TWI269809B - A plasmid for detection of genetically modified maize - Google Patents

Abstract

The present invention relates to a plasmid, which comprises a promoter, an internal control gene of maize and the transgenes of genetically modified maizes Bt11, Event176, GA21, MON810, NK603, T25 and TC1507. In addition, the primers, probes and the components of real-time PCR reagent and real-time PCR condition are also included.

Description

1269809 玖, Invention Description: [Technical Field] The present invention relates to a plastid comprising a promoter, a maize internal control gene and a genetically modified maize line Btll, Eventl76, GA21, MON810, NK6〇3, D 25 and TC15〇7 genes; in addition, it also includes primers, probes, real-time PCR reaction solution formulations and real-time PCR conditions. [Prior Art] The base Q modified k crop has become the main source of food crops in the world. Genes commonly used in transplanted crops include insect-resistant, herbicide-tolerant and antibiotic genes. However, the scientific community still has doubts about the safety of genetically modified crops. The main reason is that _(1) the effects of foods added to foreign genes are still unknown. Since many of the genes used for genetic modification come from non-human-consuming organisms (such as insects and bacteria), the effects of long-term consumption of these genetically modified foods by humans are unknown. (2) The risk of cultivating super-microorganisms. The risk of developing resistant micro-organisms is due to the incorporation of antibiotic genes in certain genetically modified foods. (3) The colonization of animal genes raises moral and religious issues for vegetarians. (4) Risk of allergies to humans. If it is a gene that causes allergies, humans may develop an allergic reaction to it and affect health. For the above reasons, countries have paid great attention to and developed the testing methods and labeling systems for genetically modified foods. At present, the most common commercial mass-produced genetically modified crops include genetic modification, large-scale, corn, cotton and Fanyou. For example, according to the statistics of the corn imported by the COA, it is estimated that there are about 6 million tons of corn imported per year in Taiwan, of which about 30% of genetically modified corn is about 88\88\88144 DOC -6- 1269809. therefore. The development of genetically modified corn testing methods has become an important issue. At present, commercialized genetically modified corn is multi-colonized with insect-resistant and herbicide-tolerant genes. In terms of insect-resistant genetically modified corn, it can be divided into four series: YieldGard (Cryl A), StarLink (Cry 9C), Herculex I (Cry 1F) and MON863 (Cry3Bbl). Herbicide-tolerant genetically modified corn can be distinguished as glyphosate, such as GA2 1, NK603, and glufosinate ammonium, Btll, Event 176' CBH-351, T25 and TC1507 . In addition to the above-mentioned genetically modified maize lines, a stacked trait hybrid has been developed, such as Monsanto's MON810XGA21 and DuPont's MON810XT25. Genetically modified food testing methods mainly include (1) nucleic acid-based methods such as PCR; (2) protein-based methods such as ELISA; and (3) methods based on enzyme activity assays. Among the above methods, the most widely used application is PCR, and the products are identified using primers of different designs. In addition to qualitative analysis, PCR can also be used for quantitative analysis, such as PCR-ELISA and Real-time PCR. At present, most of the literatures or commercial kits for quantitative detection of genetically modified corn utilize TaqMan Real-time PCR (Matsuoka T et al., J. Food Hyg. Soc. Japan 42: 24-32).

Isabel Taverniers et al. provide an event_speciHc polymerase chain reaction (PCR) for the detection and quantification of Roundup ReadySoybean (RRS) (Eur Food Res Technol (2001) 213:417- 424) 〇The former case develops a transformed event-Specific primer pair. These primers are used to uniquely identify a fragment of a uniquely joined region between the inserted DNA and soybean DNA.

The sequence of O:\88\88l44 DOC 1269809 Τ25 and TC 1 5 07 is self-sequenced and the novel plastids of the present invention are constructed using the sequences. The present invention provides a germplasm comprising a promoter, an internal control gene and the following nucleic acid sequences: sequence recognition, blunt, 1, sequence identification number: 2, sequence identification number: 3, Sequence identification, 'Bian 唬 4, sequence identification number: 5, sequence identification number: 6 and sequence identification number: 7. The above sequence identification numbers: 丨 to 7 are the genes of the genetically modified maize mouth lines Btll, Eventl76, GA21, MON810, NK603, D25 and TC1507, respectively. The plastid of the invention can be used for detecting Btu,

Eventl76, GA21, MON810, NK603, T25 and TC15 07 strains. According to a preferred embodiment of the present invention, the plastid of the present invention is a pBFDAM2 negative body comprising a CaMv 3SS promoter, an HMG (h^ mobihty group) gene and the following nucleic acid sequence: sequence identification number: ^ : 2, sequence identification number: 3, queue identification number: 4, sequence m tiger · 5, sequence identification number: 6 and sequence identification number: 7. According to the present invention, a gene widely present in the maize internal control gene line maize is used to confirm that the sample to be tested is corn. Preferably, the maize internal control gene is a maize starch synthase IIb gene, an invertase gene or a high mobility group protein (HMG) gene. Childhood mobile group protein (HMG) is named for its rapid migration in gel electrophoresis. It is widely found in a variety of organisms, such as the amine, the spermamine & L, the face acid and aspartic acid. Hmg protein is characterized by its ability to bind to DNA and bend it to promote chromatin function and gene regulation. HMG works directly with nucle〇s〇mes O:\88\88144.DOC -10- 1269809 Production (Monsanto, St. Louis , MO, USA), which colonizes the glyphosate herbicide gene. The NK603 gene includes the following fragments: the rice actin I promoter and the insert, the i/m/k and the EPSPS gene CTP2, the Agrobacterium tumefaciens CP4 strain 5-enolpyruvylshikiate ate-3-phosphate synthase (EPSPS) and

Nos 3 ’-poly A signal gene, terminator of Agrobacterium tumefaciens. The sequence of the gene into which the gene is inserted is shown in Sequence Identification No.: 5. The Ding 25 genetically modified maize line was produced by Aventis, Germany (Aventis, AgrEvo, Berlin, Germany), which was colonized with the glufosinate ammonium herbicide gene. The T25 gene includes the sputum J fragment: CaMV 3 5 S promoter, Streptomyces viridochromo genes Tu494 strain bar [phosphinothricin N-acetyl transfer as e (PAT)] and CaMV 3 5S poly (A) signal gene terminator . The sequence of the gene into which the gene is inserted is shown in Sequence Identification No.: 6. The TC1507 genetically modified maize line was produced by Mycogen and Pioneer (Mycogen c/o Dow AgroSciences; Pioneer (c/o DuPont), U.S.A.), which was cloned into the Cry IF insect-resistant gene. The TCI 507 gene includes the following fragments: the ubiquitin promoter and the first exon of Zea mays and the intron 5 Bacillus thuringiensis subsp. aizawai PS811 strain Cry IF a2 (delta-endotoxin). The sequence of the colony gene is shown as sequence identification number: 7. The invention also relates to the detection of a genetically modified maize transgenic line, a CaMV 35S promoter and a primer and probe for the high mobility group (HMG) gene of maize. Using the primers and probes of the present invention, qualitative or quantitative determination of O:\88\88144 DOC-13- 1269809 is a qualitative and quantitative test of T25. The present invention provides a primer pair that hybridizes to the TC15〇7 gene sequence, the sequence of which is Sequence Identification Number: 20 and Sequence Identification Number: 21. The present invention further provides a probe which hybridizes to the TC15〇7 gene sequence, which consists of the sequence of SEQ ID NO: 22. These primers and probes are available for qualitative and quantitative testing of the genetically modified maize line TC 1507. The present invention provides a primer pair that hybridizes to the CaMV 35S promoter, the sequence of which is Sequence Identification Number: 23 and Sequence Identification Number: Μ. The present invention further provides a probe which hybridizes to the CaMV 35S promoter, which consists of the sequence of the identification number: 25. These primers and probes are available for qualitative and quantitative testing of the 35S promoter. The present invention provides a primer pair which hybridizes to the maize high mobility group protein (9) 叻 mobUity group, HMG) gene sequence, which has the sequence identification number: 26 and the sequence identification number: 27. These primers are used for qualitative and quantitative testing of high mobi Hty gr〇up (HMG). The present invention provides a method for qualitatively and quantitatively detecting genetically modified maize transgenic lines, which uses the plastids and primers and probes of the present invention. Further, the present invention provides a vector comprising the plastid of the present invention. The invention further provides a host cell comprising the vector of the invention. According to the present invention, product-specific primers and probes suitable for quantitative detection are designed for the specific regions of the above seven gene sequences. The DNA sequence fragment obtained by the above primer is further extended on both sides according to the DNa sequence data of the gene, and a tailed primer is designed to obtain a comparison.

O:\88\88144 D0C -15- 1269809 Large fragment of product-specific DNA fragments. The DNA fragments are ligated to each other and then transferred to a suitable vector, and a large number of replicated plastids of the present invention can be obtained by a large number of cultures, extraction and purification steps. The primer and probe technologies can be designed by themselves or refer to the following literature: Va'iti ling om, M. et al., 1999, J. Agric. Food Chem. 47: 5261 - 5266. DNA; Matsuoka, T., Kawashima et al. 2000, J. Food Hyg. Soc. Japan 41: 137-143; and Matsuoka, Τ· et al., 2001, J. Food Hyg. Soc. Japan 42:24-32 o DNA transfer technology is well known to artisans Any suitable carrier and commercial kit can be used for the construction of the plastids of the present invention. Host cells suitable for large-scale cultivation of the plastids of the invention can be used in the present invention. Such vectors include, but are not limited to, microorganisms and yeasts. A preferred host cell is Escherichia coli. The quality system provided by the present invention is a standardized plastid, in addition to qualitatively detecting seven genetically modified maize transgenic lines: Btll, Event 176, GA21, MON810, NK603, T25 and TC1507, and can also be used as a quantitative reference material for quantification. Screening and testing the above genetically modified maize transgenic lines. The plastids constructed by the invention can be prepared without limitation, and can be used as a qualitative and quantitative reference material at the same time to solve the problem of lack of standard substances in the detection of transgenic maize. In addition, it is also possible to avoid the influence of the reference material on the growing area, time, strain and DNA quality of the plant containing the constructed gene.

Qualitative or quantitative detection of genetically modified maize can utilize DNA or protein methods. Preferably, the qualitative detection of the genetically modified maize line of the present invention is carried out by mass culture, extraction, purification, enzymatic cleavage into linear plastid DNA, and recovery and purification, and used as a reference material for comparison. PCR amplification technology was used to detect genetically modified maize, and then real-time PCR was performed. O:\88\88l44 DOC -16- 1269809 The probe was used to confirm the genetic modification of the genetically modified maize. Quantitative detection of the present invention The genetically modified corn is subjected to mass culture, extraction, purification, enzymatic cleavage into linear plastid DNA, recovery and purification, serial dilution and determination of DNA concentration. Then, an appropriate concentration (about 20 to 300,000 copies) <linear plastid DNA was used for real-time PCR to prepare a quantitative standard curve. Further, for the transgenic maize sample, the internal coefficient of the transgenic line must be determined, and the internal standard is quantitatively tested according to the genetically modified corn reference material of the transgenic line, and the linear plastid of the present invention is used. The standard curve prepared by DNA is calculated according to the standard curve and the following formula: Intrinsic ratio - the transgenic line genetically modified corn product specific gene / maize internal control gene After quantitative analysis of the transgenic maize sample, according to the following formula Nasal results: ' ua intermediate transgenic lines genetically modified corn content % = (product specific gene / maize internal control gene) x (l / internal standard ratio) xi 〇〇 this Mao Yue another & for a kit, including The linear plastid DNA of the present invention can be specifically hybridized to Btu, GA2b MON810, NK603, Eventl76, and TCI 507 genetically modified maize transgenic lines, promoters and maize high-mobility protein gene primers and probes, and instant pcR Reaction solution formulation. According to the present invention, all primers and probes which can be specifically hybridized to Bm, _, mon810, NK603, Eventl76, T25 and Tci5〇7 genetically modified maize transgenic lines, caMV35s promoter and maize high mobile group protein gene can be used. Qualitative or quantitative detection of Btll, GA21,

O:\88\88144 DOC -17- 1269809 minutes. The supernatant was discarded and the DN A of the obtained sink was dissolved in deionized water of 100 μl. DNA extraction and purification can also be carried out using the commercially available kits Wizard Minipreps DNA Purification Resin (Promega), DNeasy Plant Mini kit or DNeasy Plant Maxi kit (Qiagen). The procedure is based on the manufacturer's instruction manual. Example 2 Construction and Confirmation of the Platinum pBFDAM2 of the Invention This example uses the PCR method to transform the following seven genetically modified maize lines: Event 176 and Bt 11 (Syngenta, Basel, Switzerland), MON8 10, NK603 and GA21 (Monsanto, St. Louis, MO, USA), T25 (Aventis, AgrEvo, Berlin, Germany) and TC1507 (Mycogen (c/o Dow AgroSciences); Pioneer (c/o DuPont), USA) Seven-product specificity (product-speciHc) The PCR amplified DNA fragment, a 35S-promoter PCR amplified DNA fragment and a HMG (high mobility group) maize internal control gene PCR amplified DNA fragment are ligated. The nucleic acid sequences of the genes of BtU, Eventl76, GA21, MON810, NK603, T25 and TC1507 are as follows: sequence identification number: 1, sequence identification number: 2, sequence identification number: 3, sequence identification number: 4, sequence identification number : 5, sequence identification number: 6 and sequence identification number: 7. 35S-promoter screening and HMG gene DNA sequence data refer to HMG (high mobility group) (accession number: AJ13 13 73) and 35S-promoter (accession number: X53860, AY147202) o O:\88\88144.DOC - 19- 1269809 Table 1 primer/probe sequence 5f-3' specificity Amplicon (bp) Eventl76 Crytml Crytm2 Crymp gTg gAC AgC CTg gAC gAg AT TgC TgA AgC CAC TgC ggA AC 5,-(FAM)-AAC AAC AAC gTg CCA CCT CgA CAg g -(TAMRA)-3, CDPK-pro/sense c/7Z4(Z?)/anti-sense 105 MON810 YM556B CR01 PYM92 TgA CAC TAT ATT gCT TCT CTT TAC ATA CgT (Serial Identification Number: 12) gAT gTT Tgg gTT gTT gTC CAT 5,-(FAM)-CTC gAT gCC TTC TCC CTA gTg TTg ACC A-(TAMRA)-3' (SEQ ID NO: 13) hsp70/sensQ cryIA(b)/mti-SQnse 139 T25 RT5006 RT3124 GT42 TTA ggC CAg TTA CCC AgA TCT gA (Serial Identification Number: 17) ATT CCC TTA TCT ggg AAC TAC TCA C (Serial Identification Number: 18) 5,-(FAM)-CAT gCC CgC TgA AAT CAC Cag TCT CT -(TAMRA)-3' (sequence identification number: 19) pat/sensQ 35S ter./anti-sense 119 Bill Btll 1-5, crylA 1-3, 0205C CCA TTT TTC AgC TA g gAA gTT C TCg TTg ATg TTK ggg TTg TTg TCC 5,-(FAM)-TCC gCg gCT TgT TgT ggT CTT TTg -(TAMRA)-3' (SEQ ID NO: 8) adhlAS IVS6/sense cryIA(b)/ mti-SQnsQ 110 GA21 YCC-6 YCC-2 YCC-35 TTC CAg ggg CTC AAg TCC A (sequence identification number: 9) TCTCCTTgATgggCTgCA (sequence identification number: 10) 5,-(FAM)-TCg CCC gCC gCT CCT CCA gAA g -(TAMRA)-3' (sequence identification number: 11) OTP/sense m-epsps/mti~sense 136 35S 75 161S 100 CCgACAgTggTCCCAAAgAT (sequence identification number: 23) AgT ggA gAT ATC AC A TCA ATC CAC TT (sequence Identification number: 24) 5,-(FAM)-CCC ACC CAC gAg gAg CAT Cg -(TAMRA)-3' (sequence identification number: 25) CaMV-35S/sense CaM V-3 5 S/anti-sense 107 HMG Hmg-c hmg-d CLC13 TTg gCT ACA TAg ggA gCC TTg T (SEQ ID NO: 26) hmg/sense gAg TCg gTA AgC TCC ATC TTC Tg (SEQ ID NO: 27) hmg/anti-sense 55-(FAM) -CAA TCC ACA CAA Acg CAC gCg -(TAMRA)-3, TAH:TAMRA)-3, 123 NK603 0726D-5f 0712B PK06D gTgACAAATgCAgCCTCgTg (SEQ ID NO: 14) TgC gTT gAC Tgg ATT TCg (Sequence Identification No.: 15) 5,-(FAM)-TgC TAA CTT gCg CCA Tgg TTg ATC ACT T-(TAMRA)-3' (SEQ ID NO: 16) rice actin intron/sense CTP2/antisense 131 TC1507 1014Ra 1014Rb TC1014R CgA CCT gCA gCC AAg CTT (SEQ ID NO: 20) TCA TgT TAg TCg CAA CgA AAC C (SEQ ID NO: 21) 5,-(FAM)-CgA gCT CgA gAT CCC CgA CAT ATg C -(TAMRA)-3'( Sequence ID: 22) crylFa2/sQnsQ ORF25/antisense 71 O:\88\88144.DOC -20- 1269809 First use the above-mentioned Btll, Eventl76, GA21, MON810, NK603, T25 and TC1507 transgenic lines of genomic DNA (genomic DNA) is a template, and HMG and 35S-promoter are used as the template for the first PCR (see Figure 1) using the genomic DNA of the MON8 10 as the template. : Table II

Tailed primer sequence 5'-3' Amplicon (bp) Eventl76 E-GA EM Cgg ggT CCA AgT gCA ACC TgC CCA ggT AAg gAg AgC ATg AAT CCA gCT gAA 190 (G) MON810 ME MT TCA gCT ggA TTC ATg CTC TCC TTA CCT CAA CTC AAA ATC CTT Gta Cgg gAT gC 199 (H) T25 TM T25-3, CAT CCC gTA CAA ggA TTT TgA gTT gC TCA ACA CAT gAg CgAAAC CCT A 184 (I) Btll BT-NK BT-GA CgC AAATCT CCC TgA CTA ATA ACT gAT TAg T CgA ggC CAT CAA CTT CTg ggT TAC TC 210 (E) GA21 GA-BT GA-E gTA ACC CAg AAg TTg ATg gCC TCg T Tgg gCA ggT TgC ACT Tgg ACC CCg 201 (F) 35S 35- Hmg 35-TC TCg TgA CgA gTA CCT ACA AAT gCC AAg gTg AAC gCg ggT CTT gCg AA 205 (B) HMG hmg-5, hmg-35 AgC ATC AgT TTC CCT ACC TCg C ggC ATT TgT Agg TAC TCg TCA CgA 276 ( A) NK603 NK-TC NK-BT TgC AgC AAT ggA ATC CCT CAg CAT Tg ACT AAT CAg TTA TTA gTC Agg gAg ATT TgC gT 182 (D) TC1507 TC-35 TC-NK TTC gCA AgA CCC gCg TTC ACC TTT C AAT gCT gAg ggA TTC CAT TgC TgC A 279 (C) O:\88\88144.DOC -21- 1269809 The first PCR reaction solution and conditions are as follows: Reaction solution:

10 times PCR buffer solution 5.0 μl 25 mM MgCl2 4.0 μl Taq DNA polymerase (5 U/μl) 0.5 μl 2.5 mM dNTP 3.0 μl ΙΟμΜ primer F 1.0 μl ΙΟμΜ primer R 1.0 μl template DNA (total LOOng) 5.0 microliters of sterile pure water 30.5 microliters total volume 50.0 microliters PCR reaction conditions: step temperature time 1. initial denaturation 94 ° C 5 minutes 2. denaturation 94 ° C 30 seconds 3. bonding 45 ° C 30 seconds 4 Extend 72 ° C 30 seconds Step 2 to Step 4, a total of 5 cycles of reaction 5. Denaturation 94 ° C 30 seconds 6. Bonding 60 ° C 30 seconds 7. Extension 72 ° C 30 seconds Step 5 to Step 7 A total of 35 cycles of reaction 8. Final extension 72 ° C 7 minutes cooling 4 ° CO: \88\88144.DOC -22- 1269809 After the above PCR reaction, nine amplified DNA fragments (A, B, c, D, E, F, G, Η, I), the above steps are shown in Figure 1. After the DNA fragment was recovered, it was used as a template, and the second PCR was performed using the primer pair (Fig. 丨). The 'human PCR reaction> solution and conditions are as follows: PCR reaction solution: 10 times PCR buffer solution............. 25 mM MgCl2............. ........... TaqDNA polymerase (5U/μl;) 2.5 mM dNTP......................... 1 〇μΜ引引 F........................ 1〇μΜ引子 R................. ...... Purified DNA (10-fold dilution).......... Sterile pure water........................ .. Total volume................................... PCR conditions: ••5.0 μl•·4 · 0 microliters • 0.5 microliters • 3.0 microliters • 1.0 microliter • 1.0 microliter • 1 + 1 microliter 33.5 microliters 50.0 microliter steps - temperature time 1 · initial denaturation 94 ° C 5 minutes 3 Bonding 4. Extension

94°C 60°C 72°C 30 seconds 30 seconds 30 seconds 5·Final extension 72°C 7 minutes

O:\88\88144.DOC 23- 1269809 After the second PCR reaction described above, four fragments (J, Κ, L, Μ, see Fig. 1) were obtained, respectively. The recovered product was used as a template, and finally the second PCR was performed on the hmg-5' and T25-3' using the primer in the second PCR. The reaction solution and the conditions of the PCR were as follows: PCR reaction solution: 10 times PCR buffer Solution..............................5.0 microliters 25 mM MgC12.......... ..................................4.0 microliters

TaqDNA Polymerase (5U&L).....................0.5 μL 2.5 mM dNTP............. ................................3.0 microliter ΙΟμΜ primer F............ ................................1.0 microliter ΙΟμΜ primer R............ ...............................1.0 microliter template DNA (total lOOng)......... ........1+1+1+1 microliters of sterile pure water.............................. ....................31.5 microliters total volume......................... .............................50.0 μl PCR conditions: _fm_^_ 1. Initial denaturation 94 ° C 5 min 2. Denaturation 94°C 30 seconds 3. Bonding 60°C 30 seconds 4. Extending 1TC 30 seconds _Step 2 to Step 4, a total of 35 cycles of reaction _5·Final extension _7TC_7 minutes _ Cooling 4% O:\88\ 88144.DOC -24- 1269809 After the above third PCR reaction, the obtained fragment is the DNA fragment (plastid PBFDAM2) to be constructed in this experiment. This fragment (1926 bp) was then transferred into the pGEM®-T Easy vector, which was based on the promega pGEM-T vector (?1'〇11^83,%3(^3011,~1,1;3) The operation steps of the group were carried out. The colony gene was subjected to sequencing analysis to confirm that the nine-segment PCR amplification product fragment was present in the plastid in a single copy; FIG. 2 is the gene sequence in the plastid pBFDAM2 of the present invention; The sequence of each gene in the plastid PBFDAM2 of the present invention and its sequence; and Figure 4 shows the DNA sequence, primer and probe position of the plastid PBFDAM2. After confirmation, the plastid is largely replicated and cut into linear lines by a restriction enzyme such as Apal. The fragment was further recovered and purified, and diluted for use. Example 3 The plastid of the plastid pBFDAM2 of the present invention was constructed, and after extensive culture, replication, recovery and purification, it was constructed by real-time PCR (ABI PRISM 7700 Sequence Detector) test. Seven transgenic lines in the plastid (Bt 11, Event 176, GA2 1, MON8 1 0, > ^ 603, Ding 25 and Ding (: 1507) genetically modified maize product-specific genes, 358-promoter screening genes And HMG corn internal control gene, confirm the reaction is correct. 4 Qualitative detection of plastid PBFDAM2 After extensive culture, extraction, purification, enzyme cleavage into linear plastid DNA and recovery and purification, as a reference material for qualitative test comparison, refer to each group of primers listed in Table 1. The first PCR reaction solution formulation and the first PCR reaction conditions can be qualitatively tested for genetically modified maize such as Eventl76, MON810, T2 5, Btn, GA21, NK603 and TC15 07. O:\88\88144.DOC - 25- 1269809 strain, CaMV 35S promoter and maize high mobility group (HMG) gene. Introduction to Crytml and Crytm2 for qualitative detection of Eventl76 genetically modified maize transgenic lines; primers for YM5 5 6B and CR01 Qualitative detection of MON810 genetically modified maize transgenic lines; primers for RT5006 and RT3124 for qualitative detection of T25 gene-transformed maize transgenic lines; primers for Btll 1-51 and ci*yIA 1-3^ for qualitative detection of Btll genetically modified maize Colony line; YCC-6 and YCC-2 for qualitative detection of GA21 genetically modified maize transgenic lines; primers for 0726D-5' and 0712B for qualitative detection of NK603 genetically modified maize transgenics The primers pair l〇14Ra and 1014Rb can be used for qualitative detection of TC1507 genetically modified maize transgenic lines; the primers for 75 and 161S can be used for qualitative detection of CaMV 3 5S promoter; the primers for hmg-c and hmg-d can be used for qualitative detection of maize HMG gene. Furthermore, the set of primer pairs and the probe, the real-time PCR reaction solution formulation and the real-time PCR reaction conditions listed in Table 1 were confirmed. The primers Crytml, Crytm2 and probe Crymp can be used for the qualitative confirmation test of Event 176 genetically modified maize transgenic lines; the primers YM5 5 6B, CR01 and probe PYM92 can be used for qualitative confirmation test of MON8 10 genetically modified maize transgenic lines; RT5 006, RT3124 and probe GT42 can be used for qualitative confirmation test of T25 genetically modified maize transgenic lines; primers Btll 1-5', crylA 3' and probe 0205C can be used for qualitative confirmation test of Btll genetically modified maize transgenic lines The primers YCC-6, YCC-2 and probe YCC-35 can be used for qualitative confirmation test of GA21 genetically modified maize transgenic lines; primers 0726D-5', 0712B and probe PK06D can be used to transform NK603 genetically modified maize transgenic lines. 0\88\88I44.DOC -26- 1269809 Qualitative confirmation test; primers 1014Ra, 1014Rb and probe TC1014R are available for qualitative confirmation test of TC 1 5 07 genetically modified maize transgenic lines; primers 75, 161S and probe 100 are available Qualitative confirmation test of CaMV 3 5S promoter; primer hmg_c, hmg-d and probe CLC13 can be used for qualitative confirmation test of maize HMG gene. The instant PCR reaction solution formulation and reaction conditions are as follows: 5 μΜ primer F.................................... ....... ······1.25pL 5μΜIntroduction R................................ .......... ······1.25 \iL 3.3 μΜ probe.............................. ............................1·7μί TaqMan Universal PCR Master Mix ...········12·5 pL Template DNA (Total lOOng)...................................5.0 pL ageless water........... ...........................................3.3 pL total volume... ................................................. .·····25·0 μί PCR conditions: use Real-time PCR—ABI PRISM 7700 Sequence Detector Step temperature time 1. Thermal activation 50°C 2 minutes 2. Initial denaturation 95°C 10 minutes 3. Denaturation 95°C 15 seconds 4·bonding, stretching 60 °C Step 3 to step 4, 1 minute cycle reaction. 5. Cooling 35 ° C 45 seconds Example 5 quantitative detection of plastid pBFDAM2 after extensive culture, extraction, purification, enzyme cleavage into 0 \88\88144 D0C -27 · 1269809 linear plastid DNA and recovered and purified, serial dilution and Determine the DNA concentration. Next, appropriate linear concentrations (about 20 to 300,000 copies) of linear plastid DNA were taken for real-time PCR to prepare a quantitative standard curve. In addition, for the transgenic maize sample, the internal coefficient of the transgenic line must be determined. The internal standard is quantitatively tested according to the genetically modified corn reference material of the transgenic line, and the linear plastid DN of the present invention is used. The standard curve prepared by A is calculated according to the standard curve and the following formula: Internal standard ratio = genetically modified maize product specific gene / maize internal control gene After quantitative detection and analysis of transgenic maize samples, calculate according to the following formula Results: Genetically modified maize content in the sample% = (product specific gene / maize internal control gene) χ (1/internal standard ratio) χ 100 Each group of primer pairs and probes listed in Table 1, real-time PCR The reaction solution formulation was quantitatively tested with the real-time PCR reaction conditions. The primers Crytml, Cry tm2 and probe Cry mp can be used for the quantitative test of Event 1 76 genetically modified maize transgenic lines; the primers YM5 56B, CR01 and probe PYM92 can be used for the quantitative test of MON810 genetically modified maize transgenic lines; 5 000, RT3 124 and probe GT42 can be used for quantitative analysis of D-25 genetically modified maize transgenic lines; primers Btll 1-5', crylA 1-3' and probe 0205C for Btl 1 gene-modified maize transgenic lines Quantitative test; primers YCC-6, YCC-2 and probe YCC-35 can be used for quantitative testing of GA21 genetically modified maize transgenic lines; primers 0726D-5, 0712B and probe PK06D can be used to transform NK603 genetically modified maize lines. Quantitative test; primer O:\88\88144.DOC -28- 1269809 l〇14Ra, l〇l4Rb and probe TC1014R for quantitative testing of TC15 07 genetically modified maize transgenic lines; primer 75, i61S and probe 1〇 〇 Quantitative test for CaMV 35S promoter; primer hmg-c, hmg-d and probe CLC13 for quantitative detection of maize HMG gene. Example 6 Accuracy Test In addition to this, the actual measurement of the international genetically modified corn quantitative accuracy test 4 is compared with the commercial test issued by the patent method and the Japanese patent (Japanese Patent No. 2001 136983). In the kit, there were six test specimens containing six different transgenic lines of genetically modified maize (Eventl76, Btll, MON810, Ding 25, NK603 and GA21). The laboratory participated in the accuracy test. When the sample was first received, it was only informed that the sample was mixed with six different transgenic lines of genetically modified corn. The detailed lines and concentrations mixed in each sample were not known. The test must be completed within a limited time and the results returned, before being informed of the actual addition of genetically modified maize lines and concentrations for each sample. Know from the organizer,

Six different concentrations of genetically modified corn. Measured, 1.5%, 3.0% or 5.〇〇/0

1.5% or 5.0% of the four concentrations can only reach 60% to Jiguo for 0%, 100% accuracy, O:\88\88144.DOC -29· 1269809 土于〇·1/〇/辰度, deviation up to To 0. 2%. In addition, the Eventl70 strain is the only strain that deviates from the test results in all test strains. The three additions are 0.1% and 〇5% and 5%. After the test, both methods can only get about 70%. Up to 85% accuracy. Each item added to the Eventl76 strain can only detect and measure about 7 ()% to 85% of the added amount, presumably because the non-genetically modified maize line used to prepare the test sample is different from the Eventl76 genetically modified corn line. Copying the internal control gene of corn leads to the fact that the 4 wells must wait for the mutual confirmation between the laboratories and the main unit to understand the true cause of the error, but the results of this Eventl76 strain test are still within the acceptable range. 2. Testing the international genetically modified maize quantitative precision test sample with the plastid, primer, probe and method of the invention patent of the present application and the commercially available test kit issued by the patent.

Sample Genetically Modified Corn (%) No. T25 (%) MON810 (%) GA21 (%) Eventl76 (%) Bill (%) NK603 (%) SBWSBWSBWSBWSBWSBW 1 0 0 0 1.50 1.50 1.26 0.50 0.53 0.70 0.10 0.08 0.07 0 0 0 3.00 2.99 UN 2 1.50 1.50 1.74 0.10 0.10 0.07 0 0 0 5.00 3.97 3.83 0.50 0.50 0.70 1.50 1.46 UN 3 0.10 0.10 0.20 5.00 5.07 4.45 1.50 1.52 2.10 0.50 0.35 0.37 1.50 1.53 1.83 0.50 0.50 UN 4 1.50 1.44 1.72 0.10 0.11 0.12 0 0 0 5.00 4.17 4.41 0.50 0.51 0.59 1.50 1.50 UN 5 0.10 0.10 0.17 5.00 4.90 3.78 1.50 1.47 1.81 0.50 0.39 0.35 1.50 1.51 2.04 0.50 0.49 UN 6 0 0 0 1.50 1.49 1.19 0.50 0.49 0.58 0.10 0.08 0.11 0 0 0 3.00 3.05 UN S: Addition concentration of sample B: Test result by this patent method W: Test result of commercially available test kit issued by Japanese patent UN: This test kit cannot test the genetically modified corn of the transfer line O:\88\88144.DOC -30- 1269809 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a construction procedure of a PBFDAM2 plastid of the present invention. Figure 2 is a genetic sequence of the pBFDAM2 plastid of the present invention. Fig. 3 shows the sequence of each gene in the pBFDAM2 plastid of the present invention and its sequence. Fig. 4 shows the DNA sequence, primer and probe position of the pBFDAM2 plastid. O:\88\88144.DOC -31- 1269809 Sequence Listing < 11 ο > Food and Drug Administration of the Department of Health, Executive Administration <120> Detection of plastids of genetically modified maize <160> 27 <170> Patentln version 3.2 < 210> 1 < 211 > 3134 < 212 > DNA < 213 > Btll < 40〇 > 1 aattcgagct cgtcagaaga ccagagggct attgagactt ttcaacaaag ggtaatatcg 60 ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcgaaag gacagtagaa 120 aaggaaggtg gctcctacaa atgccatcat tgcgataaag gaaaggctat cgttcaagat 180 gcctctaccg acagtggtcc caaagatgga cccccaccca cgaggaacat cgtggaaaaa 240 gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgatatctc cactgacgta 300 agggatgacg cacaatccca ctatccttcg caagaccctt cctctatata aggaagttca 360 tttcatttgg agaggacacg ctgaaatcac cagtctctct ctacaaatct atctctctct 420 attttctcca taataatgtg tgagtagttc ccagataagg gaattagggt tcttataggg 480 tttcgctcac gtgttgagca tataagaaac ccttacgagc tcggtacccg gggatccgga 540 aggtgcaagg attgctcgag cgtcaaggat cattggtgtc gacctgaacc ccagcagatt 600 cgaagaaggt acagtacaca Cacatgtata ta tgtatgat gtatcccttc gatcgaaggc 660 atgccttggt ataatcactg agtagtcatt ttattacttt gttttgacaa gtcagtagtt 720 catccatttg tcccattttt 1020 accatggaca acaacccaaa tcagcttgga agtttggttg cactggcact tggtctaata 780 actgagtagt cattttatta cgttgtttcg acaagtcagt agctcatcca tctgtcccat 840 tttttcagct aggaagtttg gttgcactgg ccttggacta ataactgatt agtcatttta 900 ttacattgtt tcgacaagtc agtagctcat ccatctgtcc catttttcag ctaggaagtt 960 cggttgcact gaatttgtga acccaaaaga ccacaacaag ccgcggatcc tctagagtcg catcaacgaa tgcattccat acaactgctt gagtaaccca 1080 gaagttgaag tacttggtgg agaacgcatt gaaaccggtt acactcccat cgacatctcc 1140 ttgtccttga cacagtttct gctcagcgag ttcgtgccag gtgctgggtt cgttctcgga 1200 ctagttgaca tcatctgggg tatctttggt ccatctcaat gggatgcatt cctggtgcaa 1260 attgagcagt tgatcaacca gaggatcgaa gagttcgcca ggaaccaggc catctctagg 1320 P: \ YAN \ SPEC1F1CAT10N \ 88144_SYC \ 88144 sEQUENCE LISTING (in) DOC 1 _ 1 · 1269809 ttggaaggat tgagcaatct ctaccaaatc tatgcagaga gcttcagaga gtgggaagcc 1380 gatcctacta acccagctct ccgcgaggaa atgcgtattc aattcaacga catgaacagc 1440 gccttgacca cagctatccc attgttcgca gtccagaact accaagttcc tctcttgtcc 1500 gtgtacgttc aagcagctaa tcttcacctc agcgtgcttc gagacgttag cgtgtttggg 1560 caaaggtggg gattcgatgc tgcaaccatc aatagccgtt acaacgacct tactaggctg 1620 attggaaact acaccgacca cgctgttcgt tggtacaaca ctggcttgga gcgtgtctgg 1680 ggtcctgatt ctagagattg gattagatac aaccagttca ggagagaatt gaccctcaca 1740 gttttggaca ttgtgtctct cttcccgaac tatgactcca gaacctaccc tatccgtaca 1800 gtgtcccaac ttaccagaga aatctatact aacccagttc ttgagaactt cgacggtagc I860 ttccgtggtt ctgcccaagg tatcgaaggc tccatcagga gcccacactt gatggacatc 1920 ttgaacagca taactatcta caccgatgct cacagaggag agtattactg gtctggacac 1980 cagatcatgg cctctccagt tggattcagc gggcccgagt ttacctttcc tctctatgga 2040 actatgggaa acgccgctcc acaacaacgt atcgttgctc aactaggtca gggtgtctac 2100 agaaccttgt cttccacctt gtacagaaga cccttcaata tcggtatcaa caaccagcaa 2160 ctttccgttc ttgacggaac agagttcgcc tatggaacct cttctaactt gccatccgct 2220 gtttacagaa agagcggaac cgttgattcc tt ggacgaaa tcccaccaca gaacaacaat 2280 gtgccaccca ggcaaggatt ctcccacagg ttgagccacg tgtccatgtt ccgttccgga 2340 ttcagcaaca gttccgtgag catcatcaga gctcctatgt tctcatggat tcatcgtagt 2400 gctgagttca acaatatcat tccttcctct caaatcaccc aaatcccatt gaccaagtct 2460 actaaccttg gatctggaac ttctgtcgtg aaaggaccag gcttcacagg aggtgatatt 2520 cttagaagaa cttctcctgg ccagattagc accctcagag ttaacatcac tgcaccactt 2580 tctcaaagat atcgtgtcag gattcgttac gcatctacca caaacttgca attccacacc 2640 tccatcgacg gaaggcctat caatcagggt aacttctccg caaccatgtc aagcggcagc 2700 aacttgcaat ccggcagctt cagaaccgtc ggtttcacta ctcctttcaa cttctctaac 2760 ggatcaagcg ttttcaccct tagcgctcat gtgttcaatt ctggcaatga agtgtacatt 2820 gaccgtattg agtttgtgcc tgccgaagtt accttcgagg ctgagtacta gcagatcagg 2880 atcgttcaaa catttggcaa taaagtttct taagattgaa 3060 cgatagaaaa caaaatatag cgcgc tcctgttgcc ggtcttgcga 2940 tgattatcat ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca 3000 tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaattatac atttaatacg Aacct aggataaatt atcgcgcgcg gtgtcatcta 3120 tgttactaga tcca 3134 P:\YAN\SPECIF丨CATl〇N\88144_SYC\88144 Sequence Listing (middle).DOC 1 -2- 1269809 <210〉 2 <211> 2690 <212> DNA &lt ; 213 > Event17 6 < 400 > 2 cgcatggtcc ttatgcaatt ttgtctgcaa aacacaagcc ttaatacagc cacgcgacaa 60 tcatggaagt ggtcgtttta ggtcctcatc atgaagttca gggaaaacgc atcaaatgta 120 atgcagagaa atggtatttc ttctcttgta aatcagggag aggagtacca tcagtacaga 180 ttcagaatca gaattcagtc ttccaacgac aataatcgca gcatcttgta aaaatttgca 240 gaaacttctg tttgacttgt agccctgacc tttgcaaata tttgaagttg tgcctgctga 300 cacaacttca atctggaagt gctgttgatc agttttgcca gaaacagcaa gcagcctata 360 tatatctgtc acgagacacc ctgccgccct cttctttccc gccattccct ccctaccctt 420 caaaatctag aaaccttttt ttttcctccc gatacgcccc tccatctctc gccgttcatg 480 tccgtggctg gctgccctcc gtgggagcag gcggccgcac tcgttccccg ccgcagccgg 540 atccaacaat ggacaacaac cccaacatca acgagtgcat cccctacaac tgcctgagca 600 accccgaggt ggaggtgctg ggcggcgagc gcatcgagac cggctacacc cccatcga ca 660 tcagcctgag cctgacccag ttcctgctga gcgagttcgt gcccggcgcc ggcttcgtgc 720 tgggcctggt ggacatcatc tggggcatct tcggccccag ccagtgggac gccttcctgg 780 tgcagatcga gcagctgatc aaccagcgca tcgaggagtt cgcccgcaac caggccatca 840 gccgcctgga gggcctgagc aacctgtacc aaatctacgc cgagagcttc cgcgagtggg 900 aggccgaccc caccaacccc gccctgcgcg aggagatgcg catccagttc aacgacatga 960 acagcgccct gaccaccgcc atccccctgt tcgccgtgca gaactaccag gtgcccctgc 1020 tgagcgtgta cgtgcaggcc gccaacctgc acctgagcgt gctgcgcgac gtcagcgtgt 1080 tcggccagcg ctggggcttc gacgccgcca ccatcaacag ccgctacaac gacctgaccc 1140 gcctgatcgg caactacacc gaccacgccg tgcgctggta caacaccggc ctggagcgcg 1200 tgtggggtcc cgacagccgc gactggatca ggtacaacca gttccgccgc gagctgaccc 1260 tgaccgtgct ggacatcgtg agcctgttcc ccaactacga cagccgcacc taccccatcc 1320 gcaccgtgag ccagctgacc cgcgagattt acaccaaccc cgtgctggag aacttcgacg 1380 gcagcttccg cggcagcgcc cagggcatcg agggcagcat ccgcagcccc cacctgatgg 1440 acatcctgaa cagcatcacc atctacaccg acgcccaccg cggcgagtac tactgg agcg 1500 gccaccagat catggccagc cccgtcggct tcagcggccc cgagttcacc ttccccctgt 1560 acggcaccat gggcaacgct gcacctcagc agcgcatcgt ggcacagctg ggccagggag 1620 P: \ YAN \ SPECIFICAT10N \ 88144_SYC \ 88144 SEQUENCE LISTING (in) .DOC 1 1269809 tgtaccgcac cctgagcagc accctgtacc gtcgaccttt caacatcggc atcaacaacc 1680 agcagctgag cgtgctggac ggcaccgagt tcgcctacgg caccagcagc aacctgccca 1740 gcgccgtgta ccgcaagagc ggcaccgtgg acagcctgga cgagatcccc cctcagaaca 1800 acaacgtgcc acctcgacag ggcttcagcc accgtctgag ccacgtgagc atgttccgca I860 gtggcttcag caacagcagc gtgagcatca tccgtgcacc tatgttcagc tggattcacc 1920 gcagtgccga gttcaacaac atcatcccca gcagccaaat cacccagatc cccctgacca 1980 agagcaccaa cctgggcagc ggcaccagcg tggtgaaggg ccccggcttc accggcggcg 2040 acatcctgcg ccgcaccagc cccggccaga tcagcaccct gcgcgtgaac atcaccgccc 2100 ccctgagcca gcgctaccgc gtccgcatcc gctacgccag caccaccaac ctgcagttcc 2160 acaccagcat cgacggccgc Cccatcaacc agggcaactt cagcgccacc atgagcagcg 2220 gcagcaacct gcagagcggc agcttccgca ccgtgggctt caccaccccc t tcaacttca 2280 gcaacggcag cagcgtgttc accctgagcg cccacgtgtt caacagcggc aacgaggtgt 2340 acatcgaccg catcgagttc gtgcccgccg aggtgacctt cgaggccgag tacgacctgg 2400 agagggctca gaaggccgtg aacgagctgt tcaccagcag caaccagatc ggcctgaaga 2460 ccgacgtgac cgactaccac atcgatcagg tgtaggagct ctagatctgt tctgcacaaa 2520 gtggagtagt cagtcatcga tcaggaacca gacaccagac ttttattcat acagtgaagt 2580 gaagtgaagt gcagtgcagt gagttgctgg tttttgtaca acttagtatg tatttgtatt 2640 tgtaaaatac ttctatcaat aaaatttcta attcctaaaa ccaaaatcca 2690 < 21〇 > 3 < 211> 2318 < 212 > DNA < 213 > GA21 < 4〇〇 > 3 ggtgggcgag aggcggcttc ggtgcgcgcc cagatcggtg cgcgggaggg gcgggatctc 60 gcggctgggg ctctcgccgg cgtggatccg gcccggatct cgcggggaat ggggctctcg 120 gatgtagatc tgcgatccgc cgttgttggg ggagatgatg gggggtttaa aatttccgcc 180 atgctaaaca agatcaggaa gaggggaaaa gggcactatg gtttatattt ttatatattt 240 ctgctgcttc gtcaggctta gatgtgctag atctttcttt cttctttttg tgggtagaat 300 ttgaatccct cagcattgtt catcggtagt ttttcttttc at gatttgtg acaaatgcag 360 cctcgtgcgg agcttttttg taggtagacg ataagctggg ctgcaggaat tccgaaagac 420 aaagattatc gccatggctt cgatctcctc ctcagtcgcg accgttagcc ggaccgcccc 480 tgctcaggcc aacatggtgg ctccgttcac cggccttaag tccaacgccg ccttccccac 540 P: \ YAN \ SPEC1FICAT10N \ 88144_SYC \ 88144 SEQUENCE LISTING (in) .DOC 1 - 4 1269809 caccaagaag gcaggtgtgg gtctatggcg gctcaagtcc cagcaacggc caaggagatc cctactcgcc tgtccactac tgccaaaaga ggaagtgcag tactgctgct acccattggc tggcactgac caagctgtct ggctcttggg aatgacattg cagattctac tgatgcctca tgtggaaggt gatgatggga ggagccattt tgtcgccatg cgtggcttcc caagctggga gctgaacgtg tgccgcctgt ccccgactac gacgaatttc tgccggtctt taacatgtaa gctaacgact ccggcctacg cccaccgtga accgccagcc ggaaggatcc tccggcaccg gccctgtccg atgctcgggg gctgtagttg ctcttcttgg ggtggaaatg gacttggttg tgcccacctg ggctccatca gatgtggaga agattgatgg attaagggag agcgcaagct tgtggcacca gcgaaggtta gggaggaaac actcttgctg tggagagtaa gcatctgttg acggcgatcg Gccgaggtcc ttcgatgtgc cccgatcgtt gcgatgatta tgcatgacgt tctccaccct gcaacaagaa tgatggcct c tccccgtcgc ggtgcatggc tcaagctgcc aggggacaac ccttgaggac ttggctgtgg ggaatgctgg caacttacgt tcggattgaa ttcgtgtcaa gcagtcagta ttgaaatcat agcgttttgg gtcaaaaata atttcttggc ccagtttgca catggaccga acctcaaggc tggttgccct aggagaccga aggaagggcc acacgtacga ccgtcaccat tgagcacttt caaacatttg tcatataatt tatttatgag tcccagcaac gttcgagacg gtcggccacc ccgccgctcc cggcgccgag ggggtccaag agtggttgat tcttggtctc tggaaagttc aatcgcaatg gcttgatgga gcagcttggt tggaatcgga cttgagtgcc tgataaatta tgtgaaagca caagtcccct tggtgctgca gggtgatgtg gactagcgta gattgatgtc ctttgccgat gaggatggtt ggactactgc cgaccacagg ccgggaccct cgtcaagaat gcaataaagt tctgttgaat atgggttt ggtggaagag ctgtcgtacc gccgtcgctc tccagaagcc gagatcgtgc tcgctttcca aacctgctga tctgtcgaag ccagttgagg cggtccttga gtaccaagaa gcagatgttg gggctacctg ttgctgatgg atctccattc gagcattctg aaaaatgcct attactggag aagtttgctg actgttactg aacatgaaca ggcccgacag gcgatccgga atcatcacgc atggcgatgg gggtgcaccc taagctctag ttcttaagat tacgttaagc ttcaatgtat tgccgccgct cgttccaggg tcggcaacgt tgcagcccat accggatcct acagtgagga cggacaaagc atgctaaaga cagcagctgt tgagggagag attgtttcct gtggcaaggt ctgctccttt cgtacgtcga atagctggga atgttgaagg ggactgtgac aggtactgga gcccaccgcg agatgcctga ccatcagaga cggagctaac cgccggagaa ctttctccct ggaagacctt aagaagcttc tgaatcctgt atgtaataat 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 17 40 1800 I860 1920 1980 2040 2100 2160 2220 2280 2318 P:\YAN\SPEC1FICAT10N\88144_SYC\88144 Sequence Table (middle).DOC 1 1269809 <210> 4 <211> 1402 <212> DNA <213> MON810 <220><221> misc-feature <222> (70βΥ..(706) <223> n is a, c, g, or t <400> 4 ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag 60 tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 120 cacgtcttca Aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca 180 atcccactat ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag 240 gacacgctga caagctgact ctagcagatc taccgtcttc ggtacgcgct cactccgccc 300 tctgcctttg ttactgccac gtttctctga atgctctctt gtgtggtgat tgctgagagt 360 ggtttagctg gatctagaat tacactctga aatcgtgttc tgcctgtgct gattacttgc 420 cgtcctttgt agcagcaaaa tatagggaca tggtagtacg aaacgaagat agaacctaca 480 cagcaatacg agaaatgtgt aatttggtgc ttagcggtat ttatttaagc acatgttggt 540 gttatagggc acttggattc agaagtttgc tgttaattta ggcacaggct tcatactaca 600 tgggtcaata gtatagggat tcatattata ggcgatacta taataatttg ttcgtctgca 660 aagcttatta attgccaaat tagatatcct atcctgcttt tgttgngtgc tgttaaattg 720 ttaacgcctg aaggaataaa tataaatgac gaaattttga tggttatctc tgctccttta 780 ttgtgaccat aagtcaagat cagatgcact tgttttaaat attgttgtct gaagaaataa 840 gtactgacag tattttgatg cattgatctg cttgtttgtt gtaacaaaat ttaaaaataa 900 agagtttcct ttttgttgct ctccttacct cctgatggta tctagtatct accaactgac 960 actatattgc ttctcttaca tacgtatctt gctcgatgcc ttctccctag tgttgaccag 1020 tgttactcac atagtctttg ctcatttcat tgtaatgcag ataccaagcg gccatggaca 1080 acaacccaaa catcaacgag tgcatcccgt acaactgcct cagcaaccct gaggtcgagg 1140 tgctcggcgg tgagcgcatc gagaccggtt acacccccat cgacatctcc ctctccctca 1200 cgcagttcct gctcagcgag ttcgtgccag gcgctggctt cgtcctgggc ctcgtggaca 1260 tcatctgggg catctttggc ccctcccagt gggacgcctt cctggtgcaa atcgagcagc 1320 tcatcaacca gaggatcgag gagttcgcca ggaaccaggc catcagccgc ctggagggcc 1380 tcagcaacct gtaccaaatc ta 1402 P \ YAN \ SPECIFICATION \ 88144_SYC \ 88144 SEQUENCE LISTING (in) .DOC 1 - 6 - 1269809 < 210 > 5 < 211> 2145 < 212 > DNA < 213 > NK603 < 400 > 5 ccctctcctc tttctttctc cgtttttttt tccgtctcgg tctcgatctt tggccttggt 60 agtttgggtg ggcgagaggc ggcttcgtgc gcgcccagat cggtgcgcgg gaggggcggg 120 atctcgcggc tggggctctc gccggcgtgg atccggcccg gatctcgcgg ggaatggggc 180 tctcggatgt agatctgcga tccgccgttg ttgggggaga tgatgggggg tttaaaattt 240 ccgccgtgct aaacaagatc aggaagaggg gaaaagggca ctatggttta tatttttata 300 tatttctgct gcttcgtcag gcttagatgt gctagatctt tctttcttct ttttgtgggt 3 60 agaatttgaa tccctcagca ttgttcatcg gtagtttttc ttttcatgat ttgtgacaaa 420 tgcagcctcg tgcggagctt ttttgtag gt agaagtgatc aaccatggcg caagttagca 480 gaatctgcaa tggtgtgcag aacccatctc ttatctccaa tctctcgaaa tccagtcaac 540 gcaaatctcc cttatcggtt tctctgaaga cgcagcagca tccacgagct tatccgattt 600 cgtcgtcgtg gggattgaag aagagtggga tgacgttaat tggctctgag cttcgtcctc 660 ttaaggtcat gtcttctgtt tccacggcgt gcatgcttca cggtgcaagc agccggcccg 720 caaccgcccg caaatcctct ggcctttccg gaaccgtccg cattcccggc gacaagtcga 780 tctcccaccg gtccttcatg ttcggcggtc tcgcgagcgg tgaaacgcgc atcaccggcc 840 tcctggaagg cgaggacgtc atcaatacgg gcaaggccat gcaggcgatg ggcgcccgca 900 tccgtaagga aggcgacacc tggatcatcg atggcgtcgg caatggcggc ctcctggcgc 960 ctgaggcgcc gctcgatttc ggcaatgccg ccacgggctg ccgcctgatg atgggcctcg 1020 tcggggtcta cgatttcgac agcaccttca tcggcgacgc ctcgctcacg aagcggccga 1080 tgggccgcgt gatgaacccg ctgcgcgaat ggggcgtgca ggtgaaatcg gaagacggtg 1140 accgtcttcc cgttaccttg cgcggcccga agacgccgac gccgatcacc taccgcgtgc 1200 cgatggcctc cgcacaggtg aagtccgccg tgctgctcgc cggcctcaac acgcccggca 1260 tcacgacggt catcgagccg atcatgacg c gcgatcatac ggaaaagatg ctgcagggct 1320 ttggcgccaa ccttaccgtc gagacggatg cggacggcgt gcgcaccatc cgcctggaag 1380 gccgcggcaa gctcaccggc caagtcatcg acgtgccggg cgacccgacc tcgacggcct 1440 tcccgctggt tgcggccctg cttgttccgg gctccgacgt caccatcctc aacgtgctga 1500 tgaaccccac cccgaccggc ctcatcctga cgctgcagga aatgggcgcc gacatcgaag 1560 ttatcaaccc gcgccttgcc ggcggcgaag acgtggcgga cctgcgcgtt cgctcctcca 1620 cgctgaaggg cgtcacggtg ccggaagacc gcgcgccttc gatgatcgac gaatatccga 1680 P: \ YAN \ SPEC1F1CAT10N \ 88 Shu 44_SYC \ 88144 sEQUENCE LISTING (in) .DOC 1 1269809 tcctcgctgt cgccgccgcc ttcgcggaag gggggaccgt gatgaacggt ctggaagaac 1740 tccgcgtcaa ggaaagcgac cgcctctcgg ccgtcgccaa tggcctcaag ctcaatggcg 1800 tggattgcga tgagggcgag acgtcgctcg tcgtgcgtgg ccgccctgac ggcaaggggc I860 tcggcaacgc ctcgggcgcc gccgtcgcca cccatctcga tcaccgcatc gccatgagct 1920 tcctcgtcat gggcctcgtg tcggaaaacc ctgtcacggt Ggacgatgcc acgatgatcg 1980 ccacgagctt cccggagttt atggacctga tggccgggct gggcgcgaag atcgaactct 2040 ccgatacgaa ggctgcctga tg agctcgaa ttcccgatcg ttcaaacatt tggcaataaa 2100 gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatca 2145 < 210 > 6 < 211 > 834 < 212 > DNA < 213 > T25 < 400 > 6 attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag 60 acccttcctc tatataagga agttcttttc atttggagag gacagggtac ccggggatcc 120 tctagagtcg acatgtctcc ggagaggaga ccagttgaga ttaggccagc tacagcagct 180 gatatggccg cggtttgtga tatcgttaac cattacattg agacgtctac agtgaacttt 240 aggacagagc cacaaacacc acaagagtgg attgatgatc tagagaggtt gcaagataga 300 tacccttggt tggttgctga ggttgagggt gttgtggctg gtattgctta cgctgggccc 360 tggaaggcta ggaacgctta cgattggaca gttgagagta ctgtttacgt gtcacatagg 420 catcaaaggt tgggcctagg atccacattg tacacacatt tgcttaagtc tatggaggcg 480 caaggtttta agtctgtggt tgctgttata ggccttccaa acgatccatc tgttaggttg 540 catgaggctt Tgggatacac agcccggggt acattgcgcg cagctggata caagcatggt 600 ggatggcatg atgttggttt ttggcaaagg gattttgagt tgccagctcc tccaaggcca 660 gttaggccag ttacccagat ctgagtcgac Ctgcaggcat gcccgctgaa atcaccagtc 720 tctctctaca aatctatctc tctctataat aatgtgtgag tagttcccag ataagggaat 78〇tagggttctt atagggtttc gctcatgtgt tgagcatata agaaaccctt agta 834 <21〇> 7 <211> 2986 <212> DNA <213> TC1507 <4〇〇> Ί cgttctgatt gctaacttgc cagtgtttct ctttggggaa tcctgggatg gctctagccg 60 P: \ YAN \ SPECIF1CAT10N \ 88144_SYC \ 88144 sEQUENCE LISTING (in) .DOC 1 1269809 ttccgcagac gggatagatt tcatgatttt ttttgtttcg ttgcataggg tttggtttgc 120 ccttttcctt tatttcaata tatgccgtgc acttgtttgt cgggtcatct tttcatgctt 180 ttttttgtct tggttgtgat gatgtggtct ggttgggcgg tcgttctaga tcggagtaga 240 attctgtttc aaactacctg gtggatttat taattttgga tctgtatgtg tgtgccatac 300 atattcatag ttacgaattg aagatgatgg atggaaatat cgatctagga taggtatacg 360 tgttgatgcg ggttttactg atgcatatac agagatgctt tttgttcgct tggttgtgat 420 gatgtggtgt ggttgggcgg tcgttcattc gttctagatc ggagtagaat actgtttcaa 480 actacctggt gtatttatta attttggaac tgtatgtgtg tgtcatacat cttcatagtt 540 acgagtttaa gatggatgga aat atcgatc taggataggt atacatgttg atgtgggttt 600 tactgatgca tatacatgat ggcatatgca gcatctattc atatgctcta accttgagta 660 cctatctatt ataataaaca agtatgtttt ataattattt tgatcttgat atacttggat 720 gatggcatat acagcagcta tatgtggatt tttttagccc tgccttcata cgctatttat 780 ttgcttggta ctgtttcttt tgtcgatgct caccctgttg tttggtgtta cttctgcagg 840 tcgactctag aggatccaac aatggagaac aacatacaga atcagtgcgt cccctacaac 900 tgcctcaaca atcctgaagt agagattctc aacgaagaga ggtcgactgg cagattgcca 960 ttagacatct ccctgtccct tacacgtttc ctgttgtctg agtttgttcc aggtgtggga 1020 gttgcgtttg gcctcttcga cctcatctgg ggcttcatct ctccatctga ttggagcctc 1080 tttcttctcc agattgaaca gctgattgaa caaaggattg agaccttgga aaggaatcgg 1140 gccatcacta cccttcgtgg cttagcagac agctatgaga tctacattga agcactaaga 1200 gagtgggaag ccaatcctaa caatgcccaa ctgagagaag atgtgcgtat acgctttgct 1260 aacacagatg atgctttgat cacagccatc aacaacttca cccttaccag cttcgagatc 1320 cctcttctct cggtctatgt tcaagctgct agcctgcact tgtcactact gcgcgacgct 1380 gtgtcgtttg ggcaaggttg gg gactggac atagctactg tcaacaatca ctacaacaga 1440 ctcatcaatc tgattcatcg atacacgaaa cattgtttgg atacctacaa tcagggattg 1500 gagaacctga gaggtactaa cactcgccaa tgggccaggt tcaatcagtt caggagagac 1560 cttacactta ctgtgttaga catagttgct ctctttccga actacgatgt tcatacctat 1620 ccgattcaaa cgtcatccca acttacaagg gagatctaca ccagttcagt cattgaagac 1680 tctccagttt ctgcgaacat acccaatggt ttcaacaggg ctgagtttgg agtcagacca 1740 ccccatctca tggacttcat gaactctttg tttgtgactg cagagactgt tagatcccaa 1800 actgtgtggg gaggacactt agttagctca Cgcaacacgg ctggcaatcg tatcaacttt 1860 cctagttacg gggtcttcaa tcccgggggc gccatctgga ttgcagatga agatccacgt 1920

P \ YAN \ SPECIFICATION \ 88144 SYC \ 88I44 sequence table (in) .DOC I 1269809 cctttctatc ggaccttgtc agatcctgtc ttcgtccgag gaggctttgg caatcctcac 1980 tatgtactcg gtcttagggg agtggccttt caacaaactg gtacgaatca cacccgcaca 2040 ttcaggaact ccgggaccat tgactctcta gatgagatac cacctcaaga caacagcggc 2100 gcaccttgga atgactactc ccatgtgctg aatcatgtta cctttgtgcg ctggccaggt 2160 gagatctcag gttccgactc atggagagca ccaatgttct cttggacgca tcgtagcgct 2220 acccccacaa acaccattga tccagagaga atcactcaga ttcccttggt gaaggcacac 2280 acacttcagt caggaactac agttgtaaga gggccggggt tcacgggagg agacattctt 2340 cgacgcacta gtggaggacc attcgcgtac accattgtca acatcaatgg gcaacttccc 2400 caaaggtatc gtgccaggat acgctatgcc tctactacca atctaagaat ctacgttacg 2460 gttgcaggtg aacggatctt tgctggtcag ttcaacaaga caatggatac cggtgatcca 2520 cttacattcc aatctttctc ctacgccact atcaacaccg cgttcacctt tccaatgagc 2580 cagagcagtt tcacagtagg tgctgatacc ttcagttcag Gcaacgaagt gtacattgac 2640 aggtttgagt tgattccagt tactgccaca ctcgagtaag gatccgtcga cctgcagcca 270 0 agctttcgcg agctcgagat ccccgacata tgccccggtt tcgttgcgac taacatgagt 2760 tcttggacaa atttgattgg acctgatgag atgatccaac ccgaggatat agcaaagctc 2820 gttcgtgcag caatggaacg gccaaaccgt gcttttgtcc ccaagaatga ggtgctatgc 2880 atgaaggaat ctacccgttg atgtccaaca gtctcagggt taatgtctat gtatcttaaa 2940 taatgttgtc ggtattttgt aatctcatat agattttcac tgtgcg 2986 < 210 > 8 < 211 > 24 < 212 > DNA <213> 0205C <4〇〇> 8 tccgcggctt gttgtggtct tttg 24 <21〇> 9 <211>19 <212> DNA <213> YCC-6 <400> 9 ttccaggggc tcaagtcca 19 &lt ;210> 10 <211> 18 <212> DNA <213> YCC-2 P:\YAN\SPECIFICATI0N\88I44_SYC\88M4 Sequence Listing (middle) D〇C 1 -10- 181269809 <4〇〇&gt 10 tctccttgat gggctgca <210> 11 <211> 22 <212> DNA <213> YCC-35 <4〇〇> 11 tcgcccgccg ctcctccaga ag 22

<21〇> 12 <211> 30 <212> DNA <213> YM556B <4〇〇> 12 tgacactata ttgcttctct ttacatacgt 30 <21〇> 13 <211> 28 <212> DNA <213> PYM92 <4〇〇> 13 ctcgatgcct tctccctagt gttgacca 28 <21〇> 14 <211> 20 <212> DNA <213> 0726D-5, <4〇〇> 14 gtgacaaatg cagcctcgtg 20 >>>> 0 12 3 i - I 1 - I - - I τ - I 2 2 2 2 <<<<

15 18 DNA 0712B <400〉 15 tgcgttgact ggatttcg 18

<21〇> 16 <211> 28 <212> DNA <213> PK06D <4〇〇> 16 tgctaacttg cgccatggtt gatcactt

<210> 17 <211> 23 <212> DNA P:\YAN\SPEC1F1CATI0N\88144_SYC\88144 Sequence Listing (middle).DOC 1 -11 - 28 1269809 <213> RT5006 <400> 17 ttaggccagt tacccagatc Tga <210><211><212><213> 18 25 DNA RT3124 <400> 18 attcccttat ctgggaacta ctcac <21〇><211><212><213> 19 26 DNA GT42 <400> 19 catgcccgct gaaatcacca gtctct <210> <211><212><213> 20 18 DNA l〇14Ra <400> 20 cgacctgcag ccaagctt <21〇><211><212><213> 21 22 DNA l〇14Rb <4〇〇> 21 tcatgttagt cgcaacgaaa cc <21〇><211><212><213> 22 25 DNA TC1014R <4〇〇 > 22 cgagctcgag atccccgaca tatgc <210><211><212><213> 23 20 DNA 7 5 <400> 23 ccgacagtgg tcccaaagat <210> 24 P:\YAN\SPECIFICAT10N\88144_SYC\88144 List (middle).DOC 1 1269809 <211> 26 <212> DNA <213> 161S <400> 24 agtggagata tcacatcaat <;210> 25 <211> 20 <212> DNA <213> 100 <4〇〇> 25 cccacccacg aggagcatcg ccactt <210> 26 <211> 22 <212> DNA <213> hmg -c <4〇〇> 26 ttggctacat agggagcctt gt <210> 27 <211> 23 <212> DNA <213> hmg-d <400> 27 gagtcggtaa gctccatctt ctg P:\YAN\SPEC1F1CATI0N\ 88144_SYC\88144 Sequence Listing (middle).DOC 1

Claims (1)

1269809 Picking up, patent application scope: Announcement of the peri-negative negative body's including a promoter, a maize internal control gene and the following nucleic acid sequences: sequence identification number: 1, sequence identification number: 2, serial number J identification number: 3, Sequence identification number: 4, sequence identification number: 5, sequence identification number: 6 and sequence identification number: 7. 2_ According to the plastid of claim 1, wherein the promoter is a CaMV 3 5 S promoter. 3. 4. 5. According to the application. The plastid of the third aspect of the patent, wherein the maize internal control gene is a maize starch synthase lib gene, an invertase gene or a high mobility group (HMG) gene. According to the plastid of the patent application scope, the internal control gene of the corn is the South Mobile Group Protein (HMG) gene. The plastid according to the scope of the patent application, wherein the promoter is the CaMV35S promoter and the maize internal control gene is hmg. 6. A vector comprising the plastid of claim 2 or 2. 8. A host cell comprising a carrier of claim 6 in accordance with the scope of the patent application k corn lines Btll, T25 and TC1507. The plastid of item 1, which can be used to detect the gene mutation Eventl76, GA21, MON810, NK603, which consists of a sequence of a probe that hybridizes to the Btll gene sequence. 10. A pair of primers that hybridize to the GA2 1 gene sequence. Identification number: 9 and sequence identification number·· 1 〇. The sequence is the sequence O:\88\88144-950704.DOC 1269809 11. 12. 13. 14. 16. 16. 17. 18. 19. 20. 21. 22. A hybrid to the GA21 gene sequence, It consists of a sequence of sequence numbers: 11. The recognition is based on sequence identification. The sequence is identified by sequence. The sequence is respectively sequenced. The sequence of one hybridization to the MON810 gene sequence is composed of 12 sequences. A probe that hybridizes to the MON810 gene sequence. Sequence number of 13: composition. A primer pair that hybridizes to the NK603 gene sequence, column identification number: 14 and sequence identification number: 丄$. A probe that hybridizes to the NK603 gene sequence, consisting of a sequence of 16: ..., is a sequence pair identification of the hybrid i T25 gene sequence, its sequence identification number: 17 and sequence identification number: 18. A probe that hybridizes to the T25 gene sequence, 1Q, "" "Hibiscus is identified by sequence identification number: 1 9 < sequence composition. A hybrid to TC1 507 gene sequence" < 5 scorpion pair, The sequences are: shore identification number: 20 and sequence identification number: 2i. A hybrid to the TC 1507 gene sequence consisting of a sequence identification number: 22. A hybrid to CaMV 3 5S promoter The sequence of the early feed and the 5 丨 pair is the sequence identification number·· 23 and the sequence identification number: 24. A hybrid to the CaMV 35 S promoter is a privately-friendly ι 于 彳 彳 彳 , It consists of a sequence of sequence identification number: 25. The hybridization of the high-growth group of maize (high mGb called (tetra) 叩, HMG) gene sequence primer pair, the sequence is respectively _ ID: O:\88\88144 -950704.DOC -2- 1269809 26 and sequence identification number: 27. 23 24. 25. 26. A method of detecting a genetically modified maize line using a plastid as claimed in claim i and one or more Patent application number 9 Leads and probes listed in item 22. - A kit for the detection of genetically modified maize lines, including one or more specific hybrids to bui, GA21, MON810, NK603 according to the plastids of the scope of the patent application; , Eventl76, Ding 25 and Tcl5〇7 gene to U-transgenic w-type primers and probes, such as the towel, please refer to the second article of the patent scope, such as the probe of the scope of claim 21, such as the scope of patent application The introduction of item 22, and the formula of the instant pcR reaction solution.... According to the kit of the scope of the patent application, the specific hybridization to BtU, GA21, MON81〇, NK6〇3, D5 and tci5〇7 genes is changed to k. The maize transgenic foot probes are listed in the scope of application for patents Nos. 9, 1, 13, 15, 17 and 19. According to the 24th article of the patent application, the specific hybridization to GA21, MON810, NK603 The introductions of the T25 and TC1507 genetically modified maize transgenic lines are listed in the patent scope 帛1G, 12, 14, and 18, respectively. O:\88\88144-950704.DOC