CN110527737B - Positive plasmid molecule pYCID-1905 identified by transgenic rape and transformant of transgenic rape product and application thereof - Google Patents

Abstract

The invention discloses a design and application of positive plasmid molecule pYCID-1905 for identifying transgenic rape and a transformant of the transgenic rape, and relates to the safety supervision and transformant identification technology of transgenic rape in the field of transgenic safety. The base sequence of pYCID-1905 is shown as SEQ ID NO. 1. The pYCID-1905 is used as a positive control and quality control sample, so that the ordinary PCR and real-time fluorescence PCR identification and detection of transformants of rape and products thereof can be carried out, and the coverage rate of the approved transgenic rape varieties is 100% for China; the invention solves the technical problem of lack of positive control or standard in the identification and detection of transgenic rape transformants, avoids the problem of setting positive control for each detection target in strain identification and detection, and reduces the labor cost and economic cost for preparing positive control of each rape transformant.

Description

Positive plasmid molecule pYCID-1905 identified by transgenic rape and transformant of transgenic rape product and application thereof

Technical Field

The invention relates to a safety supervision and transformant identification technology of transgenic rape in the field of transgenic safety, in particular to a design and application of a positive plasmid molecule pYCID-1905.

Background

Over 20 years, the development and commercialization of transgenic organisms worldwide has rapidly progressed, and since 1992, the regulatory approval of 4133 transgenic products is globally approved, 476 transformants of 26 transgenic crops are involved, the planting area is accumulated to be more than 23 hundred million hectares, and huge economic benefits are generated. Meanwhile, safety problems of transgenic organisms have been paid attention to by various societies, and for this reason, regulations for safety management of transgenic organisms are promulgated to be implemented in more than 50 countries and regions including china, the european union, the united states, japan, korea, etc., to standardize development and industrial application of transgenic organisms.

In the transgenic biosafety supervision work, on one hand, the situation of planting and application of approved imported or commercialized transgenic products is to be supervised, and on the other hand, the situation of illegal planting or application of unauthorized transgenic products is to be supervised. Unauthorized transgenic products refer to transgenic products released to the market without approval, including transgenic materials approved in another country but not approved in our country, transgenic materials illegally planted in the field that are only approved for import as processing materials, and transgenic materials that are still in the testing stage. Transgenic rape is one of four transgenic crops, is an important source of edible oil and feed meal, and has been commercially planted on a large scale in Canadian, U.S. and Australia. In order to meet the supply of edible oil and rapeseed meal, the imported rapeseeds from abroad are needed in China every year, the imported quantity exceeds 500 ten thousand tons in 2015, and more than 90% of the rapeseeds are transgenic rapeseeds. China is also a large country for rape planting, transgenic research is active, and varieties enter a field test stage continuously. Because of the potential environmental and edible safety risks of the transgenic products, once illegal diffusion events of unauthorized transgenic rape occur, normal trade order is disturbed, panic emotion is induced in consumers, and a series of adverse effects are generated on national economy and life. The international report about unauthorized transgenic crops is frequent, and the detection and supervision technical system for unauthorized transgenic rape is urgently needed to be established in China.

The screening detection of transgenic rape is that after the sample containing transgenic components is identified in the first step of monitoring and detecting unauthorized transgenic rape, the specific detection of transformant is further carried out; through the specific detection of the transformant, if the transgene component contained in the sample is not derived from the authorized transformant, the sample is proved to contain unauthorized transgene component, and an emergency plan for transgene safety supervision is started immediately, so that safety risks are prevented. Through construction of a transgenic detection technology system for more than ten years, china establishes a perfect transformant specific detection technology standard system aiming at transgenic rape which is wholesale and applies for security certificates, a series of standards including national standards, bulletins of the agricultural department, import and export inspection and quarantine standards and the like are issued, and corresponding standards are also issued by European Union. However, due to the limitation of raw material supply and the like, the development of the standard substance or the standard sample of the rape transformant is seriously lagged behind the development of the detection method, so that the difficulty of lacking the standard substance or the standard sample is often faced in the identification and detection process of the transgenic rape transformant, and the implementation of the detection standard is influenced. The standard sample for identifying and detecting the transgenic rape transformant is developed, so that the technical standard system for detecting and monitoring the transgene in China is further perfected, and the function of the standard method in the safety supervision of the transgenic organism is effectively exerted. By developing and using the transgenic rape transformant to identify positive plasmid molecules and a detection method matched with the positive plasmid molecules, the detection and monitoring level of illegal diffusion of transgenic rape products in China is greatly improved, and an effective technical support is provided for the safety supervision of transgenic organisms in China.

In combination with the above consideration, the problem of lack of a standard sample for identifying and detecting the transgenic rape transformants is taken as an entry point, the transgenic rape varieties approved and imported in China and applied for transgenic biological safety certificates are researched, the detection sequences for identifying the transformants in the standard method of the transgenic rape are analyzed, targets and target sequences for identifying the transgenic rape transformants are determined, and the targets and target sequences are polymerized with internal rape standard genes to construct a common vector pUC18 vector skeleton, so that plasmid molecules for identifying and detecting the transgenic rape transformants are developed.

Through the search of existing patents and other documents, no report has been found on the identification of universal positive plasmid molecules and applications by constructing transgenic rape transformants using the design of the present invention or similar designs.

Disclosure of Invention

The invention aims to solve the problem that the transgenic rape transformant identification detection standard substance is lacking in the transgenic detection work as an entry point, research the transgenic rape variety approved for import in China and applying for biological safety certificate, and determine the transformant identification detection target of the transgenic rape; various standards issued by rape transformants are investigated, and the nucleotide sequence of each detection target is determined; the fusion sequence of each transformant identification detection target and rape internal standard gene is artificially synthesized and constructed into pUC18, so that positive control and quality control samples are provided for transgenic rape transformant identification detection.

In one aspect, the invention provides a positive plasmid molecule pYCID-1905 identified by transgenic rape and its product transformant, which is characterized in that: the base sequence is shown in SEQ ID NO. 1:

in another aspect, the invention provides a positive plasmid molecule pYCID-1905 identified by transgenic rape and its product transformant, which contains 11 rape transformant targets and 2 rape internal standard genes, in order:

the 3' end sequence of GT73 is shown as SEQ ID NO.2;

MS1 3' end sequence, SEQ ID NO.3;

MS1 5' end sequence, SEQ ID NO.4;

oxygen 235 3' end sequence, see SEQ ID NO.5;

oxygen 235 5' end sequence, see SEQ ID NO.6;

MS 85' end sequence, SEQ ID NO.7;

MS8 3' end sequence, SEQ ID NO.8;

RF3 3' end sequence, SEQ ID NO.9;

RF3 5' end sequence, SEQ ID NO.10;

t45 3' end sequence, see SEQ ID NO.11;

RF1 5' end sequence, SEQ ID NO.12;

topas19/2 3' end sequence, SEQ ID NO.13;

RF2 5' end sequence, SEQ ID NO.14;

73496 A 5' end sequence shown in SEQ ID NO.15;

MON88302 5' end sequence, see SEQ ID NO.16

The HMG I/Y sequence of the rape internal standard gene is shown in SEQ ID NO.17;

the sequence of the internal standard gene CruA of rape is shown in SEQ ID NO.18.

In another aspect, the present invention provides a transgenic rape and its product transformant to identify positive plasmid molecule pYCID-1905, which is a plasmid molecule formed by inserting fusion sequence SEQ ID No.19 into pUC18 vector.

In another aspect, the invention provides the plasmid molecules of the invention as positive control and/or quality control samples for the identification of transformants of transgenic canola and products thereof.

In another aspect, the invention provides a kit for detecting transgenic rape and identifying products thereof by using common PCR, which comprises the plasmid molecules, the transgenic rape transformant and detection primers of rape internal standard genes.

In the technical scheme of the invention, the detection primers of the transgenic rape transformant and the rape internal standard gene are as follows:

MS1 5′：

primer F5 '-CGGTGAGTAATATTGTACGGCTAA-3' SEQ ID NO.20

R 5′—ATCTCTGGTTAAACATTCCATCTTTG—3′SEQ ID NO.21,

RF1 5′：

Primer F5 '-CCTGTGGTCTCAAGATGGATCA-3' SEQ ID NO.22

R 5′—GGTGACTACACGCGACTCAT—3′SEQ ID NO.23；

RF2 5′：

Primer F5 '-TTGGTGGACCCTTGAGGAAAC-3' SEQ ID NO.24

R 5′—CCATCTAATAGGGTGAGACAAT—3′SEQ ID NO.25；

MS8 5′：

Primer F5 '-CCTTTTCTTATCGACCATGTACTC-3' SEQ ID NO.26

R 5′—AATTTTAAAAACTTGTGGGATGCT—3′SEQ ID NO.27；

RF3 3′：

Primer F5 '-CAATAACTTTGTTGGGCTTATGG-3' SEQ ID NO.28

R 5′—CTCGTCTCGGACCTCCGAAAACC—3′SEQ ID NO.29；

OXY235 3′：

F 5′—TTTGTTTATTGCTTTCGCC—3′SEQ ID NO.30

R 5′—CCAGGGGATTCAGTTGGA—3′SEQ ID NO.31；

Topas 19/2 3′：

Primer F5 '-CGGCCTTAATCCCACCCCAG-3' SEQ ID NO.32

R 5′—AGTTCCAAACGTAAAACGGCTT—3′SEQ ID NO.33；

T45 3′：

Primer F5 '-TCCCATTTATTTACGGTCAC-3' SEQ ID NO.34

R 5′—CCATGGGAATTCATTTACAA—3′SEQ ID NO.35；

GT73 3′：

Primer F5 '-AATAACGCTGCGGACATCTA-3' SEQ ID NO.36

R 5′—CAGCAAGATTCTCTGTCAACAA—3′SEQ ID NO.37；

73496 5′：

Primer F5 '-ACTTGTTCGTTGGAGTTGTC-3' SEQ ID NO.38

R 5′—TCGTGTTCAGTCCAATGACC—3′SEQ ID NO.39；

MON88302 5′：

Primer F5 '-CTCCTCAAGTTGTACAGTCTTGAAGAGA-3' SEQ ID NO.40

R 5′—CAGGACCTGCAGAAGCTTGATAAC—3′SEQ ID NO.41；

Rape internal standard gene CruA:

CruAF398 5′—GGCCAGGGCTTCCGTGAT—3′SEQ ID NO.42

CruAR547 5′—CTGGTGGCTGGCTAAATCGA—3′SEQ ID NO.43；

rape internal standard gene HMG I/Y:

hmg-F 5′—TCCTTCCGTTTCCTCGCC—3′SEQ ID NO.44

hmg-R 5′—TTCCACGCCCTCTCCGCT—3′SEQ ID NO.45。

in another aspect, the invention provides a kit for identifying transgenic rape and products thereof by real-time fluorescence PCR, which comprises the plasmid molecule, the transgenic rape transformant and a detection primer pair and a probe of a rape internal standard gene.

In the technical scheme of the invention, the detection primers and probes of the transgenic rape transformant and the rape internal standard gene are as follows:

RT73 transformant:

primer RT73F5 '-CCATATTGACCATCATACTCATTGCT-3' SEQ ID NO.46

RT73R 5′—GCTTATACGAAGGCAAGAAAAGGA—3′SEQ ID NO.47，

Probe RT73P 5 '-TTCCCGGACATGAAGATCATCCTCCTT-3' SEQ ID NO.48;

MS1 transformant 5';

primer MS1-5F5 '-ATCTCTGGTTAAACATTCCATCTTTG-3' SEQ ID NO.49

MS1-5R 5′—CGAGCTTTCTAATTTCAAACTATTCGG—3′SEQ ID NO.50，

Probe MS1-5p 5 '-TGGATAGGTTCTTCAGCATCATCACACC-3' seq ID No.51;

MS1 transformant 3':

primer MS1-MLD025 5 '-ACGCTGCGGACATCTACATT-3' SEQ ID NO.52

MS1-MDB175 5′—CTAGATCGGAAGCTGAAGATGG—3′SEQ ID NO.53，

Probe MS1-TM030 5 '-CTCATTGCTGATCCACCTAGCCGACTT-3' SEQ ID NO.54;

OXY235 transformant 5':

primer OXY235-5F 5 '-CTAACTTTTGGTGTGATGATGCTGA-3' SEQ ID NO.55

OXY235-5R 5′—CGATAGATGGTGGTGTGAGTCTTG—3′SEQ ID NO.56，

Probe OXY235-5P 5 '-AGCTGATGGCAAGTTAATCTCCCCGAAGTCG-3' SEQ ID NO.57;

OXY235 transformant 3':

primer QOXY235-3LG 5 '-AGAGAATCGTGAAATTATCTCTACCG-3' SEQ ID NO.58

QOXY235-3LV 5′—ATTGACCATCATACTCATTGCTGA—3′SEQ ID NO.59，

Probe QOXY235-3L 5 '-CCATGTAGATTTCCCGGACATGAAGCC-3' SEQ ID NO.60;

MS8 transformant 5';

primer QMS8RG 5 '-CCTTGAGGACGCTTTGATCATATT-3' SEQ ID NO.61

QMS8RV 5′—CCTTTTCTTATCGACCATGTACTC—3′SEQ ID NO.62，

The probe QMS8RFAM 5 '-CCGAGTTCGACGGCCGAGTACTG-3' SEQ ID NO.63;

MS8 transformant 3';

primer MS8L-KVM085 '-GTTAGAAAAAGTAAACAATTAATATAGCCGG-3' SEQ ID NO.64

MS8L-HCA048 5′—GGAGGGTGTTTTTGGTTATC—3′SEQ ID NO.65，

Probe MS8L-TM011 5 '-AATATAATCGACGGATCCCCGGGAATTC-3' SEQ ID NO.66;

RF3 transformant 3';

primer QRF3RG 5 '-TTTGTACAAAACTTGGACCCCTAGGT-3' SEQ ID NO.67

QRF3RV 5′—TTCTTTCAAGATGGGAATTAACATCT—3′SEQ ID NO.68，

Probe QRF3P5 '-TGCCTTTTCTTATCGCGAGATGAAAAAGGC-3' SEQ ID NO.69;

RF3 transformant 5':

RF3DPA165 5′—CATAAAGGAAGATGGAGACTTGAG—3′SEQ ID NO.70

RF3KVM084AGCATTTAGCATGTACCATCAGACA—3′SEQ ID NO.71，

probe RF3TM010 5 '-CGCACGCTTATCGACCATAAGCCCA-3' SEQ ID NO.72;

t45 transformant:

primer T45KVM172 5 '-CAATGGACACATGAATTATGC-3' SEQ ID NO.73

T45MDB599 5′—GACTCTGTATGAACTGTTCGC—3′SEQ ID NO.74，

Probe T45TM026 5 '-TAGAGGACCTAACAGAACTCGCCGT-3' SEQ ID NO.75;

RF1 transformant:

primer RF1-MDB118 5 '-CTAAGGGAGGTCAAGATGTAGC-3' SEQ ID NO.76

RF1-KVM170 5′—CGGGCCTAACTTTTGGTGTG—3′SEQ ID NO.77，

Probe RF1-TM022 5 '-CTCATCATCCTCACCCAGTCAGCATCA-3' SEQ ID NO.78;

topas transformants:

primer TOPAS19/2 MDB685 '-GTTGCGGTTCTGTCAGTTCC-3' SEQ ID NO.79

TOPAS 19/2KVM180CGACCGGCGCTGATATATGA—3′SEQ ID NO.80，

Probe TOPAS19/2 TM029 5 '-TCCCGCGTCATCGGCGG-3' SEQ ID NO.81;

RF2 transformants:

primer RF2-MDB207 5 '-GGGTGAGACAATATATCGACG-3' SEQ ID NO.82

RF2-KVM171 5′—GGGCATCGCACCGGTGAG—3′SEQ ID NO.83，

Probe RF2-TM024 5 '-CACCGGCCAAATTCGCTCTTAGCCGT-3' SEQ ID NO.84;

73496 transformants:

primer 73496-2824 5 '-GTTCTTCTCTTCATAGCTCATTACAGTTTT-3' SEQ ID NO.85

73496-2825 5′—CAAACCTCCATAGAGTTCAACATCTTAA—3′SEQ ID NO.86，

Probe 73496-QP-83 5 '-TTAGTTAGATCAGGATATTCTTG-3' SEQ ID NO.87;

MON88302 transformant:

primer 88302QF 5 '-TCCTTGAACCTTATTTTATAGTGCACA-3' SEQ ID NO.88

88302QR 5′—TCAGATTGTCGTTTCCCGCCTTCA—3′SEQ ID NO.89，

Probe 88302QP 5 '-TAGTCATCATGTTGTACCACTTCAAACACT-3' SEQ ID NO.90;

rape internal standard gene

CruA：qCruAF 5′—GGCCAGGGCTTCCGTGAT—3′SEQ ID NO.91

qCruAR 5′—CCGTCGTTGTAGAACCATTGG—3′SEQ ID NO.92，

Probe qCruAP 5 '-AGTCCTTATGTGCTCCACTTTCTGGTGCA-3' SEQ ID NO.93;

rape internal standard gene HMG I/Y: qhmg-F5 '-GGTCGTCCTCCTAAGGCGAAAG-3' SEQ ID NO.94 and qhmg-R5 '-CTTCTTCGGCGGTCGTCCAC-3' SEQ ID NO.95,

the probe qhmg-P5 '-CGGAGCCACTCGGTGCCGCAACTT-3' SEQ ID NO.96.

In a further aspect, the invention provides a method for identifying transgenic rape and products thereof by common PCR, which comprises the following steps:

1) The total DNA of the sample is extracted,

2) The positive plasmid molecules and the sample genome DNA of the invention are subjected to PCR amplification by utilizing the transgenic rape identification unit and the detection primer of the rape internal standard gene;

3) Separating by agarose gel electrophoresis, and identifying whether amplification products exist after EB staining; under the condition that the positive plasmid molecules are amplified normally and the standard genes in the rape in the sample are amplified normally, if amplification products exist, the rape sample contains corresponding rape transformants.

In a further aspect, the invention provides a method for real-time fluorescent PCR identification of transgenic rape and products thereof, comprising the steps of:

1) Extracting total DNA of a sample;

2) PCR amplification is carried out on the positive plasmid molecules and the sample genome DNA by utilizing the detection primer pair and the probe pair of the transgenic rape transformant and the rape internal standard gene;

3) Judging whether a corresponding rape transformant exists or not according to whether a typical amplification curve exists in the real-time fluorescence PCR product; under the condition that the positive plasmid molecules are amplified normally and the standard genes in the rape in the sample are amplified normally, if amplification products exist, the rape sample contains rape transformants.

In a further aspect, the present invention provides a method for preparing the plasmid molecule of the present invention, which comprises inserting the fusion sequence SEQ ID No.19 into pUC18 vector; or SEQ ID No.2-18 are sequentially connected to form a fusion sequence, and then the fusion sequence is inserted into a pUC18 vector.

The purpose of the invention is realized in the following way:

1. design of the Positive plasmid molecule pYCID-1905

The positive plasmid molecule pYCID-1905 is a positive plasmid molecule polymerized with 11 rape transformants and 2 rape internal standard genes.

Design (one)

1. Identification of transgenic rape transformants the positive plasmid molecule pYCID-1905 contains 11 rape transformants, 9 rape transformants GT73, MS1, OXY235, MS8, RF3, T45, RF1, topas19/2, RF2, which were approved to be imported by our country, and 2 rape transformants 73496, MON88302, which are applying for security certificates in our country. All transgenic rape varieties approved for import and under the application of security certificates in China can be fully covered by identifying the 11 transformants.

2. Identification of transgenic rape transformants Positive plasmid molecule pYCID-1905 contains 2 rape internal standard genes, HMG I/Y gene and CruA gene respectively.

3. Identification of transgenic rape transformants the target sequences of 11 transformants in the positive plasmid molecule pYCID-1905 were determined in accordance with the standards of the department of agriculture (bulletin of the Ministry of agriculture), the national standards (GB/T), the import and export inspection and quarantine standards (SN/T) and the European Union standards published for these transformants, and the plasmid molecule pYCID-1905 could be used in combination with the respective standards.

4. Identification of transgenic rape transformants the positive plasmid molecule pYCID-1905 can be used as a quality control for both the conventional qualitative PCR detection and the real-time fluorescent PCR detection of each transformant.

(II) construction

The target sequences of 11 transformants and the target sequences of 2 rape internal standard genes are spliced together for artificial synthesis, and the artificial synthesized fusion sequence is constructed on a vector pUC18 to construct a positive plasmid molecule pYCID-1905 (figure 1) for polymerizing 11 transformants and 2 rape internal standard genes.

(III) features

1. The base sequence is shown as SEQ ID NO.1, and the DNA region of the base sequence contains 11 transformant specific sequences and 2 rape internal standard gene sequences;

2. the bases at positions 437 to 3175 are fusion sequences of 9 approved rape transformants, in order, (1) GT73, (2) MS1, (3) OXY235, (4) MS8, (5) RF3, (6) T45, (7) RF1, (8) Topas19/2, (9) RF2.

3. The bases from 3176 to 3531 are fusion sequences of 2 rape internal standard genes HMG I/Y and CruA qualitative and quantitative detection targets.

4. The bases at positions 3532 to 4065 are 2 fusion sequences of the rape transformant for which the safety certificate is being applied, and are (1) 73496, (2) MON88302 in this order.

5. The sequence is the characteristic sequence of positive plasmid molecule pYCID-1905 identified by the transgenic rape transformant, and can be used as positive control and quality control samples in the identification and detection of the transgenic rape transformant.

2. Application of positive plasmid molecule pYCID-1905

Ordinary PCR and real-time fluorescent PCR screening detection for transformant identification were performed on canola samples using pYCID-1905 as positive control and quality control samples:

synthesizing a primer combination (primer/probe combination) of a rape internal standard gene HMG I/Y (CruA) and a rape transformant; extracting total DNA of the sample, and respectively carrying out PCR amplification on positive plasmid molecules and genomic DNA of the sample by using primer combinations (primer/probe combinations) of internal standard genes and rape transformants; separating the common PCR product by agarose gel electrophoresis, and identifying whether an amplification product exists after EB dyeing; judging whether the real-time fluorescent PCR product is an amplification product according to whether the typical amplification curve is present or not; under the condition that the positive plasmid molecules are amplified normally and the standard genes in the rape in the sample are amplified normally, if amplification products exist, the rape sample contains corresponding rape transformants.

Compared with the prior art, the invention has the following advantages and positive effects:

(1) The positive plasmid molecule pYCID-1905 universal for identifying and detecting the transgenic rape transformant is provided;

(2) pYCID-1905 polymerized 11 transformant identification targets for identification and detection of transgenic rape transformants and 2 internal standard gene sequences of rape;

(3) pYCID-1905 can be used as identification detection of 9 authorized rape transformants and 2 rape transformants to be authorized;

(4) The technical problem that positive control or standard substance is lacking in identification and detection of transgenic rape transformants is solved, the problem that positive control is set for each transformant in the identification and detection of the transformants is avoided, and the labor cost and the economic cost for preparing a plurality of positive controls are reduced;

(5) Is suitable for the identification, detection, monitoring and safety supervision of the transformant of the transgenic rape and products thereof.

Drawings

FIG. 1 is a block diagram of the positive plasmid molecule pYCID-1905;

FIG. 2 shows the detection targets of 11 rape transformants and 2 rape internal standard genes in positive plasmid molecules by using a qualitative PCR method. Target order: (1) GT73 transformant, (2) MS1 transformant, (3) OXY235 transformant, (4) MS8 transformant, (5) RF3 transformant, (6) T45 transformant, (7) RF1 transformant, (8) Topas19/2 transformant, (9) RF2 transformant, (10) 73496 transformant, (11) MON88302 transformant, (12) rape internal standard gene HMG I/Y, (13) rape internal standard gene CruA.

FIG. 3 shows the detection targets of 11 rape transformants and 2 rape internal standard genes in positive plasmid molecules verified by using a real-time fluorescence PCR method.

FIG. 4 shows the identification and detection of a part of rape varieties by ordinary PCR transformants using the plasmid molecule pYCID-1905 as a positive control. The results showed that only pYCID-1905 and the corresponding transformant had amplified products for each target tested.

FIG. 5 shows the identification and detection of real-time fluorescent PCR transformants for a portion of canola varieties using the plasmid molecule pYCID-1905 as a positive control. The results showed that only pYCID-1905 and the corresponding transformants had amplification curves for each target tested.

Detailed Description

The invention is described in detail below with reference to the attached drawings and examples:

1. construction of the Positive plasmid molecule pYCID-1905

Firstly, determining identification and detection targets of transgenic rape transformants

Referring to related data of transgenic rape industrialization, 9 rape transformants approved to be imported in China are GT73, MS1, OXY235, MS8, RF3, T45, RF1, topas19/2 and RF2 respectively; there are 2 rape transformants in our country that are applying for security certificates, 73496 and MON88302, respectively. And determining whether the transgenic components in the sample are derived from authorized transgenic rape or unauthorized transgenic rape by determining the identification and detection of the transgenic rape transformants by using 11 rape transformant targets.

(II) determination of target sequences of 11 transformants

Methods and primer/probe sequences for detection of these 11 transformants were collected in standards issued by the Ministry of agriculture (bulletin of the Ministry of agriculture), national standards (GB/T), import and export inspection and quarantine standards (SN/T) and European Union standards. The amplified sequences of each primer/probe combination and binding sites on the transformant specific sequences were analyzed to determine the target sequences to be constructed on the plasmid. The 3' -end sequence of GT73 is 207bp long, and SEQ ID NO.2 is shown; MS1 3' end sequence is 191bp long, see SEQ ID NO.3; MS1 5' end sequence is 200bp long, see SEQ ID NO.4; the 3' -end sequence of OXY235 is 344bp long, and is shown in SEQ ID NO.5; the length of the OXY235 5' end sequence is 129bp, and SEQ ID NO.6; MS 85' end sequence is 164bp long, see SEQ ID NO.7; the MS8 3' end sequence is 134bp long and is shown in SEQ ID NO.8; the RF3 3' end sequence is 361bp long, and is shown in SEQ ID NO.9; the length of the RF3 5' end sequence is 256bp, and SEQ ID NO.10 is shown; the sequence of the T45' end is 304bp long, and SEQ ID NO.11 is shown; the RF1 5' end sequence is 254bp long, and is shown in SEQ ID NO.12; the Topas19/2 3' end sequence is 134bp long and is shown in SEQ ID NO.13; the RF2 5' end sequence is 206bp long, and is shown in SEQ ID NO.14;73496 The 5' end sequence is 284bp long, and is shown as SEQ ID NO.15; the MON88302 5' end sequence is 250bp long and is shown in SEQ ID NO.16.

(III) determining target sequences of 2 rape internal standard genes

The two internal standard genes which are most widely used in transgenic rape detection are HMG I/Y and CruA genes respectively. The general PCR method for these two genes and the detection method for performing the fluorescent PCR method and primer/probe sequences were referred to. The amplified sequences of each primer/probe combination and the binding sites on the screening element are analyzed to determine the target sequence to be constructed on the plasmid. The length of the HMG I/Y sequence of the rape internal standard gene is 206bp, and SEQ ID NO.17 is shown; the internal standard gene CruA sequence of rape is 150bp long, and SEQ ID NO.18 is shown.

(IV) construction of plasmid molecule pYCID-1905

The nucleotide sequences of 15 transformant-specific targets (the nucleotide sequences of MS1, OXY235, MS8 and RF 34 transformants contained the 5 'and 3' transformant-specific sequences) of the 11 rape transformants and the nucleotide sequences of 2 rape internal standard genes were spliced together to form one fusion sequence (SEQ ID NO. 19). The spliced fusion sequence is entrusted to Shanghai engineering synthesis, and is inserted into pUC18 vector after synthesis to construct plasmid molecule pYCID-1905.

2. Verification of the Positive plasmid molecule pYCID-1905

Sequencing verification

The plasmid molecule pYCID-1905 was subjected to full-molecular sequencing, and the accuracy of the target sequence was verified.

(II) verification of ordinary PCR method

According to published standards, common PCR primers for 11 transformants and 2 rape internal standard genes were synthesized. MS1 5': primers F5 '-CGGTGAGTAATATTGTACGGCTAA-3' SEQ ID NO.20 and R5 '-ATCTCTGGTTAAACATTCCATCTTTG-3' SEQ ID NO.21, amplification product 194bp; RF1 5': primers F5 '-CCTGTGGTCTCAAGATGGATCA-3' SEQ ID NO.22 and R5 '-GGTGACTACACGCGACTCAT-3' SEQ ID NO.23, amplified 200bp; RF2 5': primers F5 '-TTGGTGGACCCTTGAGGAAAC-3' SEQ ID NO.24 and R5 '-CCATCTAATAGGGTGAGACAAT-3' SEQ ID NO.25, amplified 200bp; MS8 5': primers F5 '-CCTTTTCTTATCGACCATGTACTC-3' SEQ ID NO.26 and R5 '-AATTTTAAAAACTTGTGGGATGCT-3' SEQ ID NO.27, amplification product 159bp; RF3 3': primers F5 '-CAATAACTTTGTTGGGCTTATGG-3' SEQ ID NO.28 and R5 '-CTCGTCTCGGACCTCCGAAAACC-3' SEQ ID NO.29, amplification product 284bp; OXY235 3': f5 '-TTTGTTTATTGCTTTCGCC-3' SEQ ID NO.30 and R5 '-CCAGGGGATTCAGTTGGA-3' SEQ ID NO.31, amplification product 331bp; topas19/2 3': primers F5 '-CGGCCTTAATCCCACCCCAG-3' SEQ ID NO.32 and R5 '-AGTTCCAAACGTAAAACGGCTT-3' SEQ ID NO.33, amplification product 110bp; t45' 3: primers F5 '-TCCCATTTATTTACGGTCAC-3' SEQ ID NO.34 and R5 '-CCATGGGAATTCATTTACAA-3' SEQ ID NO.35, amplification product 233bp; GT 73': primers F5 '-AATAACGCTGCGGACATCTA-3' SEQ ID NO.36 and R5 '-CAGCAAGATTCTCTGTCAACAA-3' SEQ ID NO.37, amplification product 204bp;73496 5': primers F5 '-ACTTGTTCGTTGGAGTTGTC-3' SEQ ID NO.38 and R5 '-TCGTGTTCAGTCCAATGACC-3' SEQ ID NO.39, amplification product 281bp; MON88302 5': primers F5 '-CTCCTCAAGTTGTACAGTCTTGAAGAGA-3' SEQ ID NO.40 and R5 '-CAGGACCTGCAGAAGCTTGATAAC-3' SEQ ID NO.41, amplified product 150bp; rape internal standard gene CruA: cruAF3985 '-GGCCAGGGCTTCCGTGAT-3' SEQ ID NO.42 and CruAR547 '-CTGGTGGCTGGCTAAATCGA-3' SEQ ID NO.43, amplified products 150bp; rape internal standard gene HMG I/Y: hmg-F5 '-TCCTTCCGTTTCCTCGCC-3' SEQ ID NO.44 and hmg-R5 '-TTCCACGCCCTCTCCGCT-3' SEQ ID NO.45, amplified 206bp.

And (3) performing PCR amplification by using the synthesized common PCR primer and taking the constructed positive plasmid molecule as a template. The PCR reaction used a 25ul reaction system containing 1. Mu.L of positive plasmid molecule, 1 XPCR Buffer (containing 10mM Tris HCl pH8.3,KCl 50mM), 200uM dNTPs,2.5mM MgCl2, 250nM forward and reverse primers, DNATAq enzyme 1u. The reaction procedure was 94℃for 2 minutes, 94℃for 15 seconds, 60℃for 30 seconds, 72℃for 30 seconds, 35 cycles, and 72℃for 2 minutes. The PCR products were separated by agarose gel electrophoresis, and the presence of amplified products was identified after EB staining.

(III) verification of real-time fluorescence PCR method

Real-time fluorescent PCR primers/probes were synthesized for 11 transformants and 2 canola internal standard genes. RT73 transformant: primers RT73F5 '-CCATATTGACCATCATACTCATTGCT-3' SEQ ID NO.46 and RT73R 5 '-GCTTATACGAAGGCAAGAAAAGGA-3' SEQ ID NO.47, probe RT73P 5 '-TTCCCGGACATGAAGATCATCCTCCTT-3' SEQ ID NO.48; MS1 transformant 5'; primers MS1-5F5 '-ATCTCTGGTTAAACATTCCATCTTTG-3' SEQ ID NO.49 and MS1-5R 5 '-CGAGCTTTCTAATTTCAAACTATTCGG-3' SEQ ID NO.50, probes MS1-5P 5 '-TGGATAGGTTCTTCAGCATCATCACACC-3' SEQ ID NO.51; primers MS1-MLD025 5 '-ACGCTGCGGACATCTACATT-3' SEQ ID NO.52 and MS1-MDB175 5 '-CTAGATCGGAAGCTGAAGATGG-3' SEQ ID NO.53, probe MS1-TM030 5 '-CTCATTGCTGATCCACCTAGCCGACTT-3' SEQ ID NO.54; OXY235 transformant 5': primers OXY235-5F 5 '-CTAACTTTTGGTGTGATGATGCTGA-3' SEQ ID NO.55 and OXY235-5R 5 '-CGATAGATGGTGGTGTGAGTCTTG-3' SEQ ID NO.56, probe OXY235-5P 5 '-AGCTGATGGCAAGTTAATCTCCCCGAAGTCG-3' SEQ ID NO.57; OXY235 transformant 3': primers QOXY235-3LG 5 '-AGAGAATCGTGAAATTATCTCTACCG-3' SEQ ID NO.58 and QOXY235-3LV 5 '-ATTGACCATCATACTCATTGCTGA-3' SEQ ID NO.59, probe QOXY235-3L 5 '-CCATGTAGATTTCCCGGACATGAAGCC-3' SEQ ID NO.60; MS8 transformant 5'; primers QMS8RG 5 '-CCTTGAGGACGCTTTGATCATATT-3' SEQ ID NO.61 and QMS8RV 5 '-CCTTTTCTTATCGACCATGTACTC-3' SEQ ID NO.62, probe QMS8RFAM 5 '-CCGAGTTCGACGGCCGAGTACTG-3' SEQ ID NO.63; MS8 transformant 3'; primers MS8L-KVM0855 '-GTTAGAAAAAGTAAACAATTAATATAGCCGG-3' SEQ ID NO.64 and MS8L-HCA048 5 '-GGAGGGTGTTTTTGGTTATC-3' SEQ ID NO.65, probe MS8L-TM011 5 '-AATATAATCGACGGATCCCCGGGAATTC-3' SEQ ID NO.66; RF3 transformant 3'; primers QRF3RG 5 '-TTTGTACAAAACTTGGACCCCTAGGT-3' SEQ ID NO.67 and QRF3RV 5 '-TTCTTTCAAGATGGGAATTAACATCT-3' SEQ ID NO.68, probe QRF3P5 '-TGCCTTTTCTTATCGCGAGATGAAAAAGGC-3' SEQ ID NO.69; RF3 transformant 5': RF3DPA165 5 '-CATAAAGGAAGATGGAGACTTGAG-3' SEQ ID NO.70 and RF3KVM084

AGCATTTAGCATGTACCATCAGACA-3 ' SEQ ID NO.71, probe RF3TM010 5' -CGCACGCTTATCGACCATAAGCCCA-3 ' SEQ ID NO.72; t45 transformant: primers T45KVM172 5 '-CAATGGACACATGAATTATGC-3' SEQ ID NO.73 and T45MDB5995 '-GACTCTGTATGAACTGTTCGC-3' SEQ ID NO.74, probe T45TM026 5 '-TAGAGGACCTAACAGAACTCGCCGT-3' SEQ ID NO.75; RF1 transformant: primers RF1-MDB118 5 '-CTAAGGGAGGTCAAGATGTAGC-3' SEQ ID NO.76 and RF1-KVM1705 '-CGGGCCTAACTTTTGGTGTG-3' SEQ ID NO.77, probe RF1-TM022 5 '-CTCATCATCCTCACCCAGTCAGCATCA-3' SEQ ID NO.78; topas transformants: primers TOPAS19/2 MDB685 ' -GTTGCGGTTCTGTCAGTTCC-3 ' SEQ ID NO.79 and TOPAS19/2KVM180 CGACCGGCGCTGATATATGA-3 ' SEQ ID NO.80, probe TOPAS19/2 TM029 5' -TCCCGCGTCATCGGCGG-3 ' SEQ ID NO.81; RF2 transformants: primers RF2-MDB207 5 '-GGGTGAGACAATATATCGACG-3' SEQ ID NO.82 and RF2-KVM171 5 '-GGGCATCGCACCGGTGAG-3' SEQ ID NO.83, probe RF2-TM024 5 '-CACCGGCCAAATTCGCTCTTAGCCGT-3' SEQ ID NO.84;73496 transformants: primers 73496-2824 5 '-GTTCTTCTCTTCATAGCTCATTACAGTTTT-3' SEQ ID NO.85 and 73496-2825 5 '-CAAACCTCCATAGAGTTCAACATCTTAA-3' SEQ ID NO.86, probe 73496-QP-83 5 '-TTAGTTAGATCAGGATATTCTTG-3' SEQ ID NO.87; MON88302 transformant: primers 88302QF 5 '-TCCTTGAACCTTATTTTATAGTGCACA-3' SEQ ID NO.88 and 88302QR 5 '-TCAGATTGTCGTTTCCCGCCTTCA-3' SEQ ID NO.89, probe 88302QP 5 '-TAGTCATCATGTTGTACCACTTCAAACACT-3' SEQ ID NO.90. Rape internal standard gene CruA: qCruAF 5 '-GGCCAGGGCTTCCGTGAT-3' SEQ ID NO.91 and qCruAR 5 '-CCGTCGTTGTAGAACCATTGG-3' SEQ ID NO.92, probe qCruAP 5 '-AGTCCTTATGTGCTCCACTTTCTGGTGCA-3' SEQ ID NO.93; rape internal standard gene HMG I/Y: qhmg-F5 DEG-GGTCGTCCTCCTAAGGCGAAAG-3 ' SEQ ID NO.94 and qhmg-R5' -CTTCTTCGGCGGTCGTCCAC-3 ' SEQ ID NO.95, probe qhmg-P5 ' -CGGAGCCACTCGGTGCCGCAACTT-3 ' SEQ ID NO.96.

And (3) carrying out real-time fluorescence PCR amplification by using the synthesized real-time fluorescence PCR primer and taking the constructed positive plasmid molecules as templates. Real-time fluorescence PCR analysis was performed on a CFX96PCR instrument with 20. Mu.L of PCR reaction containing 1. Mu.L of plasmid template, 1 XPCR buffer,1U Taq DNA polymerase, 4.5mM MgCl2, 300. Mu.M dNTPs,200nM primers, and 100nM probe. PCR reaction procedure: after 2 min at 50℃and 10 min at 95℃pre-denaturation, 50 PCR cycles were performed: denaturation at 95℃for 15 seconds, 60 ℃

Annealing and extension for 1 min, and collecting fluorescence signals.

3. Application method

Positive control and quality control samples for common PCR screening detection of transgenic rape by using pYCID-1905

Transgenic component screening detection was performed on the extracted DNA samples using transgenic canola GT73, topas19/2, T45, MS1, RF2, MS8, RF3, OXY235, MON88302 and 73496 as test samples, using synthetic common PCR primers, and plasmid molecule pYCID-1905 as positive control.

The PCR reaction was carried out using a 25ul reaction system containing 1. Mu.L of DNA template and 1 XPCR Buffer (containing 10mM Tris HCl pH8.3,KCl 50mM), 200uM dNTPs,2.5mM MgCl ₂ Forward primer and reverse primer of 250nM1U DNA Taq enzyme. The reaction procedure was 94℃for 2 minutes, 94℃for 15 seconds, 60℃for 30 seconds, 72℃for 30 seconds, 35 cycles, and 72℃for 2 minutes. The PCR products were separated by agarose gel electrophoresis, and the presence of amplified products was identified after EB staining.

(II) pYCID-1905 is used as positive control and quality control sample for real-time fluorescence PCR screening detection of transgenic rape

And (3) utilizing a synthesized real-time fluorescent PCR primer, and taking a plasmid molecule pYCID-1905 as a positive control to screen and detect the transgene component of the extracted transgenic rape DNA sample.

Real-time fluorescence PCR analysis was performed on a CFX96PCR instrument with 20. Mu.L of PCR reaction system containing 1. Mu.L of DNA template, 1 XPCR buffer,1U Taq DNA polymerase, 4.5mM MgCl2, 300. Mu.M dNTPs,200nM forward and reverse primers, 100nM probe. PCR reaction procedure: after 2 min at 50℃and 10 min at 95℃pre-denaturation, 50 PCR cycles were performed: denaturation at 95℃for 15 seconds, annealing at 60℃for 1 minute and extension, and fluorescence signals were collected.

4. Experimental results

Construction and sequencing analysis of (one) pYCID-1905

The plasmid molecule pYCID-1905 is subjected to full-molecular sequencing, and the sequencing result shows that the synthetic DNA target sequence is completely consistent with the expected DNA sequence (SEQ ID NO. 19), and the constructed plasmid molecule contains 11 expected rape transformant specific targets and 2 rape internal standard genes.

(II) verification of detection targets for transformants on pYCID-1905

PCR amplification was performed using the synthesized 11 transformants and 2 common PCR primers for the rape internal standard gene, and the plasmid molecule pYCID-1905 as a template. The PCR products were electrophoretically detected, and the PCR products of the expected sizes were amplified for each gene element (FIG. 2), indicating that the plasmid molecule pYCID-1905 contained the common PCR detection targets for each transformant. The plasmid molecule pYCID-1905 can be used as a positive control and quality control sample for identifying common PCR of transgenic rape transformant.

Real-time fluorescence PCR amplification was performed using the synthesized 11 transformants and 2 rape internal standard genes with real-time fluorescence PCR primers and plasmid molecule pYCID-1905 as a template. Each transformant target was found to generate a typical amplification curve (FIG. 3), indicating that the plasmid molecule pYCID-1905 contained a real-time fluorescent PCR detection target for each transformant. The plasmid molecule pYCID-1905 can be used as a positive control and quality control sample for identifying real-time fluorescence PCR by transgenic rape transformant.

(III) pYCID-1905 is used as positive control and quality control sample for common PCR identification and detection of transgenic rape

The plasmid molecule pYCID-1905 was used as a positive control, and the synthetic 2 rape internal standard gene primers and 11 transformant primers were used for ordinary PCR amplification, and 11 transgenic rape (GT 73, topas19/2, T45, MS1, RF2, MS8, RF3, OXY235, MON88302 and 73496) were subjected to transformant identification detection. The PCR products were examined and each primer combination was found to have amplified products only in the plasmid molecule pYCID-1905 and the corresponding transformant (FIG. 4). The result shows that the plasmid molecule pYCID-1905 can be used as a control sample for common PCR amplification of rape internal standard genes and 11 transformants, and the extraction quality of sample DNA and whether a PCR amplification system is normal can be judged by the amplification of the control sample.

4.4, use pYCID-1905 as positive control and quality control sample for real-time fluorescence PCR identification and detection of transgenic rape

The plasmid molecule pYCID-1905 was used as a positive control, and the synthesized 2 rape internal standard gene primers and 11 transformant primers were used for real-time fluorescence PCR amplification, and the 11 transgenic rapes (GT 73, topas19/2, T45, MS1, RF2, MS8, RF3, OXY235, MON88302 and 73496) were subjected to real-time fluorescence PCR identification analysis. The internal standard genes of 2 rapes have good amplification in the plasmid molecule pYCID-1905 and all rapes; the 11 transformant primer/probe combinations only have typical amplification curves in plasmid molecule pYCID-1905 and the corresponding transformants (FIG. 5). The result shows that the plasmid molecule pYCID-1905 can be used as a control sample when the rape internal standard gene and 11 transformants are subjected to real-time fluorescence PCR amplification, and the extraction quality of sample DNA and whether a PCR amplification system is normal can be judged through the amplification of the control sample.

SEQUENCE LISTING

<110> institute of oil crop and oil crop at national academy of agricultural sciences

<120> transgenic rape and positive plasmid molecule pYCID-1905 identified by transformant of transgenic rape and application thereof

<130> CP11903925C

<160> 96

<170> PatentIn version 3.5

<210> 1

<211> 6314

<212> DNA

<213> pYCID-1905

<400> 1

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg 420

actctagagg atcccctaat aacgctgcgg acatctacat ttttgaattg aaaaaaaatt 480

ggtaattact ctttcttttt ctccatattg accatcatac tcattgctga tccatgtaga 540

tttcccggac atgaagatca tcctccttcc tttccttgcc tttccttcct tttcttgcct 600

tcgtataagc ttgtgtcaat tgttgacaga gaatcttgct gaataacgct gcggacatct 660

acatttttga attgaaaaaa aattggtaat tactctttct ttttctccat attgaccatc 720

atactcattg ctgatccacc tagccgactt ttttaaataa ccatatccca ctatccatca 780

ggcataaata accatatccc gtcgatgctt ccatcttcag cttccgatct agaccaccgg 840

tgagtaatat tgtacggcta agagcgaatt tggcctgtag acctcaattg cgagctttct 900

aatttcaaac tattcgggcc taacttttgg tgtgatgatg ctgaagaacc tatccatgaa 960

actcacaaaa acatcatcac ctgagaattc tctggaatct aagtccaaag atggaatgtt 1020

taaccagaga tttctttgtt tattgctttc gcctataaat acgacggatc gtaatttgtc 1080

gttttatcaa aatgtacttt cattttataa taacgctgcg gacatctaca tttttgaatt 1140

gaaaaaaaat tggtaattac tctttctttt tctccatatt gaccatcata ctcattgctg 1200

atccatgtag atttcccgga catgaagcca tttacaattg aatatatcct catcgccggt 1260

agagataatt tcacgattct cttaattcaa ttctctcatc gccactgaat ggtttactag 1320

atttgtctta gaattcagat ttaccagccc gggccgtcga ccacgcgtgc cctatagtgc 1380

ctaacttttg gtgtgatgat gctgactggc aagttaatct agtttccggt tatgaagcac 1440

ggcgtgtcag ctgatggcaa gttaatctcc ccgaagtcga caagactcac accaccatct 1500

atcgaaggca aattttaaaa acttgtggga tgctgtggcc ataaaccttg aggacgcttt 1560

gatcatattc tattaactac agtacgaata tgattcgacc tttgcaattt tctcttccag 1620

tactcggccg tcgaactcgg ccgtcgagta catggtcgat aagaaaaggc aatggttaga 1680

aaaagtaaac aattaatata gccggctatt tgtgtaaaaa tccctaatat aatcgacgga 1740

tccccgggaa ttccggggga agcttagatc catggatttg ttatgataac caaaaacacc 1800

ctcctttcat aaaggaagat ggagacttga gaagtttttt ttggactttg tttagctttg 1860

ttgggcgttt tttttttttg atcaataact ttgttgggct tatggtcgat aagcgtgcgc 1920

atgtctgatg gtacatgcta aatgctatat ttctgtttaa agtgttaaaa tcattttctg 1980

atggaactaa atccagtttt aagagtaact gacaagtaca attaagcaca acaataaaat 2040

agtagtaatt ggcatctttg attgttaaat atcaaaacaa taaagttaca aaaaaaaata 2100

ccaaaccaat aatgaagact tggcggagac agtgccgtgc gaaggttttc ggaggtccga 2160

gacgagtttt ttgtaattag aaaccctgaa atttgtacaa aacttggacc cctaggtaaa 2220

tgcctttttc atctcgcgat aagaaaaggc aatttgtaga tgttaattcc catcttgaga 2280

ctctgtatga actgttcgcc agtctttacg gcgagttctg ttaggtcctc tatttgaatc 2340

tttgactcca tgggaattca tttacaactg tattccatat gcataattca tgtgtccatt 2400

gattttaccc cttattttat cattcaaaat acttcaagat atttagattt catgtaatgt 2460

agagaataaa ccgatttgct tatggtttct ttatcttttc tctaatccat attgtcgtgt 2520

tgtcatatat atctctgtag tgactagtga agaatttata gtgaccgtaa ataaatggga 2580

gggcctgtgg tctcaagatg gatcattaat ttccaccttc acctacgatg gggggcatcg 2640

caccggtgag taatattgta cggctaagag cgaatttggc ctgtagacct caattgcgag 2700

ctttctaatt tcaaactatt cgggcctaac ttttggtgtg atgatgctga ctgggtgagg 2760

atgatgagtc gcgtgtagtc accggaaaag atggaaaagg gttcttcgcc tgctacatct 2820

tgacctccct tagcccttca acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt 2880

cccgcgtcat cggcgggggt cgtaacgtga ctcccggtca tatatcagcg ccggtcggcc 2940

ccgggcctgg ggtgggatta aggccgaagt tttggtggac ccttgaggaa actggtagct 3000

gttgtgggcc tgtggtctca agatggatca ttaatttcca ccttcaccta cgatgggggg 3060

catcgcaccg gtgagtaata ttgtacggct aagagcgaat ttggccggtg agtaatattg 3120

tactggcaca gctatatata cgtcgatata ttgtctcacc ctattagatg ggaagtcctt 3180

ccgtttcctc gccgaggcct agaggtcgtc ctcctaaggc gaaaggacct tcctcggagg 3240

tggagacgaa agttgcggca ccgagtggct ccgggaggcc acgtggacga ccgccgaaga 3300

agcagaagac ggaatccgag gcggttaaag ccgatgttga acctgcggag gctccggctg 3360

gggagcggag agggcgtgga aggccagggc ttccgtgata tgcaccagaa agtggagcac 3420

ataaggactg gggacaccat cgctacacat cccggtgtag cccaatggtt ctacaacgac 3480

ggaaaccaac cacttgtcat cgtttccgtc ctcgatttag ccagccacca gcacttgttc 3540

gttggagttg tctactgctt gctgagctgg tccgtgggcc ttcctaaacg tgccgtaagt 3600

tcttctcttc atagctcatt acagttttca ttagttagat caggatattc ttgtttaaga 3660

tgttgaactc tatggaggtt tgtatgaact gatgatctag gaccggataa gttcccttct 3720

tcatagcgaa cttattcaaa gaatgttttg tgtatcattc ttgttacatt gttattaatg 3780

aaaaaatatt attggtcatt ggactgaaca cgagtatcct cctcaagttg tacagtcttg 3840

aagagattgt aacacacggt ttcctacatt taaatactta attaatgtct cagtatttgt 3900

attatcagtt ccttgaacct tattttatag tgcacaaaac cttttagtca tcatgttgta 3960

ccacttcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctga tccccatcaa 4020

gctctagcta gagcggccgc gttatcaagc ttctgcaggt cctgcggtac cgagctcgaa 4080

ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 4140

caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 4200

cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 4260

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 4320

ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 4380

ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4440

agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4500

taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4560

cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4620

tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4680

gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4740

gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4800

tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4860

gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4920

cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4980

aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 5040

tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 5100

ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5160

attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 5220

ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 5280

tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 5340

aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 5400

acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 5460

aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 5520

agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 5580

ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 5640

agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 5700

tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 5760

tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 5820

attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 5880

taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 5940

aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 6000

caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 6060

gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 6120

cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 6180

tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 6240

acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac 6300

gaggcccttt cgtc 6314

<210> 2

<211> 207

<212> DNA

<213> GT73 3' end sequence

<400> 2

taataacgct gcggacatct acatttttga attgaaaaaa aattggtaat tactctttct 60

ttttctccat attgaccatc atactcattg ctgatccatg tagatttccc ggacatgaag 120

atcatcctcc ttcctttcct tgcctttcct tccttttctt gccttcgtat aagcttgtgt 180

caattgttga cagagaatct tgctgaa 207

<210> 3

<211> 191

<212> DNA

<213> MS1 3' end sequence

<400> 3

taacgctgcg gacatctaca tttttgaatt gaaaaaaaat tggtaattac tctttctttt 60

tctccatatt gaccatcata ctcattgctg atccacctag ccgacttttt taaataacca 120

tatcccacta tccatcaggc ataaataacc atatcccgtc gatgcttcca tcttcagctt 180

ccgatctaga c 191

<210> 4

<211> 200

<212> DNA

<213> MS1 5' end sequence

<400> 4

caccggtgag taatattgta cggctaagag cgaatttggc ctgtagacct caattgcgag 60

ctttctaatt tcaaactatt cgggcctaac ttttggtgtg atgatgctga agaacctatc 120

catgaaactc acaaaaacat catcacctga gaattctctg gaatctaagt ccaaagatgg 180

aatgtttaac cagagatttc 200

<210> 5

<211> 344

<212> DNA

<213> OXY235 3' end sequence

<400> 5

tttgtttatt gctttcgcct ataaatacga cggatcgtaa tttgtcgttt tatcaaaatg 60

tactttcatt ttataataac gctgcggaca tctacatttt tgaattgaaa aaaaattggt 120

aattactctt tctttttctc catattgacc atcatactca ttgctgatcc atgtagattt 180

cccggacatg aagccattta caattgaata tatcctcatc gccggtagag ataatttcac 240

gattctctta attcaattct ctcatcgcca ctgaatggtt tactagattt gtcttagaat 300

tcagatttac cagcccgggc cgtcgaccac gcgtgcccta tagt 344

<210> 6

<211> 129

<212> DNA

<213> OXY235 5' end sequence

<400> 6

gcctaacttt tggtgtgatg atgctgactg gcaagttaat ctagtttccg gttatgaagc 60

acggcgtgtc agctgatggc aagttaatct ccccgaagtc gacaagactc acaccaccat 120

ctatcgaag 129

<210> 7

<211> 164

<212> DNA

<213> MS 85' end sequence

<400> 7

caaattttaa aaacttgtgg gatgctgtgg ccataaacct tgaggacgct ttgatcatat 60

tctattaact acagtacgaa tatgattcga cctttgcaat tttctcttca ggtactcggc 120

cgtcgaactc ggccgtcgag tacatggtcg ataagaaaag gcaa 164

<210> 8

<211> 164

<212> DNA

<213> MS8 3' end sequence

<400> 8

caaattttaa aaacttgtgg gatgctgtgg ccataaacct tgaggacgct ttgatcatat 60

tctattaact acagtacgaa tatgattcga cctttgcaat tttctcttca ggtactcggc 120

cgtcgaactc ggccgtcgag tacatggtcg ataagaaaag gcaa 164

<210> 9

<211> 361

<212> DNA

<213> RF3 3' end sequence

<400> 9

tcataaagga agatggagac ttgagaagtt ttttttggac tttgtttagc tttgttgggc 60

gttttttttt tttgatcaat aactttgttg ggcttatggt cgataagcgt gcgcatgtct 120

gatggtacat gctaaatgct atatttctgt ttaaagtgtt aaaatcattt tctgatggaa 180

ctaaatccag ttttaagagt aactgacaag tacaattaag cacaacaata aaatagtagt 240

aattggcatc tttgattgtt aaatatcaaa acaataaagt tacaaaaaaa aataccaaac 300

caataatgaa gacttggcgg agacagtgcc gtgcgaaggt tttcggaggt ccgagacgag 360

t 361

<210> 10

<211> 110

<212> DNA

<213> RF3 5' end sequence

<400> 10

tttttgtaat tagaaaccct gaaatttgta caaaacttgg acccctaggt aaatgccttt 60

ttcatctcgc gataagaaaa ggcaatttgt agatgttaat tcccatcttg 110

<210> 11

<211> 304

<212> DNA

<213> T45 3' end sequence

<400> 11

agactctgta tgaactgttc gccagtcttt acggcgagtt ctgttaggtc ctctatttga 60

atctttgact ccatgggaat tcatttacaa ctgtattcca tatgcataat tcatgtgtcc 120

attgatttta ccccttattt tatcattcaa aatacttcaa gatatttaga tttcatgtaa 180

tgtagagaat aaaccgattt gcttatggtt tctttatctt ttctctaatc catattgtcg 240

tgttgtcata tatatctctg tagtgactag tgaagaattt atagtgaccg taaataaatg 300

ggag 304

<210> 12

<211> 254

<212> DNA

<213> RF1 5' end sequence

<400> 12

ggcctgtggt ctcaagatgg atcattaatt tccaccttca cctacgatgg ggggcatcgc 60

accggtgagt aatattgtac ggctaagagc gaatttggcc tgtagacctc aattgcgagc 120

tttctaattt caaactattc gggcctaact tttggtgtga tgatgctgac tgggtgagga 180

tgatgagtcg cgtgtagtca ccggaaaaga tggaaaaggg ttcttcgcct gctacatctt 240

gacctccctt agcc 254

<210> 13

<211> 134

<212> DNA

<213> Topas19/2 3' terminal sequence

<400> 13

cttcaacgtt gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc gtcatcggcg 60

ggggtcgtaa cgtgactccc ggtcatatat cagcgccggt cggccccggg cctggggtgg 120

gattaaggcc gaag 134

<210> 14

<211> 206

<212> DNA

<213> RF2 5' end sequence

<400> 14

ttttggtgga cccttgagga aactggtagc tgttgtgggc ctgtggtctc aagatggatc 60

attaatttcc accttcacct acgatggggg gcatcgcacc ggtgagtaat attgtacggc 120

taagagcgaa tttggccggt gagtaatatt gtactggcac agctatatat acgtcgatat 180

attgtctcac cctattagat gggaag 206

<210> 15

<211> 284

<212> DNA

<213> 73496 5' end sequence

<400> 15

cacttgttcg ttggagttgt ctactgcttg ctgagctggt ccgtgggcct tcctaaacgt 60

gccgtaagtt cttctcttca tagctcatta cagttttcat tagttagatc aggatattct 120

tgtttaagat gttgaactct atggaggttt gtatgaactg atgatctagg accggataag 180

ttcccttctt catagcgaac ttattcaaag aatgttttgt gtatcattct tgttacattg 240

ttattaatga aaaaatatta ttggtcattg gactgaacac gagt 284

<210> 16

<211> 250

<212> DNA

<213> MON88302 5' terminal sequence

<400> 16

atcctcctca agttgtacag tcttgaagag attgtaacac acggtttcct acatttaaat 60

acttaattaa tgtctcagta tttgtattat cagttccttg aaccttattt tatagtgcac 120

aaaacctttt agtcatcatg ttgtaccact tcaaacactg atagtttaaa ctgaaggcgg 180

gaaacgacaa tctgatcccc atcaagctct agctagagcg gccgcgttat caagcttctg 240

caggtcctgc 250

<210> 17

<211> 206

<212> DNA

<213> HMG I/Y sequence of internal marker gene of rape

<400> 17

tccttccgtt tcctcgccga ggcctagagg tcgtcctcct aaggcgaaag gaccttcctc 60

ggaggtggag acgaaagttg cggcaccgag tggctccggg aggccacgtg gacgaccgcc 120

gaagaagcag aagacggaat ccgaggcggt taaagccgat gttgaacctg cggaggctcc 180

ggctggggag cggagagggc gtggaa 206

<210> 18

<211> 150

<212> DNA

<213> CruA sequence of internal control gene of rape

<400> 18

ggccagggct tccgtgatat gcaccagaaa gtggagcaca taaggactgg ggacaccatc 60

gctacacatc ccggtgtagc ccaatggttc tacaacgacg gaaaccaacc acttgtcatc 120

gtttccgtcc tcgatttagc cagccaccag 150

<210> 19

<211> 3629

<212> DNA

<213> fusion sequence

<400> 19

taataacgct gcggacatct acatttttga attgaaaaaa aattggtaat tactctttct 60

ttttctccat attgaccatc atactcattg ctgatccatg tagatttccc ggacatgaag 120

atcatcctcc ttcctttcct tgcctttcct tccttttctt gccttcgtat aagcttgtgt 180

caattgttga cagagaatct tgctgaataa cgctgcggac atctacattt ttgaattgaa 240

aaaaaattgg taattactct ttctttttct ccatattgac catcatactc attgctgatc 300

cacctagccg acttttttaa ataaccatat cccactatcc atcaggcata aataaccata 360

tcccgtcgat gcttccatct tcagcttccg atctagacca ccggtgagta atattgtacg 420

gctaagagcg aatttggcct gtagacctca attgcgagct ttctaatttc aaactattcg 480

ggcctaactt ttggtgtgat gatgctgaag aacctatcca tgaaactcac aaaaacatca 540

tcacctgaga attctctgga atctaagtcc aaagatggaa tgtttaacca gagatttctt 600

tgtttattgc tttcgcctat aaatacgacg gatcgtaatt tgtcgtttta tcaaaatgta 660

ctttcatttt ataataacgc tgcggacatc tacatttttg aattgaaaaa aaattggtaa 720

ttactctttc tttttctcca tattgaccat catactcatt gctgatccat gtagatttcc 780

cggacatgaa gccatttaca attgaatata tcctcatcgc cggtagagat aatttcacga 840

ttctcttaat tcaattctct catcgccact gaatggttta ctagatttgt cttagaattc 900

agatttacca gcccgggccg tcgaccacgc gtgccctata gtgcctaact tttggtgtga 960

tgatgctgac tggcaagtta atctagtttc cggttatgaa gcacggcgtg tcagctgatg 1020

gcaagttaat ctccccgaag tcgacaagac tcacaccacc atctatcgaa ggcaaatttt 1080

aaaaacttgt gggatgctgt ggccataaac cttgaggacg ctttgatcat attctattaa 1140

ctacagtacg aatatgattc gacctttgca attttctctt ccagtactcg gccgtcgaac 1200

tcggccgtcg agtacatggt cgataagaaa aggcaatggt tagaaaaagt aaacaattaa 1260

tatagccggc tatttgtgta aaaatcccta atataatcga cggatccccg ggaattccgg 1320

gggaagctta gatccatgga tttgttatga taaccaaaaa caccctcctt tcataaagga 1380

agatggagac ttgagaagtt ttttttggac tttgtttagc tttgttgggc gttttttttt 1440

tttgatcaat aactttgttg ggcttatggt cgataagcgt gcgcatgtct gatggtacat 1500

gctaaatgct atatttctgt ttaaagtgtt aaaatcattt tctgatggaa ctaaatccag 1560

ttttaagagt aactgacaag tacaattaag cacaacaata aaatagtagt aattggcatc 1620

tttgattgtt aaatatcaaa acaataaagt tacaaaaaaa aataccaaac caataatgaa 1680

gacttggcgg agacagtgcc gtgcgaaggt tttcggaggt ccgagacgag ttttttgtaa 1740

ttagaaaccc tgaaatttgt acaaaacttg gacccctagg taaatgcctt tttcatctcg 1800

cgataagaaa aggcaatttg tagatgttaa ttcccatctt gagactctgt atgaactgtt 1860

cgccagtctt tacggcgagt tctgttaggt cctctatttg aatctttgac tccatgggaa 1920

ttcatttaca actgtattcc atatgcataa ttcatgtgtc cattgatttt accccttatt 1980

ttatcattca aaatacttca agatatttag atttcatgta atgtagagaa taaaccgatt 2040

tgcttatggt ttctttatct tttctctaat ccatattgtc gtgttgtcat atatatctct 2100

gtagtgacta gtgaagaatt tatagtgacc gtaaataaat gggagggcct gtggtctcaa 2160

gatggatcat taatttccac cttcacctac gatggggggc atcgcaccgg tgagtaatat 2220

tgtacggcta agagcgaatt tggcctgtag acctcaattg cgagctttct aatttcaaac 2280

tattcgggcc taacttttgg tgtgatgatg ctgactgggt gaggatgatg agtcgcgtgt 2340

agtcaccgga aaagatggaa aagggttctt cgcctgctac atcttgacct cccttagccc 2400

ttcaacgttg cggttctgtc agttccaaac gtaaaacggc ttgtcccgcg tcatcggcgg 2460

gggtcgtaac gtgactcccg gtcatatatc agcgccggtc ggccccgggc ctggggtggg 2520

attaaggccg aagttttggt ggacccttga ggaaactggt agctgttgtg ggcctgtggt 2580

ctcaagatgg atcattaatt tccaccttca cctacgatgg ggggcatcgc accggtgagt 2640

aatattgtac ggctaagagc gaatttggcc ggtgagtaat attgtactgg cacagctata 2700

tatacgtcga tatattgtct caccctatta gatgggaagt ccttccgttt cctcgccgag 2760

gcctagaggt cgtcctccta aggcgaaagg accttcctcg gaggtggaga cgaaagttgc 2820

ggcaccgagt ggctccggga ggccacgtgg acgaccgccg aagaagcaga agacggaatc 2880

cgaggcggtt aaagccgatg ttgaacctgc ggaggctccg gctggggagc ggagagggcg 2940

tggaaggcca gggcttccgt gatatgcacc agaaagtgga gcacataagg actggggaca 3000

ccatcgctac acatcccggt gtagcccaat ggttctacaa cgacggaaac caaccacttg 3060

tcatcgtttc cgtcctcgat ttagccagcc accagcactt gttcgttgga gttgtctact 3120

gcttgctgag ctggtccgtg ggccttccta aacgtgccgt aagttcttct cttcatagct 3180

cattacagtt ttcattagtt agatcaggat attcttgttt aagatgttga actctatgga 3240

ggtttgtatg aactgatgat ctaggaccgg ataagttccc ttcttcatag cgaacttatt 3300

caaagaatgt tttgtgtatc attcttgtta cattgttatt aatgaaaaaa tattattggt 3360

cattggactg aacacgagta tcctcctcaa gttgtacagt cttgaagaga ttgtaacaca 3420

cggtttccta catttaaata cttaattaat gtctcagtat ttgtattatc agttccttga 3480

accttatttt atagtgcaca aaacctttta gtcatcatgt tgtaccactt caaacactga 3540

tagtttaaac tgaaggcggg aaacgacaat ctgatcccca tcaagctcta gctagagcgg 3600

ccgcgttatc aagcttctgc aggtcctgc 3629

<210> 20

<211> 24

<212> DNA

<213> artificial sequence

<400> 20

cggtgagtaa tattgtacgg ctaa 24

<210> 21

<211> 26

<212> DNA

<213> artificial sequence

<400> 21

atctctggtt aaacattcca tctttg 26

<210> 22

<211> 22

<212> DNA

<213> artificial sequence

<400> 22

cctgtggtct caagatggat ca 22

<210> 23

<211> 20

<212> DNA

<213> artificial sequence

<400> 23

ggtgactaca cgcgactcat 20

<210> 24

<211> 21

<212> DNA

<213> artificial sequence

<400> 24

ttggtggacc cttgaggaaa c 21

<210> 25

<211> 22

<212> DNA

<213> artificial sequence

<400> 25

ccatctaata gggtgagaca at 22

<210> 26

<211> 24

<212> DNA

<213> artificial sequence

<400> 26

ccttttctta tcgaccatgt actc 24

<210> 27

<211> 24

<212> DNA

<213> artificial sequence

<400> 27

aattttaaaa acttgtggga tgct 24

<210> 28

<211> 23

<212> DNA

<213> artificial sequence

<400> 28

caataacttt gttgggctta tgg 23

<210> 29

<211> 22

<212> DNA

<213> artificial sequence

<400> 29

ctcgtctcgg acctccgaaa ac 22

<210> 30

<211> 19

<212> DNA

<213> artificial sequence

<400> 30

tttgtttatt gctttcgcc 19

<210> 31

<211> 18

<212> DNA

<213> artificial sequence

<400> 31

ccaggggatt cagttgga 18

<210> 32

<211> 20

<212> DNA

<213> artificial sequence

<400> 32

cggccttaat cccaccccag 20

<210> 33

<211> 22

<212> DNA

<213> artificial sequence

<400> 33

agttccaaac gtaaaacggc tt 22

<210> 34

<211> 20

<212> DNA

<213> artificial sequence

<400> 34

tcccatttat ttacggtcac 20

<210> 35

<211> 20

<212> DNA

<213> artificial sequence

<400> 35

ccatgggaat tcatttacaa 20

<210> 36

<211> 20

<212> DNA

<213> artificial sequence

<400> 36

aataacgctg cggacatcta 20

<210> 37

<211> 22

<212> DNA

<213> artificial sequence

<400> 37

cagcaagatt ctctgtcaac aa 22

<210> 38

<211> 20

<212> DNA

<213> artificial sequence

<400> 38

acttgttcgt tggagttgtc 20

<210> 39

<211> 20

<212> DNA

<213> artificial sequence

<400> 39

tcgtgttcag tccaatgacc 20

<210> 40

<211> 28

<212> DNA

<213> artificial sequence

<400> 40

ctcctcaagt tgtacagtct tgaagaga 28

<210> 41

<211> 24

<212> DNA

<213> artificial sequence

<400> 41

caggacctgc agaagcttga taac 24

<210> 42

<211> 18

<212> DNA

<213> artificial sequence

<400> 42

ggccagggct tccgtgat 18

<210> 43

<211> 20

<212> DNA

<213> artificial sequence

<400> 43

ctggtggctg gctaaatcga 20

<210> 44

<211> 18

<212> DNA

<213> artificial sequence

<400> 44

tccttccgtt tcctcgcc 18

<210> 45

<211> 18

<212> DNA

<213> artificial sequence

<400> 45

ttccacgccc tctccgct 18

<210> 46

<211> 26

<212> DNA

<213> artificial sequence

<400> 46

ccatattgac catcatactc attgct 26

<210> 47

<211> 24

<212> DNA

<213> artificial sequence

<400> 47

gcttatacga aggcaagaaa agga 24

<210> 48

<211> 27

<212> DNA

<213> artificial sequence

<400> 48

ttcccggaca tgaagatcat cctcctt 27

<210> 49

<211> 26

<212> DNA

<213> artificial sequence

<400> 49

atctctggtt aaacattcca tctttg 26

<210> 50

<211> 27

<212> DNA

<213> artificial sequence

<400> 50

cgagctttct aatttcaaac tattcgg 27

<210> 51

<211> 28

<212> DNA

<213> artificial sequence

<400> 51

tggataggtt cttcagcatc atcacacc 28

<210> 52

<211> 20

<212> DNA

<213> artificial sequence

<400> 52

acgctgcgga catctacatt 20

<210> 53

<211> 22

<212> DNA

<213> artificial sequence

<400> 53

ctagatcgga agctgaagat gg 22

<210> 54

<211> 27

<212> DNA

<213> artificial sequence

<400> 54

ctcattgctg atccacctag ccgactt 27

<210> 55

<211> 25

<212> DNA

<213> artificial sequence

<400> 55

ctaacttttg gtgtgatgat gctga 25

<210> 56

<211> 24

<212> DNA

<213> artificial sequence

<400> 56

cgatagatgg tggtgtgagt cttg 24

<210> 57

<211> 31

<212> DNA

<213> artificial sequence

<400> 57

agctgatggc aagttaatct ccccgaagtc g 31

<210> 58

<211> 26

<212> DNA

<213> artificial sequence

<400> 58

agagaatcgt gaaattatct ctaccg 26

<210> 59

<211> 24

<212> DNA

<213> artificial sequence

<400> 59

attgaccatc atactcattg ctga 24

<210> 60

<211> 27

<212> DNA

<213> artificial sequence

<400> 60

ccatgtagat ttcccggaca tgaagcc 27

<210> 61

<211> 24

<212> DNA

<213> artificial sequence

<400> 61

ccttgaggac gctttgatca tatt 24

<210> 62

<211> 24

<212> DNA

<213> artificial sequence

<400> 62

ccttttctta tcgaccatgt actc 24

<210> 63

<211> 23

<212> DNA

<213> artificial sequence

<400> 63

ccgagttcga cggccgagta ctg 23

<210> 64

<211> 31

<212> DNA

<213> artificial sequence

<400> 64

gttagaaaaa gtaaacaatt aatatagccg g 31

<210> 65

<211> 20

<212> DNA

<213> artificial sequence

<400> 65

ggagggtgtt tttggttatc 20

<210> 66

<211> 28

<212> DNA

<213> artificial sequence

<400> 66

aatataatcg acggatcccc gggaattc 28

<210> 67

<211> 26

<212> DNA

<213> artificial sequence

<400> 67

tttgtacaaa acttggaccc ctaggt 26

<210> 68

<211> 26

<212> DNA

<213> artificial sequence

<400> 68

ttctttcaag atgggaatta acatct 26

<210> 69

<211> 30

<212> DNA

<213> artificial sequence

<400> 69

tgccttttct tatcgcgaga tgaaaaaggc 30

<210> 70

<211> 24

<212> DNA

<213> artificial sequence

<400> 70

cataaaggaa gatggagact tgag 24

<210> 71

<211> 25

<212> DNA

<213> artificial sequence

<400> 71

agcatttagc atgtaccatc agaca 25

<210> 72

<211> 25

<212> DNA

<213> artificial sequence

<400> 72

cgcacgctta tcgaccataa gccca 25

<210> 73

<211> 21

<212> DNA

<213> artificial sequence

<400> 73

caatggacac atgaattatg c 21

<210> 74

<211> 21

<212> DNA

<213> artificial sequence

<400> 74

gactctgtat gaactgttcg c 21

<210> 75

<211> 25

<212> DNA

<213> artificial sequence

<400> 75

tagaggacct aacagaactc gccgt 25

<210> 76

<211> 22

<212> DNA

<213> artificial sequence

<400> 76

ctaagggagg tcaagatgta gc 22

<210> 77

<211> 20

<212> DNA

<213> artificial sequence

<400> 77

cgggcctaac ttttggtgtg 20

<210> 78

<211> 27

<212> DNA

<213> artificial sequence

<400> 78

ctcatcatcc tcacccagtc agcatca 27

<210> 79

<211> 20

<212> DNA

<213> artificial sequence

<400> 79

gttgcggttc tgtcagttcc 20

<210> 80

<211> 20

<212> DNA

<213> artificial sequence

<400> 80

cgaccggcgc tgatatatga 20

<210> 81

<211> 17

<212> DNA

<213> artificial sequence

<400> 81

tcccgcgtca tcggcgg 17

<210> 82

<211> 21

<212> DNA

<213> artificial sequence

<400> 82

gggtgagaca atatatcgac g 21

<210> 83

<211> 18

<212> DNA

<213> artificial sequence

<400> 83

gggcatcgca ccggtgag 18

<210> 84

<211> 26

<212> DNA

<213> artificial sequence

<400> 84

caccggccaa attcgctctt agccgt 26

<210> 85

<211> 30

<212> DNA

<213> artificial sequence

<400> 85

gttcttctct tcatagctca ttacagtttt 30

<210> 86

<211> 28

<212> DNA

<213> artificial sequence

<400> 86

caaacctcca tagagttcaa catcttaa 28

<210> 87

<211> 23

<212> DNA

<213> artificial sequence

<400> 87

ttagttagat caggatattc ttg 23

<210> 88

<211> 27

<212> DNA

<213> artificial sequence

<400> 88

tccttgaacc ttattttata gtgcaca 27

<210> 89

<211> 24

<212> DNA

<213> artificial sequence

<400> 89

tcagattgtc gtttcccgcc ttca 24

<210> 90

<211> 30

<212> DNA

<213> artificial sequence

<400> 90

tagtcatcat gttgtaccac ttcaaacact 30

<210> 91

<211> 18

<212> DNA

<213> artificial sequence

<400> 91

ggccagggct tccgtgat 18

<210> 92

<211> 21

<212> DNA

<213> artificial sequence

<400> 92

ccgtcgttgt agaaccattg g 21

<210> 93

<211> 29

<212> DNA

<213> artificial sequence

<400> 93

agtccttatg tgctccactt tctggtgca 29

<210> 94

<211> 22

<212> DNA

<213> artificial sequence

<400> 94

ggtcgtcctc ctaaggcgaa ag 22

<210> 95

<211> 20

<212> DNA

<213> artificial sequence

<400> 95

cttcttcggc ggtcgtccac 20

<210> 96

<211> 24

<212> DNA

<213> artificial sequence

<400> 96

cggagccact cggtgccgca actt 24

Claims (2)

1. A method for identifying transgenic rape and products thereof by common PCR, which comprises the following steps:

1) The total DNA of the sample is extracted,

2) Carrying out PCR amplification on positive plasmid molecules and sample genome DNA by using detection primers of transgenic rape transformants and rape internal standard genes;

3) Separating by agarose gel electrophoresis, and identifying whether amplification products exist after EB staining; under the conditions that the positive plasmid molecules are amplified normally and the standard genes in the rape in the sample are amplified normally, if amplification products exist, the rape sample contains corresponding rape transformants;

the detection primers of the transgenic rape transformant and the rape internal standard gene are as follows:

MS1 5′：

primer F5 '-CGGTGAGTAATATTGTACGGCTAA-3' SEQ ID NO.20

R 5′—ATCTCTGGTTAAACATTCCATCTTTG—3′SEQ ID NO.21,

RF1 5′：

Primer F5 '-CCTGTGGTCTCAAGATGGATCA-3' SEQ ID NO.22

R 5′—GGTGACTACACGCGACTCAT—3′SEQ ID NO.23；

RF2 5′：

Primer F5 '-TTGGTGGACCCTTGAGGAAAC-3' SEQ ID NO.24

R 5′—CCATCTAATAGGGTGAGACAAT—3′SEQ ID NO.25；

MS8 5′：

Primer F5 '-CCTTTTCTTATCGACCATGTACTC-3' SEQ ID NO.26

R 5′—AATTTTAAAAACTTGTGGGATGCT—3′SEQ ID NO.27；

RF3 3′：

Primer F5 '-CAATAACTTTGTTGGGCTTATGG-3' SEQ ID NO.28

R 5′—CTCGTCTCGGACCTCCGAAAACC—3′SEQ ID NO.29；

OXY235 3′：

F 5′—TTTGTTTATTGCTTTCGCC—3′SEQ ID NO.30

R 5′—CCAGGGGATTCAGTTGGA—3′SEQ ID NO.31；

Topas 19/2 3′：

Primer F5 '-CGGCCTTAATCCCACCCCAG-3' SEQ ID NO.32

R 5′—AGTTCCAAACGTAAAACGGCTT—3′SEQ ID NO.33；

T45 3′：

Primer F5 '-TCCCATTTATTTACGGTCAC-3' SEQ ID NO.34

R 5′—CCATGGGAATTCATTTACAA—3′SEQ ID NO.35；

GT73 3′：

Primer F5 '-AATAACGCTGCGGACATCTA-3' SEQ ID NO.36

R 5′—CAGCAAGATTCTCTGTCAACAA—3′SEQ ID NO.37；

73496 5′：

Primer F5 '-ACTTGTTCGTTGGAGTTGTC-3' SEQ ID NO.38

R 5′—TCGTGTTCAGTCCAATGACC—3′SEQ ID NO.39；

MON88302 5′：

Primer F5 '-CTCCTCAAGTTGTACAGTCTTGAAGAGA-3' SEQ ID NO.40

R 5′—CAGGACCTGCAGAAGCTTGATAAC—3′SEQ ID NO.41；

Rape internal standard gene CruA:

CruAF398 5′—GGCCAGGGCTTCCGTGAT—3′SEQ ID NO.42

CruAR547 5′—CTGGTGGCTGGCTAAATCGA—3′SEQ ID NO.43；

rape internal standard gene HMG I/Y:

hmg-F 5′—TCCTTCCGTTTCCTCGCC—3′SEQ ID NO.44

hmg-R 5′—TTCCACGCCCTCTCCGCT—3′SEQ ID NO.45；

the base sequence of the positive plasmid molecule is shown as SEQ ID NO. 1;

the transgenic rape variety is selected from GT73, topas19/2, T45, MS1, RF2, MS8, RF3, OXY235, MON88302 and 73496.

2. A method for identifying transgenic rape and products thereof by fluorescence real-time PCR, which comprises the following steps:

1) The total DNA of the sample is extracted,

2) Carrying out PCR amplification on positive plasmid molecules and sample genome DNA by using a transgenic rape screening unit and a detection primer pair and a probe pair of rape internal standard genes;

3) Judging whether a corresponding rape transformant exists or not according to whether a typical amplification curve exists in the real-time fluorescence PCR product; under the conditions that the positive plasmid molecules are amplified normally and the standard genes in the rape in the sample are amplified normally, if amplification products exist, the rape sample contains rape transformants;

the detection primers and probes of the transgenic rape transformant and the rape internal standard gene are as follows:

RT73 transformant:

primer RT73F5 '-CCATATTGACCATCATACTCATTGCT-3' SEQ ID NO.46

RT73R 5′—GCTTATACGAAGGCAAGAAAAGGA—3′SEQ ID NO.47，

Probe RT73P 5 '-TTCCCGGACATGAAGATCATCCTCCTT-3' SEQ ID NO.48;

MS1 transformant 5';

primer MS1-5F5 '-ATCTCTGGTTAAACATTCCATCTTTG-3' SEQ ID NO.49

MS1-5R 5′—CGAGCTTTCTAATTTCAAACTATTCGG—3′SEQ ID NO.50，

Probe MS1-5p 5 '-TGGATAGGTTCTTCAGCATCATCACACC-3' seq ID No.51;

MS1 transformant 3':

primer MS1-MLD025 5 '-ACGCTGCGGACATCTACATT-3' SEQ ID NO.52

MS1-MDB175 5′—CTAGATCGGAAGCTGAAGATGG—3′SEQ ID NO.53，

Probe MS1-TM030 5 '-CTCATTGCTGATCCACCTAGCCGACTT-3' SEQ ID NO.54;

OXY235 transformant 5':

primer OXY235-5F 5 '-CTAACTTTTGGTGTGATGATGCTGA-3' SEQ ID NO.55

OXY235-5R 5′—CGATAGATGGTGGTGTGAGTCTTG—3′SEQ ID NO.56，

Probe OXY235-5P 5 '-AGCTGATGGCAAGTTAATCTCCCCGAAGTCG-3' SEQ ID NO.57;

OXY235 transformant 3':

primer QOXY235-3LG 5 '-AGAGAATCGTGAAATTATCTCTACCG-3' SEQ ID NO.58

QOXY235-3LV 5′—ATTGACCATCATACTCATTGCTGA—3′SEQ ID NO.59，

Probe QOXY235-3L 5 '-CCATGTAGATTTCCCGGACATGAAGCC-3' SEQ ID NO.60;

MS8 transformant 5';

primer QMS8RG 5 '-CCTTGAGGACGCTTTGATCATATT-3' SEQ ID NO.61

QMS8RV 5′—CCTTTTCTTATCGACCATGTACTC—3′SEQ ID NO.62，

The probe QMS8RFAM 5 '-CCGAGTTCGACGGCCGAGTACTG-3' SEQ ID NO.63;

MS8 transformant 3';

primer MS8L-KVM085 '-GTTAGAAAAAGTAAACAATTAATATAGCCGG-3' SEQ ID NO.64

MS8L-HCA048 5′—GGAGGGTGTTTTTGGTTATC—3′SEQ ID NO.65，

Probe MS8L-TM011 5 '-AATATAATCGACGGATCCCCGGGAATTC-3' SEQ ID NO.66;

RF3 transformant 3';

primer QRF3RG 5 '-TTTGTACAAAACTTGGACCCCTAGGT-3'SEQ ID NO.67QRF3RV 5 '-TTCTTTCAAGATGGGAATTAACATCT-3' SEQ ID NO.68, probe QRF3P5 '-TGCCTTTTCTTATCGCGAGATGAAAAAGGC-3' SEQ ID NO.69; RF3 transformant 5':

RF3DPA165 5' -CATAAAGGAAGATGGAGACTTGAG-3'SEQ ID NO.70RF3KVM084 AGCATTTAGCATGTACCATCAGACA-3 ' SEQ ID NO.71, probe RF3TM010 5' -CGCACGCTTATCGACCATAAGCCCA-3 ' SEQ ID NO.72;

t45 transformant:

primer T45KVM172 5 '-CAATGGACACATGAATTATGC-3'SEQ ID NO.73T45MDB599 5 '-GACTCTGTATGAACTGTTCGC-3' SEQ ID NO.74, probe T45TM026 5 '-TAGAGGACCTAACAGAACTCGCCGT-3' SEQ ID NO.75; RF1 transformant:

primer RF1-MDB118 5 '-CTAAGGGAGGTCAAGATGTAGC-3'SEQ ID NO.76RF1-KVM170 5 '-CGGGCCTAACTTTTGGTGTG-3' SEQ ID NO.77, probe RF1-TM022 5 '-CTCATCATCCTCACCCAGTCAGCATCA-3' SEQ ID NO.78; topas transformants:

primer TOPAS19/2 MDB685 ' -GTTGCGGTTCTGTCAGTTCC-3'SEQ ID NO.79TOPAS 19/2KVM180 CGACCGGCGCTGATATATGA-3 ' SEQ ID NO.80, probe TOPAS19/2 TM029 5' -TCCCGCGTCATCGGCGG-3 ' SEQ ID NO.81;

RF2 transformants:

the primer RF2-MDB207 5 '-GGGTGAGACAATATATCGACG-3'SEQ ID NO.82RF2-KVM171 5 '-GGGCATCGCACCGGTGAG-3' SEQ ID NO.83,

probe RF2-TM024 5 '-CACCGGCCAAATTCGCTCTTAGCCGT-3' SEQ ID NO.84;73496 transformants:

primers 73496-2824 5 '-GTTCTTCTCTTCATAGCTCATTACAGTTTT-3'SEQ ID NO.8573496-2825 5 '-CAAACCTCCATAGAGTTCAACATCTTAA-3' SEQ ID NO.86, probe 73496-QP-83 5 '-TTAGTTAGATCAGGATATTCTTG-3' SEQ ID NO.87;

MON88302 transformant:

primer 88302QF 5 '-TCCTTGAACCTTATTTTATAGTGCACA-3' SEQ ID NO.88

88302QR 5 '-TCAGATTGTCGTTTCCCGCCTTCA-3' SEQ ID NO.89, probe 88302QP 5 '-TAGTCATCATGTTGTACCACTTCAAACACT-3' SEQ ID NO.90;

rape internal standard gene

CruA：qCruAF 5′—GGCCAGGGCTTCCGTGAT—3′SEQ ID NO.91

qCruAR 5′—CCGTCGTTGTAGAACCATTGG—3′SEQ ID NO.92，

Probe qCruAP 5 '-AGTCCTTATGTGCTCCACTTTCTGGTGCA-3' SEQ ID NO.93;

rape internal standard gene HMG I/Y: qhmg-F5 '-GGTCGTCCTCCTAAGGCGAAAG-3' SEQ ID NO.94 and qhmg-R5 '-CTTCTTCGGCGGTCGTCCAC-3' SEQ ID NO.95,

the probe qhmg-P5 '-CGGAGCCACTCGGTGCCGCAACTT-3' SEQ ID NO.96;

the base sequence of the positive plasmid molecule is shown as SEQ ID NO. 1;

the transgenic rape variety is selected from GT73, topas19/2, T45, MS1, RF2, MS8, RF3, OXY235, MON88302 and 73496.