CN103278632B - ELISA kit for fast detection of corn phosphoenolpyruvate carboxylase (PEPC) gene transgenic crop and use thereof - Google Patents
ELISA kit for fast detection of corn phosphoenolpyruvate carboxylase (PEPC) gene transgenic crop and use thereof Download PDFInfo
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Abstract
The invention discloses an ELISA kit for fast detection of a corn phosphoenolpyruvate carboxylase (PEPC) gene transgenic crop and a use thereof, and belongs to the technical field of agricultural science. Through specific monoclonal and polyclonal antibodies of a corn PEPC protein, the ELISA kit for fast detection of the corn PEPC protein and a detection method are designed. The ELISA kit built based on the detection method has the characteristics of use convenience, simple operation, fast and sensitive detection and accurate and reliable result. The ELISA kit can fast detect if the corn PEPC protein exists under the laboratory conditions, can be used for qualitative analysis of a PEPC protein in a corn PEPC gene transgenic crop such as paddy rice, wheat and tobacco, and still has the effect after being preserved at a temperature of -20 DEG C for 12 months.
Description
Technical field
The present invention relates to a kind of detect corn phosphoric acid enol pyruvic acid carboxylase (PEPC) fast ELISA kit and in application agriculturally, belong to technical field of agriculture science.
Background technology
Phosphoric acid enol pyruvic acid carboxylase gene is the C such as corn
4plant can carry out efficient photosynthesis, especially at low CO
2one of photosynthetic key gene is carried out under concentration and intense light irradiation.Can obtain at photosynthetic efficiency, light saturation point, low CO after utilizing the phosphoric acid enol pyruvic acid carboxylase gene of corn (PEPC) rice transformation
2the physical signs aspects such as concentration offsets point are greatly improved, and in transfer-gen plant (KuMSB, etal, 1999,2001 that the Major Economics such as the effective fringe of individual plant, total grain panicle number, mass of 1000 kernel and single plant yield increase substantially than original parent; Chi Wei etc., 2001; JiaoDM, etal, 2001,2002,2003,2007; ZhangF, 2003; Chen Xu is clear, and 2004; Li Jihang etc., 2006; He Libin etc., 2006; Li Xia etc., 2003,2005,2007; Yuan Dingyang etc., 2007; BandyopadhyayA, etal, 2007; Zhang Bianjiang etc., 2008,2009; LauraM, etal, 2008; Li Yan, 2009).There are some researches show, utilize C
4one of photosynthesis key gene of type (as corn, Chinese sorghum etc.)---phosphoric acid enol pyruvic acid carboxylase gene (PEPC) improves the C such as paddy rice, wheat
3plant has potential important economy and social effect.
After foreign gene proceeds to receptor species, three kinds are mainly contained to the detection method of target gene: (1) biological test method; (2) DNA detects; (3) immune detection of albumen.In these three kinds of detection methods, first method is more loaded down with trivial details and workload large, and labor cost is higher, and measurement result deviation is large, is difficult to carry out horizontal and vertical comparison; Second method is whether testing goal gene exists, and whether do not represent gene and express, actual directive significance is little.Immunologic detection method is convenient, simple, practical, economical, and sensitivity is higher, and can the expression of Direct Identification gene outcome, and the whole operating process of double fastener heart euzymelinked immunosorbent assay (ELISA) only needs 5 hours.
Along with more and more to concern and the further investigation of pepc gene (GenBank accession number E17154), the biological information of gene itself is utilized just to seem more and more important to improve seed selection transgenic progeny material.But, the research of relevant pepc genes all at present mainly concentrates on the detection of the physiological and biochemical index of transgenic progeny and utilizes the nucleotide information of pepc gene design primer to carry out the integration and expression of analysis purpose gene, yet there are no the method report detecting this gene expression fast.Utilize the expressing protein of pepc gene to obtain narrow spectrum antibody as antigen to make ELISA kit and can detect in transgenic progeny plant, whether pepc gene is expressed, for breeding man provides testing result fast.
Summary of the invention
Technical matters
The object of this invention is to provide a kind of ELISA kit detecting corn phosphoric acid enol pyruvic acid carboxylase gene (PEPC) expressing protein fast.
Technical scheme
Corn phosphoric acid enol pyruvic acid carboxylase gene (PEPC) expressing protein ELISA kit of the present invention comprises:
1) 96 hole ELISA Plate: wrap by the ELISA Plate of corn PEPC protein polyclone antibody (wrapping by concentration 1:1600-1:3200); The preparation process of corn PEPC protein polyclone antibody: the new zealand white rabbit at corn PEPC protein immunization 3 monthly age, Culling heart blood after 4 immunity, centrifugal, get serum.
2) corn PEPC protein monoclonal antibody: the female BAl BIc/c mouse in 8 week age of corn PEPC protein immunization, obtains the hybridoma 4E5 that preserving number is CCTCCNO:C2012199; Corn PEPC protein immunization BALB/c mouse obtains hybridoma 4E5 monoclonal antibody (work tire 1:400-1:800)
3) ELIAS secondary antibody: sheep anti-mouse igg that horseradish peroxidase (HRP) marks (work tire 1:5000-1:10000);
4) negative controls: the rice protein extract not turning corn pepc gene.
5) sample treatment liquid: 50mmolL
-1tris-HCL, pH7.5,1mmolL
-1mgCl
2, 5mmolL
-1dTT, 2%PVP, 10% glycerine;
6) dilution: phosphate buffer (PBS) (0.01M, pH7.5);
7) cleansing solution: phosphate Tween buffer (PBST) (adding 0.5mLTween-20 in 1000mL0.01MPBS);
8) substrate solution:
Substrate buffer solution: Na
2hPO
412H
2o0.2g; NaH
2pO
42H
2o6.84g, distilled water 500mL; 4 DEG C save backup;
TMB mother liquor: TMB40mg; DMSO5mL; Lucifuge 4 DEG C saves backup;
The volume ratio 1%H of distilled water preparation
2o
2.
The using method of above-mentioned corn pepc gene expressing protein ELISA kit is:
1) by the plant sample of 0.1g be added with precooling 1mL sample treatment liquid mortar in grind, ambient temperatare puts the centrifugal 5min of about 5min, 10000rpm, gets supernatant 100 μ L and adds in a hole of ELISA Plate, hatch 60min for 37 DEG C;
2) cleansing solution washes plate 3 times, each 3-5min;
3) add the monoclonal antibody of 100 μ L working concentrations, hatch 90min for 37 DEG C;
4) cleansing solution washes plate 3 times, each 3-5min;
5) add the ELIAS secondary antibody of 100 μ L working concentrations, hatch 90min for 37 DEG C;
6) cleansing solution washes plate 5 times, each 3-5min;
7) substrate solution of new preparation is added: the volume ratio 1%H of 12mL substrate buffer solution+120 μ LTMB mother liquor+48 μ L distilled water preparation
2o
2, lucifuge effect 10min;
8) 2mol/LH is added
2sO
4stop buffer 50 μ L, puts into microplate reader Gen5 software and carries out OD by ELISA Plate
450nmreading Analysis;
9) result judges: the OD of measuring samples
450nmvalue is P, negative control OD
450nmvalue is N, and be judged to the positive when P/N>=2.1, be judged to suspicious during 1.5≤P/N < 2.1, P/N < 1.5 is judged to feminine gender.
Accompanying drawing illustrates:
Fig. 1 carries out specificity analyses result for utilizing Western-blot to monoclonal antibody.Show that the monoclonal anti physical efficiency obtained is combined with the PEPC protein-specific that pepc gene (GenBank accession number E17154) expresses 109kD.(Fig. 1) 50kD place is the fragment of PEPC protein degradation.
Fig. 2 carries out individual plant PCR testing result for utilizing pepc gene feature SSR primer (being numbered MP35).Marker, 117209,117267,117272,117275,9311 is from left to right followed successively by 6 swimming lanes.SSR primer MP35(wherein used is shown in " rice in China science ", 2007,21(1): 25-30) sequence is: 5 ' AAGCAGGGAAGCGAGACG3 ' and 5 ' GATTGCCGCCAGCAGTAG3 ' (synthesis of the handsome biological company limited in Shanghai, PAGE purifying); PCR response procedures is: 94 DEG C, 5min; 94 DEG C of 40s, 58 DEG C of 40s, 72 DEG C of 40s, 32 circulations; 72 DEG C, 10min.Pcr amplification product adopts 3% agarose (the original-pack reagent of Amerisco company) gel electrophoresis, often pipe pcr amplification product adds the bromophenol blue mixing of 5 μ L, click and enter 3% Ago-Gel containing ethidium bromide (EB), in 1 × tbe buffer liquid, constant voltage 120V electrophoresis is about 60min, take out gel, imaging, photo archive on BIO-RADQuantity One gel imaging system.
Beneficial effect
The present invention utilizes the expressing protein of pepc gene to obtain narrow spectrum antibody as antigen and makes ELISA kit and can detect in transgenic progeny plant, whether pepc gene is expressed, for breeding man provides testing result fast.
When within 2012, utilizing this kit to carry out ELISA kit analysis to the 325 strain positive plants that pepc gene PCR detects, find that there is 252 strains and show as the positive, the doubtful positive strain of 6 strain and the negative strain of 67 strains; Its positive rate is that 77.54%(the results are shown in embodiment 2 and table 3), be wherein 44.68% in the positives rate of the colony being numbered 56, and be 100% in the positives rate of the colony being numbered 78,82 and 84.
Biological deposits
Hybridoma cell strain 4E5, China typical culture collection center, preserving number is CCTCCNO:C2012199, and the date is on Dec 26th, 2012, address: Wuhan University's preservation center, Wuchang District, Wuhan City, Hubei Province.
Specific implementation method
Corn phosphoric acid enol pyruvic acid carboxylase gene (PEPC) expressing protein ELISA kit of the present invention comprises:
1) 96 hole ELISA Plate: wrap by the ELISA Plate of corn PEPC protein polyclone antibody (wrapping by concentration 1:1600-1:3200);
The preparation process of corn PEPC protein polyclone antibody: corn PEPC albumen is (purchased from Nanjing Hua Ze Bioisystech Co., Ltd, No. CAS is 9067-77-0) new zealand white rabbit at immune 3 monthly ages, get PEPCase and equivalent Freund's complete adjuvant fully mixes emulsification, the subcutaneous multi-point injection of neck, every 1mg; Second time immunity after 2 weeks, get PEPCase and equivalent not formula Freund's incomplete adjuvant fully mix emulsification, the subcutaneous multi-point injection of neck, every 1mg; Third time immunity after 2 weeks again, get PEPCase and equivalent not formula Freund's incomplete adjuvant fully mix emulsification, the subcutaneous multi-point injection of neck, every 1mg; 4th immunity after 2 weeks, gets the injection of PEPCase auricular vein, every 2mg.After last immunity, 7d Culling heart blood obtains antiserum.
2) corn PEPC protein monoclonal antibody: corn PEPC protein immunization BALB/c mouse obtains hybridoma 4E5, the monoclonal antibody prepared with hybridoma 4E5 (work tire 1:400-1:800).
The preparation process of corn PEPC monoclonal antibody:
1. female BAl BIc/c the mouse in 8 week age of corn PEPC protein immunization, first immunisation, gets PEPCase and equivalent Freund's complete adjuvant fully mixes emulsification, subcutaneous abdomen multi-point injection, every only 50 μ g; Second time immunity after 2 weeks, get PEPCase and equivalent not formula Freund's incomplete adjuvant fully mix emulsification, subcutaneous abdomen multi-point injection, every 50 μ g; Third time immunity after 2 weeks again, get PEPCase and equivalent not formula Freund's incomplete adjuvant fully mix emulsification, subcutaneous abdomen multi-point injection, every 50 μ g; 4th immunity after 2 weeks, gets PEPCase lumbar injection again, every only 100 μ g.To dock after last immunity blood sampling, detect antiserum titre.After 3 days, select high mouse of tiring to get spleen cell, merge through 50%PEG with murine myeloma cell, obtain hybridoma 4E5;
2.: after 7d, to swap out 1/2 nutrient culture media with the HT nutrient culture media of 15%FCS.After 14d, use the HT complete medium of 15%FCS instead, when Growth of Cells to hole floorage 1/5 and supernatant turn yellow time, adopt indirect ELISA method detect hybridoma supernatant;
3.: select the hybridoma cell line with positive reaction, utilize limiting dilution assay through 3 subclones, monoclonal antibody hybridoma cell strain 4E5 is obtained.By BALB/c mouse lumbar injection whiteruss, 10d pneumoretroperitoneum inoculation monoclonal antibody hybridoma cell 4E5, gather ascites after about 10d, every mouse can adopt 5-10mL ascites usually, centrifugal, removes cell component and other sediments, gets supernatant.
3) ELIAS secondary antibody: sheep anti-mouse igg that horseradish peroxidase (HRP) marks (work tire 1:5000-1:10000);
4) negative controls: the rice protein extract not turning corn pepc gene.
5) sample treatment liquid: 50mmolL
-1tris-HCL, pH7.5,1mmolL
-1mgCl
2, 5mmolL
-1dTT, 2%PVP, 10% glycerine;
6) dilution: phosphate buffer (PBS) (0.01M, pH7.5);
7) cleansing solution: phosphate Tween buffer (PBST) (adding 0.5mL Tween-20 in 1000mL0.01MPBS);
8) substrate solution:
Substrate buffer solution: Na
2hPO
412H
2o0.2g; NaH
2pO
42H
2o6.84g, distilled water 500mL; 4 DEG C save backup;
TMB mother liquor: TMB40mg; DMSO5mL; Lucifuge 4 DEG C saves backup;
The volume ratio 1%H of distilled water preparation
2o
2.
The using method of corn pepc gene expressing protein ELISA kit is:
1) by the plant sample of 0.1g be added with precooling 1mL sample treatment liquid mortar in grind, ambient temperatare puts the centrifugal 5min of about 5min, 10000rpm, gets supernatant 100 μ L and adds in a hole of ELISA Plate, hatch 60min for 37 DEG C;
2) cleansing solution washes plate 3 times, each 3-5min;
3) add the monoclonal antibody of 100 μ L working concentrations, hatch 90min for 37 DEG C;
4) cleansing solution washes plate 3 times, each 3-5min;
5) add the ELIAS secondary antibody of 100 μ L working concentrations, hatch 90min for 37 DEG C;
6) cleansing solution washes plate 5 times, each 3-5min;
7) substrate solution of new preparation is added: the volume ratio 1%H of 12mL substrate buffer solution+120 μ LTMB mother liquor+48 μ L distilled water preparation
2o
2, lucifuge effect 10min;
8) lucifuge effect 10min;
9) 2mol/LH is added
2sO
4stop buffer 50 μ L, puts into microplate reader Gen5 software and carries out OD by ELISA Plate
450nmreading Analysis;
10) result judges: the OD of measuring samples
450nmvalue is P, negative control OD
450nmvalue is N, and be judged to the positive when P/N>=2.1, be judged to suspicious during 1.5≤P/N < 2.1, P/N < 1.5 is judged to feminine gender.
Embodiment 1
First carry out Western-blot analysis to PEPC albumen, result shows being combined with the PEPC protein-specific that pepc gene (GenBank accession number E17154) expresses 109kD by monoclonal anti physical efficiency prepared by hybridoma 4E5 of acquisition.(Fig. 1) 50kD place is the fragment of PEPC protein degradation.
Utilize the positive kind 06D351(Yuan Ding sun of the paddy rice containing corn pepc gene marker-free introduced from Hunan Research Centre for Hybrid Rice etc., cotransformation method obtain screening marker-free turn PEPC, PPDK trans-genetic hybrid rice restorer homozygote, " hybrid rice ", 2007,22(2): 57-63) be donor, 9311(has another name called and raises rice No. 6 " hybrid rice ", 2001,16(6): 13-15) for acceptor carries out backcross transformation, wherein 117209,117267,117272 and 117275 is backcross transformation BC
4f
3the stable individual plant through PCR test positive (PCR testing result is shown in Fig. 2) in colony.Pepc gene paddy rice ELISA kit testing result (table 1) shows, and 117209,117267 is positive, and 117272,117275 is negative.Designing the electrophoresis result that SSR primer carries out pcr analysis to compare with utilizing pepc gene, in the rice varieties of 117272 and 117275 turns of pepc genes, not expressing PEPC albumen.Table 1 is for utilizing the result of ELISA kit detecting portion backcross transformation pepc gene of the present invention.
Table 1 part PEPC transgenic rice ELISA kit testing result
Embodiment 2
First corn pepc gene characteristic primer MP35(" rice in China science " is utilized, 2007,21(1): 25-30) to different generations (F
2, BC
1f
2, F
3and F
4colony, these colonies utilize the sun of the transfer-gen plant 06D351(Yuan Ding containing corn pepc gene etc. introduced from Hunan Research Centre for Hybrid Rice, cotransformation method obtain screening marker-free turn PEPC, PPDK trans-genetic hybrid rice restorer homozygote, " hybrid rice " 2007,22(2): 57-63) be male parent; 9311(has another name called and raises rice No. 6 " hybrid rice ", 2001,16(6): 13-15), peaceful extensive 108(" Chinese rice ", 2006,6:19-20) and Xu rice No. 7 (" North Japonica Rice ", 2010,40(4): 66-67) for maternal carry out hybridizing backcross after colony's individual plant of building carry out pcr analysis, result has 325 individual plants to be positive strain, then utilizes this ELISA kit to carry out detecting and judging according to standard.Wherein " numbering " is expressed as the field numbering of this individual plant; " mean value " measures 3 times average for same sample; The ratio that " P/N " is sample and negative control.
To the hybridization of the PCR Molecular Detection positive, the F of backcross transformation pepc gene
2, BC
1f
2, F
3and F
4colony's individual plant (totally 325 individual plant samples) carries out elisa assay, and result shows, and in 325 strain samples, has 252 strains to be positive individual plant, the doubtful positive strain of 6 strain and the negative strain of 67 strains; Its positive rate is that 77.54%(the results are shown in Table 3), be wherein 44.68% in the positives rate of the colony being numbered 56, and be 100% in the positives rate of the colony being numbered 78,82 and 84.
Table 3 detects the ELISA kit testing result of positive individual plant through PCR
Embodiment 3
Kit storage life is tested
Kit preservation condition is-20 DEG C, and measure after preserving 1,2,4,6 and 12 month, kit still can obtain obvious testing result (the results are shown in Table 2).
Table 2 storage life is tested
P is the OD of measuring samples 2 parallel holes
450nmmean value; N is the OD of negative control 93112 parallel holes
450nmmean value.
Claims (2)
1., for detecting an ELISA kit for corn pepc gene expressing protein fast, comprising:
1) 96 hole ELISA Plate: wrap by the ELISA Plate of corn phosphoric acid enol pyruvic acid carboxylase gene PEPC protein polyclone antibody, wrap by concentration 1:1600-1:3200;
2) corn PEPC protein monoclonal antibody: monoclonal antibody prepared by the mouse hybridoma 4E5 being CCTCC2012199 by preserving number;
3) ELIAS secondary antibody: the sheep anti-mouse igg that horseradish peroxidase HRP marks, work the 1:5000-1:10000 that tires;
4) negative controls: the rice protein extract not turning corn pepc gene;
5) sample treatment liquid: pH7.5,50mmolL
-1tris-HCL, 1mmolL
-1mgCl
2, 5mmolL
-1dithiothreitol (DTT) DTT, mass ratio 2% polyvinylpyrrolidone PVP, mass ratio 10% glycerine;
6) dilution is phosphate buffer PBS0.01M, pH7.5;
7) cleansing solution is add 0.5mLTween-20 in phosphate Tween buffer PBST:1000mL0.01MPBS;
8) substrate solution:
Substrate buffer solution: Na
2hPO
412H
2o0.2g; NaH
2pO
42H
2o6.84g, distilled water 500mL; 4 DEG C save backup;
TMB mother liquor: TMB40mg; DMSO5mL; Lucifuge 4 DEG C saves backup;
The volume ratio 1%H of distilled water preparation
2o
2.
2. the using method of corn pepc gene expressing protein ELISA kit described in claim 1, comprising:
1) by the plant sample of 0.1g be added with precooling 1mL sample treatment liquid mortar in grind, ambient temperatare puts the centrifugal 5min of about 5min, 10000rpm, gets supernatant 100 μ L and adds in a hole of ELISA Plate, hatch 60min for 37 DEG C;
2) cleansing solution washes plate 3 times, each 3-5min;
3) add the monoclonal antibody of 100 μ L working concentrations, hatch 90min for 37 DEG C;
4) cleansing solution washes plate 3 times, each 3-5min;
5) add the ELIAS secondary antibody of 100 μ L working concentrations, hatch 90min for 37 DEG C;
6) cleansing solution washes plate 5 times, each 3-5min;
7) substrate solution of new preparation is added: the volume ratio 1%H of 12mL substrate buffer solution+120 μ LTMB mother liquor+48 μ L distilled water preparation
2o
2, lucifuge effect 10min;
8) stop buffer 2mol/LH is added
2sO
450 μ L, put into microplate reader Gen5 software and carry out OD by ELISA Plate
450nmreading Analysis;
9) result judges: the OD of measuring samples
450nmvalue is P, negative control OD
450nmvalue is N, judges that in sample, corn pepc gene expressing protein is as negative or positive by P/N value.
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| CN105063025A (en) * | 2015-07-24 | 2015-11-18 | 江苏省农业科学院 | Specific SSR marker of corn phosphoenolpyruvate carboxylase gene and application of specific SSR marker |
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| CN101492689A (en) * | 2009-03-04 | 2009-07-29 | 河南省农业科学院 | Corn C4 phosphoric acid enol pyruvic acid carboxylase gene and uses in producing wheat |
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