CN112646829B - Plasmid standard molecule capable of being used for detecting multiple crops and multiple exogenous genes - Google Patents

Abstract

The invention discloses a plasmid standard molecule pLANGUTUOU-5 for detecting various crops and exogenous genes and application thereof, wherein the base sequence of the plasmid standard molecule is shown as SEQ ID NO. 1. The pLANGUTUOU-5 is used as a positive control or a positive standard substance, can detect 13 species and 39 exogenous genes, solves the problems of difficult acquisition of transgenic positive materials and the like, greatly expands the detection range of the exogenous genes, reduces the risk of missed detection and reduces the requirement for preparing a plurality of positive controls. The plasmid constructed by the method can be applied to 13 main crops and economic crops, greatly expands the variety of detected crops and is beneficial to transgene monitoring and safety supervision. The plasmid molecule provided by the method is simple and rapid in construction and low in cost, and the transgene detection cost can be greatly saved.

Description

Plasmid standard molecule capable of being used for detecting multiple crops and multiple exogenous genes

Technical Field

The invention belongs to the technical field of transgene detection, and particularly relates to a plasmid standard molecule capable of being used for detecting multiple crops and multiple exogenous genes.

Background

The safety of transgenic products is always concerned, and the molecular detection of transgenic crops and products thereof is more and more important. At present, different requirements are provided for the identification of transgenic products in various countries, the content of transgenic components must be indicated by the regulations of some countries, and the requirements of various countries on the content of identified transgenes are different. In order to solve the problems of trade disputes and the like, qualitative and quantitative detection of transgenic products becomes a key (Dong Lianhua and the like, 2012). The detection methods of the transgenic crops commonly used at present are a detection method based on sample DNA and a detection method based on sample protein. Sample protein-based detection methods include Enzyme-linked immunosorbent assay (ELISA) and transgenic rapid detection strips. Sample DNA-based detection methods are most widely used. The transgenic detection method of gene PCR can be divided into screening, gene-specific detection, vector-specific detection and event-specific detection, which respectively detect the integration sites of universal elements and screening markers, target genes, transformation vectors and foreign genes on chromosomes (segu wude, 2009). Under the condition that the screening PCR result is positive, the specificity detection of genes, vectors and events can be further carried out to judge which exogenous genes contained in the transgenic variety are from which transformation vector and belong to which transformation strain. The common PCR method based on sample DNA needs electrophoresis and ultraviolet observation processes, is complex to operate, consumes long time and is difficult to realize high-throughput detection. The DNA detection of crops by utilizing fluorescent quantitative PCR is the mainstream method for international transgene detection at present, and has the advantages of very high sensitivity and accuracy.

When performing PCR assays, transgenic crop DNA is required as a positive control to ensure the accuracy of the assay. The transgenic standard substance is a substance having sufficient stability and uniformity to contain a transgenic component. As an important positive control sample, the current home and abroad transgenic standard substances comprise 2 types: matrix standard substance and nucleic acid molecule standard substance. The matrix standard substance mainly comprises a seed standard substance and a seed powder standard substance; the nucleic acid molecule standard substance mainly comprises plant genome DNA and plasmid DNA molecule standard substances. On the other hand, in the aspect of developing matrix standard substances in China, the development of matrix standard substances in China has a large difference compared with developed countries such as European Union, united states and the like, the commercialized transgenic standard substances are only Bt63 and the like, and if other standard substances are bought, import is needed, and the procedure is complicated and the period is long. The transgenic plasmid standard molecule is one of transgenic standard products, and has the advantages of simple manufacturing process and low cost, thereby having better application prospect.

The plasmid DNA standard substance refers to a plasmid DNA molecule containing an exogenous target gene and an internal standard gene. The positive plasmid molecule has many advantages, for example, one standard molecule can simultaneously possess a plurality of exogenous target genes; fussy addition and reproduction are not needed; the microorganism can be used for mass culture, and has the advantages of easy preservation and high purity; quantitative analysis can be performed by converting the molecular weight into plant genomic DNA molecules. However, the current transgenic plasmid standard molecules also have certain limitations, for example, the transgenic plasmid standard molecules can be used for detecting limited types of crops, most of the transgenic plasmid standard molecules can only detect one species, and the few transgenic plasmid standard molecules can detect 4-5 species, and are limited to corn, rice, cotton, rape and soybean; in addition, the transgenic plasmid standard molecule contains limited foreign genes, most of which can only detect 4-5 genes, and the few of which can detect about 10 genes, and is limited to CaMV35S promoter, FMV promoter, NOS terminator, BT, HPT, NPTII, cry1Ab, EPSPS, BAR, PAT and the like. With the increase of commercial transgenic crop varieties, more and more genes are applied to the breeding of transgenic crops, so that the expansion of the range of screening genes and the expansion of the variety of monitoring crops become more and more necessary. Therefore, it is urgently needed to create a plasmid standard molecule aiming at various crops and various exogenous genes and develop a detection method aiming at more exogenous genes.

At present, transgenic lines are various and expensive, and if the transgenic lines are to be identified, positive controls are needed, so that the transgenic lines need to be purchased with standard products. There are hundreds of transgenic lines that are evaluated for safety or have been commercialized throughout the world, but many transgenic lines have not been commercially produced in China. The plant materials of the strains are difficult to obtain, the current commercialized transgenic detection standard is mainly matrix standard substances, the development process of the matrix standard substances is complex, the price is very expensive, and the powder of each gram of the matrix standard substances is about 100-200 Euros. Therefore, the purchase of standard substances is a heavy burden for the institution that wants to perform the transgenic test. In addition, the development of domestic standard products is relatively delayed, and if other standard products need to be purchased, import is needed, and the procedures are complicated and the period is long due to the transgenic process. With the increase of commercial transgenic crop varieties, more and more genes are applied to the breeding of transgenic crops, so that the expansion of the range of screening genes and the expansion of the variety of monitoring crops become more and more necessary. Therefore, it is urgently needed to create a plasmid standard molecule aiming at various crops and various exogenous genes and develop a detection method aiming at more exogenous genes. The invention aims to create and apply a positive plasmid which can be used for detecting various transgenic crops and various exogenous genes, and develop corresponding fluorescent quantitative PCR primers aiming at a promoter and a terminator which are commonly used for the transgenic crops.

Disclosure of Invention

The first object of the present invention is to provide a nucleic acid molecule.

It is a second object of the present invention to provide a plasmid molecule.

The third purpose of the invention is to provide the application of the nucleic acid molecule and the plasmid molecule in qualitative screening and detection of transgenic crops and/or exogenous genes.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided a nucleic acid molecule comprising the nucleic acid sequence of:

(1) Transgenic breeding exogenous genes;

(2) A standard gene in the crop;

wherein the transgenic breeding exogenous gene is selected from at least one of the following genes: T-G7 terminator, T-E9 terminator, T-CaMV35S terminator, T-NOS terminator, PVYep gene, P-SSuAra promoter, P-NOS promoter, PMI gene, PLRVrep gene, P-ubitin promoter, P-TA29 promoter, P-RACT promoter, NPTII gene, P-CaMV35S promoter, P-FMV35S promoter, PAT gene, HPT gene, GUS gene, EPSPS gene, cry3A gene, cry1Ac gene, cry1Ab gene, cry1A.105 gene, bt gene, barnase gene, bar gene, T-VSP terminator, T-Tr7 terminator, T-TML terminator, T-pinII terminator, T-PEPC terminator, T-OCS3 terminator, T-GLB1 terminator, UTRT-7 sP terminator, P-MTL promoter, GLOCS 1P-4 GAX promoter, and GLOCS promoter.

According to the nucleic acid molecule of the first aspect of the present invention, the standard gene in crop plants is derived from one or more of corn, rice, cotton, rape, wheat, soybean, alfalfa, papaya, tomato, sugar beet, sesame, flax or peanut.

The nucleic acid molecule according to the first aspect of the present invention, which comprises the following nucleotide fragments:

(1) One or more nucleotide fragments in any linkage order selected from SEQ ID NO 4 to SEQ ID NO 42, and

(2) One or two nucleotide fragments selected from any connection sequence in SEQ ID NO. 43-55.

The nucleic acid molecule according to the first aspect of the present invention, which comprises a 52 nucleotide fragment of SEQ ID NO. 4 to SEQ ID NO. 55.

The nucleic acid molecule according to the first aspect of the invention comprises a nucleotide fragment of SEQ ID NO 2 and SEQ ID NO 3.

Preferably, the nucleic acid molecule contains nucleotide fragments of SEQ ID NO. 2 and SEQ ID NO. 3, specifically, SEQ ID NO. 4 to 42 are spliced in series into SEQ ID NO. 2, and SEQ ID NO. 43 to 55 are spliced in series into SEQ ID NO. 3.

Preferably, the nucleic acid molecule contains a nucleotide fragment shown as SEQ ID NO. 1, and is formed by splicing SEQ ID NO. 2-SEQ ID NO. 3 in series.

In a second aspect of the invention, there is provided a plasmid comprising a nucleic acid molecule according to the first aspect of the invention.

Furthermore, the plasmid is obtained by inserting the nucleotide fragments shown in SEQ ID NO. 2 and SEQ ID NO. 3 into a plasmid vector.

Preferably, the plasmid is obtained by inserting the nucleotide fragments shown as SEQ ID NO. 2 and SEQ ID NO. 3 into an Escherichia coli vector.

Preferably, the plasmid is pUC57, which is preferred as an E.coli vector, into which the nucleotide fragments shown in SEQ ID NO. 2 and SEQ ID NO. 3 have been inserted.

Furthermore, the plasmid is a positive plasmid molecule which is constructed by inserting nucleotide fragments shown in SEQ ID NO. 2 and SEQ ID NO. 3 into an EcoRV site of pUC57 optimized by an escherichia coli vector and polymerizes 39 exogenous genes and 13 internal standard genes of large crops, and is named as pLANGUTUOU-5.

The construction process comprises the following steps: the detection target sequences of 13 internal standard genes of main crops and commercial crops are connected in series to form a sequence SEQ ID NO. 3, the detection target sequences of 39 exogenous genes with higher use frequency are connected in series to form a sequence SEQ ID NO. 2, and then the SEQ ID NO. 2 and the SEQ ID NO. 3 are spliced to form a long sequence SEQ ID NO. 1. Entrusted general biological system (Anhui) limited company synthesizes the sequence SEQ ID NO:1 by means of gene synthesis and inserts the sequence into the EcoRV site of an escherichia coli vector pUC57 to construct a positive plasmid molecule pLANGUTUOU-5 which polymerizes 39 screening elements and 13 crop internal standard genes.

Characteristics of the positive plasmid molecules: pLANGUTUOU-5 has 39 exogenous genes and 13 crop internal standard genes polymerized, and pLANGUTUOU-5 as positive control can be used to perform real-time fluorescent PCR screening detection of transgenic components in 13 transgenic crop samples.

In a third aspect of the present invention, there is provided a use of the nucleic acid molecule of the first aspect of the present invention or the plasmid of the second aspect of the present invention in qualitative screening and detecting of transgenic crops and/or exogenous genes.

According to the third aspect of the invention, the crop is one or more of corn, rice, cotton, rape, wheat, soybean, alfalfa, papaya, tomato, sugar beet, sesame, flax or peanut.

According to the third aspect of the present invention, the foreign gene is one or more of a T-G7 terminator, a T-E9 terminator, a T-CaMV35S terminator, a T-NOS terminator, a PVYep gene, a P-SSuAra promoter, a P-NOS promoter, a PMI selectable marker gene, a PLRVrep gene, a P-Ubiquitin promoter, a P-TA29 promoter, a P-RACT promoter, an NPTII resistance screening gene, a P-CaMV35S promoter, a P-FMV35S promoter, a PAT herbicide resistance screening gene, an HPT resistance screening gene, a GUS marker gene, an EPSPS herbicide resistance gene, a Cry3A gene, a Cry1Ac gene, a Cry1Ab gene, a Cry1A 105 gene, a Bt gene, a Barnase gene, a Barr gene, a T-VSP terminator, a T-Tr7 terminator, a T-TML terminator, a T-pinII terminator, a T-PEPC terminator, a T-OCS3 terminator, a MTOCS 1-GLS 1-P promoter, a GLP 4-GAP promoter.

According to the third aspect of the invention, the application is particularly used as a positive control or a positive standard substance for detecting the transgenic crops and/or exogenous genes.

According to the application of the third aspect of the invention, the plasmid or the positive standard substance used as the positive control substance for detecting the transgenic crops is used for carrying out real-time fluorescence PCR screening detection on transgenic components of crops or exogenous genes to be detected.

Furthermore, fluorescent dye method PCR detection primers aiming at promoters such as P-MTL, P-OCS, P-PepC and P-RbcS and terminator transgenic elements such as T-TML, corn T-Glb1, soybean T-7sUTR, T-PEPC, potato T-pinII, T-Tr7 and T-VSP and the like aiming at the exogenous genes are also developed:

the invention has the beneficial effects that:

the method constructs universal positive plasmids and reduces the need for preparing a plurality of positive controls. The plasmid standard molecule constructed by the invention can detect up to 39 exogenous genes, greatly expands the detection range of the exogenous genes and reduces the risk of missed detection. The plasmid constructed by the method can be applied to 13 main crops and economic crops, greatly expands the variety of detected crops and is beneficial to transgene monitoring and safety supervision. Can solve the problems of difficult acquisition of transgenic positive materials and the like. The purchase of transgenic standard substances is expensive. The plasmid molecule provided by the method is simple and rapid in construction and low in cost, and the transgene detection cost can be greatly saved.

Drawings

FIG. 1 is a diagram showing a structure of plasmid standard molecule pLANGUTUOU-5.

FIG. 2 shows the verification of 13 crop internal standard genes in plasmid standard molecule pLANGUTUOU-5 by real-time fluorescent PCR method.

FIG. 3 shows the verification of the original 39 foreign genes in plasmid standard molecule pLANGUTUOU-5 by real-time fluorescent PCR.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention provides a plasmid standard molecule, which comprises 39 exogenous genes commonly used for transgenic breeding and 13 endogenous genes of species (corn ZmzSSIIb, rice OsSPS, cotton GhACP, rape BnCruA, wheat TsWaxy, soybean Gmletin, alfalfa MsAcc, pawpaw CpChy, tomato StLAT52, beet BvGluA, sesame SiAlb, flax LuSad and peanut JrVic). When detecting whether the sample of the species contains common transgenic components, the plasmid molecule can be used as a positive control. Promoters such as P-MTL, P-OCS, P-PepC and P-RbcS, terminators such as T-TML, corn T-Glb1, soybean T-7sUTR, T-PEPC, potato T-pinII, T-Tr7 and T-VSP, and the like are widely used in transgenic crops, but detection of these elements is lacked in a corresponding method, and a fluorescent quantitative PCR detection method for these genes is also developed in the present invention.

EXAMPLE 1 construction of plasmid Standard molecules

1. Interrogation of DNA sequences of foreign genes and internal standard genes

39 exogenous genes and 13 internal standard genes of main crops and commercial crops, wherein the frequency of use of the genes in selective transgenic breeding is high, the 39 exogenous genes are shown in a table 1, the 13 internal standard genes are shown in a table 2, and DNA sequences of the genes are searched in GenBank. According to the method published by the Chinese and European Union transgenic organism detection technical standard and the verified detection method, the detection target sequence SEQ ID NO. 4-SEQ ID NO. 55 of each gene is determined.

TABLE 1 common foreign genes

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TABLE 2 internal Standard genes

Internal standard gene name	Origin of genes	Fragment sequences
			zSSIIb	Corn (maize)	SEQ ID NO:43
TsWaxy	Wheat (Triticum aestivum L.)	SEQ ID NO:44
			StLAT52	Tomato	SEQ ID NO:45
SiAlb	Sesame seed	SEQ ID NO:46
			OsSPS	Rice (Oryza sativa L.) with improved resistance to stress	SEQ ID NO:47
MsACC	Alfalfa	SEQ ID NO:48
			LuSad	Flax	SEQ ID NO:49
JrVic	Peanut	SEQ ID NO:50
			GmLectin	Soybean	SEQ ID NO:51
GhACP	Cotton	SEQ ID NO:52
			CpChy	Pawpaw	SEQ ID NO:53
BvGluA	Sugar beet	SEQ ID NO:54
			BnCruA	Rape	SEQ ID NO:55

2. Obtaining of target DNA fragment

According to the published transgenic detection standard method, determining PCR amplification fragments of the exogenous genes and the internal standard genes, connecting detection target sequences of all the exogenous genes in series to form a sequence SEQ ID NO. 2, connecting detection target sequences of all the internal standard genes in series to form a sequence SEQ ID NO. 3, and connecting the sequence SEQ ID NO. 2 and the sequence SEQ ID NO. 3 in series to form SEQ ID NO. 1. General biosystems (Anhui) Ltd was entrusted with the synthesis of SEQ ID NO:1 by means of gene synthesis.

3. Plasmid construction

The full-length DNA sequence SEQ ID NO 1 containing 39 exogenous gene elements and 13 crop internal standard genes is digested by EcoRV endonuclease and cloned to pUC57 vector, the plasmid is named pLANGUTUOU-5, and the connection sequence and molecular size are shown in figure 1. The constructed plasmid is transferred into escherichia coli, a single clone is selected and extracted, and sequencing confirms that 39 exogenous gene elements and 13 crop internal standard genes are constructed on the pUC57 vector.

Example 2 primer information

Fluorescent dye method PCR detection primers for P-MTL, P-OCS, P-PepC, P-RbcS and other promoters and T-TML, corn T-Glb1, soybean T-7sUTR, T-PEPC, potato T-pinII, T-Tr7, T-VSP and other terminator transgenic elements are also developed, and are shown in Table 3.

TABLE 3 fluorescent dye method PCR detection primers

EXAMPLE 3 use of Standard plasmid molecules

The real-time fluorescent PCR amplification is carried out by using 39 synthesized exogenous gene elements and real-time fluorescent PCR primers and Taqman probes of 13 crop internal standard genes and using a plasmid molecule pLANGUTUOU-5 as a template, wherein the amplification curve of the 13 crop internal standard genes is shown in figure 2, and the amplification curve of the 39 exogenous gene elements is shown in figure 3.

From FIGS. 2 and 3, it can be seen that each gene element generates a typical amplification curve, indicating that the plasmid molecule pLANGUTUOU-5 contains the real-time fluorescent PCR detection target of each gene element. The plasmid molecule pLANGUTUOU-5 can be used as a positive control and a quality control sample for real-time fluorescent PCR screening detection of 13 transgenic crops.

The above embodiments are merely preferred examples to illustrate the present invention, and it should be apparent to those skilled in the art that any obvious variations and modifications can be made without departing from the spirit of the present invention.

SEQUENCE LISTING

<110> Huazhi Biotechnology Limited

<120> a plasmid standard molecule for detecting various crops and various exogenous genes

<130>

<160> 73

<170> PatentIn version 3.5

<210> 1

<211> 5975

<212> DNA

<213> Artificial sequence

<400> 1

atgtattaca cataatatcg cactcagtct ttcatctacg gcaatgtacc agctgatata 60

atcagttatt gaaatatttc tgaatttaaa cttgcattga gaatgaacaa aaggaccata 120

tcattcatta actcttctcc atccatttcc atttcacagt tcgatagcga aaaccgaata 180

aaaaggggat ctggatttta gtactggatt ttggttttag gaattagaaa ttttattgat 240

agaagtattt tacaaataca aatacatact aagggtttct tatatgctca acacatgagc 300

gaaacatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 360

tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 420

atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat gaatcaaggc 480

tatcacgtcc aaaatgagaa tgcccacaag caagggagca accgtgctaa acttagaaca 540

cttgcttgag tatgctccac aacaaattga tatttcaaat actcgggcca ctcaatcaca 600

gtttgatacg tggtatgagg cagtgcggat gggcctaagg agaggtgttg agacccttat 660

cggcttgaac cgctggaata atgccacgtg gaagataatt ccatgaatct tatcgttatc 720

tatgaggaca gaaccgcaac gttgaaggag ccactcagcc gcgggtttct ggagtttaat 780

gagctaagca catacgtcag aaactgcctt tcctgttcaa agtattatgc gcagcacagc 840

cactctccat tcaggttcat ccaaacaaac acaattctga aatcggtttt gccaaagatc 900

gtcattaaac ttgacgaccc aaccaccgcc gccgcttaca gatcggagct actacgagtt 960

agttcaagct cttacatcca aaatgcggct ggattgtcaa acggttgggg acatgacatg 1020

gaggcatttg tcagaaatgc tatttgcctc ctggaactcc gtgaaagaag gagtagataa 1080

tgccagcctg ttaaacgccg tcgacgagtc taacggacac caaccagcga accagcagcg 1140

tcgcgtgctc gaagtatgca catttagcaa tgtaaattaa atcagttttt gaatcaagct 1200

aaaagcagac ttgcataagg tgggtggctg gactagaata aacatcttct ctagcacagc 1260

ttctcgaggt cattcatatg cttgagaaga gagtcgggat agtccaaaat aaaacaaagg 1320

taagattacc tggtcaaaag tgaaaacatc agttaaaact atgactgggc acaacagaca 1380

atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt 1440

gtcaagaccg acctgtccga agacgtggtt ggaacgtctt ctttttccac gatgctcctc 1500

gtgggtgggg gtccatcttt gggaccactg tcgaagacat ccaccgaaga cttaaagtta 1560

gtgggcatct ttgaaagtaa tcttgtcaac atcgagcagc tggcttgtgg ggaccagaca 1620

aaaaaggaat ggtgcagaat tgttaggcgc acctaccaaa agcatctttg cctttattgc 1680

aaagataaag cagattcctc tagtacaagt ggggaacaaa ataacgtgga aaagagctgt 1740

cctgtcgaca tgtctccgga gaggagacca gttgagatta ggccagctac agcagctgat 1800

atggccgcgg tttgtgatat cgttaaccat tacattgaga cgtctacagt gaactttagg 1860

acagagccac aaacaccaca agagtggatt gatgatctag agaggttgca agatagatac 1920

ccttggttgg ttgctgaggt tgagggtgtt gtggctggta ttgcttacgc tgggccctgg 1980

aaggctagga acgcttacga ttggacaccg ctcgtctggc taagatcggc cgcagcgatc 2040

gcatccatag cctccgcgac cggttgtaga acagcgggca gttcggtttc aggcaggtct 2100

tgcaacgtga caccctgtta actatgccgg aatccatcgc agcgtaatgc tctacaccac 2160

gccgaacacc tgggtggacg atatcaccgt ggtgacgcat gtcgcgcaag actgtaacca 2220

cgcgtctgtt gactggcagg tggtggccaa tggtgatgtc agcgttccga cgccgatcac 2280

ctaccgcgtg ccgatggcct ccgcacaggt gaagtccgcc gtgctgctcg ccggcctcaa 2340

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accagttccg ccgcgagctg accctgaccg tgctggacat cgtgagcctg ttccccatca 2640

gaggtccagg gtttacagga ggagacattc ttcgtcgcac aagtggagga ccctttgctt 2700

acactattgt taacatcaat ggccaattgc cccaaaggta tcgtgcaaga atccgctatg 2760

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gaacctaccc tatccgtaca gtgtcccaac ttaccagaga aataatcaga agcacaagcc 2880

ctcggctggg tggcatcaaa agggaacctt gcagacgtcg ctccggggaa aagcatcggc 2940

ggagacatct tctcaaacag ggaaggcaaa ctacaagcac ggtcaacttc cgtaccgagc 3000

cgcaggaacc gcaggagtgg acggacgacc tcgtccgtct gcgggagcgc tatccctggc 3060

tcgtcgccga ggtggacggc gaggtcgccg gcatcgccta cgcgggcccc tggaaggcac 3120

gcaacgccta cgactggacg gccgagtgtt taaacttatg ttatgtgggt aagtcaccta 3180

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ggccaactta attaatgtat gaaataaaag gatgcacaca tagtgacatg ctaatcacta 3540

taatgtgggc atcaaccaac atctccccgc tctactaaga gctctagatc tgttctgcac 3600

aaagtggagt agtcagtcat cgatcaggaa ccagacacca gacttttatt cataccggtc 3660

aaacctaaaa gactgattac ataaatctta ttcaaatttc aaaagtgccc caggggctag 3720

tatctacgac acaccgagcg gcgaactaat aacgctcact gaagggaact ccggttcccc 3780

ttcgcttctt catttgtccc cttgcggagt ttggcatcca ttgatgccgt tacgctgaga 3840

acagacacag cagacgaacc aaaagtgagt tcttgtatga agtatcgggc tatttaacta 3900

tgacttgagc tccatctatg aataaataaa tcagcatatg atgcttttgt tttgtgtact 3960

tcaactgtct gcttagctat ttatagcgac tgacgctgtg gcaggcacgc tatcggaggt 4020

tacgacgtgg cgggtcactc gacgcggagt tcacaggtcc tatccttgca tcgctcggcc 4080

ctctcttaca gcaaattgtc cgctgcccgt cgtccagata caatgaacgt acctagtagg 4140

aactctttta cacgctcggt cgctcgccgc ggatcggagg tcgtttattt cggcgtgtag 4200

gacatggcaa ccgggcctga atttcgcggg tattctgttt ctattccaac tttttcttga 4260

tccgcagcca ttaacgactg tcttcgtgtt gctggaaccg ttgagttcgc tggtctcact 4320

gctgctccta actggaagcg tgctcacgtt ctctacactc acgctcgtaa gttgcttcca 4380

gctctcgctc ctgccagttc ctgcttcagg atactacagg aagttgggat tcagtgagca 4440

aggagaggtg ttcgatactc ctccagttgg acctcacatc ctgatgtata agaggatcac 4500

gtttaaacat cggatcccgg gcccgtcgac tgcagagggt ttaaacatcc tttgacatct 4560

gctccgaagc aaagtcagag cgctgcaatg caaaacggaa cgagtggggg cagcagcgcg 4620

agcaccgccg cgccggtgtc cggacccaaa gctgatcatc catcagctcc tgtcaccaag 4680

agagaaatcg atgccagtgc ggtgaagcca gagcccgcag gtgatgatgc tagaccggtg 4740

gaaagcatag gcatcgctga accggtggat gctaaggctg atgcagctcc ggctacagat 4800

gcggcggcga gtgctcctta tgacagggag gataatgaac ctggcccttt gtcgcaggaa 4860

cagaggtgtt caaggcggcc gaaataggtt gccgcctgcg gcggaatcgc cacccaccgt 4920

gaagttcacc gtttcgcaat ggaggaacac cagaccacga gaacgatatt tgcgaggtga 4980

cagttgtgaa aagcccaagg gaggactgca aagagagtgt gtctggatat gaaaaggcaa 5040

gaattaccag agggctaggg accttcctcg caggtgcaac atgcgacccc agcaatgcca 5100

attccgagtt tgaacatgtc caaggctctg ggggaacagg ttagcaatgg gaagctggtc 5160

ttgcgcctga acggatatct ttcagtttgt aaccaccgga tgacgcacgg acggctcgga 5220

tcatcccgaa aagatcaacc ggatcagtga acttcgcaaa gtactcggtt agtagacagt 5280

gaatgctcct gtgatctgcc catgcactca tgttgtagtg ttcacgtcgt tggctcaacc 5340

cagtcaccac cttcccttca acacgctccc tcaacaactt ctcctccaga tctcctcgca 5400

gcgcagagaa agcagaggcc gggaagagga gcaacagagg cacaatccct actactttca 5460

ctcccagagc attaggtcga gacatgaggc cctctactcc acccccatcc acatttggga 5520

caaagaaacc ggtagcgttg ccagcttcgc cgcttccttc aacttcacct tctatgcccc 5580

tgacacaaaa aggcttgcag atgggcattg tgatgggact tgaggaagaa ttcggaatca 5640

cggtggaaga ggacaatgca caatccatca caactgttca agccatgcgg atcctcccat 5700

ttcccttcat ccattcccac tcttgagagt tagctagtgt tacaatggct acgatggacc 5760

tccatattac tgaaaggaag ccaaaaggga tcaattaagt gctctacgaa gtttaaagta 5820

tgtgccgctc tcaagactga acatggcact gtgaacagga tggagcaatt actcggccag 5880

ggtttccgtg atatgcacca gaaagtggag cacataagga ctggggacac catcgctaca 5940

catcccggtg tagcccaatg gttctacaac gacgg 5975

<210> 2

<211> 4500

<212> DNA

<213> Artificial sequence

<400> 2

atgtattaca cataatatcg cactcagtct ttcatctacg gcaatgtacc agctgatata 60

atcagttatt gaaatatttc tgaatttaaa cttgcattga gaatgaacaa aaggaccata 120

tcattcatta actcttctcc atccatttcc atttcacagt tcgatagcga aaaccgaata 180

aaaaggggat ctggatttta gtactggatt ttggttttag gaattagaaa ttttattgat 240

agaagtattt tacaaataca aatacatact aagggtttct tatatgctca acacatgagc 300

gaaacatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 360

tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 420

atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat gaatcaaggc 480

tatcacgtcc aaaatgagaa tgcccacaag caagggagca accgtgctaa acttagaaca 540

cttgcttgag tatgctccac aacaaattga tatttcaaat actcgggcca ctcaatcaca 600

gtttgatacg tggtatgagg cagtgcggat gggcctaagg agaggtgttg agacccttat 660

cggcttgaac cgctggaata atgccacgtg gaagataatt ccatgaatct tatcgttatc 720

tatgaggaca gaaccgcaac gttgaaggag ccactcagcc gcgggtttct ggagtttaat 780

gagctaagca catacgtcag aaactgcctt tcctgttcaa agtattatgc gcagcacagc 840

cactctccat tcaggttcat ccaaacaaac acaattctga aatcggtttt gccaaagatc 900

gtcattaaac ttgacgaccc aaccaccgcc gccgcttaca gatcggagct actacgagtt 960

agttcaagct cttacatcca aaatgcggct ggattgtcaa acggttgggg acatgacatg 1020

gaggcatttg tcagaaatgc tatttgcctc ctggaactcc gtgaaagaag gagtagataa 1080

tgccagcctg ttaaacgccg tcgacgagtc taacggacac caaccagcga accagcagcg 1140

tcgcgtgctc gaagtatgca catttagcaa tgtaaattaa atcagttttt gaatcaagct 1200

aaaagcagac ttgcataagg tgggtggctg gactagaata aacatcttct ctagcacagc 1260

ttctcgaggt cattcatatg cttgagaaga gagtcgggat agtccaaaat aaaacaaagg 1320

taagattacc tggtcaaaag tgaaaacatc agttaaaact atgactgggc acaacagaca 1380

atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt 1440

gtcaagaccg acctgtccga agacgtggtt ggaacgtctt ctttttccac gatgctcctc 1500

gtgggtgggg gtccatcttt gggaccactg tcgaagacat ccaccgaaga cttaaagtta 1560

gtgggcatct ttgaaagtaa tcttgtcaac atcgagcagc tggcttgtgg ggaccagaca 1620

aaaaaggaat ggtgcagaat tgttaggcgc acctaccaaa agcatctttg cctttattgc 1680

aaagataaag cagattcctc tagtacaagt ggggaacaaa ataacgtgga aaagagctgt 1740

cctgtcgaca tgtctccgga gaggagacca gttgagatta ggccagctac agcagctgat 1800

atggccgcgg tttgtgatat cgttaaccat tacattgaga cgtctacagt gaactttagg 1860

acagagccac aaacaccaca agagtggatt gatgatctag agaggttgca agatagatac 1920

ccttggttgg ttgctgaggt tgagggtgtt gtggctggta ttgcttacgc tgggccctgg 1980

aaggctagga acgcttacga ttggacaccg ctcgtctggc taagatcggc cgcagcgatc 2040

gcatccatag cctccgcgac cggttgtaga acagcgggca gttcggtttc aggcaggtct 2100

tgcaacgtga caccctgtta actatgccgg aatccatcgc agcgtaatgc tctacaccac 2160

gccgaacacc tgggtggacg atatcaccgt ggtgacgcat gtcgcgcaag actgtaacca 2220

cgcgtctgtt gactggcagg tggtggccaa tggtgatgtc agcgttccga cgccgatcac 2280

ctaccgcgtg ccgatggcct ccgcacaggt gaagtccgcc gtgctgctcg ccggcctcaa 2340

cacgcccggc atctccggtt acgaggttct tttcctcact acctatgctc aagctgccaa 2400

cacccacttg tttctcctta aggacgctca aatctatggt acttggtgga gaacgcattg 2460

aaaccggtta cactcccatc gacatctcct tgtccttgac acagtttctg ctcagcgagt 2520

tcgtgccagg tgctgggttc gttctcggac tagttgacat ccgcgactgg atcaggtaca 2580

accagttccg ccgcgagctg accctgaccg tgctggacat cgtgagcctg ttccccatca 2640

gaggtccagg gtttacagga ggagacattc ttcgtcgcac aagtggagga ccctttgctt 2700

acactattgt taacatcaat ggccaattgc cccaaaggta tcgtgcaaga atccgctatg 2760

cctctactac gaccctcaca gttttggaca ttgtgtctct cttcccgaac tatgactcca 2820

gaacctaccc tatccgtaca gtgtcccaac ttaccagaga aataatcaga agcacaagcc 2880

ctcggctggg tggcatcaaa agggaacctt gcagacgtcg ctccggggaa aagcatcggc 2940

ggagacatct tctcaaacag ggaaggcaaa ctacaagcac ggtcaacttc cgtaccgagc 3000

cgcaggaacc gcaggagtgg acggacgacc tcgtccgtct gcgggagcgc tatccctggc 3060

tcgtcgccga ggtggacggc gaggtcgccg gcatcgccta cgcgggcccc tggaaggcac 3120

gcaacgccta cgactggacg gccgagtgtt taaacttatg ttatgtgggt aagtcaccta 3180

agacactcca cgtacctact tgttgtctct taccgcggct ttaataaatc ttctgccctt 3240

gttccatatt tactaatcaa tttatgtatt acacataata tcgcactcag tctttcatct 3300

acggcaatgt accagctgat ataatcagtt attgaaatat ttctgaattt aaactaaaag 3360

caggctgtgt tttcggcaaa catcgccacc catcgctagt ttttctaaaa gtgttctaag 3420

ctagcctggt aataatctat acgagcttat atttccctag acttgtccat cttctggatt 3480

ggccaactta attaatgtat gaaataaaag gatgcacaca tagtgacatg ctaatcacta 3540

taatgtgggc atcaaccaac atctccccgc tctactaaga gctctagatc tgttctgcac 3600

aaagtggagt agtcagtcat cgatcaggaa ccagacacca gacttttatt cataccggtc 3660

aaacctaaaa gactgattac ataaatctta ttcaaatttc aaaagtgccc caggggctag 3720

tatctacgac acaccgagcg gcgaactaat aacgctcact gaagggaact ccggttcccc 3780

ttcgcttctt catttgtccc cttgcggagt ttggcatcca ttgatgccgt tacgctgaga 3840

acagacacag cagacgaacc aaaagtgagt tcttgtatga agtatcgggc tatttaacta 3900

tgacttgagc tccatctatg aataaataaa tcagcatatg atgcttttgt tttgtgtact 3960

tcaactgtct gcttagctat ttatagcgac tgacgctgtg gcaggcacgc tatcggaggt 4020

tacgacgtgg cgggtcactc gacgcggagt tcacaggtcc tatccttgca tcgctcggcc 4080

ctctcttaca gcaaattgtc cgctgcccgt cgtccagata caatgaacgt acctagtagg 4140

aactctttta cacgctcggt cgctcgccgc ggatcggagg tcgtttattt cggcgtgtag 4200

gacatggcaa ccgggcctga atttcgcggg tattctgttt ctattccaac tttttcttga 4260

tccgcagcca ttaacgactg tcttcgtgtt gctggaaccg ttgagttcgc tggtctcact 4320

gctgctccta actggaagcg tgctcacgtt ctctacactc acgctcgtaa gttgcttcca 4380

gctctcgctc ctgccagttc ctgcttcagg atactacagg aagttgggat tcagtgagca 4440

aggagaggtg ttcgatactc ctccagttgg acctcacatc ctgatgtata agaggatcac 4500

<210> 3

<211> 1475

<212> DNA

<213> Artificial sequence

<400> 3

gtttaaacat cggatcccgg gcccgtcgac tgcagagggt ttaaacatcc tttgacatct 60

gctccgaagc aaagtcagag cgctgcaatg caaaacggaa cgagtggggg cagcagcgcg 120

agcaccgccg cgccggtgtc cggacccaaa gctgatcatc catcagctcc tgtcaccaag 180

agagaaatcg atgccagtgc ggtgaagcca gagcccgcag gtgatgatgc tagaccggtg 240

gaaagcatag gcatcgctga accggtggat gctaaggctg atgcagctcc ggctacagat 300

gcggcggcga gtgctcctta tgacagggag gataatgaac ctggcccttt gtcgcaggaa 360

cagaggtgtt caaggcggcc gaaataggtt gccgcctgcg gcggaatcgc cacccaccgt 420

gaagttcacc gtttcgcaat ggaggaacac cagaccacga gaacgatatt tgcgaggtga 480

cagttgtgaa aagcccaagg gaggactgca aagagagtgt gtctggatat gaaaaggcaa 540

gaattaccag agggctaggg accttcctcg caggtgcaac atgcgacccc agcaatgcca 600

attccgagtt tgaacatgtc caaggctctg ggggaacagg ttagcaatgg gaagctggtc 660

ttgcgcctga acggatatct ttcagtttgt aaccaccgga tgacgcacgg acggctcgga 720

tcatcccgaa aagatcaacc ggatcagtga acttcgcaaa gtactcggtt agtagacagt 780

gaatgctcct gtgatctgcc catgcactca tgttgtagtg ttcacgtcgt tggctcaacc 840

cagtcaccac cttcccttca acacgctccc tcaacaactt ctcctccaga tctcctcgca 900

gcgcagagaa agcagaggcc gggaagagga gcaacagagg cacaatccct actactttca 960

ctcccagagc attaggtcga gacatgaggc cctctactcc acccccatcc acatttggga 1020

caaagaaacc ggtagcgttg ccagcttcgc cgcttccttc aacttcacct tctatgcccc 1080

tgacacaaaa aggcttgcag atgggcattg tgatgggact tgaggaagaa ttcggaatca 1140

cggtggaaga ggacaatgca caatccatca caactgttca agccatgcgg atcctcccat 1200

ttcccttcat ccattcccac tcttgagagt tagctagtgt tacaatggct acgatggacc 1260

tccatattac tgaaaggaag ccaaaaggga tcaattaagt gctctacgaa gtttaaagta 1320

tgtgccgctc tcaagactga acatggcact gtgaacagga tggagcaatt actcggccag 1380

ggtttccgtg atatgcacca gaaagtggag cacataagga ctggggacac catcgctaca 1440

catcccggtg tagcccaatg gttctacaac gacgg 1475

<210> 4

<211> 97

<212> DNA

<213> Agrobacterium

<400> 4

atgtattaca cataatatcg cactcagtct ttcatctacg gcaatgtacc agctgatata 60

atcagttatt gaaatatttc tgaatttaaa cttgcat 97

<210> 5

<211> 87

<212> DNA

<213> pea

<400> 5

tgagaatgaa caaaaggacc atatcattca ttaactcttc tccatccatt tccatttcac 60

agttcgatag cgaaaaccga ataaaaa 87

<210> 6

<211> 121

<212> DNA

<213> cauliflower mosaic virus

<400> 6

ggggatctgg attttagtac tggattttgg ttttaggaat tagaaatttt attgatagaa 60

gtattttaca aatacaaata catactaagg gtttcttata tgctcaacac atgagcgaaa 120

c 121

<210> 7

<211> 165

<212> DNA

<213> Agrobacterium tumefaciens

<400> 7

atcgttcaaa catttggcaa taaagtttct taagattgaa tcctgttgcc ggtcttgcga 60

tgattatcat ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca 120

tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaat 165

<210> 8

<211> 161

<212> DNA

<213> disease resistance Gene (PVYep)

<400> 8

gaatcaaggc tatcacgtcc aaaatgagaa tgcccacaag caagggagca accgtgctaa 60

acttagaaca cttgcttgag tatgctccac aacaaattga tatttcaaat actcgggcca 120

ctcaatcaca gtttgatacg tggtatgagg cagtgcggat g 161

<210> 9

<211> 95

<212> DNA

<213> Arabidopsis thaliana

<400> 9

ggcctaagga gaggtgttga gacccttatc ggcttgaacc gctggaataa tgccacgtgg 60

aagataattc catgaatctt atcgttatct atgag 95

<210> 10

<211> 76

<212> DNA

<213> Agrobacterium

<400> 10

gacagaaccg caacgttgaa ggagccactc agccgcgggt ttctggagtt taatgagcta 60

agcacatacg tcagaa 76

<210> 11

<211> 96

<212> DNA

<213> phosphomannose isomerase Gene (PMI)

<400> 11

actgcctttc ctgttcaaag tattatgcgc agcacagcca ctctccattc aggttcatcc 60

aaacaaacac aattctgaaa tcggttttgc caaaga 96

<210> 12

<211> 172

<212> DNA

<213> disease-resistant Gene (PLRVrep)

<400> 12

tcgtcattaa acttgacgac ccaaccaccg ccgccgctta cagatcggag ctactacgag 60

ttagttcaag ctcttacatc caaaatgcgg ctggattgtc aaacggttgg ggacatgaca 120

tggaggcatt tgtcagaaat gctatttgcc tcctggaact ccgtgaaaga ag 172

<210> 13

<211> 76

<212> DNA

<213> corn

<400> 13

gagtagataa tgccagcctg ttaaacgccg tcgacgagtc taacggacac caaccagcga 60

accagcagcg tcgcgt 76

<210> 14

<211> 117

<212> DNA

<213> tobacco

<400> 14

gctcgaagta tgcacattta gcaatgtaaa ttaaatcagt ttttgaatca agctaaaagc 60

agacttgcat aaggtgggtg gctggactag aataaacatc ttctctagca cagcttc 117

<210> 15

<211> 95

<212> DNA

<213> Rice

<400> 15

tcgaggtcat tcatatgctt gagaagagag tcgggatagt ccaaaataaa acaaaggtaa 60

gattacctgg tcaaaagtga aaacatcagt taaaa 95

<210> 16

<211> 101

<212> DNA

<213> Escherichia coli

<400> 16

ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 60

gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc g 101

<210> 17

<211> 74

<212> DNA

<213> cauliflower mosaic virus

<400> 17

aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt 60

tgggaccact gtcg 74

<210> 18

<211> 210

<212> DNA

<213> Scrophularia mosaic Virus

<400> 18

aagacatcca ccgaagactt aaagttagtg ggcatctttg aaagtaatct tgtcaacatc 60

gagcagctgg cttgtgggga ccagacaaaa aaggaatggt gcagaattgt taggcgcacc 120

taccaaaagc atctttgcct ttattgcaaa gataaagcag attcctctag tacaagtggg 180

gaacaaaata acgtggaaaa gagctgtcct 210

<210> 19

<211> 264

<212> DNA

<213> glufosinate acetyltransferase gene (PAT)

<400> 19

gtcgacatgt ctccggagag gagaccagtt gagattaggc cagctacagc agctgatatg 60

gccgcggttt gtgatatcgt taaccattac attgagacgt ctacagtgaa ctttaggaca 120

gagccacaaa caccacaaga gtggattgat gatctagaga ggttgcaaga tagataccct 180

tggttggttg ctgaggttga gggtgttgtg gctggtattg cttacgctgg gccctggaag 240

gctaggaacg cttacgattg gaca 264

<210> 20

<211> 110

<212> DNA

<213> Streptomyces hygroscopicus

<400> 20

ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tagcctccgc gaccggttgt 60

agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg 110

<210> 21

<211> 149

<212> DNA

<213> Escherichia coli

<400> 21

ttaactatgc cggaatccat cgcagcgtaa tgctctacac cacgccgaac acctgggtgg 60

acgatatcac cgtggtgacg catgtcgcgc aagactgtaa ccacgcgtct gttgactggc 120

aggtggtggc caatggtgat gtcagcgtt 149

<210> 22

<211> 87

<212> DNA

<213> Agrobacterium tumefaciens

<400> 22

ccgacgccga tcacctaccg cgtgccgatg gcctccgcac aggtgaagtc cgccgtgctg 60

ctcgccggcc tcaacacgcc cggcatc 87

<210> 23

<211> 86

<212> DNA

<213> insect-resistant Gene (Cry 3A)

<400> 23

tccggttacg aggttctttt cctcactacc tatgctcaag ctgccaacac ccacttgttt 60

ctccttaagg acgctcaaat ctatgg 86

<210> 24

<211> 122

<212> DNA

<213> Bacillus thuringiensis

<400> 24

tacttggtgg agaacgcatt gaaaccggtt acactcccat cgacatctcc ttgtccttga 60

cacagtttct gctcagcgag ttcgtgccag gtgctgggtt cgttctcgga ctagttgaca 120

tc 122

<210> 25

<211> 76

<212> DNA

<213> insect-resistant Gene (Cry 1 Ab)

<400> 25

cgcgactgga tcaggtacaa ccagttccgc cgcgagctga ccctgaccgt gctggacatc 60

gtgagcctgt tcccca 76

<210> 26

<211> 133

<212> DNA

<213> insect-resistant Gene (Cry1A.105)

<400> 26

tcagaggtcc agggtttaca ggaggagaca ttcttcgtcg cacaagtgga ggaccctttg 60

cttacactat tgttaacatc aatggccaat tgccccaaag gtatcgtgca agaatccgct 120

atgcctctac tac 133

<210> 27

<211> 93

<212> DNA

<213> insect-resistant gene (Bt)

<400> 27

gaccctcaca gttttggaca ttgtgtctct cttcccgaac tatgactcca gaacctaccc 60

tatccgtaca gtgtcccaac ttaccagaga aat 93

<210> 28

<211> 109

<212> DNA

<213> Bacillus amyloliquefaciens

<400> 28

aatcagaagc acaagccctc ggctgggtgg catcaaaagg gaaccttgca gacgtcgctc 60

cggggaaaag catcggcgga gacatcttct caaacaggga aggcaaact 109

<210> 29

<211> 175

<212> DNA

<213> glufosinate resistance gene (Bar)

<400> 29

acaagcacgg tcaacttccg taccgagccg caggaaccgc aggagtggac ggacgacctc 60

gtccgtctgc gggagcgcta tccctggctc gtcgccgagg tggacggcga ggtcgccggc 120

atcgcctacg cgggcccctg gaaggcacgc aacgcctacg actggacggc cgagt 175

<210> 30

<211> 100

<212> DNA

<213> Soybean

<400> 30

ttatgttatg tgggtaagtc acctaagaca ctccacgtac ctacttgttg tctcttaccg 60

cggctttaat aaatcttctg cccttgttcc atatttacta 100

<210> 31

<211> 100

<212> DNA

<213> Agrobacterium

<400> 31

atcaatttat gtattacaca taatatcgca ctcagtcttt catctacggc aatgtaccag 60

ctgatataat cagttattga aatatttctg aatttaaact 100

<210> 32

<211> 100

<212> DNA

<213> cauliflower mosaic virus

<400> 32

aaaagcaggc tgtgttttcg gcaaacatcg ccacccatcg ctagtttttc taaaagtgtt 60

ctaagctagc ctggtaataa tctatacgag cttatatttc 100

<210> 33

<211> 100

<212> DNA

<213> Potato

<400> 33

cctagacttg tccatcttct ggattggcca acttaattaa tgtatgaaat aaaaggatgc 60

acacatagtg acatgctaat cactataatg tgggcatcaa 100

<210> 34

<211> 100

<212> DNA

<213> terminator of phosphoenolpyruvate carboxylase gene (T-PEPC)

<400> 34

ccaacatctc cccgctctac taagagctct agatctgttc tgcacaaagt ggagtagtca 60

gtcatcgatc aggaaccaga caccagactt ttattcatac 100

<210> 35

<211> 124

<212> DNA

<213> Octopus

<400> 35

cggtcaaacc taaaagactg attacataaa tcttattcaa atttcaaaag tgccccaggg 60

gctagtatct acgacacacc gagcggcgaa ctaataacgc tcactgaagg gaactccggt 120

tccc 124

<210> 36

<211> 100

<212> DNA

<213> corn

<400> 36

cttcgcttct tcatttgtcc ccttgcggag tttggcatcc attgatgccg ttacgctgag 60

aacagacaca gcagacgaac caaaagtgag ttcttgtatg 100

<210> 37

<211> 100

<212> DNA

<213> Soybean

<400> 37

aagtatcggg ctatttaact atgacttgag ctccatctat gaataaataa atcagcatat 60

gatgcttttg ttttgtgtac ttcaactgtc tgcttagcta 100

<210> 38

<211> 100

<212> DNA

<213> Bisui brachypodium

<400> 38

tttatagcga ctgacgctgt ggcaggcacg ctatcggagg ttacgacgtg gcgggtcact 60

cgacgcggag ttcacaggtc ctatccttgc atcgctcggc 100

<210> 39

<211> 100

<212> DNA

<213> corn

<400> 39

cctctcttac agcaaattgt ccgctgcccg tcgtccagat acaatgaacg tacctagtag 60

gaactctttt acacgctcgg tcgctcgccg cggatcggag 100

<210> 40

<211> 100

<212> DNA

<213> Octopus

<400> 40

gtcgtttatt tcggcgtgta ggacatggca accgggcctg aatttcgcgg gtattctgtt 60

tctattccaa ctttttcttg atccgcagcc attaacgact 100

<210> 41

<211> 121

<212> DNA

<213> human Ochrobactrum

<400> 41

gtcttcgtgt tgctggaacc gttgagttcg ctggtctcac tgctgctcct aactggaagc 60

gtgctcacgt tctctacact cacgctcgta agttgcttcc agctctcgct cctgccagtt 120

c 121

<210> 42

<211> 100

<212> DNA

<213> herbicide resistance Gene (GAT)

<400> 42

ctgcttcagg atactacagg aagttgggat tcagtgagca aggagaggtg ttcgatactc 60

ctccagttgg acctcacatc ctgatgtata agaggatcac 100

<210> 43

<211> 304

<212> DNA

<213> corn

<400> 43

atcctttgac atctgctccg aagcaaagtc agagcgctgc aatgcaaaac ggaacgagtg 60

ggggcagcag cgcgagcacc gccgcgccgg tgtccggacc caaagctgat catccatcag 120

ctcctgtcac caagagagaa atcgatgcca gtgcggtgaa gccagagccc gcaggtgatg 180

atgctagacc ggtggaaagc ataggcatcg ctgaaccggt ggatgctaag gctgatgcag 240

ctccggctac agatgcggcg gcgagtgctc cttatgacag ggaggataat gaacctggcc 300

cttt 304

<210> 44

<211> 101

<212> DNA

<213> wheat

<400> 44

gtcgcaggaa cagaggtgtt caaggcggcc gaaataggtt gccgcctgcg gcggaatcgc 60

cacccaccgt gaagttcacc gtttcgcaat ggaggaacac c 101

<210> 45

<211> 92

<212> DNA

<213> tomato

<400> 45

agaccacgag aacgatattt gcgaggtgac agttgtgaaa agcccaaggg aggactgcaa 60

agagagtgtg tctggatatg aaaaggcaag aa 92

<210> 46

<211> 67

<212> DNA

<213> sesame

<400> 46

ttaccagagg gctagggacc ttcctcgcag gtgcaacatg cgaccccagc aatgccaatt 60

ccgagtt 67

<210> 47

<211> 81

<212> DNA

<213> Rice

<400> 47

ttgcgcctga acggatatct ttcagtttgt aaccaccgga tgacgcacgg acggctcgga 60

tcatcccgaa aagatcaacc g 81

<210> 48

<211> 91

<212> DNA

<213> alfalfa

<400> 48

gatcagtgaa cttcgcaaag tactcggtta gtagacagtg aatgctcctg tgatctgccc 60

atgcactcat gttgtagtgt tcacgtcgtt g 91

<210> 49

<211> 68

<212> DNA

<213> flax

<400> 49

gctcaaccca gtcaccacct tcccttcaac acgctccctc aacaacttct cctccagatc 60

tcctcgca 68

<210> 50

<211> 88

<212> DNA

<213> peanut

<400> 50

gcgcagagaa agcagaggcc gggaagagga gcaacagagg cacaatccct actactttca 60

ctcccagagc attaggtcga gacatgag 88

<210> 51

<211> 118

<212> DNA

<213> Soybean

<400> 51

gccctctact ccacccccat ccacatttgg gacaaagaaa ccggtagcgt tgccagcttc 60

gccgcttcct tcaacttcac cttctatgcc cctgacacaa aaaggcttgc agatgggc 118

<210> 52

<211> 76

<212> DNA

<213> Cotton

<400> 52

attgtgatgg gacttgagga agaattcgga atcacggtgg aagaggacaa tgcacaatcc 60

atcacaactg ttcaag 76

<210> 53

<211> 74

<212> DNA

<213> pawpaw

<400> 53

ccatgcggat cctcccattt cccttcatcc attcccactc ttgagagtta gctagtgtta 60

caatggctac gatg 74

<210> 54

<211> 118

<212> DNA

<213> sugar beet

<400> 54

gacctccata ttactgaaag gaagccaaaa gggatcaatt aagtgctcta cgaagtttaa 60

agtatgtgcc gctctcaaga ctgaacatgg cactgtgaac aggatggagc aattactc 118

<210> 55

<211> 101

<212> DNA

<213> oil seed rape

<400> 55

ggccagggtt tccgtgatat gcaccagaaa gtggagcaca taaggactgg ggacaccatc 60

gctacacatc ccggtgtagc ccaatggttc tacaacgacg g 101

<210> 56

<211> 21

<212> DNA

<213> Artificial sequence

<400> 56

gtcgtttatt tcggcgtgta g 21

<210> 57

<211> 19

<212> DNA

<213> Artificial sequence

<400> 57

taatggctgc ggatcaaga 19

<210> 58

<211> 19

<212> DNA

<213> Artificial sequence

<400> 58

tgcccgtcgt ccagataca 19

<210> 59

<211> 21

<212> DNA

<213> Artificial sequence

<400> 59

ccgagcgtgt aaaagagttc c 21

<210> 60

<211> 18

<212> DNA

<213> Artificial sequence

<400> 60

gctgtggcag gcacgcta 18

<210> 61

<211> 23

<212> DNA

<213> Artificial sequence

<400> 61

gcaaggatag gacctgtgaa ctc 23

<210> 62

<211> 25

<212> DNA

<213> Artificial sequence

<400> 62

aagtatcggg ctatttaact atgac 25

<210> 63

<211> 24

<212> DNA

<213> Artificial sequence

<400> 63

gctaagcaga cagttgaagt acac 24

<210> 64

<211> 19

<212> DNA

<213> Artificial sequence

<400> 64

cttcatttgt ccccttgcg 19

<210> 65

<211> 21

<212> DNA

<213> Artificial sequence

<400> 65

tcacttttgg ttcgtctgct g 21

<210> 66

<211> 19

<212> DNA

<213> Artificial sequence

<400> 66

atttcaaaag tgccccagg 19

<210> 67

<211> 23

<212> DNA

<213> Artificial sequence

<400> 67

cttcagtgag cgttattagt tcg 23

<210> 68

<211> 24

<212> DNA

<213> Artificial sequence

<400> 68

tagacttgtc catcttctgg attg 24

<210> 69

<211> 24

<212> DNA

<213> Artificial sequence

<400> 69

tgcccacatt atagtgatta gcat 24

<210> 70

<211> 25

<212> DNA

<213> Artificial sequence

<400> 70

acataatatc gcactcagtc tttca 25

<210> 71

<211> 20

<212> DNA

<213> Artificial sequence

<400> 71

tcagctggta cattgccgta 20

<210> 72

<211> 18

<212> DNA

<213> Artificial sequence

<400> 72

caaacatcgc cacccatc 18

<210> 73

<211> 26

<212> DNA

<213> Artificial sequence

<400> 73

ttattaccag gctagcttag aacact 26

Claims (5)

1. A nucleic acid molecule comprising the nucleic acid sequence of the following genes:

(1) Transgenic breeding exogenous genes;

(2) A crop internal standard gene;

wherein the content of the first and second substances,

the transgenic breeding exogenous gene comprises the following genes: T-G7 terminator, T-E9 terminator, T-CaMV35S terminator, T-NOS terminator, PVYep gene, P-SSuAra promoter, P-NOS promoter, PMI gene, PLRVrep gene, P-ubitin promoter, P-TA29 promoter, P-RACT promoter, NPTII gene, P-CaMV35S promoter, P-FMV35S promoter, PAT gene, HPT gene, GUS gene, EPSPS gene, cry3A gene, cry1Ac gene, cry1Ab gene, cry1A.105 gene, bt gene, barnase gene, bar gene, T-VSP terminator, T-Tr7 terminator, T-TML terminator, T-pinII terminator, T-PEPC terminator, T-OCS3 terminator, T-GLB1 terminator, UTRT-7 sP promoter, P-MTL promoter, GLOCS 1 promoter, GLOCS 4 GAX gene and GAOCS promoter;

the standard gene in the crops is derived from corn, rice, cotton, rape, wheat, soybean, alfalfa, pawpaw, tomato, beet, sesame, flax and peanut;

the sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

2. A plasmid comprising the nucleic acid molecule of claim 1.

3. The plasmid of claim 2, wherein the nucleic acid molecule of claim 1 is inserted into an E.coli vector.

4. Use of the nucleic acid molecule of claim 1 or the plasmid of any one of claims 2 to 3 for the qualitative screening and detection of transgenic crops and/or foreign genes.

5. The use according to claim 4, in particular as a positive control or a positive standard for the detection of transgenic crops and/or foreign genes.

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