CN102080128A - Application of methylation sensitive amplification polymorphism technique in toxicity analysis of transgenic plants - Google Patents
Application of methylation sensitive amplification polymorphism technique in toxicity analysis of transgenic plants Download PDFInfo
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Abstract
The invention relates to application of a methylation sensitive amplification polymorphism technique in toxicity analysis of transgenic plants, in particular provides a method for analyzing and predicting non-expected toxicities of the transgenic plants, which comprises the following steps of: 1 acquiring methylation sensitive amplification polymorphism difference segments of the transgenic plants; 2 recovering, cloning and sequencing methylation difference DNA segments of the transgenic plants; and 3 analyzing the non-expected toxicities of the transgenic plants by utilizing the methylation difference DNA segments of the transgenic plants. In the invention, the methylation sensitive amplification polymorphism technique is used for analyzing the non-expected toxicities of the transgenic plants so that a technical system for analyzing whether the transgenic plants have the non-expected toxicities or not is established, and therefore, the invention contributes to monitoring and generalization application of the transgenic plants and has important economic and social benefits.
Description
(1) technical field
The present invention's responsive amplification polymorphism technology that will methylate is used to analyze the unexpected toxicity of transgenic plant, belongs to molecular biology and biological technical field.
(2) background technology
The a plurality of foreign genes of normal importing are the complex character transgenic plant in a new generation transgenic plant.Between the foreign gene, may have mutual work between foreign gene and the plant endogenous gene of wild-type (acceptor), make some methylated dna sequence dna demethylations in transfer-gen plant of new generation in wild-type (acceptor) plant, and then the gene in these dna sequence dnas is expressed in transfer-gen plant of new generation, make to occur unexpected toxicity in the transgenic plant of new generation, so be necessary to set up the unexpected toxic analyzing and predicting method of transgenic plant.The responsive amplification sheet polymorphism technology that methylates is improved on the amplified fragment length polymorphism technical foundation of inventions such as nineteen ninety-five Zabeau.Since this technology in 1999 was proved a kind of reliable detection DNA Methylation in Plants method by Xiong etc., the responsive amplification polymorphism technology that methylates had been widely used in the research of various plants such as paddy rice, cotton.But the responsive amplification polymorphism technology that will not methylate as yet both at home and abroad at present is used to analyze transgenic plant and whether has unexpected toxicity.
The inventor's responsive amplification polymorphism technology that will methylate is used to analyze the unexpected toxicity of transgenic plant, set up and whether had unexpected toxic technical system in the analysis transfer-gen plant, help the monitoring of transgenic plant and apply, have important economic benefit and social benefit.
(3) summary of the invention
The present invention relates to methylate responsive amplification polymorphism technology in the application of transgenic plant oxicity analysis, specifically provide a kind of transgenic plant unexpected toxic analyzing and predicting method, this method may further comprise the steps:
1, obtains the transfer-gen plant responsive polymorphism differential fragment that methylates
Extract the genomic dna of transfer-gen plant and wild-type (acceptor) respectively.To genomic dna carry out enzyme cut and jointing after, increase in advance.With pre-expansion volume increase thing is template, carries out the selectivity pcr amplification, and selectivity PCR product is carried out denaturing polyacrylamide gel electrophoresis.On denaturing polyacrylamide gel, take the methylation differential DNA fragment of transfer-gen plant.
2, the segmental recovery of transfer-gen plant methylation differential DNA, clone and order-checking
3, utilize the unexpected toxicity of transfer-gen plant methylation differential DNA fragment analysis transfer-gen plant
According to sequencing result, utilize the related pathways metabolism of these differential fragments of KEGG database analysis, analyze in the transfer-gen plant whether have unexpected toxicity.
According to above-mentioned technical system, after extracting the genomic dna of transfer-gen plant and wild-type (acceptor), can analyze whether have unexpected toxicity in the transfer-gen plant, help the monitoring of transfer-gen plant and apply, have important economic benefit and social benefit.
(4) description of drawings
Fig. 1 is the part picture that transfer-gen plant methylates and takes after responsive polymorphic bands separates on denaturing polyacrylamide gel:
The responsive polymorphic bands of the part methylization that on behalf of the transfer-gen plant genomic dna, H1 obtain behind EcoR I+Hpa II double digestion,
The responsive polymorphic bands of the part methylization that on behalf of the transfer-gen plant genomic dna, M1 obtain behind EcoR I+Msp I double digestion,
The responsive polymorphic bands of the part methylization that on behalf of non-transgenic plant genomic dna, H2 obtain behind EcoR I+Hpa II double digestion,
The responsive polymorphic bands of the part methylization that on behalf of non-transgenic plant genomic dna, M2 obtain behind EcoR I+Msp I double digestion,
The arrow indication is the responsive polymorphism differential fragment of part methylization,
S4, S6, S7, S12, S13, S15, S17, S23 are the responsive polymorphism differential fragment of part methylization title.
Fig. 2 be the part transfer-gen plant methylate responsive polymorphism differential fragment reclaim from denaturing polyacrylamide gel after the result of amplification again:
S4, S6, S7, S12, S13, S15, S17, S23 are the responsive polymorphism differential fragment of part methylization,
M is a dna molecular amount mark.
(5) concrete invention embodiment
Embodiment 1: the methylate acquisition of susceptibility polymorphism differential fragment of transfer-gen plant
1, the extraction of transfer-gen plant genomic dna
Under identical cultivation condition, cultivate and change fhy3, gus gene Arabidopis thaliana and wild-type Arabidopis thaliana seedling, adopt traditional CTAB method to extract its genomic dna respectively.
2, the enzyme of transfer-gen plant genomic dna is cut and is connected
Entrust the synthetic following primer sequence of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd: EcoR I joint 1 (5 '-CTCGTAGACTGC GTACC-3 '), EcoR I joint 2 (5 '-AATTGGTACGCAGTC-3 '), MspI/Hpa II joint 1 (5 '-GATCATGAGTC CTGCT-3 '), Msp I/Hpa II joint 2 (5 '-CGAGCAGGACTCATGA-3 ').Behind EcoR I joint 1 and EcoR I joint 2 usefulness dissolved in distilled water and mixing, place 95 ℃ of 5min, in the room temperature cooling, make 5pmol EcoR I joint then.Behind Msp I/Hpa II joint 1 and MspI/Hpa II joint 2 usefulness dissolved in distilled water mixings, place 95 ℃ of 5min, in the room temperature cooling, make 50pmol MspI/Hpa II joint then.Choose two coordination enzyme Hpa II/Msp I and make up with restriction enzyme EcoR I respectively, to changeing fhy3, gus gene Arabidopis thaliana and wild-type arabidopsis thaliana genomic dna at 37 ℃ of double digestion 6h to the sensitivity that methylates.The 25 μ l enzyme systems of cutting comprise: 1 * buffer, 1 μ g templet gene group DNA, 1U EcoR I, 1U Hpa II/Msp I.The genomic dna endonuclease bamhi with after 10 μ l are connected the mixture mixing, is spent the night 16 ℃ of connections, then 65 ℃ of insulation 15min termination reactions.10 μ l connect mixture and comprise: 5pmol EcoR I joint, 50pmol Hpa II/Msp I joint, 1U T4 DNA ligase, 1 * buffer for T4 DNA ligase.
3, pre-amplification
Cut with the transgenic arabidopsis and the enzyme of wild-type arabidopsis thaliana genomic dna respectively that to be connected product be template, utilize EcoR I primer (5 '-GACTGCGTACCAATTCA-3 ') and the Hpa II/Msp I primer (5 '-ATCATGAGTCCTGCTCGG-3 ') that increases in advance that increases in advance to increase in advance.25 μ l reaction systems comprise 1 * PCR buffer, 0.4mMdNTP, 1.2mM MgCl
2, 2 μ l enzymes are cut the connection product, the 50ng EcoR I primer that increases in advance, the 50ng Hpa II/Msp I primer that increases in advance, 1U Taq polysaccharase.Pre-amplification condition is: behind 94 ℃ of sex change 2min, press 30 circulations of following parameter amplification: 94 ℃ of 30sec, and 56 ℃ of 30sec, 72 ℃ of 60sec, last 72 ℃ are extended 10min.With the dilution proportion pre-expansion volume increase thing of TE buffer by 1: 10.
4, selectivity pcr amplification
Pre-expansion volume increase thing with dilution is a template, utilize EcoR I selective amplification primer (at sequence 5 '-GACTGCGTACCAATTCA-3,3 ' end increase 2-3 base) and Hpa II/Msp I selective amplification primer (in sequence 5,3 ' of-ATCATGAGTCCTGCTCGG-3 ' holds 2-4 base of increase) carry out the selectivity pcr amplification.25 μ l reaction systems comprise 1 * PCR buffer, 0.4mM dNTP, 1.2mM MgCl
2, 4 μ l pre-expansions volume increase thing, 50ng EcoR I selective amplification primer, 50ng Hpa II/Msp I selective amplification primer, 1U Taq polysaccharase.By following parameter PCR circulation: behind 94 ℃ of sex change 2min, take turns circulation: 94 ℃ of 30sec, 65 ℃ of 30sec (every take turns 0.7 ℃ of circulation lapse of temperature), 72 ℃ of 60sec by the amplification 13 earlier of following parameter; Take turns circulation by following parameter amplification 23 at last: 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 60sec.
5, denaturing polyacrylamide gel electrophoresis
Use the order-checking electrophoresis apparatus (Sequi-Gen GT) of Bio-Rad company, the selectivity pcr amplification product is carried out denaturing polyacrylamide gel electrophoresis.Last sample 6 μ l, permanent power 70W electrophoresis 3h.Electrophoresis carries out silver and dyes after finishing.Endonuclease bamhi is carried out MSAP analyze methylation state and the degree that to react restriction enzyme site, the amplified band of EcoR I+Hpa II and EcoR I+Msp I enzyme being cut product is divided into 4 types: type i, EcoR I+Hpa II and two swimming lanes of EcoR I+Msp I all have band, do not have the generation that methylates; Type II, EcoR I+Hpa II swimming lane have the band and EcoR I+Msp I swimming lane does not have band, the single stranded DNA outside methylates; Type-iii, EcoR I+Hpa II swimming lane do not have the band and EcoR I+Msp I swimming lane has band, double-stranded DNA inside methylates; Type i V, EcoR I+Hpa II and two swimming lanes of EcoR I+Msp I all do not have band, and the outside of double-stranded DNA methylates.The banding pattern that compares the two swimming lanes of transgenic arabidopsis and wild-type Arabidopis thaliana EcoR I+Hpa II and EcoR I+Msp I, do not have in the selective analysis transgenic arabidopsis methylate, methylated DNA differential fragment in the wild-type Arabidopis thaliana, take the transfer-gen plant responsive polymorphism differential fragment that methylates with the disposable surgical blade from polyacrylamide gel.
Embodiment 2: the segmental recovery of transfer-gen plant methylation differential DNA, clone and order-checking
1, the segmental recovery of methylation differential DNA
The transfer-gen plant that will the take responsive polymorphism differential fragment that methylates places 30 μ l distilled waters, boils 5min.Slowly reduce to after the room temperature centrifugally, reclaim supernatant liquor.Getting 4 μ l supernatant liquors is that template is carried out pcr amplification, and the PCR reaction conditions is consistent with pre-amplification PCR reaction.Reclaim purification kit (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's product) specification sheets according to DNA the PCR product is reclaimed purifying.
2, the segmental clone of methylation differential DNA
According to connecting test kit (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's product) specification sheets, the PCR product that reclaims purifying is connected to the pUCm-T carrier, transformed into escherichia coli bacterial strain DH5 α.
3, the segmental order-checking of methylation differential DNA
Originally being operated in Shanghai Sangon Biological Engineering Technology And Service Co., Ltd carries out.
Embodiment 3: the unexpected toxic analysis of transfer-gen plant
1, opens the KEGG database, do not have in the input transgenic arabidopsis and methylate and methylated dna sequence dna in the wild-type Arabidopis thaliana, analyze whether synthetic relevant with toxicant of the related pathways metabolism of this sequence.If the related pathways metabolism of sequence is synthetic relevant with toxicant, illustrate that transfer-gen plant may have unexpected toxicity.
2, Southern hybridization checking
Adopt traditional CTAB method, extract transgenic arabidopsis, wild-type arabidopsis thaliana genomic dna respectively.If the related pathways metabolism of transfer-gen plant methylation differential DNA fragments sequence is synthetic relevant with toxicant, after this sequence is marked as probe according to digoxigenin labeled and detection kit (Roche company product) specification sheets, hybridize with transgenic arabidopsis, wild-type arabidopsis thaliana genomic dna respectively.If results of hybridization is positive, illustrate that transfer-gen plant methylation differential DNA fragment is the specificity product of pcr amplification, transfer-gen plant has unexpected toxicity.
Claims (1)
1. unexpected toxic analytical procedure of transgenic plant is characterized in that may further comprise the steps:
(1) the transfer-gen plant acquisition of responsive polymorphism differential fragment that methylates
(1.1) extraction of transfer-gen plant genomic dna
Under identical cultivation condition, cultivate transfer-gen plant and wild-type receptor, adopt traditional CTAB method to extract its genomic dna respectively;
(1.2) enzyme of transfer-gen plant genomic dna is cut and is connected
Synthetic following primer sequence:
EcoR I joint 15 '-CTCGTAGACTGCGTACC-3 ';
EcoR I joint 25 '-AATTGGTACG CAGTC-3 ';
Msp I/Hpa II joint 15 '-GATCATGAGTC CTGCT-3 ';
Msp I/Hpa II joint 25 '-CGAGCAGGAC TCATGA-3 ';
Behind EcoR I joint 1 and EcoR I joint 2 usefulness dissolved in distilled water and mixing, place 95 ℃ of 5min, in the room temperature cooling, make 5pmol EcoR I joint then; Behind Msp I/Hpa II joint 1 and Msp I/Hpa II joint 2 usefulness dissolved in distilled water and mixing, place 95 ℃ of 5min, in the room temperature cooling, make 50pmol Msp I/Hpa II joint then; Choose two the responsive coordination enzyme Hpa II/Msp I that methylate made up with restriction enzyme EcoR I respectively, to the genomic dna of transfer-gen plant and wild-type plant at 37 ℃ of double digestion 6h; The 25 μ l enzyme systems of cutting comprise: 1 * buffer, 1 μ g genomic dna, 1U EcoR I, 1U Hpa II/Msp I; The endonuclease bamhi of genomic dna with after 10 μ l are connected the mixture mixing, is spent the night 16 ℃ of connections, then 65 ℃ of insulation 15min termination reactions; 10 μ l connect mixture and comprise: 5pmol EcoR I joint, 50pmol Hpa II/Msp I joint, 1U T4 DNA ligase, 1 * buffer for T4 DNA ligase;
(1.3) pre-amplification
Cut with the enzyme of transfer-gen plant and wild-type plant genomic dna respectively that to connect product be template, utilize EcoR I primer 5 '-GACTGCGTACCAATTCA-3 ' and Hpa II/Msp I the primer 5 '-ATCATGAGTCCTGCTCGG-3 ' that increases in advance that increases in advance to increase in advance; 25 μ l reaction systems comprise 1 * PCR buffer, 0.4mM dNTP, 1.2mM MgCl
2, 2 μ l enzymes are cut the connection product, the 50ng EcoR I primer that increases in advance, the 50ng Hpa II/Msp I primer that increases in advance, 1U Taq polysaccharase; Pre-amplification condition is: behind 94 ℃ of sex change 2min, take turns circulation by following parameter amplification 30: 94 ℃ of 30sec, and 56 ℃ of 30sec, 72 ℃ of 60sec extend 10min at 72 ℃ at last; With the dilution proportion pre-expansion volume increase thing of TE buffer by 1: 10;
(1.4) selectivity pcr amplification
Increasing production thing with the pre-expansion of diluting is template, utilizes EcoR I selective amplification primer to carry out the selectivity pcr amplification at 3 ' 2-3 base of end increase and the Hpa II/Msp I selective amplification primer of sequence 5 '-GACTGCGTACCAATTCA-3 ' in 2-4 base of 3 ' end increase of sequence 5 '-ATCATGAGTCCTGCTCGG-3 '; 25 μ l reaction systems comprise 1 * PCR buffer, 0.4mM dNTP, 1.2mM MgCl
2, 4 μ l pre-expansions volume increase thing, 50ng EcoR I selective amplification primer, 50ng Hpa II/Msp I selective amplification primer, 1U Taq polysaccharase; Carry out the PCR circulation by following parameter: behind 94 ℃ of sex change 2min, take turns circulation by the amplification 13 earlier of following parameter: 94 ℃ of 30sec, 65 ℃ of 30sec, every 0.7 ℃ of circulation lapse of temperature, 72 ℃ of 60sec of taking turns; Take turns circulation by following parameter amplification 23 at last: 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 60sec;
(1.5) denaturing polyacrylamide gel electrophoresis
Use the order-checking electrophoresis apparatus Sequi-Gen GT of Bio-Rad company, the selectivity pcr amplification product is carried out denaturing polyacrylamide gel electrophoresis; Last sample 6 μ l, permanent power 70W electrophoresis 3h; Electrophoresis carries out silver and dyes after finishing; Endonuclease bamhi is carried out MSAP analyze methylation state and the degree that to react restriction enzyme site, the amplified band of EcoR I+Hpa II and EcoR I+Msp I enzyme being cut product is divided into 4 types: type i, EcoR I+HpaII and two swimming lanes of EcoR I+Msp I all have band, do not have the generation that methylates; Type II, EcoR I+Hpa II swimming lane have the band and EcoR I+Msp I swimming lane does not have band, the single stranded DNA outside methylates; Type-iii, EcoR I+Hpa II swimming lane do not have the band and EcoR I+Msp I swimming lane has band, double-stranded DNA inside methylates; Type i V, EcoR I+Hpa II and two swimming lanes of EcoR I+Msp I all do not have band, and the outside of double-stranded DNA methylates; The banding pattern that compares the two swimming lanes of transfer-gen plant and wild-type plant EcoR I+Hpa II and EcoR I+Msp I, do not have in the selective analysis transfer-gen plant methylate, methylated DNA differential fragment in the wild-type plant, take the transfer-gen plant responsive polymorphism differential fragment that methylates with the disposable surgical blade from polyacrylamide gel;
(2) the segmental recovery of transfer-gen plant methylation differential DNA, clone and order-checking
(2.1) the segmental recovery of methylation differential DNA
The transfer-gen plant that will the take responsive polymorphism differential fragment that methylates places 30 μ l distilled waters, boils 5min; Slowly reduce to after the room temperature centrifugally, collect supernatant liquor; Getting 4 μ l supernatant liquors is that template is carried out pcr amplification, and the PCR reaction conditions is consistent with pre-amplification PCR reaction; Reclaim the purification kit specification sheets according to the DNA of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd the PCR product is reclaimed purifying;
(2.2) the segmental clone of methylation differential DNA
Connect the test kit specification sheets according to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, the PCR product that reclaims purifying is connected to the pUCm-T carrier, transformed into escherichia coli bacterial strain DH5 α;
(2.3) the segmental order-checking of methylation differential DNA
(3) the unexpected toxic analysis of transfer-gen plant
(3.1) open the KEGG database, do not have in the input transfer-gen plant and methylate and methylated dna sequence dna in the wild-type plant, analyze whether synthetic relevant with toxicant of the related pathways metabolism of this sequence; If the related pathways metabolism of sequence is synthetic relevant with toxicant, illustrate that transfer-gen plant may have unexpected toxicity;
(3.2) Southern hybridization checking
Adopt traditional CTAB method, extract transfer-gen plant, wild-type plant genomic dna respectively; If the related pathways metabolism of transfer-gen plant methylation differential DNA fragments sequence is synthetic relevant with toxicant, after this sequence is marked as probe according to Roche company digoxigenin labeled and detection kit specification sheets, hybridize with transfer-gen plant, wild-type plant genomic dna respectively; If results of hybridization is positive, illustrate that transfer-gen plant methylation differential DNA fragment is the specificity product of pcr amplification, transfer-gen plant has unexpected toxicity.
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| CN104293893A (en) * | 2013-07-16 | 2015-01-21 | 北京林业大学 | Method and kit for detecting forest tree xylem DNA methylation through MSAP technology |
| CN104293890A (en) * | 2013-07-16 | 2015-01-21 | 北京林业大学 | Method and kit for detecting specific DNA methylation modification site in plant flower organ |
| CN105256379A (en) * | 2015-11-23 | 2016-01-20 | 武汉大学 | Method for preparing novel genome simplified methylation sequencing library |
| CN105734131A (en) * | 2016-03-15 | 2016-07-06 | 山东农业大学 | Analysis method of unexpected effect of transgenosis plants |
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| CN104293775B (en) * | 2013-07-16 | 2016-12-28 | 北京林业大学 | The molecular marker of degeneration-resistant willow, screening technique, test kit and application |
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| CN105734131A (en) * | 2016-03-15 | 2016-07-06 | 山东农业大学 | Analysis method of unexpected effect of transgenosis plants |
| CN108182346A (en) * | 2016-12-08 | 2018-06-19 | 杭州康万达医药科技有限公司 | Predict method for building up and its application of the siRNA for the machine learning model of the toxicity of certain class cell |
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| CN106893780A (en) * | 2017-03-07 | 2017-06-27 | 中国农业科学院棉花研究所 | A kind of method for screening cotton gene related to drought tolerance |
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