CN110229846A - General positive standard plasmid and its construction method for transgenic paddy rice screening - Google Patents
General positive standard plasmid and its construction method for transgenic paddy rice screening Download PDFInfo
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Abstract
The invention discloses the general positive standard plasmids and its construction method for transgenic paddy rice screening；The general positive standard plasmid includes carrier framework and screening element, and the screening element includes CaMV35S promoter, Ubiquitin promoter, NOS terminator, CaMV35S terminator, Bar gene, HPT gene, Bt gene, SPS gene and PLD gene.The present invention further discloses the method for constructing the general positive standard plasmid and its applications in transgenic paddy rice screening, detection and monitoring.In transgenic paddy rice screening detection, a variety of transgenic paddy rices can detect as positive reference substance and quality-control sample using general positive standard plasmid provided by the invention, have the characteristics that applicability is wide, covering surface is extensive.
Description
Technical field
The present invention relates to general positive standard plasmids, more particularly, to the general positive standard matter of transgenic paddy rice screening
Grain, the invention further relates to the general positive standard plasmid construction method and its in transgenic paddy rice screening, detection and monitoring
In application, belong to the building and application field of the general positive standard plasmid of transgenic paddy rice screening.
Background technique
With the research and development of global genetically modified organism and commercialized fast development, more and more genetically modified crops are different
Countries and regions go through commercial growth.For rice, at present there are many pest-resistant, disease-resistant, herbicide-resistant and nutrition at
The transgenic paddy rice of improvement is divided to research and develop successfully.But genetically modified crops are while bringing huge economic results in society, there is also
Potential environment and food-safety problem, endure dispute to the fullest extent all the time.
Rice is as important cereal crops, only transgenic pest-resistant rice kind China extensive No. 1 and its cenospecies Bt Shan at present
Excellent 63 obtained the production application safety certificate that the Ministry of Agriculture issues, and not granted carry out commercial growth in 2009.But
From 2005, international and China all successively discovery rice by LLRICE601, Bt Shanyou 63, section rich No. 6 and Kemingdao etc.
The phenomenon that transgene component pollutes.It is granted and in the field trial stage and with the fast development of biotechnology industry
Genetically modified crops are more and more, and genetically modified crops accident or the risk deliberately leaked also increase therewith, to environment and food safety
It threatens, also easily causes the common people panic and trade dispute.Therefore, reinforce the screening, detection and supervision to transgenic paddy rice
It is particularly important.
During routine testing, positive criteria substance is the scale in detection process, but is prepared and marked with material of vegetable origin
The operating process of quasi- substance is complicated, and the requirement to environment and instrument and equipment is also relatively high.And in transgenosis screening detection process
In, many testing agencies are to be selected to contain respective target target transformation event specific detection standard substance according to the target of detection
As positive controls, multiple positive controls are needed for multiple detection targets in such screening detection, improve testing cost,
Increase inconvenience to detection work.
2002, the positive plasmid standard substance that Japanese Scientists Kuribara H is proposed can not depend on raw material, adopt
Multiple external source target gene can be built into jointly in a plasmid molecule with gene synthesis technology, and by microorganism into
Row storage and mass propgation.Extraction of plasmid DNA is easy and purity is higher, can effectively solve positive criteria product scarcity and preparation is tired
Difficult problem offers convenience to detection work.Therefore it develops a kind of for transgenic paddy rice screening detection positive plasmid molecule
Have great importance.
Have partial monopoly and document report recombination standard plasmid of transgenic paddy rice screening, such as the small good fortune of Wang etc. at present
Utilized overlapping pcr by CaMV35S promoter, SPS gene, Bt gene, II gene of NPT, Htp gene, Bar base in 2013
Cause and NOS terminator segment clone are building up in carrier pUC-19, obtain the recombination standard plasmid pUC- of transgenic paddy rice screening
A kind of RS (denomination of invention: recombination standard plasmid and kit for transgenic paddy rice screening；Patent application publication number:
CN103215346A).And Li Xiaofei is equal to 2013 for CaMV35S promoter, NOS terminator, marker gene Bar, HPT and
II gene of NPT constructs inspection of the plasmid molecule pBS Rice for transgenic paddy rice as the screening detection target of transgenic paddy rice
Survey (the transgenic paddy rice such as Li Xiaofei detection building and application of positive plasmid molecule, 2013).
But it is existing for screening detection transgenic paddy rice positive plasmid molecule it is different degrees of there are the targets of element
The defects of sequence applicability is relatively narrow, covering surface is not extensive enough is marked, is had much room for improvement.
Summary of the invention
The object of the present invention is to provide it is a kind of can screening detection transgenic paddy rice positive plasmid molecule, in transgenic paddy rice
When screening detects, it is used to can detect a variety of transgenic paddy rices as positive reference substance and quality-control sample, covering wide with applicability
The advantages that face is more extensive.
To achieve the goals above, the present invention the following technical schemes are provided:
A kind of general positive standard plasmid for transgenic paddy rice screening, including carrier framework and screening element；Wherein,
The screening element includes CaMV35S promoter, Ubiquitin promoter, NOS terminator, CaMV35S terminator, Bar base
Cause, HPT gene, Bt gene, SPS gene and PLD gene.
As a preferred embodiment, the SPS gene is selected from shown in SEQ ID No.8 or SEQ ID No.9
Any one of nucleotide sequence or be simultaneously selected from both sequences of SEQ ID No.8 or SEQ ID No.9.
As a preferred embodiment, the nucleotides sequence of the CaMV35S promoter is classified as SEQ ID No.1 institute
Show；The nucleotides sequence of the Ubiquitin promoter is classified as shown in SEQ ID No.2；The nucleotide sequence of the NOS terminator
Shown in SEQ ID No.3；The nucleotides sequence of the CaMV35S terminator is classified as shown in SEQ ID No.4；The Bar gene
Nucleotides sequence is classified as shown in SEQ ID No.5；The nucleotides sequence of the HPT gene is classified as shown in SEQ ID No.6；The Bt base
The nucleotides sequence of cause is classified as shown in SEQ ID No.7；The nucleotides sequence of the PLD gene is classified as shown in SEQ ID No.10.
The screening element is whole by CaMV35S promoter, Ubiquitin promoter, NOS terminator sequence, CaMV35S
Only son, Bar gene, HPT gene, Bt gene, SPS gene and PLD gene order are attached obtain in any order；
As a kind of specific embodiment of the invention, by CaMV35S promoter, Ubiquitin promoter, NOS terminator sequence,
CaMV35S terminator, Bar gene, HPT gene, Bt gene, SPS gene and PLD gene order are successively attached composition company, institute
The nucleotides sequence of the fusion connect is classified as shown in SEQ ID No.11.
Invention further provides a kind of construction methods of general positive standard plasmid, comprising: starts CaMV35S
Son, Ubiquitin promoter, NOS terminator, CaMV35S terminator, Bar gene, HPT gene, Bt gene, SPS gene and
PLD gene splicing obtains fusion afterwards together；The fusion is connected on carrier framework and obtains recombinant plasmid；It will weigh
Group plasmid conversion E. coli acceptor bacterium to get.
As a preferred embodiment, by CaMV35S promoter, Ubiquitin promoter, NOS terminator,
What CaMV35S terminator, Bar gene, HPT gene, Bt gene, SPS gene and PLD gene obtained after being successively stitched together melts
The nucleotides sequence for closing gene is classified as shown in SEQ ID No.11.
As a preferred embodiment, the carrier framework is pUC18 plasmid.
General positive standard plasmid provided by the present invention can be applied to transgenic paddy rice screening, detection and monitoring；Packet
It includes: right using the general positive standard plasmid as the positive control plasmid of transgenic paddy rice detection or positive criteria product
Rice sample to be detected carries out regular-PCR and the real-time fluorescence PCR screening detection of transgene component.
Detailed technology scheme whole description of the present invention
" the GM Approval that the present invention passes through access website International Agriculture biotechnology applications Servers Organization (ISAAA)
Database " database inquires 8 transgenic paddy rice transformant.Wherein, the rice conversion body containing 2 domestic research and development: turn
The extensive No. 1/TT51-1 of Bt trans-genetic hybrid rice China and its cenospecies Bt Shanyou 63.In conjunction with the rice conversion of the country retrieved and be collected into 11
The information such as conversion elements, the marker gene of body.Select 7 rice screening element/genes: CaMV35S promoter, Ubiquitin
Promoter, NOS terminator, CaMV35S terminator, Bar gene, HPT gene, Bt gene；And 2 rice internal standard genes:
SPS (2 segments) and PLD, for constructing transgenic paddy rice screening element positive plasmid molecule.To this 7 foreign elements/genes
Detected, it can be achieved that in the present invention 19 known Transgenic Rices all standing.
It is eaten by transgenic detection method database (GMDD, http://gmdd.sjtu.edu.cn/) and European Union's transgenosis
Product are to feed with reference to related turn of method database (http://gmo-crl.jrc.ec.europa.eu/gmomethods/) retrieval
Gene tester, and combine announced agriculture ministerial standard, existing patent or diplomatic related detecting method and sequence letter
Breath, it is final to determine transgenic paddy rice screening element/gene and internal standard gene target sequence.
The sequence number and contained nucleotide fragments such as table 1 of above-mentioned transgenic paddy rice screening element/gene and internal standard gene
It is shown:
1 rice screening element of table/gene and internal standard gene sequence number
It is a kind of polymerization as a result, the present invention provides a kind of positive universal standard plasmid for transgenic paddy rice screening
Transgenic paddy rice screening element/gene and 2 common rice internal standard genes positive plasmid molecule, contains SEQ ID No.1
~SEQ ID No.7 and SEQ ID No.10 nucleotide segment, and containing any in SEQ ID No.8~SEQ ID No.9
One or two nucleotide fragments.
Preferably, the transgenic paddy rice screening element plasmid molecule is containing SEQ ID No.1~SEQ ID No.10
Totally ten nucleotide fragments；Described ten nucleotide fragments can be attached in any order.
In an embodiment of the present invention, by 7 rice screening element/genes (pCaMV35S, pUbiquitin, tNOS,
TCaMV35S, Bar, HPT, Bt) and 2 common rice internal standard genes (SPS and PLD) detection target sequence SEQ ID
No.1~SEQ ID No.10 is long to being spliced into one section together by the sequential concatenation of SEQ ID No.1~SEQ ID No.10
The sequence SEQ ID No.11 of 3791bp；It is the fusion sequence SEQ ID No.11 of splicing is artificial synthesized and be loaded into pUC18 matter
On grain, to obtain transgenic paddy rice screening element positive plasmid molecule, it is denoted as pUC18-RICE-sceen.
The present invention is verified by PCR amplification, it was demonstrated that the transgenic paddy rice screening element positive plasmid molecule of above-mentioned building has
There is screening area coverage extensively, easily largely to obtain, and prepare the features such as simple, can be used as positive when transgenic paddy rice screening detection
Control sample, for most of rice conversion body announced and in the screening, detection and supervision for grinding transgenic paddy rice.And
The selection of each screening element/gene target sequence makes every effort to it with most wide applicability, the experiment proved that, above-mentioned pUC18-
RICE-sceen plasmid is as positive control, suitable for table 13 and table 14 in listed 7 screening element/genes and 2 rice
The existing Ministry of Agriculture of standard gene and entry and exit standard, efficiently solve in transgenic paddy rice screening detection and lack positive control or mark
The technical issues of quasi- product, avoids in screening detection aiming at the problem that multiple detection targets prepare multiple positive controls, significant to drop
Low human cost and economic cost.
Detailed description of the invention
19 transgenic paddy rice foreign elements information matrixs of Fig. 1.
Covering schematic diagram of the 7 screening elements that Fig. 2 is selected to 19 rice conversion bodies.
The determination of the target sequence of Fig. 3 pCaMV 35S.
The target sequence of Fig. 4 pUbiquitin determines.
The target sequence of Fig. 5 tNOS determines.
The target sequence of Fig. 6 tCaMV 35S determines.
The target sequence of Fig. 7 Bar gene determines.
The target sequence of Fig. 8 HPT gene determines.
The target sequence of Fig. 9 Bt gene determines.
The target sequence of Figure 10 internal standard gene SPS (1) determines.
The target sequence of Figure 11 internal standard gene SPS (2) determines.
The target sequence of Figure 12 internal standard gene PLD determines.
Figure 13 is the pUC18-RICE-sceen positive plasmid molecular structure that the present invention constructs.
Figure 14 is that 1 each element/gene of obtained pUC18-RICE-sceen positive plasmid molecule of embodiment is existing common
The electrophoretogram of PCR qualitative detection standard applicability verifying.
Figure 15 is the obtained pUC18-RICE-sceen positive plasmid of embodiment 1 for each element/gene regular-PCR
The electrophoretogram of the suitable concentration test experiments of qualitative detection.
Figure 16 is the obtained pUC18-RICE-sceen positive plasmid of embodiment 1 for each element/gene real-time fluorescence
The amplification curve diagram of the suitable concentration test experiments of PCR detection.
Specific embodiment
Further describe the present invention below in conjunction with specific embodiment, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that can modify without departing from the spirit and scope of the invention to details and form of the invention or
Replacement, but these modifications and replacement are fallen within the protection scope of the present invention.
Building of the embodiment 1 for the general positive standard plasmid of transgenic paddy rice screening
1, the detection target of transgenic paddy rice is determined
By " the GM Approval for retrieving the website International Agriculture biotechnology applications Servers Organization (ISAAA)
Database " database, related patents, document, and separate, identification, acquisition rice conversion body foreign gene insertion be sequenced
Heterogenous expression frame.Finally obtain the information such as conversion elements and the marker gene of domestic and international 19 rice conversion bodies.Table 2 is the present invention
In the relevant information of transgenic rice lines that is related to and its source.
The relevant information of transgenic rice lines involved in 2 present invention of table and its source
It is counted by the element/gene contained in the heterogenous expression frame that is inserted into above-mentioned rice conversion body, draws square
The system of battle formations (Fig. 1), the frequency of use of each Genetic elements in each rice conversion body of comparative analysis, select CaMV35S promoter,
Ubiquitin promoter, NOS terminator, CaMV35S terminator, Bar gene, HPT gene and Bt gene are as transgenic paddy rice
The target of screening detection.And with 2 rice internal standard genes: SPS and PLD constructs transgenic paddy rice screening element positive matter jointly
Grain.
In covering schematic diagram (Fig. 2) from 7 screening elements of selection to 19 rice conversion bodies it can be seen that, with regard to this hair
For 19 rice conversion bodies being related in bright, above-mentioned 7 screening elements are detected, above-mentioned detection coverage rate can reach
100%.Wherein, transformant 114-7-2 and PA110-15 is covered 1 time, remaining transformant covers 2 times or more.
2, target sequence is determined
Method database is referred to by retrieving agriculture ministerial standard, entry and exit standard, European Union's GM food and feed
(http://gmo-crl.jrc.ec.europa.eu/gmomethods/) and transgenic detection method database (GMDD,
Http:// gmdd.sjtu.edu.cn/), compile the detection of screening element/gene and internal standard gene-correlation detection method
Primer sequence (table 3- table 12), and (Fig. 3-Figure 12) is compared with each element/gene nucleotide series for primer sequence.Each
The selection of screening element/gene target sequence, which follows, makes every effort to its principle with most wide applicability, final to determine transgenosis water
Rice screening element/gene and internal standard gene target sequence.
(1) Fig. 3 is shown in the target sequence determination of pCaMV 35S.
3 CaMV 35S promoter examination criteria corresponding primer of table/probe sequence
(2) Fig. 4 is shown in the target sequence determination of pUbiquitin.
4 Ubiquitin promoter detection standard corresponding primer of table/probe sequence
(3) Fig. 5 is shown in the target sequence determination of tNOS.
5 NOS terminator examination criteria corresponding primer of table/probe sequence
(4) Fig. 6 is shown in the target sequence determination of tCaMV 35S.
6 CaMV 35S promoter examination criteria corresponding primer of table/probe sequence
(5) Fig. 7 is shown in the target sequence determination of Bar gene.
7 Bar genetic test standard corresponding primer of table/probe sequence
(6) Fig. 8 is shown in the target sequence determination of HPT gene.
8 HPT genetic test standard corresponding primer of table/probe sequence
(7) Fig. 9 is shown in the target sequence determination of Bt gene.
9 Bt genetic test standard corresponding primer of table/probe sequence
(8) Figure 10 is shown in the target sequence determination of internal standard gene SPS (1) (SEQ ID No.8).
10 SPS genetic test standard corresponding primer of table/probe sequence
(9) Figure 11 is shown in the target sequence determination of internal standard gene SPS (2) (SEQ ID No.9).
11 SPS genetic test standard corresponding primer of table/probe sequence
(10) Figure 12 is shown in the target sequence determination of internal standard gene PLD.
12 PLD genetic test standard corresponding primer of table/probe sequence
3, the building and preservation of general positive standard plasmid
According to the nucleotide sequence corresponding in table 1, by target sequence SEQ ID No.1~SEQ ID No.10 by suitable
Sequence is spliced into the sequence SEQ ID No.11 of one section of long 3791bp.The fusion sequence SEQ ID No.11 of splicing is artificial synthesized simultaneously
It is loaded on pUC18 plasmid, to obtain transgenic paddy rice screening element positive plasmid, is named as pUC18-RICE-sceen.
The order of connection and plasmid molecule size are shown in Figure 13.
Obtained positive plasmid pUC18-RICE-sceen is constructed to be transformed into E. coli acceptor bacterium, culture presevation-
80 DEG C of refrigerators, with carrier amicillin resistance (Amp+) it is selection markers.
3, the extraction, purifying and sequence verification of positive plasmid
The strain of preservation is drawn into plate, 37 DEG C are incubated overnight, and picking monoclonal colonies are containing ampicillin within second day
Resistance (Amp+) LB culture solution in, 37 DEG C, 200rpm cultivate 12h, then with Axygen company small amount plasmid DNA extract reagent
Box (AxyPrepTMPlasmid Miniprep Kit) it extracts and purifies.
It will extract and the plasmid molecule of purifying complete sequence verification.Sequencing analysis is the results show that pUC18-RICE-sceen
Plasmid is to integrate sequence (target sequence) by the screening element of the pUC18 carrier framework of 2653bp and 3791bp to constitute, and sequence is total
Long 6444bp, and the exogenous array in positive plasmid is consistent with expected sequence.
The existing regular-PCR qualitative detection standard applicability verification test of each element/gene of 1 positive plasmid molecule of test example
1, the extraction and detection of positive plasmid pUC18-RICE-sceen
By the Escherichia coli containing positive plasmid molecule pUC18-RICE-sceen in 37 DEG C, 200rpm cultivates 12h, according to
Axygen company small amount plasmid DNA extraction kit (AxyPrepTMPlasmid Miniprep Kit) specification extraction plasmid
DNA.With the purity and concentration of ultramicron nucleic acid-protein detector test plasmid, OD as the result is shown260/OD280Ratio 1.8 a left side
The right side meets qualitative, quantitative PCR detection requirement.
2, positive plasmid verifying utilizes common PCR primers (table 13), reaction system and the journey in following corresponding bulletins, standard
Building matter is examined in carry out PCR verifying of the sequence (table 14) to transgenic paddy rice screening element positive plasmid pUC18-RICE-sceen
Whether grain contains expected exogenous sequences, and whether is suitable for the existing Ministry of Agriculture and entry and exit regular-PCR qualitative detection standard,
PCR amplification uses the PCR instrument of ABI company production, as a result as shown in figure 14.As shown in Figure 14, screening element/gene can be had
Effect amplification illustrates that the plasmid of building contains expected purpose gene, and is suitable for the existing Ministry of Agriculture and entry and exit examination criteria.
13 screening element of table/gene and internal standard gene regular-PCR detection primer
2 transgenic paddy rice screening element positive plasmid pUC18-RICE-sceen of test example is used for regular-PCR qualitative detection
Suitable concentration testing experiment
According to positive plasmid DNA initial concentration and size, ddH is used2It is successively diluted to 1 × 10 by O8Copies/ μ L (ladder
Degree 1), 1 × 107Copies/ μ L (gradient 2), 1 × 106Copies/ μ L (gradient 3), 1 × 105Copies/ μ L (gradient 4), 1 ×
104Copies/ μ L (gradient 5), 1 × 103Copies/ μ L (gradient 6), 1 × 102Copies/ μ L (gradient 7), 1 × 10copies/
μ L (gradient 8), 1copies/ μ L (gradient 9), 4 DEG C save backup.
With the DNA sample (1 × 10 of the positive plasmid pUC18-RICE-sceen of various concentration8、1×107、1×106、1×
105、1×104、1×103、1×102, 1 × 10,1copies/ μ L) be template, should be announced using each element/gene pairs, standard
In primer (table 13), reaction system and program (table 14) carry out regular-PCR amplification, judge that transgenic paddy rice screening element is positive
Plasmid is used for the suitable concentration of regular-PCR qualitative detection.
Experimental result shows that each screening element/gene is 1 × 10 in plasmid concentration2Copies/ μ L (gradient 7) or more
PUC18-RICE-sceen Plasmid samples in can be amplified (Figure 15) detection of respective element/gene qualitative PCR can be met
Demand.Plasmid molecule pUC18-RICE-sceen is detected as positive control for regular-PCR screening, and stablize amplification is suitable for dense
Spending range is 1 × 103~1 × 105copies/μL。
3 transgenic paddy rice screening element positive plasmid pUC18-RICE-sceen of test example is used for real-time fluorescence PCR detection
Suitable concentration testing experiment
5 gradients in Selection experiment example 2 in 9 concentration gradients: 1 × 106Copies/ μ L (gradient 3), 1 ×
105Copies/ μ L (gradient 4), 1 × 104Copies/ μ L (gradient 5), 1 × 103Copies/ μ L (gradient 6), 1 ×
102Copies/ μ L (gradient 7), 1 × 10copies/ μ L (gradient 8) transgenic paddy rice screening element positive plasmid as DNA
Template is used using fluorescent primer (table 15), reaction system (table 16) and the response procedures in following corresponding bulletins, standard
5 real-time PCR of Applied Biosystems QuantStudio is to transgenic paddy rice screening element positive plasmid
PUC18-RICE-sceen carry out real-time fluorescence PCR detection, judge transgenic paddy rice screening element positive plasmid in real time it is glimmering
The suitable concentration of light PCR detection.
Testing result shows that each element/gene pairs answers real-time fluorescent PCR testing primer in various concentration (1 × 106、1×
105、1×104、1×103、1×102, 1 × 10copies/ μ L) pUC18-RICE-sceen Plasmid samples in can detect
Fluorescence signal (Figure 16), the plasmid are suitable for the corresponding Ministry of Agriculture and entry and exit real-time fluorescence PCR detection standard, can meet corresponding
Detection demand.And plasmid molecule pUC18-RICE-sceen is used for each screening element/gene real-time fluorescence PCR as positive control
The appropriate concentration range of detection is 1 × 103~1 × 105copies/μL。
Each screening element/gene of table 15 and internal standard gene real-time fluorescent PCR testing primer
Each screening element of table 16/gene real-time fluorescence PCR detecting reaction system
| Reagent | Volume |
| TaqMan reaction buffer | 10.0μL |
| 10 μm of ol/L upstream primers (F) | 1.0μL |
| 10 μm of ol/L upstream primers (R) | 1.0μL |
| 10 μm of ol/L probes (P) | 0.5μL |
| DNA profiling | 2.0μL |
| ddH2O | 5.5μL |
| Total volume | 20.0μL |
Response procedures are as follows: 50 DEG C, 2min；95℃,2min；95℃,1s；60℃,20s；Recurring number 40；In second stage
Annealing collects fluorescence signal when extending (60 DEG C).
SEQUENCE LISTING
<110>Paddy Rice Inst., Anhui Agriculture Science Academy
<120>the general positive standard plasmid and its construction method of transgenic paddy rice screening are used for
<130> AH-2001-190410A
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 287
<212> DNA
<213> Artifical sequence
<400> 1
aaaggaaggt ggctcctaca aatgccatca ttgcgataaa ggaaaggcta tcattcaaga 60
tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa 120
agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgacatct ccactgacgt 180
aagggatgac gcacaatccc actatccttc gcaagaccct tcctctatat aaggaagttc 240
atttcatttg gagaggacag cccaagcttc gactctagag gatcccc 287
<210> 2
<211> 653
<212> DNA
<213> Artifical sequence
<400> 2
caggattcct caaagagaaa cactggcaag ttagcaatca gaacatgtct gatgtacagg 60
tcgcatccgt gtacgaacgc tagcagcacg gatctaacac aaacacggat ctaacacaaa 120
catgaacaga agtagaacta ccgggcccta accatggacc ggaacgccga tctagagaag 180
gtagagagag gggggggggg aggatgagcg gcgtaccttg aagcggaggt gccgacgggt 240
ggatttgggg gagatctggt tgtgtgtgtg tgcgctccga acgaacacga ggttggggaa 300
agagggtgtg gagggggtgt ctatttatta cggcgggcga ggaagggaaa gcgaaggagc 360
ggtgggaaag gaatcccccg taggctgccg tgccgtgaga ggaggaggag gccgcctgcc 420
gtgccgcctc acgtctgccg ctccgccacg caatttctgg atgccgacag cggagcaagt 480
ccaacggtgg agcggaactc tcgagagggg tccagaggca gcgacagaga tgccgtgccg 540
tctgcttcgc ttggcccgac gcgacgctgc tggttcgctg gttggtgtcc gttagactcg 600
tcgacggcgt ttaacaggct ggcattatct actcgaaaca agaaaaatgt ttc 653
<210> 3
<211> 257
<212> DNA
<213> Artifical sequence
<400> 3
cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 60
gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 120
catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 180
cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 240
tatgttacta gatcggg 257
<210> 4
<211> 209
<212> DNA
<213> Artifical sequence
<400> 4
gatctgtcga tcgacaagct cgagtttctc cataataatg tgtgagtagt tcccagataa 60
gggaattagg gttcctatag ggtttcgctc atgtgttgag catataagaa acccttagta 120
tgtatttgta tttgtaaaat acttctatca ataaaatttc taattcctaa aaccaaaatc 180
cagtactaaa atccagatcc cccgaatta 209
<210> 5
<211> 440
<212> DNA
<213> Artifical sequence
<400> 5
tcgtcaacca ctacatcgag acaagcacgg tcaacttccg taccgagccg caggaaccgc 60
aggagtggac ggacgacctc gtccgtctgc gggagcgcta tccctggctc gtcgccgagg 120
tggacggcga ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc aacgcctacg 180
actggacggc cgagtcgacc gtgtacgtct ccccccgcca ccagcggacg ggactgggct 240
ccacgctcta cacccacctg ctgaagtccc tggaggcaca gggcttcaag agcgtggtcg 300
ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc ggatatgccc 360
cccgcggcat gctgcgggcg gccggcttca agcacgggaa ctggcatgac gtgggtttct 420
ggcagctgga cttcagcctg 440
<210> 6
<211> 500
<212> DNA
<213> Artifical sequence
<400> 6
ccgattccgg aagtgcttga cattggggag tttagcgaga gcctgaccta ttgcatctcc 60
cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttcta 120
caaccggtcg cggaggctat ggatgcgatc gctgcggccg atcttagcca gacgagcggg 180
ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc 240
gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg 300
tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg 360
cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca 420
gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc 480
ttcttctgga ggccgtggtt 500
<210> 7
<211> 530
<212> DNA
<213> Artifical sequence
<400> 7
aggccatctc taggttggaa ggtttgagca atctctacca aatctatgca gagagcttca 60
gagagtggga agccgatcct actaacccag ctctccgcgg ggaaatgcgt attcaattca 120
acgacatgaa cagcgccttg accacagcta tcccattgtt cgcagtccag aactaccaag 180
ttcctctctt gtccgtgtac gttcaagcag ctaatcttca cctcagcgtg cttcgagacg 240
ttagcgtgtt tgggcaaagg tggggattcg atgctgcaac catcaatagc cgttacaacg 300
accttactag gctgatcgga aactacaccg accacgctgt tcgttggtac aacactggct 360
tggagcgtgt ctggggtcct gattctagag attggattag atacaaccag ttcaggagag 420
aattgaccct cacagttttg gacattgtgt ctctcttccc gaactatgac tccagaacct 480
accctatccg tacagtgtcc caacttacca gagaaatcta tactaaccca 530
<210> 8
<211> 295
<212> DNA
<213> Artifical sequence
<400> 8
ctttttattt gcgcctgaac ggatatcttt cagtttgtaa ccaccggatg acgcacggac 60
ggctcggatc atcccgaaaa gatcaaccgc ggcgcgagca cgagaccacc gtgggcccca 120
tggcccaccg acttacacaa tctctcccac tgccatgcgg gcccacacca gcaacagtcc 180
agtccagaga gccccgaact cctccaaacc cggggggcca caccctgcca cgtgtcaccc 240
gccagcctcc ctctcatcct ctctctcctc gtccagtgct tctccttctc ctcgc 295
<210> 9
<211> 310
<212> DNA
<213> Artifical sequence
<400> 9
tacagagtgg atctgtttac tcgtcaagtg tcatctcctg aagtggactg gagctatggg 60
gagcctactg aaatgttaac tccggttcca ctgacggaga gggaagcggt gagagtgctg 120
gtgcgtacat tgtgcgcatt ccgtgcggtc caagggacaa gtacctccgt aaagagccct 180
gtggccttac ctccaagagt ttgtcgacgg agctctcgcg catatctgaa catgtccaag 240
gctctggggg aacaggttag caatgggaag ctggtcttgc catatgtaat ccatggccac 300
tatgccgatg 310
<210> 10
<211> 310
<212> DNA
<213> Artifical sequence
<400> 10
atcaggcctt gtcagtggca agaacaacac cattgacagg agcatccaag atgcatacat 60
ccacgcaatt cgccgcgcca agaacttcat ctacatcgag aatcagtact tccttggcag 120
ctcatttgca tggaaagccg atggcatcag accagaagac attgaggcgt tgcatctgat 180
tcccagagag atttctctga agattgtgaa caagattgaa gctggtgagc gttttgcagt 240
ctatgttgtg ctgccaatgt ggcctgaagg acctcctgct agtggatcag tgcaggcaat 300
actggattgg 310
<210> 11
<211> 3791
<212> DNA
<213> Artifical sequence
<400> 11
aaaggaaggt ggctcctaca aatgccatca ttgcgataaa ggaaaggcta tcattcaaga 60
tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa 120
agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgacatct ccactgacgt 180
aagggatgac gcacaatccc actatccttc gcaagaccct tcctctatat aaggaagttc 240
atttcatttg gagaggacag cccaagcttc gactctagag gatcccccag gattcctcaa 300
agagaaacac tggcaagtta gcaatcagaa catgtctgat gtacaggtcg catccgtgta 360
cgaacgctag cagcacggat ctaacacaaa cacggatcta acacaaacat gaacagaagt 420
agaactaccg ggccctaacc atggaccgga acgccgatct agagaaggta gagagagggg 480
gggggggagg atgagcggcg taccttgaag cggaggtgcc gacgggtgga tttgggggag 540
atctggttgt gtgtgtgtgc gctccgaacg aacacgaggt tggggaaaga gggtgtggag 600
ggggtgtcta tttattacgg cgggcgagga agggaaagcg aaggagcggt gggaaaggaa 660
tcccccgtag gctgccgtgc cgtgagagga ggaggaggcc gcctgccgtg ccgcctcacg 720
tctgccgctc cgccacgcaa tttctggatg ccgacagcgg agcaagtcca acggtggagc 780
ggaactctcg agaggggtcc agaggcagcg acagagatgc cgtgccgtct gcttcgcttg 840
gcccgacgcg acgctgctgg ttcgctggtt ggtgtccgtt agactcgtcg acggcgttta 900
acaggctggc attatctact cgaaacaaga aaaatgtttc cgatcgttca aacatttggc 960
aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc 1020
tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat 1080
gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat 1140
agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcggggat 1200
ctgtcgatcg acaagctcga gtttctccat aataatgtgt gagtagttcc cagataaggg 1260
aattagggtt cctatagggt ttcgctcatg tgttgagcat ataagaaacc cttagtatgt 1320
atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac caaaatccag 1380
tactaaaatc cagatccccc gaattatcgt caaccactac atcgagacaa gcacggtcaa 1440
cttccgtacc gagccgcagg aaccgcagga gtggacggac gacctcgtcc gtctgcggga 1500
gcgctatccc tggctcgtcg ccgaggtgga cggcgaggtc gccggcatcg cctacgcggg 1560
cccctggaag gcacgcaacg cctacgactg gacggccgag tcgaccgtgt acgtctcccc 1620
ccgccaccag cggacgggac tgggctccac gctctacacc cacctgctga agtccctgga 1680
ggcacagggc ttcaagagcg tggtcgctgt catcgggctg cccaacgacc cgagcgtgcg 1740
catgcacgag gcgctcggat atgccccccg cggcatgctg cgggcggccg gcttcaagca 1800
cgggaactgg catgacgtgg gtttctggca gctggacttc agcctgccga ttccggaagt 1860
gcttgacatt ggggagttta gcgagagcct gacctattgc atctcccgcc gtgcacaggg 1920
tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctacaac cggtcgcgga 1980
ggctatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg 2040
accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc 2100
ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc 2160
tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc 2220
ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg 2280
gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc 2340
gtggttaggc catctctagg ttggaaggtt tgagcaatct ctaccaaatc tatgcagaga 2400
gcttcagaga gtgggaagcc gatcctacta acccagctct ccgcggggaa atgcgtattc 2460
aattcaacga catgaacagc gccttgacca cagctatccc attgttcgca gtccagaact 2520
accaagttcc tctcttgtcc gtgtacgttc aagcagctaa tcttcacctc agcgtgcttc 2580
gagacgttag cgtgtttggg caaaggtggg gattcgatgc tgcaaccatc aatagccgtt 2640
acaacgacct tactaggctg atcggaaact acaccgacca cgctgttcgt tggtacaaca 2700
ctggcttgga gcgtgtctgg ggtcctgatt ctagagattg gattagatac aaccagttca 2760
ggagagaatt gaccctcaca gttttggaca ttgtgtctct cttcccgaac tatgactcca 2820
gaacctaccc tatccgtaca gtgtcccaac ttaccagaga aatctatact aacccacttt 2880
ttatttgcgc ctgaacggat atctttcagt ttgtaaccac cggatgacgc acggacggct 2940
cggatcatcc cgaaaagatc aaccgcggcg cgagcacgag accaccgtgg gccccatggc 3000
ccaccgactt acacaatctc tcccactgcc atgcgggccc acaccagcaa cagtccagtc 3060
cagagagccc cgaactcctc caaacccggg gggccacacc ctgccacgtg tcacccgcca 3120
gcctccctct catcctctct ctcctcgtcc agtgcttctc cttctcctcg ctacagagtg 3180
gatctgttta ctcgtcaagt gtcatctcct gaagtggact ggagctatgg ggagcctact 3240
gaaatgttaa ctccggttcc actgacggag agggaagcgg tgagagtgct ggtgcgtaca 3300
ttgtgcgcat tccgtgcggt ccaagggaca agtacctccg taaagagccc tgtggcctta 3360
cctccaagag tttgtcgacg gagctctcgc gcatatctga acatgtccaa ggctctgggg 3420
gaacaggtta gcaatgggaa gctggtcttg ccatatgtaa tccatggcca ctatgccgat 3480
gatcaggcct tgtcagtggc aagaacaaca ccattgacag gagcatccaa gatgcataca 3540
tccacgcaat tcgccgcgcc aagaacttca tctacatcga gaatcagtac ttccttggca 3600
gctcatttgc atggaaagcc gatggcatca gaccagaaga cattgaggcg ttgcatctga 3660
ttcccagaga gatttctctg aagattgtga acaagattga agctggtgag cgttttgcag 3720
tctatgttgt gctgccaatg tggcctgaag gacctcctgc tagtggatca gtgcaggcaa 3780
tactggattg g 3791
Claims (10)
1. a kind of general positive standard plasmid for transgenic paddy rice screening, including carrier framework and screening element, feature
Be, the screening element include CaMV35S promoter, Ubiquitin promoter, NOS terminator, CaMV35S terminator,
Bar gene, HPT gene, Bt gene, SPS gene and PLD gene.
2. general positive standard plasmid in accordance with claim, which is characterized in that the SPS gene is selected from SEQ ID
Any one of nucleotide sequence shown in No.8 or SEQ ID No.9 or two kinds.
3. general positive standard plasmid described in accordance with the claim 1, which is characterized in that the nucleosides of the CaMV35S promoter
Acid sequence is shown in SEQ ID No.1；The nucleotides sequence of the Ubiquitin promoter is classified as shown in SEQ ID No.2；It is described
Shown in the nucleotide sequence SEQ ID No.3 of NOS terminator；The nucleotides sequence of the CaMV35S terminator is classified as SEQ ID
Shown in No.4；The nucleotides sequence of the Bar gene is classified as shown in SEQ ID No.5；The nucleotides sequence of the HPT gene is classified as
Shown in SEQ ID No.6；The nucleotides sequence of the Bt gene is classified as shown in SEQ ID No.7；The nucleotides sequence of the PLD gene
It is classified as shown in SEQ ID No.10.
4. general positive standard plasmid described in accordance with the claim 1, which is characterized in that the screening element is by CaMV35S
Promoter, Ubiquitin promoter, NOS terminator sequence, CaMV35S terminator, Bar gene, HPT gene, Bt gene, SPS
Gene and PLD gene order are successively attached composition.
5. general positive standard plasmid according to claim 4, which is characterized in that by CaMV35S promoter,
Ubiquitin promoter, NOS terminator sequence, CaMV35S terminator, Bar gene, HPT gene, Bt gene, SPS gene and
The nucleotides sequence for the fusion that PLD gene order is successively attached is classified as shown in SEQ ID No.11.
6. the construction method of general positive standard plasmid described in claim 1 characterized by comprising start CaMV35S
Son, Ubiquitin promoter, NOS terminator, CaMV35S terminator, Bar gene, HPT gene, Bt gene, SPS gene and
PLD gene splicing obtains fusion afterwards together；The fusion is connected on carrier framework and obtains recombinant plasmid；It will weigh
Group plasmid conversion E. coli acceptor bacterium to get.
7. construction method according to claim 6, which is characterized in that by CaMV35S promoter, Ubiquitin promoter,
NOS terminator, CaMV35S terminator, Bar gene, HPT gene, Bt gene, SPS gene and PLD gene are successively stitched together
The nucleotides sequence of the fusion obtained afterwards is classified as shown in SEQ ID No.11.
8. construction method according to claim 6, which is characterized in that the carrier framework is pUC18 plasmid.
9. general positive standard plasmid answering in transgenic paddy rice qualitatively screening, detection and monitoring described in claim 1-5
With.
10. applying according to claim 9, which is characterized in that the application includes: that will lead to described in claim 1-5
Use positive criteria plasmid as the positive control plasmid or positive criteria product of transgenic paddy rice detection, to rice to be detected
Sample carries out regular-PCR and the real-time fluorescence PCR screening detection of transgene component.
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Cited By (3)
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| CN110724704A (en) * | 2019-10-16 | 2020-01-24 | 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 |
| CN110885804A (en) * | 2019-12-12 | 2020-03-17 | 东北师范大学 | Method for synthesizing sucrose-6-phosphate by using recombinant high-temperature-resistant sucrose phosphate synthase |
| CN111826388A (en) * | 2020-07-07 | 2020-10-27 | 黑龙江省农业科学院农产品质量安全研究所 | Unauthorized transgenic corn screening positive plasmid molecule pYMSC-1905 |
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| CN103215346A (en) * | 2013-03-01 | 2013-07-24 | 浙江省农业科学院 | Recombinant standard plasmid and kit used in transgenic rice screening |
| CN103540665A (en) * | 2013-10-18 | 2014-01-29 | 中国农业科学院生物技术研究所 | Plasmid standard molecule capable of detecting multiple types of genetically modified rice |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724704A (en) * | 2019-10-16 | 2020-01-24 | 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 |
| CN110885804A (en) * | 2019-12-12 | 2020-03-17 | 东北师范大学 | Method for synthesizing sucrose-6-phosphate by using recombinant high-temperature-resistant sucrose phosphate synthase |
| CN110885804B (en) * | 2019-12-12 | 2021-06-29 | 东北师范大学 | Method for synthesizing sucrose-6-phosphate by using recombinant high-temperature-resistant sucrose phosphate synthase |
| CN111826388A (en) * | 2020-07-07 | 2020-10-27 | 黑龙江省农业科学院农产品质量安全研究所 | Unauthorized transgenic corn screening positive plasmid molecule pYMSC-1905 |
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