CN1385541A - Method for detecting transgenic plant and products thereof - Google Patents
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Abstract
The present invention relates to a transgenic plant and detection method of its product. Said invention uses the characteristic sequence being in transgenic plant: T-DNA left anjd right boundary sequence, CaMV 35S promotor, CaMV 35S poly A, nos promotor and nos termination sequence to design and synthesize initiator to make PCR to obtain correspondent DNA amplification product or signal of amplification product. According to the PCR result it can be used for recognizing that said detected sample is transgenic plant and its product or not.
Description
Technical field
The invention belongs to transgenic plant safety management technology field and biological technical field, be specifically related to the method for quick of transgenic plant and goods thereof.
Background technology
1, the security control of transgenic plant
Transgenic plant (transgenic plant) are meant artificially to be transferred to foreign gene in the genome of plant by certain method, and plant varient hereditary, that express, and the foreign gene that is changed over to Plant Genome is called transforming gene (transgene).
Nineteen eighty-three, human reported first transgene tobacco was born, and reached 4,420 ten thousand hectares to 2000 global transgenic plant cultivated areas.Transgenic plant from the date of birth, just the someone worries ecology and the food-safety problem that they may exist, these worry once to be subjected to the support of some results of study.In order to strengthen management to genetically modified organism, avoid its ecology that may bring, food to threaten, the protection consumer rights guarantee the transgenic plant research normal development, corresponding law, rules have all been formulated in many international organizations, countries and regions.The enforcement of these laws, rules at first requires relevant functional department, research institution when implementing management, and relevant plants and plant product is detected, identifies.It is general to set up a cover, fast, accurately, economical, detect authenticate technology easily, be a current research focus both domestic and external.
2, the problem of the development of transgenic plant detection technology and existence
So far, the detection of transgenic plant mainly is limited to goal gene and detects, and report or marker gene detect.Goal gene is promptly given the functional gene of the useful proterties of plant, and is at present ten hundreds of at the goal gene that grinds as disease-resistant, pest-resistant, anti-herbicide gene etc., and all increasing every day, relates to floristics above 100 kinds.By December calendar year 2001, only 51 of the drugs approved by FDA industrialization kinds of contained goal gene of transgenic plant are just above 65 kinds; By in December, 1999, China Ministry of Agriculture ratifies 150 multinomial transgenic plant altogether and enters commercialization production, environment release or interim test, relates to about 103 kinds of gene.Report or marker gene are screenings for convenience and differentiate transgenic plant and foreign gene that the Plant Genome that adopts does not have that about 20 kinds of report commonly used or marker gene are as no, NPTII, GUS, GFP, bar gene etc.On concrete detection method, four levels are arranged again:
(1) there are situation in the molecular hybridization of DNA (Southern hybridization), polymerase chain reaction analysis technology such as (PCR) in order to detection of a target gene, and round pcr is because of simple and easy to do and with the most use.But the past people are primarily aimed at goal gene, report or marker gene, its limitation is, if a concrete plant does not know which kind of gene the investigator imports, if a kind of goods contain the various plants composition, with aforesaid method band is born the character of much blindness, spend the long time could determine its () whether transgenic plant or goods.
(2) molecular hybridization of RNA (Northern hybridization), reverse transcription PCR analysis technology such as (RT-PCR), in order to the expression situation of detection of a target gene at rna level, its defective is same as described above, and test sample must be fresh and alive vegetable material, can't detect withered, dead material and is the various goods of raw material with the plant.
(3) analysis technology such as proteinic ELISA, Western blot, utilize the immune response between antibody-antigen to come the expression situation of analyzing gene on protein level, its deficiency is, if a concrete plant does not know which kind of gene the investigator imports, which kind of if a kind of goods contain the various plants composition, will be difficult to determine to select for use the antibody of gene product to detect.
(4) gene function analysis technology, the function of some transforming gene products (enzyme or protein) can be observed with live body, living tissue, protein crude extract and the substrate reactions of transgenic plant; And other genes encoding antibiotics resistances, Herbicid resistant etc. then can be analyzed by live tests such as simple seed germinations.Be not difficult to find out that they all must be with live body or living tissue, and occurs error in result's judgement easily.
There is no both at home and abroad and utilize T-DNA left and right sides border sequence, CaMV 35S polyA, no promotor, no terminator sequence design synthetic primer to carry out the report that PCR detects transgenic plant and goods thereof.Though have several pieces to utilize the report that the CaMV 35S promoter detects changes plant and goods thereof,, use the CaMV 35S promoter to detect separately and might cause important errors for the cress of easy infection CaMV.
Summary of the invention
The detection method that the purpose of this invention is to provide a kind of transgenic plant and goods thereof.This method is according to the universal characteristics sequence of transgenic plant: T-DNA left and right sides border sequence, CaMV 35S polyA, no promotor, no terminator sequence design synthetic primer, carry out PCR and detect transgenic plant and goods thereof, thereby overcome the existing problem of above-mentioned existing detection method.
In order to obtain transgenic plant, usually foreign gene is building up to by Agrobacterium (Agrobacterium) Ti-plasmids (tumer-inducingplasmid) or Ri plasmid (root-inducing plasmid) and derives and next carrier T-DNA district formation plant expression vector, the T-DNA left and right sides border sequence (right border sequence, the RB that on this carrier, always have 25bp; Left border sequence, LB), and the CaMV 35S promoter, CaMV 35S polyA, no promotor, the no terminator sequence that are used with foreign gene, plant expression vector is total to culture method (co-cultivation), particle bombardment (gene gun by Agrobacterium, particle bombardment) etc. method imports vegetable cell, and exogenous origin gene integrator is expressed to Plant Genome and in transgenic plant.
The inventive method is: adopt ordinary method to extract the DNA of sample, according to T-DNA left and right sides border sequence, CaMV 35S promoter, CaMV 35S polyA, no promotor, the synthetic one or more pairs of primers of no terminator sequence design, carry out PCR, obtain the signal of corresponding D NA amplified production or amplified production.
The inventive method specifically may further comprise the steps:
(1) the above primer of Synthetic 2 bar, wherein one is T-DNA left and right sides border sequence primer, length 18-25nt, other is any one or more snippets sequence in CaMV 35S promoter, CaMV 35S polyA, no promotor and the no terminator sequence, length 〉=18nt;
(2) get T-DNA left and right sides border sequence primer, match with one or more other primer or many to the PCR primer, according to a conventional method sample DNA is carried out pcr amplification reaction, do over against photograph with plasmid DNA such as pBI121 or pCAMBIA1300, so that transgenic plant or goods DNA do not do negative contrast, the PCR reaction conditions is 94 ℃ of pre-sex change 5 minutes, reaction 25-35 circulation, every circulation comprises: 94 ℃ of sex change 1 minute, annealed 1 minute for 45-65 ℃, 72 ℃ of extensions 30 seconds-10 minutes after the loop ends, appended 72 ℃ of extensions 5 minutes;
(4) pcr amplification product is observed the DNA cloning fragment of big or small 50-10000bp by agarose gel electrophoresis and bromination second pyridine dyeing under the ultraviolet lamp.
According to the invention described above method, also can carry out real-time quantitative PCR, specifically: the SYBR fluorescence dye of adding and double-stranded DNA specific combination in the PCR reaction tubes of step (2), perhaps special TaqMan fluorescent probe; Pcr amplification product observes corresponding fluorescent signal by the fluorescent scanning instrument, perhaps set by step (3) observations.
According to the invention described above method, also can carry out PCR-ELISA, specifically: in step (1), with biotin labeling synthetic primer; Pcr amplification product combines with streptavidin (streptavidin)-albumin A on being fixed on microplate, carry out the ELISA reaction with alkaline phosphatase or horseradish peroxidase mark biotin antibody again, read the light absorption value of 405-410nm, (3) observations perhaps set by step with microplate reader (microplate reader).
The inventive method has following beneficial effect:
(1) popularity.T-DNA left and right sides border sequence, no promotor and no terminator sequence are present in most transgenic plant, CaMV35S promotor and/or CaMV 35S polyA are present in the transgenic plant more than 70%, thereby have the most extensive representativeness, be applicable to the detection of current most transgenic plant and goods thereof.
(2) rapidity.Great majority detect and take about 2 hours to 2 days, can guarantee the needs of rapid detection.
(3) accuracy.Above-mentioned several sequence all is the characteristic sequence of transgenic plant, and the detected result accuracy is strong.
(4) susceptibility.Only need use the minute quantity sample, as a seed, a block organization, a slice leaf, very at least to ug (microgram) grade sample.
Embodiment
The invention will be further described below by specific embodiment.
Embodiment 1: utilize T-DNA left and right sides border sequence and no terminator sequence primer to carry out PCR and detect transgenic papaya
As previously described, T-DNA left and right sides border sequence, no terminator sequence are present in most transgenic plant, so carry out PCR with these two kinds of sequence synthesized primer things, are applicable to the detection of current most transgenic plant and goods thereof.Obtained the antiviral papaya strain of commentaries on classics prv (PRV) replicative enzyme (RP) gene in our work in the past, it has been carried out the transgenosis detection with PCR method.
(1) extracts the transgenic papaya leaf DNA with the CTAB method.
(2) synthetic a pair of primer, wherein one is T-DNA left and right sides border sequence primer (4-23nt): 5 '-caggatatattggcgggtaa-3 ', and another is no terminator sequence primer (no terminator sequence 81-100nt): 5 '-catgcttaacgtaattcaac-3 '.
(3) according to a conventional method sample DNA is carried out pcr amplification reaction, do over against photograph with pRPTW plasmid DNA (containing T-DNA left and right sides border sequence, PRV RP gene, no terminator sequence etc.), do negative contrast with not genetically modified papaya DNA, the PCR reaction conditions is 94 ℃ of pre-sex change 5 minutes, reaction 30 circulations, and every circulation comprises: 94 ℃ of sex change 1 minute, annealed 1 minute for 54 ℃, 72 ℃ of extensions 4 minutes after the loop ends, appended 72 ℃ of extensions 5 minutes.
(5) pcr amplification product is by agarose gel electrophoresis and the pyridine of bromination second dyeing, and ultraviolet lamp is observed down, sample and over against according to the DNA cloning fragment that 1 about 2500bp of size is all arranged, the negative contrast without any amplified fragments.The result conforms to design, shows that correlated series and RP gene have been integrated into the papaya genome.
Embodiment 2: utilize T-DNA left and right sides border sequence and CaMV35S promoter primer to carry out real-time quantitative PCR
Detect transgene tobacco
The CaMV 35S promoter has plant strong promoter function, no organ specificity, thereby widely adopt, the transgenic plant more than 70% are adopted this promotor so far.But minority cress easy infection CaMV, so when detecting this class plant and goods thereof, as independent use CaMV 35S promoter primer, may cause important errors, utilize respectively two primers from T-DNA left and right sides border sequence and CaMV 35S promoter to carry out PCR and then can overcome this problem.Adopt the real-time quantitative PCR method that the transgenosis resistance glyphosate tobacco that we obtain is detected.
(1) extracts the transgene tobacco leaf DNA with the CTAB method.
(2) synthetic a pair of primer, wherein one is T-DNA left and right sides border sequence primer (4-23nt complementary sequence): 5 '-ttacccgccaatatatcctg-3 ', and another is CaMV35S promoter primer (325-344 nt): 5 '-atggtggagcacgacactct-3 '.
(3) according to a conventional method sample DNA is carried out pcr amplification reaction, (contain T-DNA left and right sides border sequence with the pM12 plasmid DNA, the CaMV35S promoter sequence, EPSPS mutant gene etc.) do over against photograph, do negative contrast with not genetically modified tobacco DNA, in reaction tubes, add SYBR fluorescence dye with the double-stranded DNA specific combination, be reflected in the ABI Prism 7000 type quantitative real time PCR Instruments and carry out, the PCR reaction conditions is 94 ℃ of pre-sex change 5 minutes, reaction 35 circulations, every circulation comprises: 94 ℃ of sex change 1 minute, annealed 1 minute for 58 ℃, 72 ℃ of extensions 2 minutes after the loop ends, appended 72 ℃ of extensions 5 minutes.
(4) increase along with cycle number, transgene tobacco and over against constantly strengthening according to the pcr amplification product fluorescent signal, 30 circulation time fluorescence intensities reach the strongest, and negative contrast does not detect fluorescence; Pcr amplification product is by agarose gel electrophoresis and bromination second pyridine dyeing, ultraviolet lamp is observed down, transgene tobacco and over against according to 1 about 2000bp DNA cloning fragment of size is all arranged, negative contrast is without any amplified fragments, the result conforms to design, shows that correlated series and EPSPS mutant gene have been integrated into the tobacco gene group.
Embodiment 3: utilize CaMV35S promotor and no terminator sequence primer to carry out PCR-ELISA and detect transgenic Fructus Lycopersici esculenti
We are with saliva attachment zone (the Saliva-binding region of Streptococcus mutans albumen PAC (Streptococcus mutans surface protein), SBR) gene and with (the Cholera toxin B subunit of vibrio cholerae B subunit, CTB) form mosaic gene, cultivate altogether and transform tomato, obtain transfer-gen plant, it has been carried out the transgenosis detection with the PCR-ELISA method.
(1) extracts transgenic Fructus Lycopersici esculenti fruit DNA with conventional CTAB method.
(2) synthetic a pair of primer, wherein one is CaMV35S promoter primer (61-70nt, 5 ' end connects vitamin H): 5 '-biotin-cagcaggtctcatcaagacg-3 ', another is that another is no terminator sequence primer (no terminator sequence 81-100nt complementary sequence): 5 '-gttgaattacgttaagcatg-3 '.
(3) according to a conventional method sample DNA is carried out pcr amplification reaction, do over against photograph with pROSB plasmid DNA (containing CaMV35S promotor, SBR-CTB mosaic gene, no terminator sequence etc.), do negative contrast with not genetically modified tomato DNA, the PCR reaction conditions is 94 ℃ of pre-sex change 5 minutes, reaction 30 circulations, and every circulation comprises: 94 ℃ of sex change 1 minute, annealed 1 minute for 54 ℃, 72 ℃ of extensions 3 minutes after the loop ends, appended 72 ℃ of extensions 5 minutes.
(4) pcr amplification product combines with streptavidin (streptavidin)-albumin A on being fixed on microplate, again respectively with digoxigenin-probe (CaMV35S promotor 181-200nt, 5 '-aagatatattt ctcaagatc-3 '-Dig), enzyme mark DigiTAb carry out the ELISA reaction, read the light absorption value of 405-410nm with the BioRad450 microplate reader, sample and over against according to visible significantly absorption peak, negative contrast does not then have; Pcr amplification product is by agarose gel electrophoresis and bromination second pyridine dyeing, ultraviolet lamp is observed down, sample and over against according to the DNA cloning fragment that 1 about 2900bp of size is all arranged, negative contrast is without any amplified fragments, the result conforms to design, shows that correlated series and SBR-CTB mosaic gene have been integrated into the tomato dna group.
Claims (4)
1, the detection method of a kind of transgenic plant and goods thereof, it is characterized in that: adopt ordinary method to extract the DNA of sample, according to T-DNA left and right sides border sequence, CaMV 35S promoter, CaMV 35S polyA, no promotor and the synthetic one or more pairs of primers of no terminator sequence design, carry out PCR, obtain the signal of corresponding D NA amplified production or amplified production.
2, in accordance with the method for claim 1, it is characterized in that specifically may further comprise the steps:
(1) the above primer of Synthetic 2 bar, wherein one is T-DNA left and right sides border sequence primer, length 18-25nt, other is any one or more snippets sequence in CaMV 35S promoter, CaMV 35S polyA, no promotor and the no terminator sequence, length 〉=18nt;
(2) get T-DNA left and right sides border sequence primer, match with one or more other primer or many to the PCR primer, according to a conventional method sample DNA is carried out pcr amplification reaction, do over against photograph, with the not negative contrast of DNA do of transgenic plant or goods with pBI121 or pCAMBIA1300 plasmid DNA; The PCR reaction conditions is 94 ℃ of pre-sex change 5 minutes, reaction 25-35 circulation, and every circulation comprises: 94 ℃ of sex change 1 minute, 45-65 ℃ of annealing 1 minute, 72 ℃ of extensions 30 seconds-10 minutes after the loop ends, appended 72 ℃ of extensions 5 minutes;
(3) pcr amplification product is observed the DNA cloning fragment of big or small 50-10000bp by agarose gel electrophoresis and bromination second pyridine dyeing under the ultraviolet lamp.
3, in accordance with the method for claim 2, it is characterized in that carrying out real-time quantitative PCR: the SYBR fluorescence dye of adding and double-stranded DNA specific combination in the PCR reaction tubes of step (2), perhaps special TaqMan fluorescent probe; Pcr amplification product observes corresponding fluorescent signal by the fluorescent scanning instrument, perhaps set by step (3) observations.
4, in accordance with the method for claim 2, it is characterized in that carrying out PCR-ELISA: in step (1), with biotin labeling synthetic primer; Pcr amplification product combines with streptavidin-albumin A on being fixed on microplate, carries out the ELISA reaction with specificity digoxigenin-probe, enzyme mark DigiTAb respectively again, reads the light absorption value of 405-410nm, (3) observations perhaps set by step with microplate reader.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100372947C (en) * | 2005-03-03 | 2008-03-05 | 华南师范大学 | Fluorescence correlation spectral detection method and device for transgenosis material |
| CN103352075A (en) * | 2013-06-13 | 2013-10-16 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Transgenic component ultrasensitive detection probe and primers |
| CN103757120A (en) * | 2014-01-23 | 2014-04-30 | 宁夏林业研究所股份有限公司 | DNA (deoxyribonucleic acid) detection method for quickly distinguishing transgenic plant and product thereof |
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2002
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100372947C (en) * | 2005-03-03 | 2008-03-05 | 华南师范大学 | Fluorescence correlation spectral detection method and device for transgenosis material |
| CN103352075A (en) * | 2013-06-13 | 2013-10-16 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Transgenic component ultrasensitive detection probe and primers |
| CN103352075B (en) * | 2013-06-13 | 2015-07-29 | 深圳出入境检验检疫局动植物检验检疫技术中心 | A kind of transgene component super sensitivity detection probe and primer |
| CN103757120A (en) * | 2014-01-23 | 2014-04-30 | 宁夏林业研究所股份有限公司 | DNA (deoxyribonucleic acid) detection method for quickly distinguishing transgenic plant and product thereof |
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