CN106480216A

CN106480216A - A kind of standard plasmid for detecting transgenic regulation element and its construction method and application

Info

Publication number: CN106480216A
Application number: CN201611117850.5A
Authority: CN
Inventors: 邢宇俊; 陆丹丹; 吴季荣; 徐剑宏; 王园园; 史建荣
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2016-12-07
Filing date: 2016-12-07
Publication date: 2017-03-08

Abstract

The invention provides a kind of restructuring standard plasmid molecule for detecting controlling element in transgene agricultural product，This restructuring standard plasmid is by the CaMV35S promoter shown in SEQ ID No.1 using overlapping pcr、FMV35S promoter shown in SEQ ID No.2、Pat29 shown in SEQ ID No.3、Ubiquitin shown in SEQ ID No.4、NOS promoter shown in SEQ ID No.5、T 35S terminator shown in SEQ ID No.6、T E9 terminator gene shown in NOS terminator shown in SEQ ID No.7 and SEQ ID No.8 is sequentially connected acquisition and merges fragment，Recycle molecule clone technology that the fusion being obtained fragment is cloned into acquisition in carrier pMD 19T；The restructuring standard plasmid that the present invention provides can substitute the positive criteria material in transgene agricultural product examination detection, for blind sample transgene agricultural product qualitative PCR examination detection, solve the positives standard substance of China transgene agricultural product examination not enough the problems such as.

Description

A kind of standard plasmid for detecting transgenic regulation element and its construction method with Application

Technical field

The present invention relates to biological technical field, a kind of standard plasmid for detecting transgenic regulation element and its structure side Method and application, and comprise the test kit of this standard plasmid.

Background technology

Since the first transgenic product goes through to commercially produce, increasing genetically modified crops are in different states Family and area start commercial growth, and the transgene agricultural product of increasing approved occurs in circulation market, such as：Turn Gene corn, Semen sojae atricolor, Brassica campestris L, Fructus Chaenomeliss, Cotton Gossypii etc..But with sending out rapidly in the whole world of genetically modified crops and its Related product Exhibition, its Environmental security and food safety question are constantly subjected to the concern of people.Detection to transgene agricultural product is conducive to political affairs Mansion functional department supervises to it.

At present, the detection method of genetically modified crops is mainly the detection in nucleic acid level, and common method is to extract detection The DNA of sample, carries out qualitative PCR or quantitative PCR detection.It is necessary to setting negative control and the positive are right in PCR detection process According to it is ensured that experiment effectiveness.Therefore the reliability of testing result, accuracy, effectiveness then need positive criteria material (to include Standard plasmid) compare it is seen that positive criteria material is particularly significant indispensable to the checking process of transgenic agricultural product 's.At present, the external unit developing transgenic positive standard substance is mainly European standard material academy (IRMM) and the U.S. POL chemistry association (AOCS).The species developed mainly includes transgenic corns, Semen sojae atricolor, Brassica campestris L, Cotton Gossypii, Radix Betae, Rhizoma Solani tuber osi Deng more than 50 kinds.These standard substances are the specific strains for it, and their own only comprises one or two and specifically opens simultaneously Mover or terminator, can not cover multiple promoteres or terminator.

The structure of positive criteria plasmid molecule is to carry out specific amplification using the purpose exogenous genetic fragment of detection, restructuring On plasmid molecule.Its advantage can be by microorganism mass propgation, and plasmid DNA is easily extracted and purity is high, and with One positive plasmid molecule can comprise multiple external source genes of interest.However, this is compared with more than 190 kinds of genetically modified crops species, also Demand far can not be met.In transgene agricultural product detection, transgenic standard substance is very deficient.Therefore, according to transgenic The exogenous gene of kind is special to develop positive transgenic standard plasmid, covers the plasmid of multiple promoteres or terminator to replace standard Material is detected, it has also become this area technical barrier urgently to be resolved hurrily.

Content of the invention

For the problems referred to above, the present invention provides a kind of standard plasmid for detecting transgenic regulation element, in transgenic It is used as positive control matter during agricultural product screening controlling element, the controlling element in multiple transgene agricultural products can be detected, cover Wide, the present invention is realized in：

A kind of standard plasmid for detecting transgenic regulation element, its nucleotide sequence is as shown in SEQ ID No.9.

A kind of as described herein be used for detect transgenic regulation element standard plasmid construction method, concrete steps are such as Under：

With overlapping pcr by shown in the CaMV35S promoter gene shown in SEQ ID No.1, SEQ ID No.2 Ubiquitin gene shown in Pat29 gene shown in FMV35S promoter gene, SEQ ID No.3, SEQ ID No.4, T-35S terminator gene shown in NOS promoter gene shown in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 institute T-E9 shown in the NOS terminator gene showing and SEQ ID No.8 terminates mrna exon fragment and is sequentially connected acquisition fusion fragment, then It is cloned into merging fragment in carrier pMD-19, that is, obtain the described standard plasmid for detecting transgenic regulation element.

It is used for as described herein detecting the standard plasmid of transgenic regulation element in detection transgene agricultural product regulation and control unit Application in part.

A kind of comprise as described herein be used for detect transgenic regulation element standard plasmid test kit.

Further, in test kit of the present invention, also include following primer：

Specific primer P-35S-F and P-35S-R of CaMV35S promoter, their nucleotide sequence is respectively as SEQ Shown in ID NO.10 and SEQ ID NO.11；

Or, specific primer FMV35S-F and FMV35S-R of FMV35S promoter, their nucleotide sequence is respectively As shown in SEQ ID NO.12 and SEQ ID NO.13；

Or, specific primer PTA29-F and PTA29-R of PTA29 promoter, their nucleotide sequence is respectively such as Shown in SEQ ID NO.14 and SEQ ID NO.15；

Or, specific primer Ubiquitin-F and Ubiquitin-R of Ubiquitin promoter, their nucleotide Sequence is respectively as shown in SEQ ID NO.16 and SEQ ID NO.17；

Or, specific primer PNOS-F and PNOS-R of NOS promoter, their nucleotide sequence is respectively as SEQ ID Shown in NO.18 and SEQ ID NO.19；

Or, specific primer T35S-F and T35S-R of T35S terminator T-35S, their nucleotide sequence is respectively such as Shown in SEQ ID NO.20 and SEQ ID NO.21；

Or, specific primer T-NOS-F and T-NOS-R of NOS terminator T-NOS, their nucleotide sequence is respectively As shown in SEQ ID NO.22 and SEQ ID NO.23；

Or, specific primer T-E9-F and T-E9-R of E9 terminator T-E9, their nucleotide sequence is respectively as SEQ Shown in ID NO.24 and SEQ ID NO.25.

Further, test kit volume of the present invention is 25 μ L, including：10 × PCR buffer Mg²⁺2.5μL、 The dNTPs of 200mmol, content are the specific primer of 0.5mmol, the Taq archaeal dna polymerase of 1.25U and concentration The described standard plasmid 1.5 μ L for detecting transgenic regulation element of 25mg/L, deionized water complements to 25 μ L.

The design that it is critical only that restructuring standard plasmid and specific primer of test kit provided by the present invention, in test kit Other components, such as PCR reaction reagent, can be selected by this area routine.PCR reaction reagent includes PCR buffer, deoxidation Nucleoside triphosphate mixture and archaeal dna polymerase etc..PCR reaction kit amplimer is mixed, adds testing sample or mark Quasi- product, you can carry out pcr amplification reaction, pcr amplification product runs glue with 2% agarose, if corresponding to occurs in the PCR primer of sample 195bp, 210bp, 266bp, 314bp, 183bp, 121bp, 180bp, 262bp band, you can judge to contain corresponding transgenic Element.

The conventional controlling element in being detected according to current transgene agricultural product of the present invention：CaMV (cauliflower mosaic viruses) 35S promoter, Fmv35S promoter, Pat29, Ubiquitin, NOS promoter, T-35S terminator, NOS terminator and T-e9 Terminator, is designed corresponding primer, is merged 8 sequence fragments using overlapping pcr, these external source controlling elements Gene constructed in same plasmid molecule, recycling molecule clone technology is cloned into merging fragment in pMD-19T, obtains length Restructuring standard plasmid pMD-RG (its aminoacid sequence is as described in SEQ ID NO.9) for 1731bp.

This restructuring standard plasmid can substitute the positive criteria material in transgene agricultural product examination detection, turns for blind sample Gene agricultural product qualitative PCR examination detects, solve the positives standard substance of China transgene agricultural product examination not enough the problems such as.

Brief description

Fig. 1 builds the schematic flow sheet of standard plasmid molecule pMD-RG for the present invention.

Whole exogenous arrays of the standard plasmid molecule that Fig. 2 builds for the present invention, wherein CaMV35S promoter, FMV35S, Pat29, Ubiquitin, NOS promoter, T-35S terminator, NOS terminator and T-e9 terminator represent universal component respectively Distinguished sequence；Dash area sequence is the lap in overlapping PCR primers；Dot arrows sequence is qualitative PCR primer.

Fig. 3 is restructuring standard plasmid pMD-RG and positive criteria material contrasts electrophoretogram in PCR detection.

Fig. 4 is the electrophoretogram that restructuring standard plasmid is used for qualitative PCR sensitivity technique.

Specific embodiment

Genetically engineered soybean MON89788, the GTS40-3-2 being related in embodiment；Transgenic paddy rice KF6, TT51-1；Turn base Because of Brassica campestris L RF1, MS1, GT73；Genetically modified rape GT 73, genetically modified corn MON 863, the development of Bt176, MON810 Jun You Ministry of Agriculture Center provides；

Non-transgenic material is rice south round-grained rice 46, is extracted by Jiangsu Province Agriculture Science Institute and preserves.

The test kit instrument being related in embodiment：

PMD-19T carrier, Taq archaeal dna polymerase and its buffer, dNTPs, Marker are purchased from precious biological engineering (Dalian) Company limited；

Plant DNA extraction kit and DNA 5a competence are purchased from Beijing Tiangeng biology company limited；

PCR primer QIAquick Gel Extraction Kit, plasmid extraction reagent, biochemical reagents box and primer are purchased from the limited public affairs of Shanghai biological engineering Department；

PCR (German Biometra), gel imaging system (Tiangen), the Nandrop100 ultraviolet spectrophotometer (U.S. GE), Other Instruments includes：Constant temperature blending instrument, electronic balance, micropipettor etc. are bought by commercial sources.

DNA sequence SEQ the ID NO.1-SEQ ID NO.32 such as sequence table institute in description adnexa being related in embodiment Show.

Embodiment 1 builds standard plasmid

1st, utilize Genbank data base and pertinent literature retrieval, obtain conventional external source controlling element：35S promoter (with Lower abbreviation CaMV35S), FMV35S promoter (hereinafter referred to as FMV35S), pTA 29 promoter (hereinafter referred to as pTA 29), Ubiquitin promoter (hereinafter referred to as Ubiquitin), NOS promoter (hereinafter referred to as PNOS), (letter below of T-35S terminator Claim T-35S), the sequence of NOS terminator (hereinafter referred to as TNOS) and T-E9 terminator (hereinafter referred to as T-E9)：These controlling elements Sequence be following gene partial sequence：

CaMV35S(SEQ ID NO.1,195bp)：Cauliflower mosaic virus 35 S promoter；

Fmv35S(SEQ ID NO.2,210bp)：Radix scrophulariae mosaic virus 35 S promoter；

pTA29(SEQ ID NO.3,266bp)：Tobacco anther tissuespecific genes TA29；

Ubiquitin(SEQ ID NO.4,314bp)：Maize ubiquitin protein gene promoter；

PNOS(SEQ ID NO.5,183bp)：The promoter of rouge alkali synthetase gene NOS；

T-35S(SEQ ID NO.6,121bp)：The 35S terminator of cauliflower mosaic viruses；

TNOS(SEQ ID NO.7,180bp)：Rouge alkali synthetase gene NOS terminator；

T-E9(SEQ ID NO.8,262bp)：Semen Pisi sativi 1,5- diphosphoribulose carboxylase E9 gene terminator；

Said gene sequence is successively as shown in SEQ ID NO.1-SEQ ID NO.8 in sequence table.

2nd, according to above-mentioned controlling element：CaMV35S, Fmv35S, pTA29, Ubiquitin, PNOS, T-35S, TNOS and T- The sequence (numbering 1-8 respectively) of E9, designs overlapping PCR primers.Described overlapping PCR primers, refer to sequence identical or Complementary primer, makes each purpose fragment reach seamless connection (as shown in Figure 1), the such as table 1 of the overlapping PCR primers sequence in embodiment Shown：

Table 1 builds the overlapping PCR primers of standard plasmid molecule pMD-RG

(1) it is utilized respectively forward primer in table 1 and reverse primer is the primer (final concentration of two kinds of primers in reaction system It is 0.5mmol/L), so as to corresponding 8 transgenic standard substance, (its aminoacid sequence is successively as SEQ ID NO:1-SEQ ID NO:Shown in 7) carry out regular-PCR amplification for template：

Regular-PCR system is：10×PCR buffer(Mg²⁺Plus), the dNTPs of final concentration of 200mmol/L, final concentration For being the primer of 0.5mmol/L, the Taq archaeal dna polymerase of 1.25U and 1.5 μ LDNA template (25mg/L), deionization complements to 25μL；

Regular-PCR program is:95 DEG C, 5min denaturation；94 DEG C, 1min, 56 DEG C, 30s, 72 DEG C of 30s, 35 circulations；72 DEG C extend 7min.

Obtained 1-8 group PCR primer runs glue with 2% agarose more respectively, cuts glue and utilizes PCR primer QIAquick Gel Extraction Kit (strictly according to description operate) reclaims the fragment of correct size, obtain CaMV35S amplified production, FMV35S amplified production, PTA29 amplified production, Ubiquitin amplified production, PNOS amplified production and TNOS amplified production.

(2) respectively with above-mentioned 1-7 group amplified production as template (the 8th group of T-E9 need not expand), with corresponding forward direction in table 1 Primer and overlapping PCR primers enter performing PCR amplification (PCR system and program and step (1) regular-PCR system and regular-PCR program phase With)；

The 7 groups of PCR primer being obtained run glue with 2% agarose more respectively, cut glue and utilize PCR primer QIAquick Gel Extraction Kit (strictly being operated according to description) reclaims the fragment of correct size, obtains the CaMV35S amplified production containing junction fragment, contains The FMV35S amplified production of junction fragment, the PTA29 amplified production containing junction fragment, the Ubiquitin containing junction fragment Amplified production, the PNOS amplified production containing junction fragment and the TNOS amplified production containing junction fragment.

(3) overlapping pcr is utilized to be spliced step (2) the multiple amplified productions containing junction fragment of acquisition

Over-lap PCR system (25 μ L)：10×PCR Buffer(Mg²⁺Plus), the dNTPs of final concentration of 200mmol/L, dense Degree is each 1 μ L of template of 25mg/L, the Taq archaeal dna polymerase of 1.25U, complements to 25 μ L with deionization；

Over-lap PCR program：94 DEG C of denaturations 2min；94 DEG C of degeneration 30s, 58 DEG C of annealing 10min, 72 DEG C of extension 3min, follow Ring 15 times；72 DEG C of extension 10min.

Splicing step is as follows：

A () first, CaMV35S and FMV35S fragment is linked together：

According to as above over-lap PCR system, obtained containing the CaMV35S amplified production connecting section with step (2) and contain The FMV35S amplified production connecting even section is template, carries out over-lap PCR reaction；

After over-lap PCR amplification terminates, with over-lap PCR amplified production as template (template concentrations 25mg/L), with primer P- 35S-F (final concentration of 0.5mmol/L in 25 μ L reaction systems) and FMV35S-PTA29-R is (final concentration of in 25 μ L reaction systems 0.5mmol/L) be forward primer and overlapping PCR primers carry out regular-PCR amplification (according to the described regular-PCR system of step (1) and Regular-PCR program is expanded, similarly hereinafter), thus two fragments of CaMV35S and FMV35S are linked together, using PCR primer QIAquick Gel Extraction Kit reclaims, and obtains amplified production (CaMV35S+FMV35S)；

B () PTA29 promoter and Ubiquitin promoter fragment link together

According to over-lap PCR amplification system, the PTA29 amplified production containing junction fragment being obtained with step (2) and containing The Ubiquitin amplified production of junction fragment is template, carries out over-lap PCR reaction；

After over-lap PCR amplification terminates, with over-lap PCR amplified production as template, with primer PTA29-F (25 μ L reaction systems In final concentration of 0.5mmol/L) and Ubiquitin-PNOS-R (final concentration of 0.5mmol/L in 25 μ L reaction systems) be forward direction Primer and overlapping PCR primers carry out regular-PCR amplification, obtain amplified production, are reclaimed using PCR primer QIAquick Gel Extraction Kit, obtain Amplified production (PTA29+Ubiquitin)；

(c) amplified production CaMV35S+FMV35S and the connection of amplified production PTA29+Ubiquitin

According to over-lap PCR amplification system, the amplified production being obtained with step (a) (CaMV35S+FMV35S) and step (b) The amplified production (PTA29+Ubiquitin) obtaining is template, carries out over-lap PCR reaction；After over-lap PCR amplification terminates, with weight Folded pcr amplification product is template, with primer P-35S-F (final concentration of 0.5mmol/L in 25 μ L reaction systems) and Ubiquitin-PNOS-R (final concentration of 0.5mmol/L in 25 μ L reaction systems) is forward primer and overlapping PCR primers are carried out Regular-PCR expands, and obtains amplified production, finally utilizes PCR primer QIAquick Gel Extraction Kit to reclaim, obtains amplified production (CaMV35S+ FMV35S+PTA29+Ubiquitin)；

D () PNOS and T-35 amplified production connects

According to over-lap PCR amplification system, the PNOS amplified production containing junction fragment being obtained with step (2) and containing even The T-35 amplified production of tab segments is template, carries out over-lap PCR reaction；

After over-lap PCR amplification terminates, with over-lap PCR amplified production as template, with primer PNOS-F and T35S-TNOS-R it is Forward primer and overlapping PCR primers carry out regular-PCR amplification, obtain amplified production, are finally returned using PCR primer QIAquick Gel Extraction Kit Receive, thus obtaining amplified production (PNOS+T35S)；

E () TNOS and T-E9 amplified production connects

According to over-lap PCR amplification system, the TNOS amplified production containing junction fragment being obtained with step (2) and step (1) the TNOS amplified production obtaining is template, carries out over-lap PCR reaction；

After over-lap PCR amplification terminates, with over-lap PCR amplified production as template, drawn with primer NOS-F/T-E9-R for forward direction Thing and reverse primer enter performing PCR amplification, obtain amplified production, finally utilize PCR primer QIAquick Gel Extraction Kit to reclaim, thus obtaining expansion Volume increase thing (NOS+T-E9)；

(f) amplified fragments (PNOS+T35S) and the connection of amplified fragments (NOS+T-E9)

According to over-lap PCR amplification system, the amplified production being obtained with step (d) (PNOS+T35S) and step (e) are obtained Amplified production (NOS+T-E9) is template, carries out over-lap PCR reaction；

After over-lap PCR amplification terminates, with over-lap PCR amplified production as template, drawn with primer PNOS-F/T-E9-R for forward direction Thing and overlapping PCR primers carry out regular-PCR amplification, obtain amplified production, finally utilize PCR primer QIAquick Gel Extraction Kit to reclaim, from And obtain amplified production (PNOS+T35S+NOS+T-E9)；

G () is according to over-lap PCR amplification system, the amplified production (CaMV35S+FMV35S+PTA29+ being obtained with step (c) Ubiquitin) and the amplified production (PNOS+T35S+TNOS+T-E9) that obtains of step (f) is template, carry out over-lap PCR reaction；

After over-lap PCR amplification terminates, with over-lap PCR amplified production as template, with primer P-35S-F/T-E9-R as forward direction Primer and reverse primer carry out regular-PCR amplification, thus obtaining amplified production, finally utilize PCR primer QIAquick Gel Extraction Kit to reclaim, Obtain the overall sequence of amplified production (CaMV35S+FMV35S+PTA29+Ubiquitin+PNOS+T-35S+TNOS+T-E9), It is the plasmid molecule of the controlling element building.

(4) recombinant fragment clone

Amplified production (the CaMV35S+FMV35S+ amplification of step (3) over-lap PCR being obtained using molecular cloning method PTA29+Ubiquitin+PNOS+T-35S+TNOS+T-E9) it is connected on carrier pMD-19T：

Linked system (10 μ L)：SolutionI 5 μ L, amplified production (CaMV35S+FMV35S+PTA29+Ubiquitin+ PNOS+T-35S+TNOS+T-E9) 4.5 μ L (25mg/L), adds pMD-19T to complement to 10ul；16 DEG C of connection 4h.

Coupled reaction converts DH5a competence bacterial strain using connection product with heat shock method after terminating,

Concrete step of converting is as follows：Add DNA connection liquid in the centrifuge tube equipped with DH5 α competence bacterial strain, gently rotate Shake up, stand 30min on ice, put rapidly on ice after heat shock 90s in 42 DEG C of water-baths, standing cooling 3-5min；Add in centrifuge tube 800 μ L LB fluid medium (or SOC), mix rear 37 DEG C of vibrations (shaking table 180rpm), cultivate 45min, take 100 μ L bacterium solution to apply Cloth, on the LB flat board containing Amp, cultivates 12-16h for 37 DEG C), that is, obtain the monoclonal bacterial strain pMD- of the plasmid containing recombinant fragment RG.Using bacterium colony PCR, recombinant bacterial strain is identified, extract plasmid according to plasmid extraction kit specification, by sequencing point Analysis determines the exogenous array recombinated in standard plasmid and is contemplated to be consistent, explanation successfully constructs, that is, obtain restructuring standard plasmid PMD-RG, its DNA sequence as shown in SEQ ID NO.9, (its whole exogenous array as shown in Fig. 2 wherein CaMV35S promoter, FMV35S, Pat29, Ubiquitin, NOS promoter, T-35S terminator, NOS terminator and T-e9 terminator represent logical respectively Distinguished sequence with element；Dash area sequence is the lap in overlapping PCR primers；Dot arrows sequence is qualitative PCR Primer).

Application in actually detected for embodiment 2 standard plasmid molecule

1st, extracting genome DNA

Experiment material：Genetically engineered soybean MON89788 (its controlling element is FMV35S, T-E9), GTS40-3-2 (its regulation and control Element is CaMV35S, PNOS), transgenic paddy rice KF6 (its controlling element is CaMV35S, TNOS and T-35S), transgene rape RF1 (its controlling element is PTA29, PNOS and TNOS), MS1 (its controlling element is PTA29, PNOS and TNOS), GT73 (its tune Control element is FMV35S and T-E9)；

Genetically modified corn MON 863 (its controlling element is CaMV35S, the Ubiqutin that TNOS and Semen Maydiss have in itself), Bt176 (its controlling element is the Ubiqutin that CaMV35, T35S and Semen Maydiss have in itself)；Above-mentioned material all takes seed powder；

Weigh the ground seed powder sample of 100mg, extract above-mentioned sample according to genome extracts kit description DNA；The quality of above-mentioned DNA sample is evaluated with the method for agarose gel electrophoresiies and nucleic acid quantification detection.

2nd, the extraction of restructuring standard plasmid pMD-RG and detection

The escherichia coli containing standard plasmid molecule pMD-RG that embodiment 1 is obtained cultivate 10h in 37 DEG C, according to plasmid Extracts kit description extracts plasmid DNA.

Take 10 groups of DNA sample that 3ul step 1 obtains, detected with 1% agarose gel electrophoresiies respectively, judge that DNA is complete Property；Detect DNA purity and concentration with spectrophotometer respectively again, calculate OD₂₆₀nm/OD₂₈₀The ratio of nm 1.8 about, meets Qualitative, quantitative PCR detection requires；According to restructuring standard plasmid DNA initial concentration and size, it is diluted to 5 × 10⁵Copies/ μ L carries out subpackage, is stored in -20 DEG C of refrigerators standby.

3rd, restructuring standard plasmid pMD-RG detects the application in universal component in qualitative PCR

According to the specific sequence of universal component, carry out qualitative PCR, the detection effect of examination criteria plasmid molecule using primer Really, the primer sequence of use is shown in Table 2：

Table 2：Restructuring standard plasmid qualitative PCR detection primer

(1) respectively with the standard plasmid pMD-RG (25mg/L) that recombinates, genetically engineered soybean MON89788, GTS40-3-2, turn base Because of Oryza sativa L. KF6, transgene rape RF1, MS1, GT73, genetically modified corn MON 863, Bt176, rice south round-grained rice 46 (make by non-transgenic Thing) DNA be template, successively using the primer in table 2 enter performing PCR amplification, judge restructuring standard plasmid be used for qualitative PCR detect Specificity.

PCR system (25 μ L) is：10 × PCR buffer (Mg²⁺) 2.5 μ L, the dNTPs of final concentration of 200mmol, final concentration Forward primer for 0.5mmol, final concentration of 0.5mmol reverse primer, the template of the Taq archaeal dna polymerase of 1.25U and 1.5 μ L (25mg/L), deionization complements to 25 μ L；

PCR program is：95 DEG C, 5min denaturation；94 DEG C, 1min, 56 DEG C, 30s, 72 DEG C of 30s, 35 circulations；72 DEG C are prolonged Stretch 7min.

PCR primer runs glue with 2% agarose, and electrophoresis result is as shown in figure 3, Fig. 3 A is with plasmid pMD-RG for DNA mould Plate PCR amplification electrophoretogram, wherein, swimming lane M be Marker, swimming lane 1-8 be followed successively by with genes of interest CaMV35S, FMV35S, PTA29, Ubiquitin, PNOS, TNOS, T-E9 and T-35S corresponding primer amplification result electrophoretogram；

Fig. 3 B be respectively with standard substance (MON89788, GTS40-3-2, KF6, RF1, MS1, GT73, MON863, Bt176 it is) template PCR amplifications electrophoretogram, wherein, swimming lane M is Marker, and swimming lane 1-8 is followed successively by with genes of interest CaMV35S (transgenic line Bt176), FMV35S (transgenic line GT73), PTA29 (transgenic line RF1), Ubiquitin (turn base Because of material MON863), PNOS (transgenic line MS1), TNOS (transgenic line GTS40-3-2), T-E9 (transgenic line ) and the corresponding primer amplification result electrophoretogram of T-35S (transgenic line KF6) MON89788；

Fig. 3 C is the electrophoretogram entering performing PCR amplification with the DNA of non-transgenic material south round-grained rice 46 for template, and wherein, swimming lane M is Marker, swimming lane 1-8 be followed successively by with genes of interest CaMV35S, FMV35S, PTA29, Ubiquitin, PNOS, TNOS, T-E9 and T-35S corresponding primer amplification result electrophoretogram；

As seen from Figure 3, only in corresponding transgenic with case, recombiant plasmid just has amplification, shows that recombiant plasmid has very Good specificity, and the amplification of standard plasmid of recombinating is consistent with the amplification of corresponding positive transgenic, illustrates to recombinate Standard plasmid pMD-RG can substitute the positives transgenic sample of transgenic paddy rice screening.

(2) with the DNA sample of the restructuring standard plasmid pMD-RG of variable concentrations, (concentration is followed successively by 1.0 × 10 respectively⁶、1.0 ×10⁵、1.0×10⁴、1.0×10³、1.0×10², 1.0 × 10 and 1.0copy/ μ L) be template, respectively with purpose base in table 2 Because the corresponding specific primer of CaMV35S, FMV35S, PTA29, Ubiquitin, PNOS, T35S, TNOS and T-E9 is primer, The same step of reaction system (1), carries out qualitative PCR amplification, to judge to recombinate standard plasmid for the sensitivity of qualitative PCR detection. Its result as shown in figure 4, wherein, Fig. 4 A- Fig. 4 H be followed successively by genes of interest CaMV35S, FMV35S, PTA29, Ubiquitin, The amplification of PNOS, T35S, TNOS and T-E9, swimming lane M is Marker, and 1-8 represents the restructuring standard plasmid adding in reaction system PMD-RG concentration is 1.0 × 10⁶、1.0×10⁵、1.0×10⁴、1.0×10³、1.0×10², 1.0 × 10 and 1.0copy/ μ L；By Fig. 4 understands, the amplification sensitivity of 8 Insert Fragments has all reached 1.0 × 10copy. μ L^-1, this copy number be far below 25mg/L, It can be seen that pMD-RG meets the demand of qualitative PCR detection.

SEQUENCE LISTING

<110>Jiangsu Province Agriculture Science Institute

<120>A kind of standard plasmid for detecting transgenic regulation element and its construction method and application

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<400> 5

gccgttttac gtttggaact gacagaaccg caacgttgaa ggagccactc agccgcgggt 60

ttctggagtt taatgagcta agcacatacg tcagaaacca ttattgcgcg ttcaaaagtc 120

gcctaaggtc actatcagct agcaaatatt tcttgtcaaa aatgctccac tgacgttcca 180

taa 183

<210> 6

<211> 121

<212> DNA

<213>Synthetic

<400> 6

gtttcgctca tgtgttgagc atataagaaa cccttagtat gtatttgtat ttgtaaaata 60

cttctatcaa taaaatttct aattcctaaa accaaaatcc agtactaaaa tccagatccc 120

c 121

<210> 7

<211> 180

<212> DNA

<213>Synthetic

<400> 7

gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca 60

tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt 120

cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa 180

<210> 8

<211> 262

<212> DNA

<213>Synthetic

<400> 8

ttatggcatt gggaaaactg tttttcttgt accatttgtt gtgcttgtaa tttactgtgt 60

tttttattcg gttttcgcta tcgaactgtg aaatggaaat ggatggagaa gagttaatga 120

atgatatggt ccttttgttc attctcaaat taatattatt tgttttttct cttatttgtt 180

gtgtgttgaa tttgaaatta taagagatat gcaaacattt tgttttgagt aaaaatgtgt 240

caaatcgtgg cctctaatga cc 262

<210> 9

<211> 1731

<212> DNA

<213>Synthetic

<400> 9

gctcctacaa atgccatcat tgcgataaag gaaaggccat cgttgaagat gcctctgccg 60

acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc 120

caaccacgtc ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg 180

cacaatccca ctatcaagac atccaccgaa gacttaaagt tagtgggcat ctttgaaagt 240

aatcttgtca acatcgagca gctggcttgt ggggaccaga caaaaaagga atggtgcaga 300

attgttaggc gcacctacca aaagcatctt tgcctttatt gcaaagataa agcagattcc 360

tctagtacaa gtggggaaca aaataacgtg gaaaagagct gtccttaagg tgggtggctg 420

gactagaata aacatcttct ctagcacagc ttcataatgt aatttccata actgaaatca 480

gggtgagaca aaattttggt actttttcct cacactaagt ccatgtttgc aacaaattaa 540

tacatgaaac cttaatgtta ccctcagatt agcctgctac tccccatttt cctcgaaatg 600

ctccaacaaa agttagtttt gcaagttgtt gtgtatgtct tgtgctctat atatgccctt 660

gtggtgcaag taacactggc aagttagcaa tcagaacgtg tctgacgtac aggtcgcatc 720

cgtgtacgaa cgctagcagc acggatctaa cacaaacacg gatctaacac aaacatgaac 780

agaagtagaa ctaccgggcc ctaaccatgg accggaacgc cgatctagag aaggtagaga 840

gggggggggg gggaggacga gcggcgtacc ttgaagcgga ggtgccgacg ggtggatttg 900

ggggagatct ggttgtgtgt gtgtgcgctc cgaacaacac gaggttgggg aaagagggtg 960

tggagggggt gtctatttat tacgggccgt tttacgtttg gaactgacag aaccgcaacg 1020

ttgaaggagc cactcagccg cgggtttctg gagtttaatg agctaagcac atacgtcaga 1080

aaccattatt gcgcgttcaa aagtcgccta aggtcactat cagctagcaa atatttcttg 1140

tcaaaaatgc tccactgacg ttccataagt ttcgctcatg tgttgagcat ataagaaacc 1200

cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac 1260

caaaatccag tactaaaatc cagatccccg aatcctgttg ccggtcttgc gatgattatc 1320

atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 1380

tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 1440

aacaaaatat agcgcgcaaa ctaggataat tatggcattg ggaaaactgt ttttcttgta 1500

ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg ttttcgctat cgaactgtga 1560

aatggaaatg gatggagaag agttaatgaa tgatatggtc cttttgttca ttctcaaatt 1620

aatattattt gttttttctc ttatttgttg tgtgttgaat ttgaaattat aagagatatg 1680

caaacatttt gttttgagta aaaatgtgtc aaatcgtggc ctctaatgac c 1731

<210> 10

<211> 23

<212> DNA

<213>Synthetic

<400> 10

gctcctacaa atgccatcat tgc 23

<210> 11

<211> 24

<212> DNA

<213>Synthetic

<400> 11

gatagtggga ttgtgcgtca tccc 24

<210> 12

<211> 21

<212> DNA

<213>Synthetic

<400> 12

aagacatcca ccgaagactt a 21

<210> 13

<211> 21

<212> DNA

<213>Synthetic

<400> 13

aggacagctc ttttccacgt t 21

<210> 14

<211> 20

<212> DNA

<213>Synthetic

<400> 14

taaggtgggt ggctggacta 20

<210> 15

<211> 20

<212> DNA

<213>Synthetic

<400> 15

acttgcacca caagggcata 20

<210> 16

<211> 20

<212> DNA

<213>Synthetic

<400> 16

aacactggca agttagcaat 20

<210> 17

<211> 19

<212> DNA

<213>Synthetic

<400> 17

ccgtaataaa tagacaccc 19

<210> 18

<211> 21

<212> DNA

<213>Synthetic

<400> 18

gccgttttac gtttggaact g 21

<210> 19

<211> 20

<212> DNA

<213>Synthetic

<400> 19

ttatggaacg tcagtggagc 20

<210> 20

<211> 20

<212> DNA

<213>Synthetic

<400> 20

gtttcgctca tgtgttgagc 20

<210> 21

<211> 22

<212> DNA

<213>Synthetic

<400> 21

ggggatctgg attttagtac tg 22

<210> 22

<211> 20

<212> DNA

<213>Synthetic

<400> 22

gaatcctgtt gccggtcttg 20

<210> 23

<211> 20

<212> DNA

<213>Synthetic

<400> 23

ttatcctagt ttgcgcgcta 20

<210> 24

<211> 21

<212> DNA

<213>Synthetic

<400> 24

ttatggcatt gggaaaactg t 21

<210> 25

<211> 20

<212> DNA

<213>Synthetic

<400> 25

ggtcattaga ggccacgatt 20

<210> 26

<211> 36

<212> DNA

<213>Synthetic

<400> 26

ggtggatgtc ttgatagtgg gattgtgcgt catccc 36

<210> 27

<211> 33

<212> DNA

<213>Synthetic

<400> 27

ccacccacct taaggacagc tcttttccac gtt 33

<210> 28

<211> 32

<212> DNA

<213>Synthetic

<400> 28

cttgccagtg ttacttgcac cacaagggca ta 32

<210> 29

<211> 31

<212> DNA

<213>Synthetic

<400> 29

acgtaaaacg gcccgtaata aatagacacc c 31

<210> 30

<211> 32

<212> DNA

<213>Synthetic

<400> 30

catgagcgaa acttatggaa cgtcagtgga gc 32

<210> 31

<211> 34

<212> DNA

<213>Synthetic

<400> 31

gcaacaggat tcggggatct ggattttagt actg 34

<210> 32

<211> 32

<212> DNA

<213>Synthetic

<400> 32

ccaatgccat aattatccta gtttgcgcgc ta 32

Claims

1. a kind of standard plasmid for detecting transgenic regulation element, its nucleotide sequence is as shown in SEQ ID No.9.

2. it is used for as claimed in claim 1 detecting the construction method of the standard plasmid of transgenic regulation element it is characterised in that having Body step is as follows：

With overlapping pcr by the FMV35S shown in the CaMV35S promoter gene shown in SEQ ID No.1, SEQ ID No.2 Ubiquitin gene shown in Pat29 gene shown in promoter gene, SEQ ID No.3, SEQ ID No.4, SEQ ID NOS shown in T-35S terminator gene shown in NOS promoter gene shown in No.5, SEQ ID No.6, SEQ ID No.7 T-E9 shown in terminator gene and SEQ ID No.8 terminates mrna exon fragment and is sequentially connected acquisition fusion fragment, then will merge Fragment is cloned in carrier pMD-19, that is, obtain the described standard plasmid for detecting transgenic regulation element.

3. it is used for as claimed in claim 1 detecting the standard plasmid of transgenic regulation element in detection transgene agricultural product regulation and control unit Application in part.

4. a kind of test kit comprising the standard plasmid for detecting transgenic regulation element as claimed in claim 1.

5. test kit as claimed in claim 4 is it is characterised in that described test kit also includes following primer：

Specific primer P-35S-F and P-35S-R of CaMV35S promoter, their nucleotide sequence is respectively as SEQ ID Shown in NO.10 and SEQ ID NO.11；

Or, specific primer FMV35S-F and FMV35S-R of FMV35S promoter, their nucleotide sequence is respectively as SEQ Shown in ID NO.12 and SEQ ID NO.13；

Or, specific primer PTA29-F and PTA29-R of PTA29 promoter, their nucleotide sequence is respectively as SEQ ID Shown in NO.14 and SEQ ID NO.15；

Or, specific primer Ubiquitin-F and Ubiquitin-R of Ubiquitin promoter, their nucleotide sequence Respectively as shown in SEQ ID NO.16 and SEQ ID NO.17；

Or, specific primer T35S-F and T35S-R of T35S terminator T-35S, their nucleotide sequence is respectively as SEQ Shown in ID NO.20 and SEQ ID NO.21；

Or, specific primer T-NOS-F and T-NOS-R of NOS terminator T-NOS, their nucleotide sequence is respectively as SEQ Shown in ID NO.22 and SEQ ID NO.23；

Or, specific primer T-E9-F and T-E9-R of E9 terminator T-E9, their nucleotide sequence is respectively as SEQ ID Shown in NO.24 and SEQ ID NO.25.

6. the test kit as described in claim 5 or 6 it is characterised in that described test kit volume be 25 μ L, including：10 × PCR buffer Mg²⁺The dNTPs of 2.5 μ L, 200mmol, content are the Taq of the specific primer of 0.5 mmol, 1.25U Archaeal dna polymerase and concentration are the described standard plasmid 1.5 μ L for detecting transgenic regulation element of 25mg/L, deionized water Complement to 25 μ L.