CN103215346A - Recombinant standard plasmid and kit used in transgenic rice screening - Google Patents
Recombinant standard plasmid and kit used in transgenic rice screening Download PDFInfo
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Abstract
The invention provides a recombinant standard plasmid used in transgenic rice screening. The ecombinant standard plasmid is obtained through the steps that: with an overlapping PCR technology, CaMV35S gene represented by SEQ ID No.1, a SPS gene represented by SEQ ID No.2, a Bt gene represented by SEQ ID No.3, a NPT II gene represented by SEQ ID No.4, a Htp gene represented by SEQ ID No.5, a Bar gene represented by SEQ ID No.6, and a NOS gene represented by SEQ ID No.7 are sequentially connected, such that a fusion fragment is obtained; and the fusion fragment is cloned into a vector pUC-19 with a molecular cloning technology, such that the recombinant standard plasmid is obtained, and is recorded as pUC-RS. The recombinant standard plasmid can be used for replacing a positive standard in transgenic rice screening, and can be used in transgenic rice qualitative PCR screening test. Therefore, a problem of the lack of positive standard in the transgenic rice screening test process is solved.
Description
(1) technical field
The present invention relates to a kind of reorganization standard plasmid that is used for the transgenic paddy rice examination, and the test kit that contains this reorganization standard plasmid.
(2) background technology
From the first transgenic Fructus Lycopersici esculenti " FLAVR SAVR " in 1994 since U.S. approval is commercially produced, along with transgenic technology develops rapidly, many valuable foreign genes have imported in the plant, and increasing genetically modified crops go through in country variant and regional commercialization plantation.For paddy rice, there is the transgenic paddy rice of many pest-resistant, disease-resistant, herbicide-resistants and nutritive ingredient improvement to research and develop successfully at present, transgenic paddy rice strain: KF6, TT51 and KMD1 are the pest-resistant Bt paddy rice that China has independent intellectual property right, wherein transgenic pest-resistant rice China extensive No. 1 (TT51) was in 2009, and China Ministry of Agriculture has issued its safety certificate in Hubei Province's production application.
For ensureing the interests in the agricultural-food foreign trade, various countries set up transgenosis sign system in succession, accuracy and sensitivity to the transgenosis detection technique have proposed strict requirement, detection to genetically modified crops and products thereof at present mainly contains protein level detection and nucleic acid level detection, and because the extraction of nucleic acid, the advantage of aspects such as preservation and stability, feasible transgenosis detection technique based on detection of nucleic acids is rapidly developed, wherein get the fastest with polymerase chain reaction (PCR) technical development, genetically modified crops PCR detection method is divided into 4 levels again, being respectively examination detects, gene test, making up specific detection and specificity of transformant detects, in daily testing, because the transgenosis sample size is bigger, it is particularly important that genetically modified screening seems, and the shortage of positive criteria product can't satisfy the demand of screening in the transgenosis security control, therefore develops a kind of the detection at the transgenic paddy rice examination and has great importance with plasmid control molecule.
The notion of positive plasmid molecule equals proposition in 2002 by Japanese scientist Kuribara H, and it is meant a kind of purpose foreign gene of genetically modified crops detection and segmental linearizing recombinant plasmid molecule of specific amplification of internal standard gene of containing.The advantage of positive plasmid molecule mainly is to cultivate in a large number by microorganism, and processing ease, and same positive plasmid molecule can comprise a plurality of external source goal gene simultaneously, and the plasmid DNA extraction is easy and purity is higher.Genetically modified crops positive plasmid molecule is a kind of of transgenosis reference material.Exploitation positive plasmid molecule can effectively solve a difficult problem that lacks certified reference material and positive material in the testing.
(3) summary of the invention
The purpose of this invention is to provide a kind ofly, satisfy the demand in the transgenic paddy rice security control examination testing for the transgenic paddy rice examination detects with reorganization standard plasmid and application thereof.
The technical solution used in the present invention is:
A kind of reorganization standard plasmid that is used for the transgenic paddy rice examination, obtain by following method: utilize overlapping pcr the CaMV35S gene shown in the SEQ ID No.1, SPS gene shown in the SEQ ID No.2, Bt gene shown in the SEQ ID No.3, NPT II gene shown in the SEQ ID No.4, Htp gene shown in the SEQ ID No.5, Bar gene shown in the SEQ ID No.6 is connected acquisition successively with the NOS gene shown in the SEQ IDNo.7 and merges fragment, utilize molecule clone technology that gained is merged fragment cloning in carrier pUC-19 again, obtain described reorganization standard plasmid, be designated as pUC-RS.
CaMV35S gene order following (195bp):
Gctcctacaaatgccatcattgcgataaaggaaaggctatcgttcaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatc
SPS gene order following (290bp):
Atctgtttactcgtcaagtgtcatctcctgaagtggactggagctatggggagcctactgaaatgttaacctccgg?ttccactgacggagagggaagcggtgagagtgctggtgcgtacattgtgcgcattccgtgcggtccaagggacaagtacttccgtaaagaggccctgtggccttacctccaagagtttgtcgacggagctctcgcgcatatcctgaacatgtccaaggctctgggggaacaggttagcaatgggaagctggtcttgccatatgtaatcctaggc
Bt gene order following (301bp):
Gaaggtttgagcaatctctaccaaatctatgcagagagcttcagagagtgggaagccgatcctactaacccagctctccgcgaggaaatgcgtattcaattcaacgacatgaacagcgccttgaccacagctatcccattgttcgcagtccagaactaccaagttcctctcttgtccgtgtacgttcaagcagctaatcttcacctcagcgtgcttcgagacgttagcgtgtttgggcaaaggtggggattcgatgctgcaaccatcaatagccgttacaacgaccttactaggctgatcg
NPT II gene order following (325bp):
Actgggcacaacagacaatcgttactaggctgatcgactgggcacaacagacaatcagctgctctgatgccgcygtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcgggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgc
Htp gene order following (472bp):
Gaagtgcttgacattggggagttcagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgcagccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatct
Bar gene order following (430bp):
Accatcgtcaaccactacatcgagacaagcacggtcaacttccgtaccgagccgcaggaaccgcaggagtggacggacgacctcgtccgtctgcgggagcgctatccctggctcgtcgccgaggtggacggcgaggtcgccggcatcgcctacgcgggcccctggaaggcatgcaacgcctacgactggacggccgagtcgaccgtgtacgtctccccccgccaccagcggacgggactgggctccacgctctacacccacctgctgaagtccctggaggcacagggcttcaagagcgtggtcgctgtcatcgggctgcccaacgacccgagcgtgcgcatgcacgaggcgctcggatatgccccccgcggcatgctgcgggcggccggcttcaagcacgggaactggcatgacgtgggtttctggcagc
NOS gene order following (180bp):
Gaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataa
The present invention is according to external source element commonly used in the present transgenic paddy rice: CaMV35S, Bt, the NPT II, Htp, Bar, the sequence of NOS and paddy rice internal standard gene SPS, design corresponding primer, utilize overlapping pcr that seven sequence fragments are merged, utilize molecule clone technology will merge fragment cloning in the pUC19 carrier again, obtain reorganization standard plasmid pUC-RS.
Concrete, described fusion fragment nucleotide sequence is shown in SEQ ID No.8 (2193bp):
gctcctacaaatgccatcattgcgataaaggaaaggctatcgttcaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatcatctgtttactcgtcaagtgtcatctcctgaagtggactggagctatggggagcctactgaaatgttaacctccggttccactgacggagagggaagcggtgagagtgctggtgcgtacattgtgcgcattccgtgcggtccaagggacaagtacttccgtaaagaggccctgtggccttacctccaagagtttgtcgacggagctctcgcgcatatcctgaacatgtccaaggctctgggggaacaggttagcaatgggaagctggtcttgccatatgtaatcctaggcgaaggtttgagcaatctctaccaaatctatg?cagagagcttcagagagtgggaagccgatcctactaacccagctctccgcgaggaaatgcgtattcaattcaacgacatgaacagcgccttgaccacagctatcccattgttcgcagtccagaactaccaagttcctctcttgtccgtgtacgttcaagcagctaatcttcacctcagcgtgcttcgagacgttagcgtgtttgggcaaaggtggggattcgatgctgcaaccatcaatagccgttacaacgaccttactaggctgatcgactgggcacaacagacaatcgttactaggctgatcgactgggcacaacagacaatcagctgctctgatgccgcygtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcgggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcgaagtgcttgacattggggagttcagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgcagccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatctaccatcgtcaaccactacatcgagacaagcacggtcaacttccgtaccgagccgcaggaaccgcaggagtggacggacgacctcgtccgtctgcgggagcgctatccctggctcgtcgccgaggtggacggcgaggtcgccggcatcgcctacgcgggcccctggaaggcatgcaacgcctacgactggacggccgagtcgaccgtgtacgtctccccccgccaccagcggacgggactgggctccacgctctacacccacctgctgaagtccctggaggcacagggcttcaagagcgtggtcgctgtcatcgggctgcccaacgacccgagcgtgcgcatgcacgaggcgctcggatatgccccccgcggcatgctgcgggcggccggcttcaagcacgggaactggcatgacgtgggtttctggcagcgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataa。
Reorganization standard plasmid pUC-RS of the present invention specifically can make up as follows:
(1) utilizes the retrieval of GenBank database and pertinent literature, obtain the external source element that arrives commonly used in paddy rice internal standard gene SPS and the transgenic paddy rice; CaMV35S, Bt, NPT II, Htp, Bar, the sequence of NOS.
Described paddy rice internal standard gene SPS is meant sucrose phosphosynthase gene (GeneBankNo.U33175), and is conservative at the rice genome camber, specificity between having kind, characteristics such as non-specific and single copy number in planting.
The external source element of using always in the described transgenic paddy rice is meant the partial sequence in the following gene: CaMV35S: cauliflower mosaic virus 35S promoter; The homologous sequence of Bt:Cry1Ab gene, Cry1Ac gene, Cry1Ab/Cry1Ac fusion gene; NPT II: neomycin phosphotransferase gene; Htp: hygromycin phosphotransferase gene; Bar: anti-herbicide gene; NOS: nopaline synthase terminator.
(2) according to the external source element that arrives commonly used in SPS gene order and the transgenic paddy rice; CaMV35S, Bt, the NPT II, Htp, Bar, the sequence of NOS designs overlapping PCR primer.
Described overlapping PCR primer is meant the external source element that arrives commonly used in SPS gene order and the transgenic paddy rice; CaMV35S, Bt, the NPT II, Htp, Bar, the identical or complementary primer of the sequence of NOS makes each purpose fragment reach seamless link.
Described overlapping PCR primer sequence is as follows:
35S-F：GCTCCTACAAATGCCATCATTGC
35S-R-add：gacgagtaaacagatGATAGTGGGATTGTGCGTCATCCC
SPS-F-add：acaatcccactatcATCTGTTTACTCGTCAAGTGTCATCTC
SPS-R-add：gctcaaaccttcGCCTAGGATTACATATGGCAAGA
Bt-F-add：atgtaatcctaggcGAAGGTTTGAGCAATCTCTAC
Bt-R-add：tgttgtgcccagtCGATCAGCCTAGTAAGGTCGT
Npt-F-add：ctaggctgatcgACTGGGCACAACAGACAATCG
Npt-R-add：aatgtcaagcacttcGCATCAGCCATGATGGATACTTT
Hpt-F-add：tcatggctgatgcGAAGTGCTTGACATTGGGGAGT
Hpt-R-add：tggttgacgatggtAGATGTTGGCGACCTCGTATT
Bar-F-add：tcgccaacatctACCATCGTCAACCACTACATCG
Bar-R-add：ggcaacaggattcGCTGCCAGAAACCCACGTCAT
Nos-F-add：ggtttctggcagcGAATCCTGTTGCCGGTCTTG
Nos-R：TTATCCTAGTTTGCGCGCTA。
(3) plasmid molecule of new recombinant dna fragment clone.
Described molecular cloning refers to the above-mentioned reorganization DNA fragment specific that obtains is connected on the plasmid vector pUC19, obtains transgenosis standard positive plasmid molecule pUC-RS, and the external source complete sequence of the plasmid molecule of acquisition is seen Fig. 2.
(4) checking of the reorganization standard plasmid of Gou Jianing in transgenic paddy rice examination qualitative PCR.
Described qualitative PCR checking, be meant among the reorganization standard plasmid pUC-RS that makes up with the qualitative PCR method validation and can amplify element (CaMV35S commonly used in paddy rice internal standard gene SPS distinguished sequence and the transgenic paddy rice, Bt, the NPT II, Htp, Bar, NOS) sequence, and the detection sensitivity of each specific sequence can satisfy the requirement that qualitative PCR detects among the pUC-RS.
The invention still further relates to the application of described reorganization standard plasmid in the transgenic paddy rice examination.
The invention still further relates to a kind of detection kit that is used for the transgenic paddy rice examination, mainly comprise described reorganization standard plasmid (standard substance) and Auele Specific Primer; Described specific primer sequence is as follows:
The CaMV35S gene-specific primer:
35S-F：GCTCCTACAAATGCCATCATTGC
35S-R：GATAGTGGGATTGTGCGTCATCCC
The SPS gene-specific primer:
SP?S-F：ATCTGTTTACTCGTCAAGTGTCATCTC
SPS-R：GCCTAGGATTACATATGGCAAGA
The Bt gene-specific primer:
Bt-F：GAAGGTTTGAGCAATCTCTAC
Bt-R：CGATCAGCCTAGTAAGGTCGT
NPT II gene-specific primer:
Npt-F：ACTGGGCACAACAGACAATCG
Npt-R：GCATCAGCCATGATGGATACTTT
The Htp gene-specific primer:
Hpt-F：GAAGTGCTTGACATTGGGGAGT
Hpt-R：AGATGTTGGCGACCTCGTATT
The Bar gene-specific primer:
Bar-F：ACCATCGTCAACCACTACATCG
Bar-R：GCTGCCAGAAACCCACGTCAT
The NOS gene-specific primer:
Nos-F：GAATCCTGTTGCCGGTCTTG
Nos-R：TTATCCTAGTTTGCGCGCTA。
Be the to recombinate design of standard plasmid and Auele Specific Primer of the key of this test kit, other compositions in the test kit, for example the PCR reaction reagent can be selected by this area routine.The PCR reaction reagent comprises PCR damping fluid, deoxidation nucleoside triphosphate mixture and archaeal dna polymerase etc., and wherein the PCR damping fluid can adopt commercial commodity, also can prepare voluntarily, for example with Tris-HCl, KCl, MgCl
2Prepare Deng being mixed in proportion, when detecting, PCR reaction reagent and amplimer are mixed, add testing sample or standard substance again, can carry out pcr amplification reaction, pcr amplification product runs glue with 2% agarose, if the 290bp band appears in the pcr amplification product of sample, contains paddy rice composition (otherwise not containing the paddy rice composition) in the expression testing sample; Further, if also have the band of 195bp, 301bp, 325bp, 472bp, 430bp or 180bp, then can judge and contain corresponding transgenosis element.
The present invention utilizes overlapping PCR and molecule clone technology to make up first to contain paddy rice internal standard gene SPS and contains element (CaMV35S, Bt, NPT II, Htp, Bar, NOS) the reorganization standard plasmid pUC-RS of sequence commonly used in six transgenic paddy rices simultaneously.This reorganization standard plasmid can substitute the positive criteria product in the transgenic paddy rice examination detection, is used for the examination of transgenic paddy rice qualitative PCR is detected, and has solved the problem of positive standard substance scarcity in the transgenic paddy rice examination testing process well.
(4) description of drawings
Fig. 1 is the process and the synoptic diagram of the standard plasmid molecule of the present invention's structure
Fig. 2 is whole exogenous arrays of the standard plasmid molecule pUC-RS of the present invention's structure, wherein SPS represents the distinguished sequence of paddy rice internal standard gene SPS, and 35S, Bt, NPTII, Htp, Bar, NOS represent the distinguished sequence of some elements commonly used in the transgenic paddy rice respectively; The dash area sequence is the lap in the overlapping PCR primer; Solid line tip sequence is the qualitative PCR primer.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the structure of reorganization standard plasmid pUC-RS
One, experiment material
Transgenic paddy rice LL RICE62 and KMD1.
Two, experiment reagent
PUC19 carrier, ExTaq archaeal dna polymerase and damping fluid thereof, dNTPs, Marker are available from precious biotechnology (Dalian) company limited; Genome extraction test kit and quantitative PCR kit are available from the handsome biotechnology in Shanghai Services Co., Ltd; The PCR product reclaims test kit, plasmid extraction kit, DH5 α competence bacterial strain available from the Beijing Quanshijin Biotechnology Co., Ltd; Biochemical reagents are given birth to worker Bioisystech Co., Ltd available from Shanghai; Primer is synthetic by the handsome biotechnology in Shanghai Services Co., Ltd.
Three, key instrument equipment
PCR instrument (German Biometra), gel imaging system (U.S. GE), Ultrospec1100pro ultraviolet spectrophotometer (U.S. GE), other instruments comprise: thermostat water bath, and whizzer,
Constant incubator, electronic balance, micropipet etc.
Four, experimental technique and process
1, overlapping PCR design of primers
Element (CaMV35S, Bt, NPT II, Htp, Bar, NOS) sequence of utilizing GenBank and data of literatures inquiry acquisition paddy rice internal standard gene SPS, transgenic paddy rice to use always; Utilize PrimerPremier5 software, design overlapping PCR primer, be respectively applied for amplifying rice internal standard gene SPS, common component (CaMV35S, Bt, NPT II, Htp, Bar, NOS) sequence (SEQ ID No.1 ~ 7).Primer sequence sees Table 1.
Table 1: the overlapping PCR primer that makes up standard plasmid molecule pMD-KTK
2, the amplification of common component and paddy rice internal standard gene SPS in the transgenic paddy rice
Utilize the primer in the table 1 that corresponding transgenic paddy rice is increased, the PCR system is: 10 * PCR damping fluid (Mg2+Plus), 200mmol dNTPs, 0.5mmol forward primer, 0.5mmol reverse primer, Taq archaeal dna polymerase and the dna profiling of 1.25U.
The PCR program is 95 ℃ of pre-sex change of 5min; 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ are extended 7min.
The PCR product runs glue with 2% agarose and takes pictures.Obtain the distinguished sequence of transgenic paddy rice common component distinguished sequence and paddy rice internal standard gene SPS.
3, utilize overlapping PCR that a plurality of fragments of above-mentioned acquisition are spliced
Utilize overlapping PCR with a plurality of fragment assemblies, splicing is seen synoptic diagram 1, and specific operation process is as follows:
The amplified production of 35S and SPS is got 1 μ L respectively add in the reaction system as amplification template, amplification condition as above utilizes primer that 35S-F/SPS-R-add is carried out overlapping pcr amplification, with 35S and two fragments of SPS link together (35S+SPS).
The amplified production of Bt and NptII is got 1 μ L respectively to add in the reaction system as amplification template, amplification condition as above, utilize primer that Bt-F-add/NptII-R-add is carried out overlapping pcr amplification, with Bt and two fragments of NptII link together (Bt+NptII).
The amplified production of above-mentioned twice overlapping PCR is got 1 μ L respectively to add in the reaction system as amplification template, amplification condition as above, utilize primer that 35S-F/NptII-R-add is carried out overlapping pcr amplification, two fragments that above-mentioned overlapping PCR is obtained link together (35S+SPS+Bt+NptII).
The amplified production of Bar and Nos is got 1 μ L respectively add in the reaction system as amplification template, amplification condition as above utilizes primer that Bar-F-add/Nos-R is carried out overlapping pcr amplification, with Bar and two fragments of Nos link together (Bar+Nos).
The amplified production of Hpt and Bar+Nos is got 1 μ L respectively to add in the reaction system as amplification template, amplification condition as above, utilize primer that Hpt-F-add/Nos-R is carried out overlapping pcr amplification, with Hpt and two fragments of Bar+Nos link together (Hpt+Bar+Nos).
The amplified production of 35S+SPS+Bt+NptII and Hpt+Bar+Nos is got 1 μ L respectively to add in the reaction system as amplification template, amplification condition as above, utilize primer that 35S-F/Nos-R is carried out overlapping pcr amplification, with 35S+SPS+Bt+NptII and two fragments of Hpt+Bar+Nos link together (35S+SPS+Bt+NptII+Hpt+Bar+Nos).The junction fragment that obtains is reclaimed order-checking, and the result shows that the sequence (SEQ ID No.8) of acquisition is consistent with the amplified fragments of design.
4, recombinant fragment clone
Utilize molecular cloning method that the recombinant fragment that above-mentioned overlapping pcr amplification obtains is connected on the plasmid vector pUC19, linked system sees Table 2.To connect product and transform DH5 α competence bacterial strain.Utilize bacterium colony PCR that recombinant bacterial strain is identified, extract plasmid pUC-RS according to the plasmid extraction kit specification sheets.Determine that by sequencing analysis the exogenous array in the reorganization standard plasmid is consistent with expection.
Table 2: linked system
Embodiment 2: the application of reorganization standard plasmid pUC-RS in actual detected
One, experiment material
Transgenic paddy rice KF6, TT51, KMD1; Genetically engineered soybean GTS40-3-2, MON89788; Transgenic corns MON810, Bt11, Bt176; Transgene cotton MON15985, MON88913, transgene rape MS1, GT73; And other crops such as non-transgenic paddy rice, soybean, corn and cotton.
Two, reagent
Taq archaeal dna polymerase and damping fluid thereof, dNTPs, Marker are available from precious biotechnology (Dalian) company limited; Plasmid extraction kit is available from the Beijing Quanshijin Biotechnology Co., Ltd;
Three, key instrument equipment
PCR instrument (German Biometra), gel imaging system (U.S. GE), Ultrospec1100pro ultraviolet spectrophotometer (U.S. GE), other instruments comprise: thermostat water bath, whizzer, constant incubator, electronic balance, micropipet etc.
Four, experimental technique and process
1, plant genome DNA extracts
Get an amount of plant sample, in liquid nitrogen, fully grind, take by weighing the ground sample of 100mg (that need not grind can directly take by weighing), extract the genomic dna that the test kit specification sheets extracts plant sample according to Plant Genome.The quality of DNA sample is estimated with the method that agarose gel electrophoresis and nucleic acid quantification detect.
2, extraction and the detection of reorganization standard plasmid pUC-RS
The intestinal bacteria that will contain standard plasmid molecule pUC-RS are extracted plasmid DNA in 37 ℃ of cultivation 10h according to the plasmid extraction kit specification sheets.Get 3 μ LDNA samples, detect, judge the DNA integrity with 1% agarose gel electrophoresis; Detect DNA purity and concentration with spectrophotometer, calculate OD
260nm/ OD
280nmRatio about 1.8, meet qualitative, quantitative PCR detection requirement; According to reorganization standard plasmid DNA starting point concentration and size, it is diluted to 1.0 * 10 successively with 0.1 * TE damping fluid
6, 1.0 * 10
5, 1.0 * 10
4, 1.0 * 10
3, 1.0 * 10
2, 1.0 * 10 and 5copy μ L
-1, 4 ℃ of preservations are standby.
3, the application of reorganization standard plasmid pUC-RS in qualitative PCR detects
According to the specific sequence of paddy rice internal standard gene SPS and 6 measuring elements, use primer to carry out qualitative PCR, the examination criteria plasmid molecule detects effect.The primer sequence that uses sees Table 3.
Table 3: reorganization standard plasmid qualitative PCR detects primer
Utilize the primer in the table 3 to increase, the PCR system is: 10 * PCR damping fluid (Mg2+Plus), 200mmol dNTPs, 0.5mmol forward primer, 0.5mmol reverse primer, Taq archaeal dna polymerase and the dna profiling of 1.25U.The PCR program is 95 ℃ of pre-sex change of 5min; 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ are extended 7min.The PCR product runs glue with 2% agarose and takes pictures.
With reorganization standard plasmid pUC-RS(1.0 * 10
4Copy μ L
-1), the DNA of transgenic paddy rice KF6, TT51 and KMD1, other transgenic lines and non-transgenic crop is that template is carried out qualitative PCR and detected, and judges that the reorganization standard plasmid is used for the specificity that qualitative PCR detects.The result only shows that recombinant plasmid just has amplification in corresponding transgenosis element, show that recombinant plasmid has excellent specificity, and the amplification of reorganization standard plasmid is consistent with corresponding positive genetically modified amplification, can substitute positive transgenosis sample in the transgenic paddy rice screening.
DNA sample (1.0 * 10 with the reorganization standard plasmid pUC-RS of different concns
6, 1.0 * 10
5, 1.0 * 10
4, 1.0 * 10
3, 1.0 * 10
2, 1.0 * 10 and 5copy μ L
-1) carry out the qualitative PCR amplification for template, judge that the reorganization standard plasmid is used for the sensitivity that qualitative PCR detects.Experimental result shows that 7 segmental amplification sensitivity of insertion can both reach 1.0 * 10
2Copy μ L
-1, satisfy the demand that qualitative PCR detects.
Claims (4)
1. reorganization standard plasmid that is used for the transgenic paddy rice examination, obtain by following method: Bar gene shown in the NPT II gene shown in the SPS gene shown in the CaMV35S gene shown in the SEQ IDNo.1, the SEQ ID No.2, the Bt gene shown in the SEQ IDNo.3, the SEQ ID No.4, the Htp gene shown in the SEQ ID No.5, the SEQ ID No.6 and the NOS gene shown in the SEQ ID No.7 are connected acquisition successively merge fragment, again gained is merged fragment cloning in carrier pUC-19, obtain described reorganization standard plasmid, be designated as pUC-RS.
2. reorganization standard plasmid as claimed in claim 1 is characterized in that described fusion fragment nucleotide sequence is shown in SEQ ID No.8.
3. the application of reorganization standard plasmid as claimed in claim 1 in the transgenic paddy rice examination.
4. a detection kit that is used for the transgenic paddy rice examination mainly comprises described reorganization standard plasmid of claim 1 and Auele Specific Primer; Described specific primer sequence is as follows:
The CaMV35S gene-specific primer:
35S-F：GCTCCTACAAATGCCATCATTGC
35S-R：GATAGTGGGATTGTGCGTCATCCC
The SPS gene-specific primer:
SPSF：ATCTGTTTACTCGTCAAGTGTCATCTC
SPS-R：GCCTAGGATTACATATGGCAAGA
The Bt gene-specific primer:
Bt-F：GAAGGTTTGAGCAATCTCTAC
Bt-R：CGATCAGCCTAGTAAGGTCGT
NPT II gene-specific primer:
Npt-F：ACTGGGCACAACAGACAATCG
Npt-R：GCATCAGCCATGATGGATACTTT
The Htp gene-specific primer:
Hpt-F：GAAGTGCTTGACATTGGGGAGT
Hpt-R：AGATGTTGGCGACCTCGTATT
The Bar gene-specific primer:
Bar-F：ACCATCGTCAACCACTACATCG
Bar-R：GCTGCCAGAAACCCACGTCAT
The NOS gene-specific primer:
Nos-F：GAATCCTGTTGCCGGTCTTG
Nos-R：TTATCCTA?GTTTGCGCGCTA。
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Cited By (6)
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| CN103540665A (en) * | 2013-10-18 | 2014-01-29 | 中国农业科学院生物技术研究所 | Plasmid standard molecule capable of detecting multiple types of genetically modified rice |
| CN105603112A (en) * | 2016-03-29 | 2016-05-25 | 四川省农业科学院分析测试中心 | Sextuple PCR detection primer and detection method for quickly screening exogenous gene of genetically modified rape imported to China |
| CN106834346A (en) * | 2017-01-23 | 2017-06-13 | 中国农业科学院油料作物研究所 | Transgene rape SD rapeseed and its application of examination target are commonly used in one polymerization |
| CN107271688A (en) * | 2017-07-14 | 2017-10-20 | 无锡福阳生物科技有限公司 | A kind of colloid gold immune detection box for detecting transgene protein Cry1Ab/Ac and its application |
| CN107868844A (en) * | 2017-12-08 | 2018-04-03 | 中国农业科学院生物技术研究所 | A kind of cry1A genes qualitative PCR detection primer, detection method and detection kit |
| CN110229846A (en) * | 2019-06-20 | 2019-09-13 | 安徽省农业科学院水稻研究所 | General positive standard plasmid and its construction method for transgenic paddy rice screening |
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| CN103540665A (en) * | 2013-10-18 | 2014-01-29 | 中国农业科学院生物技术研究所 | Plasmid standard molecule capable of detecting multiple types of genetically modified rice |
| CN103540665B (en) * | 2013-10-18 | 2015-10-28 | 中国农业科学院生物技术研究所 | The plasmid control molecule of multiple transgenic paddy rice can be detected |
| CN105603112A (en) * | 2016-03-29 | 2016-05-25 | 四川省农业科学院分析测试中心 | Sextuple PCR detection primer and detection method for quickly screening exogenous gene of genetically modified rape imported to China |
| CN106834346A (en) * | 2017-01-23 | 2017-06-13 | 中国农业科学院油料作物研究所 | Transgene rape SD rapeseed and its application of examination target are commonly used in one polymerization |
| CN107271688A (en) * | 2017-07-14 | 2017-10-20 | 无锡福阳生物科技有限公司 | A kind of colloid gold immune detection box for detecting transgene protein Cry1Ab/Ac and its application |
| CN107868844A (en) * | 2017-12-08 | 2018-04-03 | 中国农业科学院生物技术研究所 | A kind of cry1A genes qualitative PCR detection primer, detection method and detection kit |
| CN110229846A (en) * | 2019-06-20 | 2019-09-13 | 安徽省农业科学院水稻研究所 | General positive standard plasmid and its construction method for transgenic paddy rice screening |
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