CN103834646A - PCR (Polymerase Chain Reaction) detection primer and qualitative detection method and kit for specificity of transgenic rice PA110-15 strain - Google Patents
PCR (Polymerase Chain Reaction) detection primer and qualitative detection method and kit for specificity of transgenic rice PA110-15 strain Download PDFInfo
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Abstract
The invention discloses a PCR (Polymerase Chain Reaction) qualitative detection primer and a qualitative detection method and a kit for specificity of a transgenic rice PA110-15 strain. The invention firstly provides a connection area sequence inserted with a carrier gene and flanking rice sequence in a high-lysine storage protein transgenic rice PA110-15 strain, and the base sequence is shown as SEQ ID No.2. The invention further provides the PCR primer and the qualitative detection method for the specificity of the high-lysine storage protein transgenic rice PA110-15 strain. The specificity verification experiment shows that the PCR qualitative detection method for the specificity of the high-lysine storage protein transgenic rice PA110-15 strain is high in specificity.
Description
Technical field
The specific PCR that the present invention relates to transgenic paddy rice strain detects primer and detection kit, relate in particular to and turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity PCR qualitative detection primer, qualitative checking method and detection kit, belong to transgenic paddy rice strain detection field.
Background technology
Along with the extensive plantation of transgenic plant, the safety issue of genetically modified food also causes that people pay close attention to greatly, and existing more than 30 country implements transgenic product mark system in succession, comprises China.The enforcement of transgenic labeling system depends on the composition detection to transgenic plant and converted products thereof.Round pcr is the most widely used method in transgenic product that detects both at home and abroad at present.In the time that this technology of application is carried out genetically modified crops qualitative detection, according to its specificity, be divided into screening detection method, gene specific method, built specificity method and strain specificity method.Event-specific detection is that the joining region sequence by detecting insertion vector and Plant Genome realizes, due to each transgenic plant strain, all there is the joining region sequence of special exogenous insertion vector and Plant Genome, with first three methods comparison, strain specificity has higher specificity and accuracy, and the most applicable transgenic product of doing detects.
At present, existing partial monopoly and bibliographical information the event-specific detection method of transgenic plant.For example: the event-specific detection method of magnifying the people such as soldier and set up in 2006 transgenosis MON863 corn; The people such as Xie Jiajian have set up a series of event-specific detection methods such as rich No. 8 of transgenic paddy rice Kemingdao, Bt Shanyou 63, section rich No. 6 and section; The people such as Lu Changming have set up the event-specific detection method of a series of transgene rapes in 2007.But, so far, not yet find any about turning high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity PCR detection method.
Summary of the invention
One of object of the present invention is to provide the PCR qualitative detection primer that turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity;
Two of object of the present invention is to set up to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity PCR qualitative checking method;
Three of object of the present invention is to provide and turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity PCR qualitative detection test kit.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention is that LRP gene (SEQ ID No.1) obtains external source insertion gene 5 ' end paddy rice flanking sequence and 3 ' end paddy rice flanking sequence by Hi-TAIL PCR according to turning the goal gene inserting in high-lysine storage protein trans-genetic hybrid rice PA110-15 strain, and the joining region nucleotides sequence of the exogenous insertion vector that contains LRP gene obtaining and both sides 5 ' end and the 3 ' distolateral wing paddy rice sequence is classified as shown in SEQ ID No.2.
The present invention further holds the joining region sequences Design 9 of flanking sequence to specificity of transformant qualitative detection primer according to inserting genophore and 3 ', and these 9 pairs of transformant primers are respectively: nucleotides sequence is classified the primer pair 1 shown in SEQ ID No.3 and SEQ ID No.4 as; Nucleotides sequence is classified the primer pair 2 shown in SEQ ID No.5 and SEQ ID No.6 as; Nucleotides sequence is classified primer pair 3 shown in SEQ ID No.7 and SEQ ID No.8 as; Nucleotides sequence is classified the primer pair 4 shown in SEQ ID No.9 and SEQ ID No.10 as; Nucleotides sequence is classified the primer pair 5 shown in SEQ ID No.11 and SEQ ID No.12 as; Nucleotides sequence is classified the primer pair 6 shown in SEQ ID No.13 and SEQ ID No.14 as; Nucleotides sequence is classified the primer pair 7 shown in SEQ ID No.15 and SEQ ID No.16 as; Nucleotides sequence is classified the primer pair 8 shown in SEQ ID No.17 and SEQ ID No.18 as; Nucleotides sequence is classified the primer pair 9 shown in SEQ ID No.19 and SEQ ID No.20 as.The present invention applies conventional PCR reaction conditions, adopts respectively 9 pairs of transformant primers to carry out pcr amplification, and 2 repetitions are set; From amplification, these 9 pairs of transformant primers all can amplify expection fragment specifically from turn high-lysine storage protein trans-genetic hybrid rice PA110-15 positive material sample, but with primer pair 8(SEQ ID No.17 and SEQ ID No.18) shown in the band (159bp) of primer amplification the most clear, amplification efficiency is higher, and primer dimer is less.
Two of object of the present invention is to provide one and turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity PCR detection method, and the method comprises: (1) extracts the DNA of paddy rice sample to be detected; (2) take the DNA that extracts as template, design Auele Specific Primer pair with Nucleotide shown in SEQ ID No.2 for target gene, set up the performing PCR amplification of going forward side by side of pcr amplification system; (3), if amplify the specific band of expection from sample to be detected, paddy rice sample to be detected is to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain; If fail to amplify the specific band of expection from sample to be detected, paddy rice sample to be detected is not to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain.
Wherein, described Auele Specific Primer is to being selected from a pair of arbitrarily in following 9 pairs of primers: nucleotides sequence is classified the primer pair 1 shown in SEQ ID No.3 and SEQ ID No.4 as; Nucleotides sequence is classified the primer pair 2 shown in SEQ ID No.5 and SEQ ID No.6 as; Nucleotides sequence is classified primer pair 3 shown in SEQ ID No.7 and SEQ ID No.8 as; Nucleotides sequence is classified the primer pair 4 shown in SEQ ID No.9 and SEQ ID No.10 as; Nucleotides sequence is classified the primer pair 5 shown in SEQ ID No.11 and SEQ ID No.12 as; Nucleotides sequence is classified the primer pair 6 shown in SEQ ID No.13 and SEQ ID No.14 as; Nucleotides sequence is classified the primer pair 7 shown in SEQ ID No.15 and SEQ ID No.16 as; Nucleotides sequence is classified the primer pair 8 shown in SEQ ID No.17 and SEQ ID No.18 as; Nucleotides sequence is classified the primer pair 9 shown in SEQ ID No.19 and SEQ ID No.20 as.
The present invention further provides a kind of PCR qualitative checking method that turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity, having comprised: (1) extracts the DNA of paddy rice sample to be detected; (2), take the DNA that extracts as template, set up the performing PCR amplification of going forward side by side of PCR reaction system with the primer pair shown in SEQ ID No.17 and SEQ ID No.18; (3), if amplify the object band of 159bp from sample to be detected, paddy rice sample to be detected is to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain; If fail to amplify the object band of 159bp from sample to be detected, paddy rice sample to be detected is not to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain
Wherein, the described method of extracting DNA from paddy rice sample to be detected, can be the various common methods of extracting DNA from vegetable material, for example, can be the whole bag of tricks such as CTAB method, guanidine isothiocyanate method or guanidine hydrochloride method.
In PCR reaction system, the final concentration of primer and annealing temperature all have a certain impact for the specificity sensitivity of detected result; The present invention has investigated the final concentration of primer in PCR reaction system and annealing temperature for the specificity detecting and the impact of sensitivity, it is that 0.2 μ M, annealing temperature are while being 58 ℃ that experimental result is found at primer final concentration, the specificity of detected result is the strongest, and sensitivity is the highest.
Pcr amplification system described in the present invention can be set up with reference to following methods: the cumulative volume of reaction system is 25.0 μ L, wherein, 10 × PCR damping fluid, 2.5 μ L, 25mmol/L magnesium chloride solution 1.5 μ L, dNTPs mixing solutions (each 2.5mmol/L) 2 μ L, 0.2 μ mol/L upstream primer 0.5 μ L, 0.2 μ mol/L downstream primer 0.5 μ L, 0.025U/ μ L Taq enzyme 1.5 μ L, 25mg/L DNA profiling 2.0 μ L, surplus is distilled water.
Described pcr amplification condition optimization is: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out altogether 35 circulations; 72 ℃ are extended 2min.
Further aim of the present invention is to provide a kind of PCR qualitative detection test kit that turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity, comprise: 10 × PCR damping fluid, magnesium chloride solution, dNTPs mixing solutions, PCR qualitative detection primer pair, Taq enzyme and distilled water; Wherein, described PCR qualitative detection primer pair is selected from a pair of arbitrarily in following 9 pairs of primers: nucleotides sequence is classified the primer pair 1 shown in SEQ ID No.3 and SEQ ID No.4 as; Nucleotides sequence is classified the primer pair 2 shown in SEQ ID No.5 and SEQ ID No.6 as; Nucleotides sequence is classified primer pair 3 shown in SEQ ID No.7 and SEQ ID No.8 as; Nucleotides sequence is classified the primer pair 4 shown in SEQ ID No.9 and SEQ ID No.10 as; Nucleotides sequence is classified the primer pair 5 shown in SEQ ID No.11 and SEQ ID No.12 as; Nucleotides sequence is classified the primer pair 6 shown in SEQ ID No.13 and SEQ ID No.14 as; Nucleotides sequence is classified the primer pair 7 shown in SEQ ID No.15 and SEQ ID No.16 as; Nucleotides sequence is classified the primer pair 8 shown in SEQ ID No.17 and SEQ ID No.18 as; Nucleotides sequence is classified the primer pair 9 shown in SEQ ID No.19 and SEQ ID No.20 as.
Be preferably nucleotides sequence and classify the primer pair 8 shown in SEQ ID No.17 and SEQ ID No.18 as.
The strain specificity PCR qualitative checking method that adopts the present invention to set up has carried out qualitative detection to rich No. 6 of transgenic paddy rice section, rich No. 8 of section, Kemingdao, M12, TT51 and other transgenic plant and non-transgenic plant material, qualitative PCR amplification shows, only, turning the band that in high-lysine storage protein trans-genetic hybrid rice PA110-15 positive material genome, amplification has obtained object clip size, in the genomes such as other transgenic paddy rice and other transgenic plant and conventional rice bright extensive 63, do not increase and obtain expecting object fragment; Experimental result shows, what the present invention set up turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity qualitative PCR detection method high specificity.
Accompanying drawing explanation
Fig. 1 goal gene upstream and downstream Hi-TAIL PCR second takes turns and third round amplified production; M:1kp plus DNA marker; 1,2 be respectively: goal gene downstream Hi-TAIL PCR second takes turns and third round amplified production; 3,4 be respectively: goal gene upstream Hi-TAIL PCR second takes turns and third round amplified production.
Fig. 2 sequencing result is analyzed.
The primer long segment amplification designing in Fig. 3 primer and isolated Gt1 gene order; M:1kp plus DNA marker; 1: blank; 2: negative control; 3:PA110-15 sample.
The sequencing result analysis of Fig. 4 long segment amplified production.
Fig. 5 Gt1 upstream region of gene Hi-TAIL PCR second takes turns and third round amplification; M:1kp plus DNA marker; 1,2 be respectively: Gt1 upstream region of gene Hi-TAIL PCR second takes turns and third round amplified production.
Fig. 6 sequencing result and with the first step splicing result.
The schematic diagram of the complete integrated structure of Fig. 7 and flanking sequence.
Figure 83 ' end flanking sequence 9 is to specificity of transformant primer screening result; Draw M:100bp marker; 1: blank; 2,3: negative control; 4,5:PA110-15 sample.
Fig. 9 primer concentration is the amplification of 0.1 μ M/L; M:100bp Marker; 1: negative control; 2: positive a:54 ℃; B:56 ℃; C:58 ℃; D:60 ℃; E:62 ℃.
Figure 10 primer concentration is the amplification of 0.2 μ M/L; M:100bp Marker; 1: negative control; 2: positive a:54 ℃; B:56 ℃; C:58 ℃; D:60 ℃; E:62 ℃.
Figure 11 primer concentration is the amplification of 0.4 μ M/L; M:100bp Marker; 1: negative control; 2: positive a:54 ℃; B:56 ℃; C:58 ℃; D:60 ℃; E:62 ℃.
Figure 12 primer concentration is the amplification of 0.8 μ M/L; M:100bp Marker; 1: negative control; 2: positive a:54 ℃; B:56 ℃; C:58 ℃; D:60 ℃; E:62 ℃.
Figure 13 specificity the result; M:100bp marker; 1: blank; 2: negative control; 3:PA110-15 sample; 4-13: be respectively sample 1, sample 2, sample 3, sample 4, sample 5, sample 6, sample 7, sample 8, sample 9, sample 10.
The obtaining of flanking sequence that embodiment 1 turns high-lysine storage protein trans-genetic hybrid rice PA110-15 external source and inserts gene
Turning high-lysine storage protein trans-genetic hybrid rice PA110-15 external source insertion goal gene is LRP gene, and its base sequence is shown in SEQ ID No.1.
Obtain integrated structure (external source is inserted gene flanking sequence) by Hi-TAIL PCR
The first step: design upstream and downstream nested primers on goal gene LRP
LRP-Sp-1TGGTCCTGGAACCATCAAGAAGATC
LRP-Sp-2ACGATGGACTCCAGTCCGGCCTGCTGGACCTTCTGGAGGCTCTGTT
LRP-Sp-3CCAAGGGTGATGCTCTCTTCAAGGC
LRP-Ap1CAGCCTTGAAGAGAGCATCACCCTT
LRP-Ap2ACGATGGACTCCAGTCCGGCCTGAGTTTCCCAACAGAGCCTCCAGAAGLRP-Ap3GCAAAGCAGCACCACCAACTATGCT
Fig. 1 is that goal gene upstream and downstream Hi-TAIL PCR second takes turns and third round amplified production.Fig. 2 is shown in sequencing result analysis.
Second step: by two kinds of methods
1. on GenBank, obtain 5 ' coupled rice genome sequence according to 3 ' the end rice genome sequence obtaining, then design the primer designing in primer and isolated Gt1 gene order and carry out long segment amplification.
RICE-5’-F:TCAGTGCATCTGCTGTGGCTGGTAG
Gt1-R：TGCTGTCATCTTAGCTTTTCCATGCAAC
Amplification is shown in Fig. 3; Fig. 4 is shown in sequencing result analysis.
2. the Gt1 sequence obtaining according to the first step, design upstream Hi-TAIL PCR nested primers
Gt1-Ap1TGGGTTGCCTACACCATGATTAACTT
Gt1-Ap2ACGATGGACTCCAGTCCGGCCCTAATTGTTTGAACTGCACGGCACCTT
Gt1-Ap3TGCTGTCATCTTAGCTTTTCCATGCAAC
Amplification is shown in Fig. 5; Sequencing result and the results are shown in Figure 6 with the first step splicing.
The 3rd step: obtain 5 ', 3 ' with regular-PCR and hold more rice genome sequences
Fig. 7 is shown in by the schematic diagram of complete integrated structure and flanking sequence, and the nucleotides sequence of complete integrated structure and flanking sequence is classified as shown in SEQ ID No.2.
Experimental example 1 turns the screening of high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity qualitative detection Auele Specific Primer and the optimization of amplification condition
One, the foundation of specificity of transformant detection method
According to 9 pairs of specificity of transformant primers of 3 ' end flanking sequence design.The joining region sequence of amplification exogenous insertion vector and rice genome, the size of amplified production is all not less than 100bp.
9 pairs of primers are set altogether, and primer sequence is respectively:
Primer 1: object fragment length is 276bp
F：TTCGCTCATGTGTTGAGCATAT(SEQ?ID?No.3)
R：GAGATTTGATTTGGCTGATCTAGG(SEQ?ID?No.4)
Primer 2: object fragment length is 314bp
F：TTCGCTCATGTGTTGAGCATAT(SEQ?ID?No.5)
R：GTTAAATGTCAACAGCCATCTGC(SEQ?ID?No.6)
Primer 3: object fragment length is 328bp
F：TTCGCTCATGTGTTGAGCATAT(SEQ?ID?No.7)
R：ATCGTTTGGTTCCTGTTAAATGTC(SEQ?ID?No.8)
Primer 4: object fragment length is 138bp
F：TTCAGTACATTAAAAACGTCCGC(SEQ?ID?No.9)
R：GAGATTTGATTTGGCTGATCTAGG(SEQ?ID?No.10)
Primer 5: object fragment length is 176bp
F：TTCAGTACATTAAAAACGTCCGC(SEQ?ID?No.11)
R：GTTAAATGTCAACAGCCATCTGC(SEQ?ID?No.12)
Primer 6: object fragment length is 190bp
F：TTCAGTACATTAAAAACGTCCGC(SEQ?ID?No.13)
R：ATCGTTTGGTTCCTGTTAAATGTC(SEQ?ID?No.14)
Primer 7: object fragment length is 121bp
F：GTCCGCAATGTGTTATTAAGTTGTC(SEQ?ID?No.15)
R：GAGATTTGATTTGGCTGATCTAGG(SEQ?ID?No.16)
Primer 8: object fragment length is 159bp
F：GTCCGCAATGTGTTATTAAGTTGTC(SEQ?ID?No.17)
R：GTTAAATGTCAACAGCCATCTGC(SEQ?ID?No.18)
Primer 9: object fragment length is 173bp
F：GTCCGCAATGTGTTATTAAGTTGTC(SEQ?ID?No.19)
R：ATCGTTTGGTTCCTGTTAAATGTC(SEQ?ID?No.20)
Primer screening the results are shown in Figure 8.From the selection result: all obtain expecting size strip positive material, and do not obtain in negative control, preliminary identification obtain 3 ' end flanking sequence exactness, the exactness of follow-up available LA-PCR method validation integrated structure is also set up specificity of transformant detection method.
From the amplification of Fig. 8, primer 8 amplification efficiencies are higher, and primer dimer is less.Therefore, the present invention preferably adopts primer shown in SEQ ID No.17 and SEQ ID No.18 to detect primer as detecting transgenic paddy rice PA110-15 strain specificity PCR.
2.PCR reaction system and condition optimizing
Adjust the final concentration of primer in SSR-PCR optimization, 0.1 μ M is set, 0.2 μ M, 0.4 μ M, 4 concentration gradients such as 0.8 μ M, and with 54 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃ are carried out the amplification of PCR reaction for annealing temperature, result shows, under set different primers concentration and different annealing temperature condition, can amplify the big or small fragment of expection, but be 0.2 μ M at primer final concentration, best (Fig. 9: primer concentration is 0.1 μ M/L of effect when annealing temperature is 58 ℃, Figure 10: primer concentration is 0.2 μ M/L, Figure 11: primer concentration is 0.4 μ M/L, Figure 12: primer concentration is 0.8 μ M/L).
Experimental example 2 turns the foundation of high-lysine storage protein trans-genetic hybrid rice PA110-15 event-specific detection system and response procedures
One, the extraction of plant genome DNA and detection
1 DNA of plants is extracted
The preparation of 1.1CTAB Extraction buffer
In 600mL water, add 81.7g NaCl and 20g CTAB, after fully dissolving, add 1mol/LTris-HCl(pH7.5) solution 100mL, 0.5mol/L EDTA(pH8.0) solution 100mL, finally adjust pH to 8.0 with HCl or NaOH solution, add water and be settled to 1000mL.Under High Temperature High Pressure (103.4kPa/121 ℃) condition, after sterilizing 20min, use.
1.2 extracting method
A.100mg sample, fully pulverizes in the last 2ml of being transferred to centrifuge tube;
B.1ml be preheated to the CTAB Extraction buffer of 65 ℃, fully mixing, suspension sample, and softly mix.
C.65 ℃ water-bath 40min, puts upside down during this time and mixes for several times;
D.12000r/min centrifugal 15min.Shift the new centrifuge tube of supernatant to, add equal-volume phenol, chloroform-primary isoamyl alcohol (24:1), fully mix;
E.12000r/min centrifugal 10min.Shift the new centrifuge tube of supernatant to, add equal-volume chloroform-primary isoamyl alcohol (24:1), fully mix;
F.12000r/min centrifugal 10min, gets supernatant, adds 2/3 volume Virahol, 1/10 volumes of acetic acid sodium.Place 2 hours or the longer time for-20 ℃;
G.12000r/min centrifugal 10min;
H. abandon supernatant, add 500 μ L, 70% ethanolic soln, and put upside down centrifuge tube for several times.The centrifugal 10min of 12000r/min;
I. abandon supernatant, centrifuge tube is stood upside down and blotted in thieving paper, and ethanol is fully volatilized, dry DNA.
G. add 100 μ l water dissolution DNA;
K. be 100ng/ μ l with double distilled water by DNA solution Concentration Modulation, be stored in-20 ℃ for subsequent use.
2DNA detects
Get the DNA solution that 3ul extracts, with 0.8% agarose gel electrophoresis, judge the quality of DNA according to its brightness and banding pattern.Adopt ultraviolet spectrophotometer to measure concentration and the purity of the DNA that puies forward.
Two, turn the foundation of high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity qualitative PCR detection system and response procedures
Through optimizing, turn the system of high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity qualitative PCR detection in table 1.
Table 1PA110-15 rice strain specificity qualitative PCR detection system
Table 2PA110-15 rice strain specificity qualitative PCR response procedures
Experimental example 3 turns the specificity analyses of high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity qualitative PCR detection method
One, experiment material
1, vegetable material
Sample 1: the lucky round-grained rice 88 of non-transgenic paddy rice, Japan be fine, raise rice No. 6, bright extensive 86, and balanced mix is made a sample;
Sample 2: the lucky list 180 of non-transgenic corn, lucky east 26, Shen Yu 21, Yu Qing 281, balanced mix is made a sample;
Sample 3: soybean feminine gender;
Sample 4: cotton feminine gender;
Sample 5: rape feminine gender;
Sample 6: rich No. 6 of transgenic paddy rice section, rich No. 8 of section, Kemingdao, M12, TT51, be mixed and made into a sample, content each 5%;
Sample 7: transgenic corns Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON89034,59122, MIR604, MON88017, be mixed and made into 1 sample, content each 5%;
Sample 8: genetically engineered soybean 356043,305423, CV127, MON89788, A5547-127, A2704-12, GTS40-3-2, be mixed and made into 1 sample, content each 5%;
Sample 9: transgene cotton MON1445, MON531, MON15985, LLCOTTON25, MON88913, be mixed and made into 1 sample, content each 5%;
Sample 10: transgene rape MS1, MS8, RF1, RF2, RF3, T45, Oxy235, Topas19/2, be mixed and made into 1 sample, content each 5%;
Sample 11: transgenic paddy rice PA110-15.
Above experiment material is provided by the biological institute in Ministry of Agriculture's development in science and technology center and the Chinese Academy of Agricultural Sciences.
Two, experimental technique and result
Take SEQ ID No.17 and SEQ ID No.18 as Auele Specific Primer pair, the PA110-15 strain rice strain specificity qualitative PCR detection method of setting up according to experimental example 2 carries out amplification to various materials and shows, only in paddy rice PA110-15 strain genome, increase and obtained the band (159bp) of object clip size, in other transgenic paddy rice (rich No. 6 of section, rich No. 8 of section, Kemingdao, M12 and TT51), other transgenic plant material (genetically engineered soybean, transgenic corns, transgene cotton and transgene rape) and non-transgenic paddy rice, in the genomes such as non-transgenic corn, do not have amplification to obtain the fragment (Figure 13) of object clip size.Show that by sequencing analysis the sequence that in PA110-15 rice genome, amplification obtains is consistent with object amplified fragments sequence.The above results shows that the transgenic paddy rice PA110-15 strain specificity qualitative PCR detection method that the present invention sets up has specificity highly.
Claims (10)
1. the joining region Nucleotide that turns insertion vector gene and flank paddy rice sequence in high-lysine storage protein trans-genetic hybrid rice PA110-15 strain, is characterized in that: its nucleotides sequence is classified as shown in SEQ ID No.2.
2. joining region claimed in claim 1 Nucleotide detects the application that turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain as target gene.
3. the PCR qualitative detection primer that turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity, is characterized in that, is selected from a pair of arbitrarily in following 9 pairs of primers: nucleotides sequence is classified the primer pair 1 shown in SEQ ID No.3 and SEQ ID No.4 as; Nucleotides sequence is classified the primer pair 2 shown in SEQ ID No.5 and SEQ ID No.6 as; Nucleotides sequence is classified primer pair 3 shown in SEQ ID No.7 and SEQ ID No.8 as; Nucleotides sequence is classified the primer pair 4 shown in SEQ ID No.9 and SEQ ID No.10 as; Nucleotides sequence is classified the primer pair 5 shown in SEQ ID No.11 and SEQ ID No.12 as; Nucleotides sequence is classified the primer pair 6 shown in SEQ ID No.13 and SEQ ID No.14 as; Nucleotides sequence is classified the primer pair 7 shown in SEQ ID No.15 and SEQ ID No.16 as; Nucleotides sequence is classified the primer pair 8 shown in SEQ ID No.17 and SEQ ID No.18 as; Nucleotides sequence is classified the primer pair 9 shown in SEQ ID No.19 and SEQ ID No.20 as.
4. a PCR qualitative checking method that turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity, is characterized in that, comprising: (1) extracts the DNA of paddy rice sample to be detected; (2) take the DNA that extracts as template, design Auele Specific Primer pair with joining region claimed in claim 1 Nucleotide for target gene, set up the performing PCR amplification of going forward side by side of PCR reaction system; (3), if amplify the object band of expection from sample to be detected, paddy rice sample to be detected is to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain; If fail to amplify the object band of expection from sample to be detected, paddy rice sample to be detected is not to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain.
5. a PCR qualitative checking method that turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity, is characterized in that, comprising: (1) extracts the DNA of paddy rice sample to be detected; (2), take the DNA that extracts as template, detect primer with PCR claimed in claim 3 and set up the performing PCR amplification of going forward side by side of PCR reaction system; (3), if amplify the object band of expection from sample to be detected, paddy rice sample to be detected is to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain; If fail to amplify the object band of expection from sample to be detected, paddy rice sample to be detected is not to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain.
6. a PCR qualitative checking method that turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity, is characterized in that, comprising: (1) extracts the DNA of paddy rice sample to be detected; (2), take the DNA that extracts as template, set up the performing PCR amplification of going forward side by side of PCR reaction system with the primer pair shown in SEQ ID No.17 and SEQ ID No.18; (3), if amplify the object band of 159bp from sample to be detected, paddy rice sample to be detected is to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain; If fail to amplify the object band of 159bp from sample to be detected, paddy rice sample to be detected is not to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain.
7. the PCR qualitative checking method described in any one according to claim 4-6, it is characterized in that, described PCR reaction system is: the cumulative volume of reaction system is 25.0 μ L, wherein, 10 × PCR damping fluid, 2.5 μ L, 25mmol/L magnesium chloride solution 1.5 μ L, dNTPs mixing solutions 2 μ L, 0.2 μ mol/L upstream primer 0.5 μ L, 0.2 μ mol/L downstream primer 0.5 μ L, 0.025U/ μ L Taq enzyme 1.5 μ L, 25mg/L DNA profiling 2.0 μ L, surplus is distilled water.
8. the PCR qualitative checking method described in any one according to claim 4-6, is characterized in that, described pcr amplification condition is: 94 ℃, and 5min; 94 ℃, 30s, 58 ℃, 30s, 72 ℃, 30s, 35 circulations; 94 ℃, 2min.
9. a strain specificity PCR qualitative detection test kit that turns high-lysine storage protein trans-genetic hybrid rice PA110-15, comprising: 10 × PCR damping fluid, and magnesium chloride solution, dNTPs mixing solutions, detects upstream and downstream primer pair, Taq enzyme and distilled water; It is characterized in that: described detection upstream and downstream primer is that PCR claimed in claim 3 detects primer.
10. according to strain specificity PCR qualitative detection test kit claimed in claim 9, it is characterized in that: described PCR detects the nucleotides sequence of primer and classifies as shown in SEQ ID No.17 and SEQ ID No.18.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132581A (en) * | 2015-10-20 | 2015-12-09 | 中国农业科学院生物技术研究所 | Genetically modified rice PA110-15 transformant specificity quantitative PCR detection primer |
| CN105132582A (en) * | 2015-10-20 | 2015-12-09 | 中国农业科学院生物技术研究所 | PCR detection primer in genetically modified rice PA110-15 foreign gene homozygous and heterozygous state |
| CN108251551A (en) * | 2017-12-21 | 2018-07-06 | 中国农业科学院生物技术研究所 | Using the joint inspection of RPA-DNA test strips to transgenic paddy rice PA110-15 event-specific detection methods |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134603A (en) * | 2010-12-21 | 2011-07-27 | 中国检验检疫科学研究院 | Primer, probe, test kit and method for testing genetically modified rice or products thereof |
| KR20120092736A (en) * | 2010-12-24 | 2012-08-22 | 대한민국(농촌진흥청장) | Primers and probes for detecting insect resistance characteristic genetically modified organism and detecting method using the same |
| CN102888398A (en) * | 2011-07-22 | 2013-01-23 | 中国农业科学院生物技术研究所 | Flanking sequence of exogenous insertion fragment of transgenic rice variety Bar68-1 and application thereof |
| CN103320437A (en) * | 2013-07-10 | 2013-09-25 | 广东省农业科学院植物保护研究所 | Gene-specific molecular marker Pi2SNP of rice blast-resistant gene Pi2 as well as preparation method and application thereof |
| CN103397094A (en) * | 2013-08-02 | 2013-11-20 | 中国检验检疫科学研究院 | Method and kit used for detection of transgenic rice strain Bt63 |
| CN103397081A (en) * | 2013-03-28 | 2013-11-20 | 湖北省农业科学院粮食作物研究所 | Primer for detecting exogenous gene in transgenic rice T1C-19, and detection method thereof |
-
2014
- 2014-03-06 CN CN201410081539.4A patent/CN103834646B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134603A (en) * | 2010-12-21 | 2011-07-27 | 中国检验检疫科学研究院 | Primer, probe, test kit and method for testing genetically modified rice or products thereof |
| KR20120092736A (en) * | 2010-12-24 | 2012-08-22 | 대한민국(농촌진흥청장) | Primers and probes for detecting insect resistance characteristic genetically modified organism and detecting method using the same |
| CN102888398A (en) * | 2011-07-22 | 2013-01-23 | 中国农业科学院生物技术研究所 | Flanking sequence of exogenous insertion fragment of transgenic rice variety Bar68-1 and application thereof |
| CN103397081A (en) * | 2013-03-28 | 2013-11-20 | 湖北省农业科学院粮食作物研究所 | Primer for detecting exogenous gene in transgenic rice T1C-19, and detection method thereof |
| CN103320437A (en) * | 2013-07-10 | 2013-09-25 | 广东省农业科学院植物保护研究所 | Gene-specific molecular marker Pi2SNP of rice blast-resistant gene Pi2 as well as preparation method and application thereof |
| CN103397094A (en) * | 2013-08-02 | 2013-11-20 | 中国检验检疫科学研究院 | Method and kit used for detection of transgenic rice strain Bt63 |
Non-Patent Citations (1)
| Title |
|---|
| 浙江农业执法网: "农业转基因生物安全评价审查（2010年）", 《WWW.ZJNYZF.GOV.CN/UPLOADFILES/2011-01/1296181711202.DOC》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132581A (en) * | 2015-10-20 | 2015-12-09 | 中国农业科学院生物技术研究所 | Genetically modified rice PA110-15 transformant specificity quantitative PCR detection primer |
| CN105132582A (en) * | 2015-10-20 | 2015-12-09 | 中国农业科学院生物技术研究所 | PCR detection primer in genetically modified rice PA110-15 foreign gene homozygous and heterozygous state |
| CN108251551A (en) * | 2017-12-21 | 2018-07-06 | 中国农业科学院生物技术研究所 | Using the joint inspection of RPA-DNA test strips to transgenic paddy rice PA110-15 event-specific detection methods |
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