, and it has been carried out the analysis (Fig. 6) of expression pattern.The synthetic protein expression trend of involved in sugar glycolysis and starch is consistent, in two kinds of bright extensive kinds, express high relatively, and D68 and in spend express in No. 10 low.Krebs cycle pathway's related protein is opposite with above-mentioned two proteinoid expression trend.In addition, it is synthetic identical with the protein expression trend of growth with cell separation to participate in protein, opposite with proteolysis expression trend.Other three major function classes (protein folding and modification, defense reaction and PPDK) have its pattern of uniqueness separately.In the function class of analyzing and subclass, change has taken place in the protein expression level between genetically modified plants and its parent contrast, but its change degree is very little, less than the protein expression intensity of variation between Different Rice Varieties.This has proved too, the protein change level that the protein change level that transgenic event causes causes well below natural variation and conventional breeding.
Claims (8)
1. a method of estimating transgenic plant safety comprises the steps:
(1) sample to be tested is extracted total protein, and carry out 2D-DIGE electrophoresis and fluoroscopic image analysis, obtain the proteomic map of each sample to be tested; Described sample to be tested is sample first, sample second, sample third and sample fourth; Described sample first is genetically modified plants to be measured; Described sample second is the mother plant of genetically modified plants to be measured; Described sample third is the different plants that are subordinate to same cultigen with sample second; Described sample fourth is the plant that is subordinate to different cultigens with sample second; The quality such as total protein of described sample first, described sample second, described sample third and described sample fourth are mixed, as the interior mark of 2D-DIGE electrophoresis;
(2) be chosen at the albumen that occurs in the protein profiling more than 70% and carry out principal component analysis (PCA);
(3) result according to described principal component analysis (PCA) judges as follows: if the otherness of described sample first and described sample second is lower than the otherness of described sample second and described sample third and is lower than the otherness of described sample second and described sample fourth, described sample first is the genetically modified plants of candidate's safety.
2. the method for claim 1, it is characterized in that: described plant is paddy rice.
3. method as claimed in claim 2, it is characterized in that: described cultigen refers to round-grained rice subspecies and Xian subspecies.
4. as claim 1 or 2 or 3 described methods, it is characterized in that: described method also comprises the steps:
Interior target with reference under, with the protein profiling of the protein profiling of described sample first and described sample second relatively, the albumen of choosing expression ratio and being more than 1.2 carries out the mass spectrum evaluation; Interior target with reference under, with the protein profiling of the protein profiling of described sample second and described sample third relatively, the albumen of choosing expression ratio and being more than 1.2 carries out the mass spectrum evaluation; Interior target with reference under, with the protein profiling of the protein profiling of described sample second and described sample fourth relatively, the albumen of choosing expression ratio and being more than 1.2 carries out the mass spectrum evaluation.
5. method as claimed in claim 4 is characterized in that: described method comprises that also the albumen after mass spectrum identified carries out the step that function classification and/or cluster analysis and/or principal component analysis (PCA) and/or expression pattern are analyzed.
6. as the described method of claim 3 to 5, it is characterized in that: described sample first, described sample second and described sample third belong to the Xian subspecies; Described sample fourth belongs to the round-grained rice subspecies.
7. method as claimed in claim 6, it is characterized in that: described sample first is transgenic paddy rice 2036-1a; Described sample second is bright extensive 86; Described sample third is bright extensive 63; Described sample fourth is to spend in the japonica rice No. 10.
8. method as claimed in claim 6, it is characterized in that: described sample first is transgenic paddy rice Bar68-1; Described sample second is D68; Described sample third is bright extensive 63; Described sample fourth is to spend in the japonica rice No. 10.
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