CN103966208B - Transgenic paddy rice PA110-15 external source Insert Fragment flanking sequence and application - Google Patents
Transgenic paddy rice PA110-15 external source Insert Fragment flanking sequence and application Download PDFInfo
- Publication number
- CN103966208B CN103966208B CN201410081893.7A CN201410081893A CN103966208B CN 103966208 B CN103966208 B CN 103966208B CN 201410081893 A CN201410081893 A CN 201410081893A CN 103966208 B CN103966208 B CN 103966208B
- Authority
- CN
- China
- Prior art keywords
- seqidno
- strain
- pcr
- storage protein
- detected
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses transgenic paddy rice PA110-15 external source Insert Fragment flanking sequence and application.The present invention provide firstly 5 ' terminal sequence and the 3 ' flanking sequence that transgenic paddy rice PA110-15 strain external source inserts gene, does is its nucleotide sequence respectively SEQ? ID? No.1 and SEQ? ID? shown in No.2.The present invention turns PCR detection primer and the strain specificity qualitative checking method of high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity for target gene provides with described flanking sequence.Specificity experiments shows, the transgenic paddy rice PA110-15 strain specificity qualitative PCR detection method that the present invention sets up has the specificity of height.Sensitivity test result shows, the detection method sensitivity that the present invention sets up reaches 0.1%.
Description
Technical field
The present invention relates to the external source Insert Fragment flanking sequence of transgenic rice lines, particularly relate to the joining region sequence turning high-lysine storage protein trans-genetic hybrid rice PA110-15 exogenous sequences and flank rice sequences, the invention further relates to and turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity PCR detection primer, qualitative checking method and detection kit, belong to and turn high-lysine storage protein trans-genetic hybrid rice PA110-15 event-specific detection field.
Background technology
Along with the extensive plantation of transgenic plant, the safety issue of genetically modified food also causes people to pay close attention to greatly, and existing more than 50 countries and regions implement transgenic product mark system in succession, wherein also comprise China.The enforcement of transgenic labeling system depends on the composition detection to transgenic plant and converted products thereof.Round pcr detects most widely used method in transgenic product both at home and abroad at present.When applying this technology and carrying out genetically modified crops qualitative detection, according to its specificity, be divided into selective mechanisms 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 Lines, all there is the joining region sequence of special exogenous insertion vector and Plant Genome, compare with first three methods, strain specificity has higher specificity and accuracy, the most applicablely does GMO detection.
At present, partial monopoly and the bibliographical information event-specific detection method of transgenic plant is had.Such as: magnify the people such as soldier established transgenosis MON863 corn event-specific detection method in 2006; The people such as Xie Jiajian establish a series of event-specific detection method such as transgenic paddy rice Kemingdao, Bt Shanyou 63, section rich No. 6 and rich No. 8 of section etc.; The people such as Lu Changming established the event-specific detection method of a series of transgene rape in 2007.
PA110-15 is temp-sensing sterile line Peiai 64S(PA64S with two) for acceptor, turn high-lysine storage protein trans-genetic hybrid rice by agriculture bacillus mediated acquisition.Up to now, lack a kind of high specificity, highly sensitive turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity PCR detection method.
Summary of the invention
An object of the present invention is to provide the joining region sequence turning high-lysine storage protein trans-genetic hybrid rice PA110-15 exogenous sequences and flank rice sequences;
Two of object of the present invention is to provide the PCR turning high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity and detects primer;
Three of object of the present invention sets up to turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity PCR qualitative checking method;
Four 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 (SEQIDNo.19) obtains by Hi-TAILPCR the joining region sequence (SEQIDNo.1) that external source inserts gene and 5 ' end paddy rice flanking sequence according to turning the goal gene inserted in high-lysine storage protein trans-genetic hybrid rice PA110-15 strain, the length of this joining region sequence is 1451bp, wherein, 1-1144 position is that Rice Genome Sequence (is positioned at chromosomal 1063000 – 1064144 of paddy rice the 4th, GenBank registration number: NC_008397), 1146-1336 position is plasmid pSBLRP584 frame sequence, 1337-1451 position is the promoter sequence of Rice Glutelin gene Gt1.
The present invention obtains the joining region sequence (SEQIDNo.2) that external source inserts gene and 3 ' end paddy rice flanking sequence further, the length of this joining region sequence is 783bp, wherein, 1-262 position is NOS terminator sequence, for 263-569 position is plasmid pSBLRP584 frame sequence, 570-783 position is Rice Genome Sequence (being positioned at the chromosomal 1064179-1064392 position of paddy rice the 4th, GenBank registration number: NC_008397).
5 ' the end flanking sequence of the present invention according to SEQIDNo.1 is that target gene devises 4 to detection primer, and 4 pairs of primers are respectively: primer pair 1:PA110-5-F1/R1(SEQIDNo.3 and SEQIDNo.4); Primer pair 2:PA110-5-F2/R2(SEQIDNo.5 and SEQIDNo.6); Primer pair 3:PA110-5-F3/R3(SEQIDNo.7 and SEQIDNo.8); Primer pair 4:PA110-5-F4/R4(SEQIDNo.9 and SEQIDNo.10); The present invention sets up PCR amplification system with above-mentioned 4 pairs of primers respectively and carries out augmentation detection to PA110-15, and amplification finds primer PA110-5-F1/R1(SEQIDNo.3 and SEQIDNo.4) and PA110-5-F2/R2(SEQIDNo.5 and SEQIDNo.6) specificity is good, amplified band is clear; The present invention is further by system optimization, the orthogonal experiment of design primer final concentration and annealing temperature, it is best that final discovery combination of primers PA110-5-F1/R1 detects primer specificity as 5 ' end specificity of transformant, and amplification efficiency is the highest, and primer dimer is also minimum.
The present invention holds flanking sequence to be that target gene devises 4 to detection primer with 3 ' shown in SEQIDNo.2 further, and 4 pairs of primers are respectively: primer pair 5:PA110-3-F1/R1(SEQIDNo.11 and SEQIDNo.12); Primer pair 6:PA110-3-F2/R2(SEQIDNo.13 and SEQIDNo.14); Primer pair 7:PA110-3-F3/R3(SEQIDNo.15 and SEQIDNo.16); Primer pair 8:PA110-3-F4/R4(SEQIDNo.17 and SEQIDNo.18); The present invention sets up PCR amplification system with above-mentioned four pairs of primers respectively and carries out augmentation detection to PA110-15, and amplification finds primer PA110-3-F1/R1 and PA110-3-F3/R3 specificity is good, amplified band is clear; The present invention is further by system optimization, and the orthogonal experiment of design primer final concentration and annealing temperature, the final combination of primers PA110-3-F3/R3 of discovery detects primer as 3 ' end specificity of transformant and has best Detection results.
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) with the DNA extracted for template, with shown in SEQIDNo.1 5 ' end flanking sequence design Auele Specific Primer pair for target gene, set up PCR amplification system go forward side by side performing PCR increase; (3) if amplify the specific band of expection from sample to be detected, then paddy rice sample to be detected turns 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, then paddy rice sample to be detected is not turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain.
Wherein, described Auele Specific Primer is to any pair that is selected from following 4 pairs of primers: nucleotides sequence is classified as the primer pair 1 shown in SEQIDNo.3 and SEQIDNo.4; Nucleotides sequence is classified as the primer pair 2 shown in SEQIDNo.5 and SEQIDNo.6; Nucleotides sequence is classified as primer pair 3 shown in SEQIDNo.7 and SEQIDNo.8; Nucleotides sequence is classified as the primer pair 4 shown in SEQIDNo.9 and SEQIDNo.10.
Preferably, a kind of PCR qualitative checking method turning high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity, comprising: (1) extracts the DNA of paddy rice sample to be detected; (2) with the DNA extracted for template, with the primer pair shown in SEQIDNo.3 and SEQIDNo.4 set up PCR reaction system go forward side by side performing PCR amplification; (3) if amplify the object band of 262bp from sample to be detected, then paddy rice sample to be detected turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain; If fail to amplify the object band of 262bp from sample to be detected, then paddy rice sample to be detected is not turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain.
The present invention also provides another kind to turn 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) with the DNA extracted for template, with shown in SEQIDNo.2 3 ' end flanking sequence design Auele Specific Primer pair for target gene, set up PCR amplification system go forward side by side performing PCR increase; (3) if amplify the specific band of expection from sample to be detected, then paddy rice sample to be detected turns 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, then paddy rice sample to be detected is not turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain.
Wherein, described Auele Specific Primer is to any pair that is selected from following 4 pairs of primers: nucleotides sequence is classified as the primer pair 5 shown in SEQIDNo.11 and SEQIDNo.12; Nucleotides sequence is classified as the primer pair 6 shown in SEQIDNo.13 and SEQIDNo.14; Nucleotides sequence is classified as primer pair 7 shown in SEQIDNo.15 and SEQIDNo.16; Nucleotides sequence is classified as the primer pair 8 shown in SEQIDNo.17 and SEQIDNo.18.
Preferably, one 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) with the DNA extracted for template, with the primer pair shown in SEQIDNo.15 and SEQIDNo.16 set up PCR amplification system go forward side by side performing PCR amplification; (3) if amplify the band of 376bp from sample to be detected, then paddy rice sample to be detected turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain; If fail to amplify the band of 376bp from sample to be detected, then paddy rice sample to be detected is not turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain.
Wherein, the described method extracting DNA from paddy rice sample to be detected can be the various common methods extracting DNA from vegetable material, such as, can be the various methods 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 of detected result and sensitivity; The present invention has investigated the impact for the specificity detected and sensitivity of the final concentration of primer in PCR reaction system and annealing temperature, experimental result find primer final concentration be 0.2 μM, annealing temperature be 58 DEG C time, 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, 10mmol/LdNTPs mixing solutions (each 2.5mmol/L) 2 μ L, 10 μm of ol/L upstream primer 0.5 μ L, 10 μm of ol/L downstream primer 0.5 μ L, 5U/ μ LTaq enzyme 0.125 μ L, 25mg/LDNA template 2.0 μ L, surplus is distilled water.
Described pcr amplification condition optimization is: 95 DEG C of sex change 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s, carry out 35 circulations altogether; 72 DEG C extend 7min.
Further aim of the present invention is to provide a kind of PCR qualitative detection test kit turning 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, preferably in following 8 pairs of primers any pair of described PCR qualitative detection primer pair: nucleotides sequence is classified as the primer pair 1 shown in SEQIDNo.3 and SEQIDNo.4; Nucleotides sequence is classified as the primer pair 2 shown in SEQIDNo.5 and SEQIDNo.6; Nucleotides sequence is classified as primer pair 3 shown in SEQIDNo.7 and SEQIDNo.8; Nucleotides sequence is classified as the primer pair 4 shown in SEQIDNo.9 and SEQIDNo.10; Nucleotides sequence is classified as the primer pair 5 shown in SEQIDNo.11 and SEQIDNo.12; Nucleotides sequence is classified as the primer pair 6 shown in SEQIDNo.13 and SEQIDNo.14; Nucleotides sequence is classified as the primer pair 7 shown in SEQIDNo.15 and SEQIDNo.16; Nucleotides sequence is classified as the primer pair 8 shown in SEQIDNo.17 and SEQIDNo.18.
The strain specificity PCR qualitative checking method adopting 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, TT51-1 and other transgenic plant and non-transgenic plant material, qualitative PCR amplification shows, only turning in high-lysine storage protein trans-genetic hybrid rice PA110-15 positive material genome amplification and obtain the object fragment band of expection size, in other transgenic paddy rice and the genome such as other transgenic plant and conventional rice PA64S, amplification is not had to obtain expection object fragment;
The present invention uses 5 ' specific detection primer (PA110-5-F1/R1) and 3 ' specific detection primer (PA110-3-F3/R3) to transgenic paddy rice PA110-15 respectively, other transgenic paddy rice materials (M12, TT51-1, rich No. 6 of section, section rich No. 8 and Kemingdao) and non-transgenic paddy rice PA64S carry out pcr amplification, result shows, these 2 pairs of primer energy specificitys distinguish transgenic paddy rice PA110-15, and without any amplification in other transgenic paddy rices and non-transgenic paddy rice, 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.
Sensitivity test result shows, 5 ' end of the present invention detects primer PA110-5-F1/R1 or 3 ' end and detects primer PA110-3-F3/R3 and effectively can be increased from the PA110-15 transgenic sample of 0.05% content, and detection sensitivity of the present invention can be stablized and reaches 0.1%.
Accompanying drawing explanation
Fig. 1 Hi-TAILPCR amplification.
Figure 25 ' holds specificity of transformant to detect primer screening result; M:100bpMarker; 1-3:PA110-15; 4: acceptor material-CK; 5: blank.
Fig. 3 primer system is optimized; M:100bpMarker; 1-6:PA110-15; 7: acceptor material-CK; 8: blank.
Figure 43 ' holds specificity of transformant to detect primer screening; M:100bpMarker; 1-3:PA110-15; 4: acceptor material-CK; 5: blank.
Fig. 5 primer system is optimized; M:100bpMarker; 1-6:PA110-15; 7: acceptor material-CK; 8: blank.
The test of Fig. 6 transformant 5 ' end detection method specificity; M:100bpMarker; 1-3:PA110-15; 4:M12; 5:TT51-1; 6: rich No. 6 of section; 7: rich No. 8 of section; 8: Kemingdao; 9: transgenic corns compound sample; 10: transgene rape compound sample; 11: genetically engineered soybean compound sample; 12: transgene cotton compound sample; 13,14: non-transgenic paddy rice PA64S; 15: blank.
The test of Fig. 7 transformant 3 ' end detection method specificity; M:100bpMarker; 1-3:PA110-15; 4:M12; 5:TT51-1; 6: rich No. 6 of section; 7: rich No. 8 of section; 8: Kemingdao; 9: transgenic corns compound sample; 10: transgene rape compound sample; 11: genetically engineered soybean compound sample; 12: transgene cotton compound sample; 13,14: non-transgenic paddy rice PA64S; 15: blank
Fig. 8 PA110-5-F1/R1 detection sensitivity; M:100bpMarker; 1,2:PA110-15 massfraction is 10%; 3,4:PA110-15 massfraction is 1%; 5,6:PA110-15 massfraction is 0.5%; 7,8:PA110-15 massfraction is 0.1%; 9,10:PA110-15 massfraction is 0.05%; 11,12:PA110-15 massfraction is 0.01%; 13,14: acceptor material-CK; 15,16: blank.
Fig. 9 PA110-3-F3/R3 detection sensitivity; M:100bpMarker; 1,2:PA110-15 massfraction is 10%; 3,4:PA110-15 massfraction is 1%; 5,6:PA110-15 massfraction is 0.5%; 7,8:PA110-15 massfraction is 0.1%; 9,10:PA110-15 massfraction is 0.05%; 11,12:PA110-15 massfraction is 0.01%; 13,14: acceptor material-CK; 15,16: blank.
Embodiment 1 turn of high-lysine storage protein trans-genetic hybrid rice PA110-15 external source inserts the acquisition of the flanking sequence of 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 for shown in SEQIDNo.19.
Obtain external source by Hi-TAILPCR and insert the joining region sequence (SEQIDNo.1) of gene and 5 ' end paddy rice flanking sequence and the joining region sequence (SEQIDNo.2) of external source insertion gene and 3 ' end paddy rice flanking sequence.
The ultimate principle of TAIL-PCR technology is that the known array utilizing target sequence other designs 3 nested Auele Specific Primer (specialprime, be called for short spl, sp2, sp3, about 20bp), with they respectively with 1 short (14bp) arbitrary degenerate primer (Arbitrarydegenerateprimes with low T value, be called for short AD) combined, using genomic dna as template, design asymmetric temperature cycle according to the length of primer and specific difference, carry out amplifying specific primer by fractional order reaction; TAIL-PCR is divided into 3 secondary responses.1st PCR reaction is made up of 5 high specific reactions, 1 low specific reaction, 10 lower specific reactions and 12 asymmetric super circulations of heat.By the reaction of 5 high specifics, make the sequence anneals of LRP on sp1 and carrier and extend, improving the concentration of target sequence.1 low specific reaction makes degenerated primer be attached on more target sequence, and 10 lower specific reactions make two kinds of primers all energy and template annealings, carries out 12 TAIL circulations subsequently.Through the above-mentioned product being obtained by reacting 3 types of different concns: specific product (I type) and nonspecific products (II type and III type).The product dilution 40 that the first step is then reacted by the 2nd secondary response, doubly as template, by 10 asymmetric super circulations of heat, specific product is optionally increased, and nonspecific product is pressed into extremely low content.3rd secondary response again using the product dilution 40 of the 2nd secondary response doubly as template, be set to the common super circulation of hot asymmetric PCR.The target sequence contiguous with known array can be obtained by above-mentioned 3 PCR reaction.The more satisfactory band of picking directly checks order by PCR primer, finally obtain LRP gene promoter flanking sequence on one side by SP3 and AD1, DNA sequencing is carried out to the target sequence obtained, and sequencing result is carried out Blast comparison on NCBI, then can learn the particular location that LRP gene inserts.
The checking of terminator on one side flanking sequence a: primer is located at NOS(terminator) in sequence, one is located on the left side flanking sequence measured, and expection length is 777bp, and primer is 6F/6R.Primer has all increased the product (Fig. 1) of expection length, and the PCR primer of this fragment checked order, sequencing result proves that the flanking sequence that the present invention obtains is correct.Concrete PCR primer sequence is in table 1.
Table 1PCR primer sequence
| Auele Specific Primer |
| SP1：5-AACCAACCCAAGCAACCATAG-3 |
| SP2：5-GTGAGTGGTGACCCAGAAGCA-3 |
| SP3:5-CAAATCCAAGGCACTGAACACG-3 |
| Degenerated primer |
| AD:5-TGAGNAGTANCAGAGA-3 |
| Checking primer |
| 6F:5-CCGATCGTTCAAACATTTGG-3 |
| 6R:5-CGTCTTGTGGGTGATTCTGC-3 |
Concrete PCR program is in table 2.
Table 2PCR program
The screening of experimental example 1 turn 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, 5 ' end specificity of transformant detects the screening of primer
Hold flanking sequence information design 4 to detection primer (see table 3) according to 5 ', augmentation detection is carried out to PA110-15, filter out that the 2 pairs of specificitys are good, amplified band primer PA110-5-F1/R1 and PA110-5-F2/R2(Fig. 2 clearly), further by system optimization, the orthogonal experiment of design primer final concentration and annealing temperature, it is most effective that final discovery combination of primers PA110-5-F1/R1 detects primer amplification as 5 ' end specificity of transformant, primer dimer minimum (Fig. 3).
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 impact for the specificity detected and sensitivity of the final concentration of primer in PCR reaction system and annealing temperature; The present invention arranges 4 concentration gradients such as 0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, and with 54 DEG C, 56 DEG C, 58 DEG C, 60 DEG C, 62 DEG C for annealing temperature carries out the amplification of PCR reaction, result shows, can amplify under set different primers concentration and different annealing temperature condition expection size fragment, but primer final concentration be 0.2 μM, annealing temperature be 58 DEG C time expanding effect best.
Table 35 ' end design of primers information table
Two, 3 ' end specificity of transformant detects the screening of primer
4 to detection primer (see table 4) according to 3 ' end flanking sequence information design, carries out augmentation detection to PA110-15, filter out that the 2 pairs of specificitys are good, amplified band primer PA110-3-F1/R1 and PA110-3-F3/R3(Fig. 4 clearly); The orthogonal experiment of further design primer final concentration and annealing temperature, it is most effective that the final combination of primers PA110-3-F3/R3 of discovery detects primer amplification as 3 ' end specificity of transformant, and primer dimer is minimum.The final concentration of primer in adjusting and optimizing PCR reaction system, 4 concentration gradients such as 0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM are set, and with 54 DEG C, 56 DEG C, 58 DEG C, 60 DEG C, 62 DEG C for annealing temperature carries out the amplification of PCR reaction, result shows, can amplify under set different primers concentration and different annealing temperature condition expection size fragment, but primer final concentration be 0.2 μM, annealing temperature be 58 DEG C time effect best (Fig. 5).
Table 43 ' end design of primers information table
The foundation of experimental example 2 turns of high-lysine storage protein trans-genetic hybrid rice PA110-15 event-specific detection systems 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
Add 81.7gNaCl and 20gCTAB in 600mL water, after fully dissolving, add 1mol/LTris-HCl(pH7.5) solution 100mL, 0.5mol/LEDTA(pH8.0) solution 100mL, finally adjusts pH to 8.0 by HCl or NaOH solution, adds water and be settled to 1000mL.Use after sterilizing 20min under High Temperature High Pressure (103.4kPa/121 DEG C) condition.
1.2 extracting method
A.100mg sample, last being transferred in 2mL centrifuge tube of fully pulverizing.
B.1mL be preheated to the CTAB Extraction buffer of 65 DEG C, fully mixing, suspension sample, and softly mix.
C.65 DEG C water-bath 40min, period puts upside down mixing for several times.
D.12000r/min centrifugal 15min.The new centrifuge tube of transfer supernatant to, adds equal-volume phenol, chloroform-isoamyl alcohol (24:1), fully mixes.
E.12000r/min centrifugal 10min.The new centrifuge tube of transfer supernatant to, adds equal-volume chloroform-isoamyl alcohol (24:1), fully mixes.
F.12000r/min centrifugal 10min, gets supernatant, adds 2/3 volume isopropanol, 1/10 volumes of acetic acid sodium.Place 2 hours or the longer time for-20 DEG C.
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 in thieving paper and blot, and ethanol is fully volatilized, dry DNA.
G. 100 μ L water dissolution DNA are added.
K. be 100ng/ μ L with double distilled water by DNA solution Concentration Modulation, be stored in-20 DEG C for subsequent use.
2DNA detects
Get the DNA solution that 3 μ L extract, with the agarose gel electrophoresis of 0.8%, judge the quality of DNA according to its brightness and banding pattern.Adopt ultraviolet spectrophotometer to measure put forward concentration and the purity of DNA.
Two, the foundation of high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity qualitative PCR detection system and response procedures is turned
Through optimizing, turn the system of high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity qualitative PCR detection in table 5.
Table 5PA110-15 specificity of transformant detects PCR reaction system
| Reagent | Final concentration | Volume |
| Water | —— | |
| 10 × PCR damping fluid | 1× | 2.5μL |
| 25mmol/L magnesium chloride solution | 1.5mmol/L | 1.5μL |
| DNTPs mixing solutions (each 2.5mmol/L) | Each 0.2mmol/L | 2μL |
| 10 μm of ol/L upstream primers | 0.2μmol/L | 0.5μL |
| 10 μm of ol/L downstream primers | 0.2μmol/L | 0.5μL |
| Taq enzyme (5.0U/ μ L) | 0.025U/μL | —— |
| 25mg/L DNA profiling | 2mg/L | 2.0μL |
| Cumulative volume | 25.0μL |
Response procedures:
95 DEG C of sex change 5min;
95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s, carry out 35 circulations altogether;
72 DEG C extend 7min.
The specificity analyses of experimental example 3 turns of high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity qualitative PCR detection methods
Use 5 ' end detection primer (PA110-5-F1/R1) and 3 ' specific detection primer (PA110-3-F3/R3) to transgenic paddy rice PA110-15 respectively, other transgenic paddy rice material (M12, TT51-1, rich No. 6 of section, section rich No. 8 and Kemingdao), transgenic corns compound sample (Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON88017, MIR604), transgene rape compound sample (MS1, MS8, RF1, RF2, RF3, T45, Oxy235, Topas19/2), genetically engineered soybean compound sample (GTS40-3-2, MON89788, A2704-12, A5547-127, 356043, 305423, CV127), transgene cotton compound sample (MON1445, MON531, MON15985, LLCOTTON25, MON88913) and non-transgenic paddy rice PA64S(PA110-15 acceptor material) carry out pcr amplification, the survey system that the amplification system of pcr amplification and amplification condition are set up with experimental example 2 and response procedures carry out.
Specificity test result shows, 5 ' end detects primer (PA110-5-F1/R1) and 3 ' specific detection primer (PA110-3-F3/R3) specificity can distinguish transgenic paddy rice PA110-15, and without any amplification (Fig. 6, Fig. 7) in other transgenic paddy rice and non-transgenic paddy rice.
The sensitivity test of experimental example 4 turns of high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity qualitative PCR detection methods
The transgenosis PA110-15 sample arranging different mass mark carries out sensitivity test, 5 ' end detects primer PA110-5-F1/R1 and 3 ' end and detects primer PA110-3-F3/R3 and effectively can be increased (Fig. 8 and Fig. 9) from the PA110-15 transgenic sample of 0.05% content, and therefore detection sensitivity can be stablized and reaches 0.1%.
Claims (10)
1. turn the joining region sequence that high-lysine storage protein trans-genetic hybrid rice PA110-15 strain external source inserts gene and 5 ' end paddy rice flanking sequence, it is characterized in that: it is the nucleotide sequence shown in SEQIDNo.1.
2. turn the joining region sequence that high-lysine storage protein trans-genetic hybrid rice PA110-15 strain external source inserts gene and 3 ' end paddy rice flanking sequence, it is characterized in that: it is the nucleotide sequence shown in SEQIDNo.2.
3. joining region according to claim 1 sequence is as detecting the application turned in high-lysine storage protein trans-genetic hybrid rice PA110-15 strain target gene.
4. joining region according to claim 2 sequence is as detecting the application turned in high-lysine storage protein trans-genetic hybrid rice PA110-15 strain target gene.
5. the PCR turning high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity detects primer, it is characterized in that, is selected from any pair in following 8 pairs of primers: nucleotides sequence is classified as the primer pair 1 shown in SEQIDNo.3 and SEQIDNo.4; Nucleotides sequence is classified as the primer pair 2 shown in SEQIDNo.5 and SEQIDNo.6; Nucleotides sequence is classified as primer pair 3 shown in SEQIDNo.7 and SEQIDNo.8; Nucleotides sequence is classified as the primer pair 4 shown in SEQIDNo.9 and SEQIDNo.10; Nucleotides sequence is classified as the primer pair 5 shown in SEQIDNo.11 and SEQIDNo.12; Nucleotides sequence is classified as the primer pair 6 shown in SEQIDNo.13 and SEQIDNo.14; Nucleotides sequence is classified as the primer pair 7 shown in SEQIDNo.15 and SEQIDNo.16; Nucleotides sequence is classified as the primer pair 8 shown in SEQIDNo.17 and SEQIDNo.18.
6. turn a PCR qualitative checking method for high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity, it is characterized in that, comprising: (1) extracts the DNA of paddy rice sample to be detected; (2) with the DNA extracted for template, with described in claim 5 PCR detect primer pair set up PCR reaction system go forward side by side performing PCR amplification; (3) if amplify the object band of expection from sample to be detected, then paddy rice sample to be detected turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain or its Derivative line; Under the same terms, if fail to amplify the object band of expection from sample to be detected, then paddy rice sample to be detected is not turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain or its Derivative line.
7. turn a PCR qualitative checking method for high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity, comprising: (1) extracts the DNA of paddy rice sample to be detected; (2) with the DNA extracted for template, with the primer pair shown in SEQIDNo.3 and SEQIDNo.4 set up PCR reaction system go forward side by side performing PCR amplification; (3) if amplify the object band of 262bp from sample to be detected, then paddy rice sample to be detected turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain or its Derivative line; Under the same terms, if fail to amplify the object band of 262bp from sample to be detected, then paddy rice sample to be detected is not turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain or its Derivative line.
8. turn a PCR qualitative checking method for high-lysine storage protein trans-genetic hybrid rice PA110-15 strain specificity, the method comprises: (1) extracts the DNA of paddy rice sample to be detected; (2) with the DNA extracted for template, with the primer pair shown in SEQIDNo.15 and SEQIDNo.16 set up PCR amplification system go forward side by side performing PCR amplification; (3) if amplify the band of 376bp from sample to be detected, then paddy rice sample to be detected turns high-lysine storage protein trans-genetic hybrid rice PA110-15 strain or its Derivative line; Under the same terms, if fail to amplify the band of 376bp from sample to be detected, then paddy rice sample to be detected is not turn high-lysine storage protein trans-genetic hybrid rice PA110-15 strain or its Derivative line.
9. according to the PCR qualitative checking method of claim 6-8 described in any one, 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, 10mmol/LdNTPs mixing solutions 2 μ L, 10 μm of ol/L upstream primer 0.5 μ L, 10 μm of ol/L downstream primer 0.5 μ L, 5U/ μ LTaq enzyme 0.125 μ L, 25mg/LDNA template 2.0 μ L, surplus is distilled water; Wherein, in described dNTPs mixing solutions, each 2.5mmol/L of dATP, dTTP, dGTP, dCTP;
Described pcr amplification condition is: 95 DEG C of sex change 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s, carry out 35 circulations altogether; 72 DEG C extend 7min.
10. turn a strain specificity PCR qualitative detection test kit of high-lysine storage protein trans-genetic hybrid rice PA110-15, comprising: 10 × PCR damping fluid, magnesium chloride solution, dNTPs mixing solutions, detect upstream and downstream primer pair, Taq enzyme and distilled water; It is characterized in that: described detection upstream and downstream primer pair is that PCR according to claim 5 detects primer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410081893.7A CN103966208B (en) | 2014-03-06 | 2014-03-06 | Transgenic paddy rice PA110-15 external source Insert Fragment flanking sequence and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410081893.7A CN103966208B (en) | 2014-03-06 | 2014-03-06 | Transgenic paddy rice PA110-15 external source Insert Fragment flanking sequence and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103966208A CN103966208A (en) | 2014-08-06 |
| CN103966208B true CN103966208B (en) | 2016-04-20 |
Family
ID=51236215
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410081893.7A Active CN103966208B (en) | 2014-03-06 | 2014-03-06 | Transgenic paddy rice PA110-15 external source Insert Fragment flanking sequence and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103966208B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154572A (en) * | 2015-10-20 | 2015-12-16 | 中国农业科学院生物技术研究所 | Specific 5'-terminal quantitative PCR (polymerase chain reaction) detection primers for transgenic rice PA110-15 line |
| 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 |
| CN110724704B (en) * | 2019-10-16 | 2020-12-22 | 安徽省农业科学院水稻研究所 | Positive plasmid for identifying transgenic rice transformant and construction method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005061720A2 (en) * | 2003-12-11 | 2005-07-07 | Monsanto Technology Llc | High lysine maize compositions and methods for detection thereof |
| CN101824411A (en) * | 2010-04-29 | 2010-09-08 | 中国水稻研究所 | Flanking sequence of transgenic rice Kefeng No. 6 and qualitative PCR detection method |
| CN102827838A (en) * | 2012-08-29 | 2012-12-19 | 中国检验检疫科学研究院 | Flanking sequence of human serum albumin transgenic rice strain 114-7-2 and identification method of flanking sequence |
| 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 |
| CN103014136A (en) * | 2011-09-21 | 2013-04-03 | 中国农业科学院生物技术研究所 | Homozygous Ke-Ming-Dao 1 identification method established based on exogenous gene flanking sequence |
-
2014
- 2014-03-06 CN CN201410081893.7A patent/CN103966208B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005061720A2 (en) * | 2003-12-11 | 2005-07-07 | Monsanto Technology Llc | High lysine maize compositions and methods for detection thereof |
| CN1950509A (en) * | 2003-12-11 | 2007-04-18 | 孟山都技术有限公司 | High lysine maize compositions and methods for detection thereof |
| CN101824411A (en) * | 2010-04-29 | 2010-09-08 | 中国水稻研究所 | Flanking sequence of transgenic rice Kefeng No. 6 and qualitative PCR detection method |
| 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 |
| CN103014136A (en) * | 2011-09-21 | 2013-04-03 | 中国农业科学院生物技术研究所 | Homozygous Ke-Ming-Dao 1 identification method established based on exogenous gene flanking sequence |
| CN102827838A (en) * | 2012-08-29 | 2012-12-19 | 中国检验检疫科学研究院 | Flanking sequence of human serum albumin transgenic rice strain 114-7-2 and identification method of flanking sequence |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103966208A (en) | 2014-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103966208B (en) | Transgenic paddy rice PA110-15 external source Insert Fragment flanking sequence and application | |
| Yang et al. | Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5 ‘-transgene integration sequence | |
| ES2531664T3 (en) | Event detection of transgenic plants | |
| CA2915902C (en) | Resistance gene against rhizomania | |
| CN103923999B (en) | The qualitative PCR detection method of transgenic pest-resistant herbicide-resistant paddy rice G6H1 and derived varieties thereof | |
| CN106048010A (en) | RPA (recombinase polymerase amplification) technology based method for detecting phomopsis helianthi, RPA primers and kit | |
| Wang et al. | Event-specific qualitative and quantitative detection of transgenic rice Kefeng-6 by characterization of the transgene flanking sequence | |
| CN107236733A (en) | The transformation event foreign insertion vector flanking sequences of genetically engineered soybean W82 HAL1 8062 and its application | |
| RU2712730C2 (en) | Corn plant dbn9858 resistant to herbicide, and nucleotide sequence and method for detection thereof | |
| EP3638795A1 (en) | Corn elite event mzir098 | |
| Shin et al. | Event-specific detection system of stacked genetically modified maize by using the multiplex-PCR technique | |
| Yang et al. | Screening and construct‐specific detection methods of transgenic Huafan No 1 tomato by conventional and real‐time PCR | |
| CN104419708A (en) | Nucleic acid, method for detecting transgenic rice EB7001S and derived line thereof, kit and application thereof | |
| CN105177172B (en) | 11 event-specific detection methods of transgenic corns T4 and kit | |
| TWI537390B (en) | Nucleic acids and methods for detecting turfgrass pathogenic fungi | |
| Tomar et al. | RNAi-based transgene conferred extreme resistance to the geminivirus causing apical leaf curl disease in potato | |
| WO2019243370A2 (en) | Balanced resistance and avirulence gene expression | |
| CN103834646B (en) | Transgenic paddy rice PA110-15 strain specificity PCR detects primer and qualitative checking method and test kit | |
| CN102888398A (en) | Flanking sequence of exogenous insertion fragment of transgenic rice variety Bar68-1 and application thereof | |
| KR101278208B1 (en) | Multiplex-PCR detection method of stacked genetically modified maize and its primers used event-specifically | |
| CN103834737B (en) | Transgenic rice Kefeng No.2 line specific PCR detection primer as well as qualitative detection method and kit | |
| CN103834645A (en) | Connecting region sequence of exogenous inserting gene and flanking rice sequence of transgenic rice kefeng No.2 and application thereof | |
| CN104388579B (en) | A kind of Arm-PCR detection methods of genetically engineered soybean and its derived varieties | |
| Gan et al. | Application of RNA interference methodology to investigate and develop SCMV resistance in maize | |
| KR100723069B1 (en) | Qualitative and quantitative detection methods using specific primers probes and standard plasmid for GM corns |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |