CN101063171A - Standard plasmid molecule for detection of genetic improved soybean strain GTS40-3-2 and constructing method thereof - Google Patents
Standard plasmid molecule for detection of genetic improved soybean strain GTS40-3-2 and constructing method thereof Download PDFInfo
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- CN101063171A CN101063171A CN 200710041121 CN200710041121A CN101063171A CN 101063171 A CN101063171 A CN 101063171A CN 200710041121 CN200710041121 CN 200710041121 CN 200710041121 A CN200710041121 A CN 200710041121A CN 101063171 A CN101063171 A CN 101063171A
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Abstract
The invention discloses a standard plasmid molecule to check genetic improved soybean strain GTS40-3-2 and constructing method, which comprises the following steps: incorporating strain special fragment of the genetic improved soybean strain GTS40-3-2 and special fragment of soybean internal standard gene Lectin; analyzing exogenesis inserting carrier by-pass ortho gene sequence of genetic improved soybean strain GTS40-3-2; designing strain special PCR primer; augmenting; getting strain special fragment of genetic improved soybean strain GTS40-3-2 and soybean internal standard gene special sequence; constructing into a plasmid molecule with molecular cloning method; getting artificial retooling plasmid molecule pMD-RRS. This standard plasmid molecule can be fit for strain special quantitative PCR analysis and check of genetic improved soybean strain GTS40-3-2 sample.
Description
Technical field
What the present invention relates to is a kind of plasmid molecule of technical field of bioengineering, specifically, relates to a kind of genetic improved soybean strain GTS 40-3-2-3-2 and detects with standard plasmid molecule and construction process thereof.
Background technology
Brand-new development epoch have been opened up in the appearance of genetic engineering technique and the fields such as agricultural, medical science that are applied as.1994, Calgene company utilized the genetic improvement tomato strain " FLAVR SAVR " of genetic engineering technique development to go through first to enter to commercially produce in the U.S..By 2006, global genetic improvement crops planting area reached more than 100,000,000 hectare.Meanwhile, a large amount of genetic improvement agricultural-food pour in China market.China agricultural genetically modified organism security control office was respectively overseas 18 kinds of genetic improvement crops and has issued the safety in production license licensed licenser licence in 2004 and 2005, process raw material to be used for import, comprising genetic improved soybean strain GTS-40-3-2.Extensive plantation along with the genetic improvement crop, its safety issue has caused the global public's generally attention, international organization and country formulate corresponding security control rules one after another, strengthen the standardized administration of genetic improvement crop, and the genetic improvement plants and plant product is implemented the sign system.
In order to tackle genetic improvement crop management relevant laws and regulations and system, foundation is efficient, quick, specific detection technology is very necessary, and it is the strong technical support of each international organization and national management genetic improvement crop.Because of having the advantage of highly sensitive and high specificity, become most important at present, most widely used genetic improvement crop and products thereof detection technique based on polymerase chain reaction (PCR) detection technique of nucleic acid.The main foundation that the PCR detection method is set up is the exogenous genetic fragment of specific amplification genetic improvement plant.At the exogenous dna fragment difference of amplification, PCR detection strategy can be divided into four kinds, i.e. universal component screening PCR detects, gene specific PCR detects, make up the specific PCR detection and strain specificity PCR detects.It is by detecting the joining region sequence realization of exogenous insertion vector and Plant Genome that strain specificity PCR detects.Because each genetic improvement crop strain, the joining region sequence that all has special exogenous insertion vector and Plant Genome, and the joining region sequence is single copy, therefore the strain specificity detection method has specificity and the accuracy higher than screening, gene specific and structure PCR method for detecting specificity, the genetic improvement crop strain that detection that can be special is single-minded.Strain specificity PCR detection method has become the emphasis of various countries' research at present.
In actual detected, reference material is very important.Yet, since the restriction of patent right and prior art, the acquisition relative difficult of genetic improvement crop reference material, and it lacks has become the bottleneck that detection method is set up and used, has hindered the smooth implementation of transgenic product sign system.And existing reference material all utilizes the production of genetic improvement crop material development, and it is difficult to preserve, and material source is also more limited, up to the present only obtains the reference material of tens kinds of genetic improvement crops.
Find through literature search prior art, " the Novel Reference Molecules for Quantitation of GeneticallyModified Maize and Soybean (the novel standard molecule that is used for heredity modified corn and soybean detection by quantitative) " that Japan scientist Hino etc. delivers on 2002 the 85th volumes of " Journal of AOACInternational " (chemurgy scholar association of international official magazine) 1077-1089 page or leaf, propose to utilize plasmid control molecule to replace reference material to be used for theory and method that the genetic improvement crop detects in this article, and made up the plasmid molecule that contains external source testing goal fragment and endogenous standard gene, the detection that is used for genetic improved soybean and corn of success.The empirical tests standard plasmid molecule is good reference material surrogate, have genome preparation easily, amplification efficiency is high and the advantage of good stability.Yet, its weak point is that this plasmid molecule makes up at the external source target gene fragment, the gene specific that can only be used for the genetic improvement crop detects, and disconnects mutually with the trend of detection method development, therefore can not satisfy the requirement of transgenic product sign system.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of genetic improved soybean strain GTS 40-3-2-3-2 strain specificity to detect, make its positive criteria material that can replace genetic improved soybean strain GTS 40-3-2-3-2 be used for qualitative, the quantitative PCR detection of genetic improved soybean strain specificity with standard plasmid molecule and construction process.Strain specificity sequence and internal standard gene order according to genetic improved soybean strain GTS 40-3-2-3-2, utilize the principle design specific PCR primer of overlapping PCR reaction, obtain the strain specificity sequence of genetic improved soybean strain GTS 40-3-2-3-2 and the recombinant dna fragment of one section sequence of internal standard gene Lectin through pcr amplification.Utilize molecule clone technology that recombinant dna fragment is cloned in the pMD18-T carrier, obtain new plasmid molecule pMD-RRS.The standard plasmid molecule that the present invention makes up can replace the original positive criteria material of genetic improved soybean strain GTS 40-3-2-3-2, is used for qualitative and quantitative PCR detection, thoroughly solves the difficult problem of standard material want in genetic improved soybean strain GTS 40-3-2-3-2 detection.
The present invention is achieved by the following technical solutions, and standard plasmid molecule of the present invention to substitute the positive criteria material of genetic improved soybean strain GTS 40-3-2-3-2, is used for qualitative and quantitative PCR detection.This standard plasmid molecule contains the strain specificity sequence of genetic improved soybean strain GTS 40-3-2-3-2 and one section sequence of internal standard gene Lectin simultaneously.
Standard plasmid molecule pMD-RRS construction process of the present invention comprises the steps:
1. utilize database such as GenBank to carry out bioinformatic analysis, obtain the strain specificity sequence of soybean internal standard gene Lectin, genetic improved soybean strain GTS 40-3-2-3-2.
Described soybean internal standard gene Lectin refers to the soybean lectin plain gene, and it is conservative at soybean gene group camber, specificity between having kind, the characteristics of non-specific and single copy number in planting.
The strain specificity sequence of described genetic improved soybean strain GTS 40-3-2-3-2 refers to the dna sequence dna of genetic improved soybean strain GTS 40-3-2-3-2 exogenous insertion vector and soybean gene group adjoining region.
2. design the PCR Auele Specific Primer.
Described PCR Auele Specific Primer refers to the oligonucleotide chain that length is 28 ± 10nt, and the strain specificity sequence of itself and soybean Lectin gene or genetic improved soybean strain GTS 40-3-2-3-2 is identical or complementary.
3. the strain specificity sequence of specific amplification soybean internal standard gene Lectin and genetic improved soybean strain GTS 40-3-2-3-2.
Described specific amplification refers to the one section sequence of the accurate gene Lectin of PCR primer specificity amplification interior label that utilizes design and the strain specificity sequence of genetic improved soybean strain GTS 40-3-2-3-2.
4. the splicing of soybean internal standard gene Lectin and genetic improved soybean strain GTS 40-3-2-3-2 strain specificity sequence.
Described splicing, refer to utilize overlapping pcr with two of soybean internal standard gene Lectin and genetic improved soybean strain GTS 40-3-2s-3-2 strain specificity sequence independently dna fragmentation be connected the Novel DNA fragments that is fused into a reorganization.
5. the plasmid molecule clone who contains new recombinant dna fragment.
Described molecular cloning refers to and the restriction enzyme digestion sites of the above-mentioned reorganization DNA fragment specific that obtains according to design is connected on the plasmid vector pMD18-T acquisition standard plasmid molecule pMD-RRS.
6. the qualitative and quantitative PCR detecting method of standard plasmid molecule pMD-RRS checking.
Described qualitative PCR detection method checking, be meant that the strain specificity fragment that is used for standard plasmid molecule pMD-RRS structure can only obtain in genetic improved soybean strain GTS 40-3-2-3-2 genomic dna amplification, obtain and can not in other soybean lines and other genetic improvement crop gene groups, increase.
Described quantitative PCR detecting method checking, be meant the characteristics such as specificity, sensitivity, repeatability and repeatability of examination criteria plasmid molecule pMD-RRS when carrying out quantitative PCR analysis, identifying that this standard molecule substitutes the ability of genetic improved soybean strain GTS 40-3-2-3-2 positive criteria material, and the mensuration validity that is applied to quantitative PCR detection genetic improved soybean strain GTS 40-3-2-3-2.
Beneficial effect of the present invention is, utilizes the method for overlapping PCR and molecular subcloning to make up the standard plasmid molecule pMD-RRS that contains soybean internal standard gene Lectin specific sequence and genetic improved soybean strain GTS 40-3-2-3-2 strain specificity sequence first.Clearly propose the positive criteria material that this standard molecule can replace genetic improved soybean strain GTS 40-3-2-3-2 among the present invention and be used for this strain specificity quantitative PCR detection, well solved the problem that positive reference material lacks in this strain testing process.This standard plasmid molecule pMD-RRS is more accurate to the result that actual sample is analyzed, and the deviation of detected result is in the 0-0.25 scope that ISO genetic improvement food inspection standard allows.Therefore, the standard plasmid molecule that makes up among the present invention is applicable to fully the genetic improvement composition in genetic improved soybean strain GTS 40-3-2-3-2 sample and the converted products thereof is carried out quantitative analysis.
Description of drawings
Fig. 1 is the signal collection of illustrative plates of the standard plasmid molecule pMD-RRS of the present invention's structure.
Embodiment
Below embodiments of the invention are elaborated: present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually operate according to normal condition, as writing molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press with reference to Sambrook etc., 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the structure of standard molecule
One, experiment reagent
Restriction enzyme Nco I, Xba I, and the corresponding damping fluid of restriction enzyme is available from the white good development in science and technology in Shanghai company limited;
The pMD18-T carrier, T
4Dna ligase and damping fluid thereof are available from the white good development in science and technology in Shanghai company limited;
DNTPs, Taq archaeal dna polymerase and damping fluid thereof, DL2000 Marker are available from the precious biotechnology in Dalian company limited;
TaqMan probe and primer are synthetic by the rich inferior biotechnology in Shanghai company limited.
Other biochemical reagents are import packing or homemade analytical pure.
Two, laboratory apparatus
DY-501 type nucleic acid electrophoresis apparatus (Shanghai Precision Scientific Apparatus Co., Ltd)
PTC-100 type pcr amplification instrument (MJ Research Inc.)
DNA electrophoretic analysis system comprises camera bellows, digital camera, computer, scanner, ink-jet printer, photosensitive printer, Tanon UV-2000 uv analyzer, Gis gel images analysis software (sky, Shanghai energy company)
Other instruments comprise: whizzer, thermostat water bath, incubator, day equality.
Three, test method and process
1, search soybean internal standard gene Lectin in GenBank; By consulting the related data of genetic improvement crop, obtain concrete inserted mode and the copy number of genetic improved soybean strain GTS 40-3-2-3-2 exogenous insertion vector in the genetic improvement crop, and consult the sequence information of main element in the exogenous insertion vector.
2, make up the PCR primer sequence design of standard plasmid molecule pMD-RRS
According to the other adjacent gene order of the exogenous insertion vector of the genetic improved soybean strain GTS 40-3-2-3-2 that obtains, utilize two pairs of qualitative PCR primers of software Primer 5.0 designs, exogenous insertion vector and the soybean gene group adjoining region specific sequence of a pair of genetic improved soybean strain GTS 40-3-2-3-2 that is used to increase, article two, one of primer is positioned on the soybean gene group, and another is positioned on the exogenous insertion vector of genetic improved soybean strain GTS 40-3-2-3-2; Another is to the primer soybean internal standard gene specific sequence that is used to increase.Concrete primer sequence sees Table 1.
Table 1. makes up the PCR primer sequence of standard plasmid molecule pMD-RRS
| Target sequence | Direction | Primer sequence (5 '--3 ') | Clip size (bp) |
| Lectin strain specificity sequence | Justice antisense justice antisense | AAAggatcc ACCAAACATGATCCTCCAAG AAAtctaga CAAACTCAACAGCGACGACT CAACAAATGACCCTG AAActgcag TCGCTGTTGAGTTTG TTTATCAGGGTCATTTGTTG AAAccatgg ACTACCTTCTCACCGCATT | 378 349 |
3, the amplification of endogenous standard gene sequence of genetic improved soybean strain GTS 40-3-2-3-2 and strain specificity sequence
Primer according to table 1, with genetic improved soybean strain GTS 40-3-2-3-2 genomic dna is template, soybean internal standard gene Lectin and strain specificity sequence are carried out pcr amplification (reaction system and condition see Table 2,3), obtain one section 378bp fragment of soybean internal standard gene and the other adjacent gene order fragment of exogenous insertion vector of a 349bp.The band that amplification is obtained reclaims purifying, shows that by sequencing analysis the sequence of acquisition is consistent with purpose amplified fragments sequence.
Table 2. makes up the pcr amplification system of standard plasmid molecule pMD-RRS
| Reaction reagent | Volume (μ l) | Final concentration |
| 10 * PCR buffer solution dNTPs upstream primer downstream primer Taq enzyme dna template ddH2O | 3311 1.5 1 complement to 30 μ l | 1×(50mM KCl，10mM Tris-HCl，PH8.3， 1.5mM MgCl 2) 200 μ M 400nM 400nM, 2.5 unit/reactions |
Table 3. makes up the pcr amplification condition of standard plasmid molecule pMD-RRS
| Cycle number | Temperature (℃) | Time |
| 1 35 1 | 95 95 58 72 72 | 7 minutes 30 seconds 30 seconds 30 seconds 7 minutes |
4, utilize overlapping PCR method that above-mentioned two fragments are connected
The endogenous standard gene and the strain specificity sequence that obtain with above-mentioned PCR are template, and be right as the primer of overlapping PCR with the downstream primer of the upstream primer of Lectin gene and strain specificity sequence, carries out pcr amplification, realizes two segmental reorganization splicings.
5, the recombinant dna fragment clone enters the pMD18-T carrier
Utilize the method for molecular cloning that the recombinant fragment that above-mentioned connection obtains is connected on the plasmid vector pMD18-T, the ligation system sees Table 4.42 ℃ of 90 seconds heat shock transformed into escherichia coli DH5 α competent cells obtain plasmid molecule.
Table 4. target DNA fragment ligation system
| Reaction reagent | Volume (μ l) |
| 10 * ligase enzyme damping fluid T4 ligase enzyme pcr amplification product pMD 18-T carrier | 1 1 7.5 0.5 |
The concise and to the point collection of illustrative plates of the standard plasmid molecule pMD-RRS that makes up as shown in Figure 1.PMD-18T represents to make up the carrier of standard plasmid molecule among the figure; Xba I represents that this site contains the restriction enzyme site of restriction enzyme Xba I; Nco I represents that this site contains the restriction enzyme site of restriction enzyme Nco I; One section specific sequence of the endogenous standard gene Lectin of " Lectin " expression soybean; The section of DNA sequence of " strain specificity sequence " expression genetic improved soybean strain GTS 40-3-2-3-2 exogenous insertion vector and soybean gene group adjoining region; After two sections sequences realized splicing, Xba I and Nco I restriction enzyme digestion sites by the recombination sequence two ends were connected on the pMD-18T carrier, constitute standard plasmid molecule pMD-RRS.
Embodiment 2: the application of the plasmid control molecule of structure in actual detected
One, experiment material
Genetic improved soybean strain GTS 40-3-2-3-2.
Conventional soybean varieties.
Other genetic improvement crop strains: kind of No. one tomato of modified rape GT 73, cotton MON531, cotton MON1445, corn GA21, China, corn NK603 strain.
Two, enzyme and reagent
Plant genome DNA extraction and purifying adopt the food DNA extraction agent box of Academy of Agricultural Sciences, Shanghai City and Shanghai Entry-Exit Inspection and Quarantine bureau's joint research and development.
Plasmon DNA extraction and purifying adopt the plasmid of JaRa biotechnology (Shanghai) Co., Ltd. development to prepare test kit in a small amount.
The pMD18-T carrier, T
4Dna ligase and damping fluid thereof are available from the white good development in science and technology in Shanghai company limited;
DNTPs, Taq archaeal dna polymerase and damping fluid thereof, DL2000 Marker are available from the precious biotechnology in Dalian company limited;
TaqMan probe and primer are synthetic by the rich inferior biotechnology in Shanghai company limited.
Other biochemical reagents are import packing or homemade analytical pure.
Three, laboratory apparatus
DY-501 type nucleic acid electrophoresis apparatus (Shanghai Precision Scientific Apparatus Co., Ltd)
PTC-100 type pcr amplification instrument (MJ Research Inc.)
DUR 640 nucleic acid and the protein analyzer of BECKMAN company
DNA electrophoretic analysis system comprises camera bellows, digital camera, computer, scanner, Tanon UV-2000 uv analyzer, Gis gel images analysis software (sky, Shanghai energy company)
Other instruments comprise: whizzer, thermostat water bath, incubator, day equality.
Four, experimental technique and process
1, extracting genome DNA and detection
1) plant genome DNA extracts
A, get an amount of soybean sample, add liquid nitrogen grind into powder in mortar, take by weighing about 70-100mg ground sample and change in the 1.5ml centrifuge tube;
B, look what of quantity of material, add the 600-700 μ l Buffer A of preheating, gently behind the mixing, 65 ℃ of water bath heat preservations 1 hour, during between or the vibration mixing;
C, add equal-volume phenol/chloroform (600-700 μ l) solution in pipe, the abundant mixing that turns upside down, normal temperature left standstill extracting 10 minutes;
Centrifugal 10 minutes of d, 12000rpm draw in the new centrifuge tube of supernatant to;
The isopyknic Virahol of e, adding and supernatant (about 600 μ l), mixing, normal temperature was placed after 10 minutes, and centrifugal 10 minutes of 12000rpm removes supernatant, keeps precipitation;
F, in precipitation, add 60 μ l TE, place after 2 minutes with the rifle head its abundant mixing, placed about 1 hour for 37 ℃ in 37 ℃;
Add 140 μ l TE after g, the taking-up, add 200 μ l phenol/chloroforms again, mixing leaves standstill a moment;
Centrifugal 5 minutes of h, 12000rpm get supernatant (about 180 μ l), add 1/10 volume 3M NaAc and 2 times of volume dehydrated alcohols, place 30 minutes for-20 ℃;
Centrifugal 10 minutes of i, 12000rpm remove supernatant, add 75% ethanol, 200 μ l, and centrifugal 5 minutes of 12000rpm abandons supernatant, be deposited under the room temperature and dry, after be dissolved in the aseptic ddH of 50 μ l
2Among the O.
2) plasmon DNA extraction
A, with 37 ℃ of 1-2ml bacterium cultures that spend the night centrifugal 1 minute in 6000rpm, thoroughly abandon supernatant;
The precooling STE solution of b, 0.25 times of volume of adding bacterium liquid fully suspends thalline again, and centrifugal 1 minute of back 6000rpm thoroughly abandons supernatant;
C, add 100 μ l Solution I, with rifle head or the vibrator bacterium that fully suspends;
D, adding 200 μ l Solution II, the gentle mixing that turns upside down immediately, room temperature was placed 2 minutes, made the abundant cracking of bacterium;
E, add 350 μ l Solution III, turn upside down immediately for several times, make it abundant mixing, neutralization, room temperature was placed after 2 minutes 13000rpm centrifugal 5 minutes;
F, supernatant is transferred to cover be put in the GenClean post in the 2ml collection tube, centrifugal 15 seconds in room temperature 8000rpm;
G, take out the GenClean post, outwell waste liquid in the collection tube, the GenClean post is relay reclaim in the collector, add 500 μ l Wash Solution, centrifugal 30 seconds in room temperature 10000rpm;
H, repeating step g wash 1-2 time with Wash Solution;
I, abandon waste liquid, in the 12000rpm room temperature centrifugal 1 minute, with thorough removal Wash Solution;
J, the GenClean post is put into clean 1.5ml centrifuge tube, open pipe and cover room temperature and place and made remaining ethanol volatilization in 5 minutes, the back adds 50 μ l Elution Buffer in column film central authorities, 37 ℃ of placements 2 minutes;
K, the DNA on centrifugal 2 minutes wash-out films of room temperature 12000rpm.
3) DNA detection
Get the DNA sample that 3 μ l extract, the agarose gel electrophoresis with 1% detects, according to the quality of its brightness and diffusion judgement DNA.
Utilize concentration and the purity of DNA that determined by ultraviolet spectrophotometry is put forward.
2, the specificity analyses of genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantification PCR primer
Optimize genetic improved soybean strain GTS 40-3-2-3-2 strain specificity PCR detection architecture and reaction conditions, and serve as the purpose fragment that detects the qualitative amplification genetic improved soybean strain GTS 40-3-2 of template-3-2 strain specificity with other genetic improvement plants respectively, determine quantification PCR primer specificity at genetic improved soybean strain GTS 40-3-2-3-2 design.
3, standard plasmid molecule pMD-RRS is used for the limit of detection (LOD) and the quantitation limit (LOQ) of quantitative PCR detection
Optimize genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR detection system and reaction conditions, respectively with the standard plasmid molecule genome DNA sample of different concns (as 2 * 10
9Copies/ μ l, 2 * 10
8Copies/ μ l, 2 * 10
7Copies/ μ l, 2 * 10
6Copies/ μ l, 2 * 10
5Copies/ μ l, 2 * 10
4Copies/ μ l, 2 * 10
3Copies/ μ l, 2 * 10
2Copies/ μ l, 2 * 10
1Copies/ μ l and 2 * 10
0Copies/ μ l) LOD and the LOQ of the quantitative PCR detecting method that test is set up as standard substance.Each reacts triplicate, according to the typical curve of quantitative pcr amplification and the linear relationship between the amplification fluorescent signal, when determining to utilize the standard plasmid molecule pMD-RRS replacing positive reference material that makes up, the LOD and the LOQ of genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR detecting method.
4, the repeatability of standard plasmid molecule pMD-RRS quantitative PCR detection system and repeatability
Optimize genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR detection system and reaction conditions, respectively with different concns standard plasmid molecule pMD-RRS genome DNA sample (2 * 10
6Copies/ μ l, 2 * 10
5Copies/ μ l, 2 * 10
4Copies/ μ l, 2 * 10
3Copies/ μ l, 2 * 10
2Copies/ μ l and 2 * 10
1Copies/ μ l) carry out the test of repeatability and repdocutbility, each reacts triplicate.According to the typical curve of quantitative pcr amplification, determine the repeatability and the repeatability of genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR reaction according to standard plasmid molecule pMD-RRS foundation.
5, standard plasmid molecule pMD-RRS quantitative PCR detection correction coefficient is measured
When utilizing standard molecule to carry out quantitative analysis, must consider plasmid DNA and the soybean gene group DNA efficiency variance in the PCR reaction, this difference can be passed through correction coefficient, and (Coefficient Values CVs) proofreaies and correct.The measuring method of CVs value is to set up quantitative PCR detection system with standard plasmid molecule, genetic improved soybean strain GTS 40-3-2-3-2 standard positive the material that isozygotys is carried out quantitative analysis, by the correction coefficient CVs of the plasmid molecule of the base of calculation as a result pMD-RRS of quantitative analysis relatively.The CVs value can calculate by formula 1.After obtaining the CVs value, can analyze the content that obtains genetic improvement composition in the genetic improvement sample to be measured by formula 2.
The plant endogenous gene copy number of genetic improvement of the genetic improvement plant external source testing goal gene copy number that formula 1:CVs=isozygotys/isozygoty
Formula 2: genetic improvement component content %=(sample external source testing goal gene copy number * 100)/(sample native gene copy number * CVs)
6, the application standard plasmid molecule is to the quantitative analysis of actual genetic improved soybean strain GTS 40-3-2-3-2 sample
According to the correction coefficient of the standard plasmid molecule pMD-RRS that measures and the genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR typical curve of drafting, respectively 4 genetic improved soybean strain GTS 40-3-2s-3-2 biased sample (the genetic improvement component content is respectively 0.5%, 1.0%, 3.0% and 5.0%) is analyzed, determined whether the genetic improved soybean strain GTS 40-3-2-3-2 standard plasmid molecule that makes up can be effectively applied to the detection by quantitative of actual genetic improved soybean strain GTS 40-3-2-3-2 sample.
Five, experimental result
1, genetic improved soybean strain GTS 40-3-2-3-2 strain specificity PCR detection architecture and reaction conditions
Through optimizing, the strain specificity PCR detection architecture of genetic improved soybean strain GTS 40-3-2-3-2 and pcr amplification condition see Table 5 and table 6 respectively.
The strain specificity PCR detection architecture of table 5. genetic improved soybean strain GTS 40-3-2-3-2
| Reaction reagent | Volume (μ l) | Final concentration |
| 10 * PCR buffer solution dNTPs primer, 1 primer 2 Taq enzyme dna template ddH2O | 3311 1.5 1 complement to 30 μ l | 1×(50mM KCl，10mM Tris-HCl PH8.3，1.5mM MgCl 2) 200 μ M 400nM 400nM, 2.5 unit/reactions |
The strain specificity qualitative PCR amplification condition of table 6. genetic improved soybean strain GTS 40-3-2-3-2
| Cycle number | Temperature (℃) | Time |
| 1 | 95 | 7 minutes |
| 35 1 | 95 58 72 72 | 30 seconds 30 seconds 30 seconds 7 minutes |
2, the specificity analyses of genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantification PCR primer
According to the primer sequence in the table 1, genetic improved soybean strain GTS 40-3-2-3-2 and other genetic improvement crop such as genome samples such as GT73 rape and MON531 cotton are carried out the qualitative PCR amplification.The result shows that only in genetic improved soybean strain GTS 40-3-2-3-2, amplification has obtained size and has been the purpose fragment band of 85bp, and does not detect this band in other genetic improvement crop.Therefore, the quantification PCR primer of the GTS40-3-2 soybean line of the present invention's design has high degree of specificity.
3, the optimization and the foundation of genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR detection system
According to the optimization method of quantitative PCR reaction system, seek the concentration of optimum genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantification PCR primer and probe, guarantee that the reaction efficiency of quantitative PCR reaches more than 90%.Through optimization, when the final concentration of primer and probe is 400nM and 200nM, pcr amplification curve most effective, fluorescent signal is the strongest.Therefore this concentration is used to set up the quantitative PCR reaction system of genetic improved soybean strain, and other becomes component to see table 7 for details, and the pcr amplification condition sees Table 8.
The strain specificity quantitative PCR detection system of table 7. genetic improved soybean strain GTS 40-3-2-3-2
| Reaction reagent | Volume (μ l) | Final concentration |
| 5 * PCR damping fluid MgCl 2DNTPs primer 1 primer 2 probe Hotstar Taq enzyme UNG enzyme dna template | 5 6 3 0.6 0.6 0.9 1.5 0.2 1 | 1 * (50mM KCl, 10mM Tris-HCl, PH8.3) 6mM 400 μ M dATP, dGTP, dCTP; 800 μ M dUTP---1.5 units/reaction 0.2 unit/reaction |
| ddH 2O | Complement to 25 μ l |
Table 8. genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR detection amplification condition
| Cycle number | Temperature (℃) | Time |
| 1 1 45 1 | 50 95 95 58 72 | 2 minutes 10 minutes 30 seconds 60 seconds 7 minutes |
4, standard plasmid molecule pMD-RRS carries out the limit of detection (LOD) and the quantitation limit (LOQ) of quantitative PCR detection
Utilize to optimize good genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR reaction system, respectively with the standard plasmid molecule genome DNA sample of different concns (as 2 * 10
9Copies/ μ l, 2 * 10
8Copies/ μ l, 2 * 10
7Copies/ μ l, 2 * 10
6Copies/ μ l, 2 * 10
5Copies/ μ l, 2 * 10
4Copies/ μ l, 2 * 10
3Copies/ μ l, 2 * 10
2Copies/ μ l, 2 * 10
1Copies/ μ l and 2 * 10
0Copies/ μ l) LOD and the LOQ of the quantitative PCR detection system of setting up as standard model test the present invention.Determine that through 3 repeated experiments the LOD of this system is 2 copies, LOQ is 20 copies.Illustrate that the pMD-RRS standard plasmid molecule that makes up can replace genetic improved soybean strain GTS 40-3-2-3-2 positive criteria product detection by quantitative genetic improved soybean strain GTS 40-3-2-3-2 and converted products genetic improvement component content thereof.
With concentration is 2 * 10
9Copies/ μ l, 2 * 10
8Copies/ μ l, 2 * 10
7Copies/ μ l, 2 * 10
6Copies/ μ l, 2 * 10
5Copies/ μ l, 2 * 10
4Copies/ μ l, 2 * 10
3Copies/ μ l, 2 * 10
2Copies/ μ l, 2 * 10
1Copies/ μ l and 2 * 10
0The pMD-RRS soybean standard plasmid molecule genome DNA sample of copies/ μ l is that standard substance are drawn genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR typical curve.LOD and LOQ result according to quantitative criterion curve and this system can think, genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR detection system that the present invention sets up by the standard plasmid molecule that makes up has very high accuracy and sensitivity, can be used for the actual detected of genetic improved soybean strain GTS 40-3-2-3-2 and converted products thereof fully.
5, utilize the repeatability and the repeatability of the quantitative PCR detection system of standard plasmid molecule pMD-RRS foundation to measure
Under the genetic improved soybean strain specificity quantitative PCR reaction conditions of optimizing, respectively with different concns standard plasmid molecule pMD-RRS genome DNA sample (2 * 10
6Copies/ μ l, 2 * 10
5Copies/ μ l, 2 * 10
4Copies/ μ l, 2 * 10
3Copies/ μ l, 2 * 10
2Copies/ μ l and 2 * 10
1Copies/ μ l) carries out the repeatability and the test of reproducibility, between 3 parallel reactors with 3 different repeated experiments between the Ct value standard deviation that obtains basically all less than 0.2, the repeatability and the repeatability of the genetic improved soybean strain GTS 40-3-2 that explanation is set up according to the pMD-RRS standard plasmid molecule that makes up-3-2 strain specificity quantitative PCR reaction are good, can be used for the further quantitative analysis of actual genetic improved soybean strain GTS 40-3-2-3-2 sample.
6, the quantitative PCR detection system correction coefficient of utilizing soybean standard plasmid molecule pMD-RRS to set up is measured
Replace genetic improved soybean strain GTS 40-3-2-when 3-2 positive criteria product are used for quantitative PCR detection with standard plasmid molecule pMD-RRS, set up quantitative PCR detecting method by standard plasmid molecule, genetic improved soybean strain GTS 40-3-2-3-2 standard positive the material that isozygotys is carried out quantitative analysis, by the correction coefficient of the plasmid molecule of the base of calculation as a result pMD-RRS of quantitative analysis relatively, be 0.92, see Table 9.
Table 9. standard plasmid molecule pMD-RRS is used for the correction coefficient of quantitative PCR analysis and measures
| Goal gene | Copy number | Average copy number | Average Cv value | SD | ||
| Mean value 1 | Mean value 2 | Mean value 3 | ||||
| Strain specificity sequence Lectin | 1210.88 1208.86 | 1151.86 1126.82 | 1064.86 1394.77 | 1142.54 1243.48 | 0.92 | 0.14 |
7, use of the quantitative analysis of pMD-RRS standard plasmid molecule to actual genetic improved soybean strain GTS 40-3-2-3-2 sample
According to the correction coefficient of the standard plasmid molecule pMD-RRS that measures and the genetic improved soybean strain GTS 40-3-2-3-2 strain specificity quantitative PCR typical curve of drafting, (the genetic improvement component content is respectively 0.5% to 4 genetic improved soybean strain GTS 40-3-2s-3-2 biased sample respectively, 1.0%, 3.0% and 5.0%) analyzes, quantitative analysis results shows that the content of 4 genetic improved soybean strain GTS 40-3-2-3-2 biased samples is respectively 0.429%, 1.168%, 3.327% and 5.246%, be respectively 14.2% with the deviation of actual value, 16.8%, 10.9% and 4.92%, the standard deviation of quantitative result is (concrete data see Table 10) in the 0.12-0.18 scope.Detected result shows that the result who utilizes standard plasmid molecule pMD-RRS that actual sample is analyzed is more accurate, the deviation of detected result is in the 0-0.25 scope that ISO genetically modified food examination criteria allows, show that genetic improved soybean strain GTS 40-3-2-3-2 standard plasmid molecule pMD-RRS that the present invention makes up can replace the positive criteria product of this strain fully, the standard plasmid molecule of structure is applicable to the strain specificity quantitative PCR analysis and the detection of genetic improved soybean strain GTS 40-3-2-3-2 sample fully.
The quantitative analysis results of table 10. genetic improved soybean strain GTS 40-3-2-3-2 sample
| Sample | Actual value (%) | Accuracy rate | Accuracy | ||
| Average content (%) | Deviation (%) | SD | RSD | ||
| S1 S2 S3 S4 | 0.5 1.0 3.0 5.0 | 0.429 1.168 3.327 5.246 | -14.2 16.8 10.9 4.92 | 0.18 0.14 0.12 0.13 | 42.00 11.986 3.607 2.478 |
Claims (9)
1, a kind of genetic improved soybean strain GTS 40-3-2-3-2 detects and uses standard plasmid molecule, it is characterized in that, contains the strain specificity sequence of genetic improved soybean strain GTS 40-3-2-3-2 and the specific fragment of soybean internal standard gene Lectin.
2, genetic improved soybean strain GTS 40-3-2 according to claim 1-3-2 detects and uses standard plasmid molecule, it is characterized in that described soybean internal standard gene Lectin refers to the soybean lectin plain gene.
3, genetic improved soybean strain GTS 40-3-2 according to claim 1-3-2 detects and uses standard plasmid molecule, it is characterized in that, the strain specificity sequence of described genetic improved soybean strain GTS 40-3-2-3-2 refers to the dna sequence dna of genetic improved soybean strain GTS 40-3-2-3-2 exogenous insertion vector and soybean gene group adjoining region.
4, a kind of genetic improved soybean strain GTS 40-3-2 as claimed in claim 1-3-2 detects the construction process with standard plasmid molecule, it is characterized in that, comprises the steps:
1. utilize database to carry out bioinformatic analysis, obtain the strain specificity sequence of soybean internal standard gene Lectin, genetic improved soybean strain GTS 40-3-2-3-2;
2. design the PCR Auele Specific Primer;
3. the strain specificity sequence of specific amplification soybean internal standard gene Lectin and genetic improved soybean strain GTS 40-3-2-3-2;
4. the splicing of soybean internal standard gene Lectin and genetic improved soybean strain GTS 40-3-2-3-2 strain specificity sequence;
5. contain the plasmid molecule clone of new recombinant dna fragment, obtain standard plasmid molecule pMD-RRS.
5, genetic improved soybean strain GTS 40-3-2 according to claim 4-3-2 detects the construction process with standard plasmid molecule, it is characterized in that, described soybean internal standard gene Lectin, refer to the soybean lectin plain gene, it is conservative at soybean gene group camber, specificity between having kind, the characteristics of non-specific and single copy number in planting; The strain specificity sequence of described genetic improved soybean strain GTS 40-3-2-3-2 refers to the section of DNA sequence of genetic improved soybean strain GTS 40-3-2-3-2 exogenous insertion vector and soybean gene group adjoining region.
6, genetic improved soybean strain GTS 40-3-2 according to claim 4-3-2 detects the construction process with standard plasmid molecule, it is characterized in that, described PCR Auele Specific Primer, refer to the oligonucleotide chain that length is 28 ± 10nt, the strain specificity sequence of itself and soybean Lectin gene or genetic improved soybean strain GTS 40-3-2-3-2 is identical or complementary.
7, genetic improved soybean strain GTS 40-3-2 according to claim 4-3-2 detects the construction process with standard plasmid molecule, it is characterized in that, described specific amplification refers to the one section sequence of the accurate gene Lectin of PCR primer specificity amplification interior label that utilizes design and the strain specificity sequence of genetic improved soybean strain GTS 40-3-2-3-2.
8, genetic improved soybean strain GTS 40-3-2 according to claim 4-3-2 detects the construction process with standard plasmid molecule, it is characterized in that, described splicing, refer to utilize overlapping pcr with two of soybean internal standard gene Lectin and genetic improved soybean strain GTS 40-3-2s-3-2 strain specificity sequence independently dna fragmentation be connected the Novel DNA fragments that is fused into a reorganization.
9, genetic improved soybean strain GTS 40-3-2 according to claim 4-3-2 detects the construction process with standard plasmid molecule, it is characterized in that, described molecular cloning, refer to and the restriction enzyme digestion sites of the above-mentioned reorganization DNA fragment specific that obtains according to design is connected on the plasmid vector pMD18-T acquisition standard plasmid molecule pMD-RRS.
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