CN102967643A - Method for screening related candidate plant drought resisting gene by adopting proteomic technology - Google Patents
Method for screening related candidate plant drought resisting gene by adopting proteomic technology Download PDFInfo
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Abstract
The invention discloses a method for screening a related candidate plant drought resisting gene by adopting a proteomic technology. The method comprises the following steps of A. respectively taking plant leaves of a drought treatment group and a normal watering processing group, and firstly searching differential proteins by utilizing a two-dimensional gel electrophoresis technology, and preliminarily obtaining candidate genes related to plant drought resistance; and B screening the obtained differential proteins by a mass spectrum identification technology again, and further screening and obtaining the candidate genes related to the plant drought resistance. The method for screening the related candidate drought resisting gene of the plant (especially white mustard), which is provided by the invention, can efficiently overcome the problem that in the prior art, the screen efficiency is not high.
Description
Technical field
The present invention relates to plant biotechnology field, be specifically related to the especially screening technique of sinapsis alba drought resisting correlation candidate gene of plant gene.
Background technology
The drought resistance of plant is the controlled by multiple genes system of a complexity, and the transcription factor of drought resisting can be regulated and control the expression of a series of gene related to drought tolerance, is a kind of approach of highly effective raising drought tolerance.At present, existing research is found from each Plants and has been identified tens kinds of functional genes relevant with drought resistance.
Sinapsis alba is the important breeding gene resource of crucifer, is subject to American-European and more domestic scholars' great attention.By distant hybridization, in the other plant bodies such as resistant gene resource importing crops that can sinapsis alba is important, so sinapsis alba has become the important original material of initiative crop new germ plasm.Sinapsis alba has stronger drought resistance (Wang Yanping etc., 2001; Tang Daocheng, 2000), be important germplasm resourses with drought resistance, the relevant anti-drought gene of screening is significant to carrying out the anti-drought gene engineering research
For these species of sinapsis alba, Dong etc. (2012) have reported sinapsis alba in drought and rewatering condition lower blade gene differential expression situation, find because drought stress causes 309 down regulation of gene expression in the blade, and 248 gene expressions are raised.Zhang Ningning (2012) adopt PEG simulating drought Study on processing method the sinapsis alba seedling under drought stress the Physiology and biochemistry response and the citrate synthase expression under the reply drought stress condition.But both all inquire into less than concrete sinapsis alba is correlated with anti-drought gene and screening or cloning process thereof.
Genescreen carries out according to the differential expression in its plant usually.More different cells or the different genotype difference in gene expression, separate and clone's different tissues or the same gene of organizing the different developmental phases differential expression, being not only the basis of research life process molecular mechanism, also is the prerequisite of carrying out the functional genomics area research.At present, the method for screening difference expression gene mainly contains differential screening technology, Subtractive Hybridization Technique, mRNA differential display technique, serial analysis of gene expression, Using Suppression Subtractive Hybridization, biochip technology, Gene Calling technology etc.But existing triage techniques is too dependent on these biology of gene functions and biology phenotype, but up to the present, most of biology phenotypes and biology of gene function are unknown, and therefore, the utilization of these candidate gene screening methods is limited by very large.
What really play a role in the plant is protein, and protein is being played the part of " fragment of brick " role who constructs the life mansion.The constituent that proteomics (Proteomics) research relates to the protein group is the research of protein group expression pattern, and the function that relates to simultaneously the protein group is the research of protein group functional mode.The Analysis and Identification that protein is formed is the proteomics main contents corresponding with genomics, the collection of illustrative plates that its requirement separates, identifies protein.The technology path of Study on Protein group mainly contains two, article one, be the relative variation that adds various protein expressions spectrums in the method identification of organism system of biological mass spectrometry and each protein expression degree with dielectrophoresis, another route is exactly the technology path that is referred to as shotgun that multi-dimensional chromatograph combines with biological mass spectrometry.Wherein great majority research both at home and abroad is as Main Means take dielectrophoresis.
Two dimensional gel electrophore-sis (2-DE) technology is the one preferred technique of protein high-throughput isolation, comprises " one to " electrophoresis of protein being carried out isoelectric focusing according to the isoelectric point principle and " two to " electrophoresis according to the different SDS-PAGE of carrying out of molecular weight.Need the image analysis through polyacrylamide gel dyeing and protein site behind the electrophoresis.The target protein that obtains through bidimensional electrophoretic separation is at first processed through trypsin digestion and is formed the peptide section, then can identify with mass-spectrometric technique and analyzes the peptide section that obtains
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method of screening plant (especially sinapsis alba) drought resisting correlation candidate gene, in order to overcome the not high problem of prior art screening effeciency.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows.
Use the method for proteomic techniques screening plant drought correlation candidate gene, its step comprises:
A, get arid processed group and the processed group plant plant leaf that normally waters respectively, at first utilize two dimensional gel electrophoresis to seek differential protein, tentatively obtain the candidate gene relevant with plant drought;
B, by the Mass Spectrometric Identification technology gained differential protein is carried out multiple sieve again, further the candidate gene relevant with plant drought obtained in screening.
As a kind of optimal technical scheme of the present invention, the concrete operation step of steps A comprises:
A-1, vegetable material and leaf protein extract: get respectively arid processed group and the processed group plant plant leaf that normally waters, the protein example in the blade is obtained in liquid nitrogen grinding, centrifugal, cracking processing;
A-2, two dimensional gel electrophore-sis are processed: gained protein example of upper step is carried out respectively two dimensional gel electrophore-sis process, isoelectric focusing adopts the prefabricated IPG adhesive tape of pH4-7 to carry out, after finishing, electrophoresis takes off gel until second, put into the immobile liquid room temperature fixedly more than the 4h, then put into coomassie brilliant blue staining liquid and carry out saturated dyeing, utilize clear water to clean gel and namely carry out gel images scanning afterwards, obtain arid and process blade and the albumen two dimensional gel electrophore-sis figure that normally waters wiper blade;
A-3, interpretation of result: gained two dimensional gel electrophore-sis figure carried out statistical study to the upper step, utilize one-way analysis of variance, to the ratio of control group and processed group protein expression amount carry out take 2 as the end to number conversion, under Xuan Ze Zai ∣ log2expression ratio ∣＞=1, P＜0.05 condition control group and processed group protein expression profile are carried out statistical analysis, namely obtain differential protein; And further determine to process in arid the protein of high expressed in the blade, namely preliminary screening goes out the candidate gene relevant with plant drought.
As a kind of optimal technical scheme of the present invention, the concrete operation step of steps A-1 is:
A, get vegetable seeds and in culturing room, carry out vernalization after, the PEG6000 concentration for the treatment of is 20%, be treated to contrast with clear water, choosing through concentration is that plant seedlings blade that 20% PEG6000 processed the 9th day is testing for control group carries out 0 protein differential expression of experimental group and the plant seedlings blade that is left intact;
B, plant seedlings get experimental group and control group plant leaf simultaneously after the processing of PEG6000 simulating drought finishes, after distilled water is rinsed well, sop up excessive moisture with filter paper, place rapidly liquid nitrogen cooling packing mark after, place-80 ℃ of refrigerators to save backup;
C, in liquid nitrogen, fully grind blade, the extract that adds 3 times of sample volumes spends the night under-20 ℃ condition, then 4 ℃ of 8000rpm above centrifugal 1 hour, abandon supernatant, add isopyknic ice bath acetone that contains 0.07% beta-mercaptoethanol, shook up rear 4 ℃ of 8000rpm above centrifugal 1 hour, then the vacuum drying precipitation is for subsequent use;
Add lysate before d, the loading, room temperature was placed 30 minutes, made albumen fully be dissolved in the lysate, then above centrifugal 1 hour of 15 ℃ of 8000rpm or longer time, not to be precipitated as standard, be kept at 4 ℃ temporarily, with Brandford standard measure albumen, then packing put into-80 ℃ for subsequent use;
As a kind of optimal technical scheme of the present invention, the extract among the step c is: the acetone that contains the beta-mercaptoethanol of 10%TCA and 0.07%; The configuration proportion of lysate is in the steps d: 2.7g urea 0.2gCHAPS is dissolved in the deionized water of 3ml sterilization, and final volume is 5ml, adds the DTT65ul/ml of 1M before the use again.
As a kind of optimal technical scheme of the present invention, the protein sample that steps A obtains has carried out respectively dielectrophoresis and coomassie brilliant blue staining to be processed, and by analyzing acquisition differential protein particle, on this basis, the concrete operation step of step B is: at first downcut the differential protein particle from gel, then carry out mass spectrophotometry and Identification of Fusion Protein.
As a kind of optimal technical scheme of the present invention, the concrete operation step of described mass spectrophotometry comprises:
The gel that 1) will carry out mass spectrophotometry cleans 5min/ time * 5 times with ultrapure water;
2) cut 1000 μ l Eppondorf rifle heads tip according to the protein spots concentration that will analyze and size, use the rifle head that cuts from above-mentioned gel, to cut the protein spots that to analyze and put into 1.5ml EP pipe;
3) add 100 μ l ultrapure waters in every pipe, utilize shaking table concussion washing 15min under the room temperature, suck ultrapure water; Repeat this step twice;
4) add 100 μ l in-gel digestion destainers in every pipe, utilize shaking table concussion decolouring 30min under the room temperature, suck the in-gel digestion destainer; Repeat this step 4 time to micelle and be the water white transparency shape;
5) add 100 μ l acetonitriles in every pipe, shaking table concussion dehydration 10min sucks acetonitrile under the room temperature;
6) vacuumize dry 25min to the micelle graininess that is white in color;
7) add 10 μ l enzyme liquid in every pipe, place 4 ℃ to keep 1h, suck unnecessary enzyme liquid;
8) the EP pipe is inverted, 37 ℃ keep 12-14h, make protein carry out complete degestion;
9) add 30 μ l peptide section extract I in every pipe, 37 ℃ keep 1h, get supernatant to new pipe;
10) add 30 μ l peptide section extract II in the EP pipe that contains micelle, 30 ℃ keep 1h, get in the pipe that contains supernatant of supernatant to the step 9;
11) the resulting extract that contains the peptide section is carried out about vacuum drying 1h, extremely remaining liquid is 10 μ l;
12) 0.8 μ l peptide section extract is added on the sample target, behind sample drying, repeats application of sample once;
13) add 0.5 μ l matrix at the nearly sample of doing;
14) clean target position with the 1 ‰ TFA washing lotions of 1 μ l, keep 30S; Repeat twice;
15) behind the sample bone dry, place Bruker Daltonics Autoflex to analyze sample panel, obtain the peptide mass fingerprinting of protein sample, be i.e. the quality distribution diagram of all peptide sections of enzymolysis protein.
As a kind of optimal technical scheme of the present invention, the specific operation process of described Identification of Fusion Protein is: the MASCOT software by localization carries out the protein retrieval in NCBInr 20100116 databases, mate with all known in database protein fingerprints, setup parameter is: Max Mwassed Cleavages:1; Fixed Modifications; Carboxymethyl(C); Vaviable Modifications:Oxidation(M); Mass values:MH+, Monowasotopic; Peptide Mass Tolerance: ± 0.2Da; If the qualification result score was greater than 85 minutes, namely P＜0.05 illustrates that Search Results is credible.
As a kind of optimal technical scheme of the present invention, further utilize RT one round pcr that the drought resisting candidate gene that filters out is verified after the step B.
As a kind of optimal technical scheme of the present invention, further utilize the WesternBlot technology that the drought resisting candidate gene that filters out is verified after the step B.
As a kind of optimal technical scheme of the present invention, described plant is sinapsis alba.
The beneficial effect that adopts technique scheme to produce is:
The invention provides the method that can efficiently screen with plant (especially sinapsis alba) drought resistance correlation candidate gene, the embodiment of the invention utilizes the method to identify the relevant candidate gene of some sinapsis alba drought resistings; The inventive method provides a method to use for reference to the Fineness gene of clone sinapsis alba drought-resistant character; Utilizing transgenic technology cultivation drought resistant plant variety to have very important reference value.Its advantage and effect are described below: 1. can be fast and effectively screen the candidate gene that is associated with the sinapsis alba drought resisting; 2. the present invention utilizes this high flux of 2-DE well, the proteomics research means are studied the candidate gene relevant with the degeneration-resistant proterties of sinapsis alba accurately; 3. can be widely used in the research and development of the degeneration-resistant proterties such as plant drought and plant (crop) new varieties of cultivating strong stress resistance in conjunction with transgenic technology.
Description of drawings
Sinapsis alba seedling leaves (B) when processing the 9th day that Fig. 1 normally waters water treatment seedling leaves (A) and 20%PEG6000.
Fig. 2 sinapsis alba arid is processed and the seedling leaves albumen two dimensional gel electrophore-sis figure that normally waters; Among the figure, A: control group sinapsis alba seedling leaves 2-DE collection of illustrative plates; B:PEG6000 processed group sinapsis alba seedling leaves 2-DE collection of illustrative plates.
Embodiment
Following examples describe the present invention in detail.Various raw material used in the present invention and items of equipment are conventional commercially available prod, all can buy directly by market to obtain.
Embodiment 1
The screening of sinapsis alba gene related to drought tolerance
One, utilize 2-DE technical Analysis arid to process and normally water water treatment (contrast) sinapsis alba seedling leaves differential protein
1. vegetable material and leaf protein extract
The sinapsis alba seed is preserved for this laboratory.The sinapsis alba seed carries out vernalization in culturing room after, the PEG6000 concentration for the treatment of is 20%, is treated to contrast with clear water.Choosing through concentration is that sinapsis alba seedling leaves that 20% PEG6000 processed the 9th day is testing for control group carries out the protein differential expression of experimental group and the sinapsis alba seedling leaves that is left intact.
The sinapsis alba seedling is got experimental group and control group plant leaf simultaneously after the processing of PEG6000 simulating drought finishes, after distilled water is rinsed well, sop up excessive moisture with filter paper, place rapidly liquid nitrogen cooling packing mark after, place-80 ℃ of refrigerators to save backup.
In liquid nitrogen, fully grind blade.The extract that adds 3 times of sample volumes spends the night under-20 ℃ condition, and then centrifugal (4 ℃ 8000rpm above 1 hour) abandon supernatant.Add isopyknic ice bath acetone (containing 0.07% beta-mercaptoethanol), shake up rear centrifugal (4 ℃ 8000rpm above 1 hour), then the vacuum drying precipitation is for subsequent use.Add lysate before the loading, room temperature was placed 30 minutes, and albumen fully is dissolved in the lysate, then centrifugal (above 1 hour of 15 ℃ of 8000rpm or longer time be not to be precipitated as standard), can be kept at temporarily 4 ℃ stand-by.With Brandford standard measure albumen, but then packing put into-80 ℃ for subsequent use.
Medicine: extract: the acetone that contains the beta-mercaptoethanol of 10%TCA and 0.07%.Lysate: 2.7g urea 0.2gCHAPS is dissolved in the deionized water of 3ml sterilization (final volume is 5ml), adds the DTT65ul/ml of 1M before the use again.
2. the mensuration of protein concentration
Measure according to Bradford method (Coomassie brilliant blue method): get 5 EP pipe, according to the form below adds respectively after the mentioned reagent fully mixing in each pipe, leave standstill 2min after, within the 1h, take ultrapure water as blank, measure the A595nm value.Take protein content as horizontal ordinate, the A595nm value is ordinate, the drawing standard curve.Adopt spectrophotometer to measure sample albumen light absorption value at the 595nm place.
3. two dimensional gel electrophore-sis (2D-PAGE)
Dielectrophoresis is with reference to the method for sweet dew etc., and isoelectric focusing adopts the prefabricated IPG adhesive tape (long 17cm) of pH4-7 to carry out; After electrophoresis finishes, take off gel until second, put into the immobile liquid room temperature fixedly more than the 4h, then put into Coomassie brilliant blue G-250 dyeing liquor and carry out saturated dyeing, utilize clear water can carry out gel images scanning after cleaning gel 30min.What obtain processes and the seedling leaves albumen two dimensional gel electrophore-sis figure that normally waters for the sinapsis alba shown in the accompanying drawing 2 is arid.
The graphical analysis result shows, detected 135 protein spots in control group; 162 protein spots in processed group, have been detected.By the control group in the comparison diagram and processed group protein expression collection of illustrative plates, find between them to exist 120 total protein, these total protein account for the nearly 89% of total protein prime number that control group expresses, account for nearly 74% of number that processed group expresses.This illustrates that these protein play a role as the conservative protein in the sinapsis alba body, by the normal physiological activity of sinapsis alba provides necessary.
Utilize one-way analysis of variance (one-way ANOVA) and to the ratio of control group and processed group protein expression amount carry out take 2 as the end to number conversion, be chosen in | under log2expression ratio ∣＞=1, P＜0.05 condition control group and processed group protein expression profile are carried out statistical analysis, found altogether 58 differential proteins.Wherein, 16 peculiar protein are arranged in the control group; 42 peculiar protein are arranged in the processed group.
In processed group, there are 21 protein expression amounts significantly to be lower than control group; There are 22 protein expression amounts to be significantly higher than control group.These differential proteins comprise that it may be because the difference that the difference of extraneous drought environment condition causes that peculiar protein expression and expression fluctuate.Therefore, the protein that 20%PEG6000 processes high expressed in 9 days rear blades may be conducive to strengthen the drought resistance of sinapsis alba, so that it more can adapt to the variation of drought environment.
Two, the Mass Spectrometric Identification of differential protein spot
(wherein 5 obtain identifying to choose above-mentioned percentage bulking value (vol%) and be 10 special differential protein spots more than 2 times, 5 do not search the data result that is complementary with it) carry out mass spectrophotometry, numbering is respectively: G12, G13, G14, G15, G16, G17, G18, G19, G20, G21.
Mass spectrophotometry:
The gel that 1) will carry out mass spectrophotometry cleans 5min/ time * 5 times with ultrapure water.
2) according to want analyzing proteins particle concentration and the big or small 1000 μ lEppondorf rifle heads tip that cuts certain-length, use the rifle head that cuts from above-mentioned gel, to cut the protein spots that to analyze and put into 1.5ml EP pipe.
3) add 100 μ l ultrapure waters in every pipe, utilize shaking table concussion washing 15min under the room temperature, suck ultrapure water.Repeat this step twice.
4) add 100 μ l in-gel digestion destainers in every pipe, utilize shaking table concussion decolouring 30min under the room temperature, suck the in-gel digestion destainer.Repeat this step 4 time to micelle and be the water white transparency shape.
5) add 100 μ l acetonitriles in every pipe, shaking table concussion dehydration 10min sucks acetonitrile under the room temperature.
6) vacuumize dry 25min to the micelle graininess that is white in color.
7) add 10 μ l enzyme liquid in every pipe, place 4 ℃ to keep 1h, suck unnecessary enzyme liquid.
8) the EP pipe is inverted, 37 ℃ keep 12-14h, make protein carry out complete degestion.
9) add 30 μ l peptide section extract I in every pipe, 37 ℃ keep 1h, get supernatant to new pipe.
10) add 30 μ l peptide section extract II in the EP pipe that contains micelle, 30 ℃ keep 1h, get in the pipe that contains supernatant of supernatant to the step 9.
11) the resulting extract that contains the peptide section is carried out about vacuum drying 1h, extremely remaining liquid is 10 μ l.
12) 0.8 μ l peptide section extract is added on the sample target, behind sample drying, repeats application of sample once.
13) add 0.5 μ l matrix at the nearly sample of doing.
14) clean target position with the 1 ‰ TFA washing lotions of 1 μ l, keep 30S.Repeat twice.
15) behind the sample bone dry, place Bruker Daltonics Autoflex to analyze sample panel, obtain the peptide mass fingerprinting of protein sample, be i.e. the quality distribution diagram of all peptide sections of enzymolysis protein.
Identification of Fusion Protein: according to the peptide mass fingerprinting of the protein sample that obtains (quality (or molecular weight) distribution plans of all peptide sections of the enzymolysis protein that namely obtains by Mass Spectrometer Method), then the MASCOT software by localization carries out the protein retrieval in database [NCBInr 20100116] and SP, and setup parameter is: Max Mwassed Cleavages:1; FixedModifications; Carboxymethyl (C); Vaviable Modifications:Oxidation(M); Mass values:MH+, Monowasotopic; Peptide MassTolerance: ± 0.2Da; Criterion: each albumen has 10 result for retrieval, and when albumen mark (Protein Score C.I.%) numerical value reaches more than 95, score was greater than 85 minutes as a result, and namely P＜0.05 illustrates that Search Results is credible.
The protein that table 1 sinapsis alba arid is processed high expressed in the seedling leaves tissue and obtained identifying
In addition, G12, G14, G18, G19, G21 do not search the data result that is complementary with it, therefore infer that these may be some new relevant albumen of undiscovered and plant drought resistance; This has just established important basis for finding that finally relevant excellent genes and agricultural thereof are used.
Three, RT one PCR of differentially expressed protein and WesternBlot checking
The reliability of the differential protein that filters out for preliminary identification 2-DE-MS, the present invention has carried out preliminary sxemiquantitative PCR checking, and its result is consistent with mass spectral:mass spectrographic result basically.Choose several albumen and carried out western Blot checking, the relevant differentially expressed protein of its presentation of results 2-DE-MS screening sinapsis alba drought resisting is feasible, reliable results.
Foregoing description only proposes as the enforceable technical scheme of the present invention, not as the Single restriction condition to its technical scheme itself.
Claims (10)
1. use the method for proteomic techniques screening plant drought correlation candidate gene, it is characterized in that:
A, get arid processed group and the processed group plant plant leaf that normally waters respectively, at first utilize two dimensional gel electrophoresis to seek differential protein, tentatively obtain the candidate gene relevant with plant drought;
B, by the Mass Spectrometric Identification technology gained differential protein is carried out multiple sieve again, further the candidate gene relevant with plant drought obtained in screening.
2. application proteomic techniques according to claim 1 screens the method for plant drought correlation candidate gene, and it is characterized in that: the concrete operation step of steps A comprises:
A-1, vegetable material and leaf protein extract: get respectively arid processed group and the processed group plant plant leaf that normally waters, the protein example in the blade is obtained in liquid nitrogen grinding, centrifugal, cracking processing;
A-2, two dimensional gel electrophore-sis are processed: gained protein example of upper step is carried out respectively two dimensional gel electrophore-sis process, isoelectric focusing adopts the prefabricated IPG adhesive tape of pH4-7 to carry out, after finishing, electrophoresis takes off gel until second, put into the immobile liquid room temperature fixedly more than the 4h, then put into coomassie brilliant blue staining liquid and carry out saturated dyeing, utilize clear water to clean gel and namely carry out gel images scanning afterwards, obtain arid and process blade and the albumen two dimensional gel electrophore-sis figure that normally waters wiper blade;
A-3, interpretation of result: gained two dimensional gel electrophore-sis figure carried out statistical study to the upper step, utilize one-way analysis of variance, to the ratio of control group and processed group protein expression amount carry out take 2 as the end to number conversion, under Xuan Ze Zai ∣ log2expression ratio ∣＞=1, P＜0.05 condition control group and processed group protein expression profile are carried out statistical analysis, namely obtain differential protein; And further determine to process in arid the protein of high expressed in the blade, namely preliminary screening goes out the candidate gene relevant with plant drought.
3. application proteomic techniques according to claim 2 screens the method for plant drought correlation candidate gene, and it is characterized in that: the concrete operation step of steps A-1 is:
A, get vegetable seeds and in culturing room, carry out vernalization after, the PEG6000 concentration for the treatment of is 20%, be treated to contrast with clear water, choosing through concentration is that plant seedlings blade that 20% PEG6000 processed the 9th day is testing for control group carries out the protein differential expression of experimental group and the plant seedlings blade that is left intact;
B, plant seedlings get experimental group and control group plant leaf simultaneously after the processing of PEG6000 simulating drought finishes, after distilled water is rinsed well, sop up excessive moisture with filter paper, place rapidly liquid nitrogen cooling packing mark after, place-80 ℃ of refrigerators to save backup;
C, in liquid nitrogen, fully grind blade, the extract that adds 3 times of sample volumes spends the night under-20 ℃ condition, then 4 ℃ of 8000rpm above centrifugal 1 hour, abandon supernatant, add isopyknic ice bath acetone that contains 0.07% beta-mercaptoethanol, shook up rear 4 ℃ of 8000rpm above centrifugal 1 hour, then the vacuum drying precipitation is for subsequent use;
Add lysate before d, the loading, room temperature was placed 30 minutes, made albumen fully be dissolved in the lysate, then above centrifugal 1 hour of 15 ℃ of 8000rpm or longer time, not to be precipitated as standard, be kept at 4 ℃ temporarily, with Brandford standard measure albumen, then packing put into-80 ℃ for subsequent use;
4. application proteomic techniques according to claim 3 screens the method for plant drought correlation candidate gene, and it is characterized in that: the extract among the step c is: the acetone that contains the beta-mercaptoethanol of 10%TCA and 0.07%; The configuration proportion of lysate is in the steps d: 2.7g urea 0.2gCHAPS is dissolved in the deionized water of 3ml sterilization, and final volume is 5ml, adds the DTT65ul/ml of 1M before the use again.
5. application proteomic techniques according to claim 1 screens the method for plant drought correlation candidate gene, it is characterized in that: the protein sample that steps A obtains has carried out respectively dielectrophoresis and coomassie brilliant blue staining to be processed, and by analyzing acquisition differential protein particle, on this basis, the concrete operation step of step B is: at first downcut the differential protein particle from gel, then carry out mass spectrophotometry and Identification of Fusion Protein.
6. application proteomic techniques according to claim 5 screens the method for plant drought correlation candidate gene, and it is characterized in that: the concrete operation step of described mass spectrophotometry comprises:
The gel that 1) will carry out mass spectrophotometry cleans 5min/ time * 5 times with ultrapure water;
2) cut 1000 μ l Eppondorf rifle heads tip according to the protein spots concentration that will analyze and size, use the rifle head that cuts from above-mentioned gel, to cut the protein spots that to analyze and put into 1.5ml EP pipe;
3) add 100 μ l ultrapure waters in every pipe, utilize shaking table concussion washing 15min under the room temperature, suck ultrapure water; Repeat this step twice;
4) add 100 μ l in-gel digestion destainers in every pipe, utilize shaking table concussion decolouring 30min under the room temperature, suck the in-gel digestion destainer; Repeat this step 4 time to micelle and be the water white transparency shape;
5) add 100 μ l acetonitriles in every pipe, shaking table concussion dehydration 10min sucks acetonitrile under the room temperature;
6) vacuumize dry 25min to the micelle graininess that is white in color;
7) add 10 μ l enzyme liquid in every pipe, place 4 ℃ to keep 1h, suck unnecessary enzyme liquid;
8) the EP pipe is inverted, 37 ℃ keep 12-14h, make protein carry out complete degestion;
9) add 30 μ l peptide section extract I in every pipe, 37 ℃ keep 1h, get supernatant to new pipe;
10) add 30 μ l peptide section extract II in the EP pipe that contains micelle, 30 ℃ keep 1h, get in the pipe that contains supernatant of supernatant to the step 9;
11) the resulting extract that contains the peptide section is carried out about vacuum drying 1h, extremely remaining liquid is 10 μ l;
12) 0.8 μ l peptide section extract is added on the sample target, behind sample drying, repeats application of sample once;
13) add 0.5 μ l matrix at the nearly sample of doing;
14) clean target position with the 1 ‰ TFA washing lotions of 1 μ l, keep 30S; Repeat twice;
15) behind the sample bone dry, place Bruker Daltonics Autoflex to analyze sample panel, obtain the peptide mass fingerprinting of protein sample, be i.e. the quality distribution diagram of all peptide sections of enzymolysis protein.
7. application proteomic techniques according to claim 5 screens the method for plant drought correlation candidate gene, it is characterized in that: the specific operation process of described Identification of Fusion Protein is: the MASCOT software by localization carries out the protein retrieval in NCBInr 20100116 databases, mate with all known in database protein fingerprints, setup parameter is: MaxMwassed Cleavages:1; Fixed Modifications; Carboxymethyl (C); Vaviable Modifications:Oxidation (M); Mass values:MH+, Monowasotopic; Peptide Mass Tolerance: ± 0.2Da; If the qualification result score was greater than 85 minutes, namely P＜0.05 illustrates that Search Results is credible.
8. the method for application proteomic techniques screening plant drought correlation candidate gene according to claim 1 is characterized in that: further utilize RT one round pcr that the drought resisting candidate gene that filters out is verified after the step B.
9. the method for application proteomic techniques screening plant drought correlation candidate gene according to claim 1 is characterized in that: further utilize the WesternBlot technology that the drought resisting candidate gene that filters out is verified after the step B.
10. the method for each described application proteomic techniques screening plant drought correlation candidate gene according to claim 1-9, it is characterized in that: described plant is sinapsis alba.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103884765A (en) * | 2014-02-21 | 2014-06-25 | 杭州市农业科学研究院 | Method for obtaining two-dimensional electrophoresis difference protein map of chili pepper anther |
| CN105486561A (en) * | 2016-01-14 | 2016-04-13 | 青海省农林科学院 | Broad bean leaf proteomics analysis sample preparation method |
| CN112655502A (en) * | 2020-12-31 | 2021-04-16 | 新疆农垦科学院 | Method for screening cotton drought-resistant related gene |
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| CN103884765B (en) * | 2014-02-21 | 2015-12-09 | 杭州市农业科学研究院 | The acquisition methods of pepper anther dielectrophoresis differential protein collection of illustrative plates |
| CN105486561A (en) * | 2016-01-14 | 2016-04-13 | 青海省农林科学院 | Broad bean leaf proteomics analysis sample preparation method |
| CN112655502A (en) * | 2020-12-31 | 2021-04-16 | 新疆农垦科学院 | Method for screening cotton drought-resistant related gene |
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