CN1618960A - Method of rejecting marking gene in trans gene plant - Google Patents

Abstract

A method for rejecting the marker gene from transgenic plant in order to increase its safety includes using lambda bacteriophage attp-specific recombinant site and its recombinant enzyme gene INT to configure the plant transfer carriers pMFC containing INT and the pMFB containing target gene and marker gene, preparing their transferred plants respectively, crossing for specifically cutting out the marker gene, screening the transgenic plants without said marker gene, and determinating.

Description

A kind of method of rejecting marker gene in the transgenic plant

Technical field

The invention belongs to plant genetic engineering field.Be specifically related to a kind of method of rejecting marker gene in the transgenic plant, the separating clone, vector construction, plant genetic that it comprises genes involved and structural element transforms and aspects such as the field screening of marker-free transfer-gen plant and Molecular Identification.

Background technology

In the plant transgene process, because foreign DNA is absorbed by vegetable cell and to be integrated into the frequency of Plant Genome very low, for effective choice, identification and evaluation cell transformed and plant, when importing goal gene, need to introduce selectable marker gene to give recipient plant specific resistance.At present, used marker gene mainly is the gene of coding microbiotic or weedicide.Along with the acquisition of transgenic plant, marker gene no longer has utility value, but it still constantly instructs the synthetic of enzyme in plant, consume the cell resource, and because the Biosafety problem, it also forms certain pressure to consumer psychology.The transgenosis system of exploitation marker-free or rejecting marker gene, the transgenic plant of creating marker-free are significant.

At present, obtain the method for marker-free transgenic plant both at home and abroad, can reduce avoidance strategy and reject totally 4 kinds of strategy two big classes, be i.e. marker-free conversion method, physiological metabolism gene Selection method, goal gene carrier and marker gene carrier cotransformation method and marker gene excision method.First method needs directly the non-selection regrowth that obtains to be detected evaluation by pcr amplification, and later stage selection workload is big, expense is high.The second method limitation is big, and the physiological metabolism genes involved that changes over to influences plant individual and grows, and should not adopt.Comparatively practical is back two kinds of methods, promptly two carrier cotransformation methods and marker gene excision method, and the latter is divided into transposon method, site-specific recombination method etc. again.

The site-specific recombination method is that marker gene is placed between the specific recombination site, by the recombinase catalysis in this site of corresponding specific recognition, produces special reorganization or special excision, utilizes this characteristic marker gene can be rejected.In the rejecting process of transgenic plant marker gene, can utilize the characteristics of recombination site and recombinase to make up special plant conversion carrier system.Being about to marker gene places between two special recombination sites on the T-DNA fragment; Simultaneously, corresponding recombinase gene is building up on another plant conversion carrier, with the transformed plant hybridization that above two kind of plant conversion carriers obtain, recombinase is expressed the special excision that can realize marker gene in the hybrid.The five class site-specific recombination systems of microorganism have at present mainly been found to derive from, the R/RS system of the Cre-loxP system of bacteriophage P1, yeast plasmid FLP-FRT system, lambda particles phage attB-P system, Zygosaccharomyces rouxii and the Gin-gix recombinase system of Mu phage.The two kinds of methods in back yet there are no the report of application in plant.

The lox site of Cre-loxP system is made of the 34bp distinguished sequence, and it is core sequence that 8bp is wherein arranged, and the Cre recombinase is the protein of a 38.5KD, the reorganization of sequence between narrow spectrum identification and the catalysis two loxP sites.Utilized the marker gene in the Cre-loxP specific site recombination system excision transgenic plant on plants such as tobacco, Arabidopis thaliana, to succeed.But the excision of the Cre of this system recombinase is not thorough, efficient is very low, also need a tissue culture regeneration process of taking turns again, relatively poor (the Gleave of feasibility, A.P., Mitra, D.S.and Morris, B.A.M., 1999, Selectable marker-free transgenic plants without sexual crossing:transientexpression of Cre recombinase and use of a conditional lethal dominant gene.Plant Mol.Biol.40,223-235) (Zuo J, Niu Q.W., 2001, Chemical-regulated, site-specific DNA excision in transgenic plants.Nature Biotech.19:157-161).

The FRT site also is to be made of the 34bp distinguished sequence, and wherein 8bp is a core sequence, and FLP is its single-minded recombinase protein of 48KD.Lloyd etc. (1994) are applied to tobacco with zymic FLP-FRT site recombination system, experiment is found, the FLP recombinase has expression activity in plant tissue, can excise the GUS marker gene, but the recombinase expression efficiency is lower, (Lloyd, A.M., Davis, R.W., 1994, Functional expression of the yeast FLP/FRTsite-specific recombination system in Nicotiana tabacum.Mol.Gen.Genet.242:653-657).

The present invention has proposed to utilize lambda particles phage attB-P system to realize the rejecting of marker gene first, can overcome the inefficient deficiency of other elimination methods.At present, patent (a kind of method of position-point recombination counter-cloning genes and utilization thereof, the application number: 01130855.9), different with the present invention that utilizes the att specific site to carry out vector construction and gene clone arranged.The document that utilizes the att specific site independently to recombinate to realize the rejecting of marker gene is also arranged, but very low (the Zubko E of its efficient, Scutt C, Meyer P., 2000, and do not apply for a patent Intrachromosomal recombination between attP regionsas a tool to remove selectable marker genes from tobacco transgenes.NatureBiotech.18:442-445).

The research of introducing INT recombinase identification att site excision marker gene yet there are no relevant report and patent.The tract in attB site is lacked (21bp), core sequence 7bp, and the attP site is made of the 352bp distinguished sequence, and core sequence also is 7bp.λ-att recombinase gene (INT), but specific recognition att-B and att-P site and carry out the special excision of sequence between the site.

Summary of the invention

The objective of the invention is to, utilize the special recombination site of lambda particles phage recognition sequence (attP) with and recombinase gene INT, make up special plant conversion carrier, reject the marker gene in the transgenic plant, obtain only to contain the transfer-gen plant of goal gene, improve the biological safety of transgene agricultural product.

The present invention is achieved through the following technical solutions:

A kind of method of rejecting marker gene in the transgenic plant is utilized special recombination site of lambda particles phage recognition sequence (attP) and recombinase gene (INT) thereof, makes up plant conversion carrier pMFC and pMFB; Said pMFC contains INT, and said pMFB contains goal gene and selectable marker gene, and this selectable marker gene is between two attP sites; Obtain the transformed plant of pMFC and two kinds of carriers of pMFB respectively by Agrobacterium tumefaciens mediated or other transgenic methods; Said transformed plant is hybridized, filter out the transfer-gen plant of rejecting marker gene then from the filial generation segregating population, this plant is identified further by PCR or Southern hybrid molecule and confirms.

The present invention implements according to the following step:

(1) according to lambda particles phage genome sequence design primer, obtains recombinase gene (INT) and recognition sequence (attP) thereof respectively;

(2) make up plant conversion carrier pMFC and pMFB respectively;

(3) by the method for agriculture bacillus mediated or particle gun, utilize the carrier that makes up that plant is carried out genetic transformation, obtain transfer-gen plant, further the selfing purifying obtains the individual plant or the strain system of this gene pure;

(4) with above-mentioned two kinds of transgenosis homozygous lines or hybrid material hybridization, reject marker gene, separating the individual plant that the offspring screens does not have resistance marker;

(5) from the plant that step (4) is obtained, extract total DNA, carry out pcr amplification simultaneously with the special primer of marker gene and goal gene and detect, or utilize the label probe of marker gene and goal gene to carry out Southern hybridization and detect.

The detailed description of invention is as described in the following steps:

1. the clone of the required element of lambda particles phage locus specificity recombination system:

According to lambda particles phage genome sequence (U.S. state-run biotechnology information center, gene number of registration: NC 001416) design primer, obtain recombinase gene (INT) and recognition sequence (attP) thereof respectively.The positive anti-primer of amplification INT is respectively: INTF:5 '-ATGGGAAGAAGGCGAAGTC-3 ' INTB:5 '-TTATTTGATTTCAATTTTGTCCCAC-3 ', the positive anti-primer in amplification attP site is respectively: attPF:5 '-GATTGCGAGGCTTTGTGCTT-3 ' attPB:5 '-GGCAGGGAGTGGGACAAAAT-3 '

The PCR reaction system is: in 20 μ L reaction systems, add 13.65 μ L ddH respectively ₂O, 1.5mM/L MgCl ₂, 0.2mM/L dNTPs, 0.5 μ M/L forward and reverse primer, 0.25 μ L lambda particles phage lysate, 0.5U TqDNA polysaccharase.The reaction cycle parameter of amplification INT gene is: 94 ℃ of pre-sex change 3min, and 94 ℃ of 1min then, 62 ℃ of 1min, 72 ℃ of 1min30s reacts 30 circulations, last 72 ℃ of extension 10min.Amplification attP reaction cycle parameter: 94 ℃ of pre-sex change 3min, 94 ℃ of 1min then, 56 ℃ of 1min, 72 ℃ of 40s reacts 30 circulations, last 72 ℃ of extension 10min.

The PCR specific fragment of INT and attP reclaims through cutting glue.The former fragment reclaims product and is connected with pMD18-T carrier (Takara company product), and reclaiming product, the latter is connected with pGEM -T Easy carrier (Promega company product), difference transformed into escherichia coli DH5 α, the extracting plasmid carries out enzyme and cuts the rear electrophoresis Analysis and Identification, obtain to insert the segmental recombinant clone of purpose, respectively called after pMDTINT and pGTP.Further sequential analysis proves, inserts fragment among recombinant clone pMDT INT and the pGTP and is respectively INT and attP site.Its sequence is as follows respectively:

1. INT sequence (1071bp):

ATGGGAAGAAGGCGAAGTCATGAGCGCCGGGATTTACCCCCTAACCTTTATATAAGAAACAATGGATATTAC

TGCTACAGGGACCCAAGGACGGGTAAAGAGTTTGGATTAGGCAGAGACAGGCGAATCGCAATCACTGAAGCTATA

CAGGCCAACATTGAGTTATTTTCAGGACACAAACACAAGCCTCTGACAGCGAGAATCAACAGTGATAATTCCGTT

ACGTTACATTCATGGCTTGATCGCTACGAAAAAATCCTGGCCAGCAGAGGAATCAAGCAGAAGACACTCATAAAT

TACATGAGCAAAATTAAAGCAATAAGGAGGGGTCTGCCTGATGCTCCACTTGAAGACATCACCACAAAAGAAATT

GCGGCAATGCTCAATGGATACATAGACGAGGGCAAGGCGGCGTCAGCCAAGTTAATCAGATCAACACTGAGCGAT

GCATTCCGAGAGGCAATAGCTGAAGGCCATATAACAACAAACCATGTCGCTGCCACTCGCGCAGCAAAATCAGAG

GTAAGGAGATCAAGACTTACGGCTGACGAATACCTGAAAATTTATCAAGCAGCAGAATCATCACCATGTTGGCTC

AGACTTGCAATGGAACTGGCTGTTGTTACCGGGCAACGAGTTGGTGATTTATGCGAAATGAAGTGGTCTGATATC

GTAGATGGATATCTTTATGTCGAGCAAAGCAAAACAGGCGTAAAAATTGCCATCCCAACAGCATTGCATATTGAT

GCTCTCGGAATATCAATGAAGGAAACACTTGATAAATGCAAAGAGATTCTTGGCGGAGAAACCATAATTGCATCT

ACTCGTCGCGAACCGCTTTCATCCGGCACAGTATCAAGGTATTTTATGCGCGCACGAAAAGCATCAGGTCTTTCC

TTCGAAGGGGATCCGCCTACCTTTCACGAGTTGCGCAGTTTGTCTGCAAGACTCTATGAGAAGCAGATAAGCGAT

AAGTTTGCTCAACATCTTCTCGGGCATAAGTCGGACACCATGGCATCACAGTATCGTGATGACAGAGGCAGGGAG

TGGGACAAAATTGAAATCAAATAA。

2. the attP sequence is (353bp):

GATTGCGAGGCTTTGTGCTTCTCTGGAGTGCGACAGGTTTGATGACAAAAAATTAGCGCAAGAAGACAAAAA

TCACCTTGCGCTAATGCTCTGTTACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGACTGCATATGTTG

TGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGT

TTCTCGTTCAGCTTTTTTATACTAAGTTGGCATTATAAAAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGT

CACTATCAGTCAAAATAAAATCATTATTTGATTTCAATTTTGTCCCACTCCCTGCC。

2. construction of expression vector:

Make up plant conversion carrier pMFC respectively, its structure as shown in Figure 2, pMFB, its structure as shown in Figure 3.

The T-DNA district of pMFC contains lambda particles phage recombinase gene (INT) and selectable marker gene (BAR, NPTII or HPT etc.).Its building process is as follows: with SalI and SacI pMDT INT is carried out enzyme and cut, recovery INT fragment is connected with the big fragment that XhoI, SacI enzyme are cut pMV (being transformed by pBI121), thereby INT is building up in the plant expression vector, names to be pMFC.

Goal gene and selectable marker gene (can be any selectable marker gene) are contained in the T-DNA district of pMFB, and selectable marker gene is between lambda particles phage locus specificity recombination site attP forward tumor-necrosis factor glycoproteins.It is as follows that it makes up concrete steps: with the pBI121 plasmid (U.S. state-run biotechnology information center, gene number of registration: AF485783) be underlying carrier, it is cut with the PmeI enzyme and handles with the CIAP dephosphorylation, cut pGTP with the EcoRI enzyme and obtain the attP fragment, and with T4DNA polysaccharase end-filling.Thereby both reclaim product and connect the PmeI site of fragment attP being inserted pBI121, name to be pPBIP.With HindIII the pPBIP enzyme is cut, T4 archaeal dna polymerase end-filling is also handled with the CIAP dephosphorylation, and cut pGTP with the EcoRI enzyme and obtain the attP fragment, and with T4 archaeal dna polymerase end-filling.Thereby both reclaim product and connect the HindIII site that attP is inserted pPBIP, are built into carrier pMFB.Its goal gene is a gus gene, utilizes any goal gene of the replaceable one-tenth of restriction enzyme site when specifically implementing.

3. the genetic transformation of plant:

By agriculture bacillus mediated or other transgenic methods (for example particle gun method) pMFC and pMFB are changed over to plant (for example tomato) respectively,, obtain the transfer-gen plant or the strain system of isozygotying by Screening and Identification and purifying.The pMFC plant transformed is universal hybrid strain, can hybridize with the plant that contains the various objectives gene that the pMFB carrier transforms, thus the marker gene of excising each transgenic line.

Adopt Agrobacterium tumefaciens mediated genetic transformation step as follows: to prepare aseptic seedling cotyledon or hypocotyl explant, go up tobacco suspension cell nurse overnight incubation (also can nurse cultivation) in KCMS.Be diluted to the During Agrobacterium explant 5 minutes of O.D. ≈ 0.3 in order to MS0.2, filter paper blots, and tieback was cultivated 2 days altogether to pre-culture medium (nurse substratum), shifted and selected induced bud regeneration in the regeneration culture medium.Treat that regeneration bud grows to about 1cm, is transferred to root media.Acclimatization and transplants after taking root.

4. the rejecting of marker gene:

Two kinds of transgenosis homozygous lines (also can be hybrid material) hybridization, INT expresses in half-blood seed or the plant, discerns the attP site and sequence between two sites is carried out special excision, rejects marker gene.The hybrid selfing its offspring separate, by (for example: kantlex 100mg/L seedling being sprayed certain density selective marker microbiotic, perhaps the Basta weedicide 1%), screening does not have the individual plant of resistance marker in separating the offspring, and what obtain mostly is the transfer-gen plant of rejecting token-based thereby containing goal gene.

5. Molecular Identification:

According to carrier T-DNA plot structure, at marker gene and goal gene design special primer or synthetic specific mark probe, carry out respectively pcr amplification or (with) Southern hybridizes evaluation, can further confirm the verity of the transfer-gen plant of the rejecting marker gene that obtains.The visible schema of concrete steps as shown in Figure 1.

Description of drawings

Fig. 1. be schema of the present invention.

Fig. 2 is the plant conversion carrier pMFC of the INT of containing of the present invention;

Fig. 3 is the plant conversion carrier pMFB that rejects marker gene of the present invention, and its marker gene and goal gene are replaceable

Fig. 4 is the plant conversion carrier pMFB1 that can reject marker gene BAR among one of them embodiment of the present invention, and its goal gene is BT;

Fig. 5 is the plant conversion carrier pMFB2 that can reject marker gene BAR among one of them embodiment of the present invention, and its goal gene is IPT;

Fig. 6 is the embodiment of the invention 1 Molecular Identification result.M among the figure: λ/HindII molecular weight marker, 1,2: reject the marker gene plant, 3: the homozygous plants of not rejecting marker gene; 1.8kb fragment: BT, 1.5kb fragment: the fragment that contains marker gene BAR.

Fig. 7 is the embodiment of the invention 2 Molecular Identification results.Among the figure 1: do not reject the homozygous plants of marker gene, 2: reject the marker gene plant; 1200bp fragment: P _SAG12-IPT mosaic gene fragment, 540bp fragment: marker gene BAR fragment.

Embodiment

Embodiment 1: marker-free changes the acquisition of the pest-resistant tomato of BT

BT derives from bacillus thuringiensis, and the special poisoning lepidopteran class insect of its expressing protein energy is harmless to people and higher animal.It is significant for the breeding for pest resistance of tomato that BT imports tomato.Plant conversion carrier and the method for utilizing the present invention to make up have obtained the commentaries on classics BT tomato plant of marker-free, and step is as follows:

1. vector construction:

With the pMFB carrier is underlying carrier, and it is carried out double digestion with SmaI and EcoRI, with connecting certainly behind the T4 archaeal dna polymerase end-filling, constitutes pMFO.Plasmid pUBC2 is carried out BamHI, reclaim small segment, wherein comprise BT, be inserted into the BamHI site of pMFO, thereby BT is imported carrier pMFB.BT replaces goal gene wherein, forms new plant conversion carrier, names into pMFB1 (Fig. 4).This marker gene is between two attP sites.

2. plant genetic transforms:

Utilize agriculture bacillus mediated genetic transformation method, carrier pMFC and carrier pMFB1 are changed over to the stable tomato strain system of proterties respectively A53, obtain transfer-gen plant.According to special primer and the specific probe of marker gene and BT,, the T0 that obtains has been carried out Molecular Identification for transfer-gen plant, further transfer-gen plant is determined by PCR and Southern hybridizing method.

3. the purifying of plant:

For with the pMFB1 plants transformed, results first-generation seed is T1 generation.Because T1 generation separates, and utilizes the weedicide spraying method that the T1 of transfer-gen plant is screened for plant, the plant with this Herbicid resistant is pressed individual plant from T1 for resistant plant and gathers in the crops seed for containing genetically modified isozygotying and heterozygous plant, i.e. T2 generation.Continue to utilize the weedicide spraying method to select T2 for plant, in no longer isolating strain of T2 generation system, prove that its corresponding T1 is the individual plant that isozygotys for plant, this strain is a homozygous lines; Equally, to carrying out same screening with pMFC carrier plants transformed, obtained the transgenosis homozygous plants, screening of medicaments is a kantlex, spraying concentration 100mg/L.

4. the rejecting of marker gene:

The homozygous lines that transforms with carrier pMFC and carrier pMFB is hybridized, obtain first generation of hybrid F1.The F1 seed form early stage, the INT recombinase starts expresses, identification attP recombination site, and the marker gene BAR between this two site of specific excision.Because the BT fragment of excision marker gene is positioned on the different karyomit(e) with the INT fragment, according to the law of segregation, they will be at F2 for separation, thereby screen in for plant at F2, eliminate and to have the plant of kalamycin resistance and the plant that weedicide is had resistance, remaining basic for rejecting token-based thereby only possessing the transfer-gen plant of BT;

5. Molecular Identification:

Extract plant leaf DNA to be identified, respectively with the special primer of BT, marker gene NPTII and BAR, identify by pcr amplification, or carry out Southern hybridization with the specific probe of BT, NPTII and BAR gene fragment and identify, BT is accredited as the positive and the negative plant of marker gene can be defined as rejecting the commentaries on classics BT tomato plant of marker gene.Through identifying, obtained to reject the commentaries on classics BT tomato plant of marker gene.The PCR qualification result as shown in Figure 6.

Embodiment 2: the anti-leaf senile of marker-free changes the acquisition of IPT tomato

The IPT gene is from the IPT (claiming the tmr gene again) in agrobacterium tumefaciens T-DNA district, sequences encoding isopentenyl transferase, a most important step rate-limiting reaction in the biosynthesizing of catalysis phytokinin, promptly 5 '-adenosine phosphate and Δ 2-isopentenyl pyrophosphate chemical combination generate N6-[Δ 2-isopentene]-5 '-adenosine phosphate (isopentenyl gland purine, reaction iPA).The old and feeble specific promoter P of this gene and Arabidopsis leaf _SAG12Be built into mosaic gene P _SAG12-ITP has the self regulating and control deferring senility, has been changed over to crops such as paddy rice, tomato, has tangible anti-leaf senile effect.The step of the anti-leaf senile transgenic Fructus Lycopersici esculenti of acquisition marker-free is as follows:

1. vector construction:

With the pMFB carrier is underlying carrier, with HindIII and SacI plasmid pMFB is carried out double digestion, reclaims big fragment.Plasmid pSG529 is carried out same double digestion obtain mosaic gene P _SAG12-IPT, thus both connections build up to pMFB with mosaic gene, form new plant conversion carrier, and called after pMFB2 (Fig. 5), selection markers gene are anti-herbicide gene BAR, can resist weedicide Basta.This marker gene is between two attp sites.

2. plant genetic transforms:

Utilize Agrobacterium tumefaciens mediated genetic transformation method, carrier pMFC and carrier pMFB2 are changed over to the stable tomato strain of proterties respectively A53In, obtain genetically modified plant.With old and feeble specific promoter P _SAG12Special primer carry out PCR and detect, further proof has obtained the conversion tomato plant of this carrier.

3. the purifying of plant:

For the tomato plant that transforms with pMFB2, results first-generation seed is T1 generation.Because T1 generation separates, and utilizes the weedicide spraying method that the T1 of transfer-gen plant is screened for plant, the plant with this Herbicid resistant is pressed individual plant from T1 for resistant plant and gathers in the crops seed for containing genetically modified isozygotying and heterozygous plant, i.e. T2 generation.Continue to utilize the weedicide spraying method to select T2 for plant, in no longer isolating strain of T2 generation system, prove that its corresponding T1 is the individual plant that isozygotys for plant, this strain is a homozygous lines; Equally, to carrying out same screening with pMFC carrier plants transformed, obtained the homozygous plants of this gene, screening of medicaments is the 100mg/L kantlex;

4. the rejecting of marker gene:

The homozygous lines that transforms with carrier pMFC and carrier pMFB2 is hybridized, obtain first generation of hybrid F1.The F1 seed form early stage, INT starts expression, discern two attP sites, and specificity is excised the marker gene BAR between this two site.The P of excision marker gene _SAG12-IPT fragment is positioned on the different karyomit(e) with the INT fragment, they separated in F2 generation, utilize to select microbiotic to screen in for plant at F2, eliminate out plant with kalamycin resistance and the plant that weedicide is had resistance, remaining basic for rejecting token-based thereby only possessing P _SAG12The transfer-gen plant of-IPT mosaic gene;

5. Molecular Identification

Extract plant leaf DNA to be identified, respectively with promotor P _SAG12The special primer of (identify IPT), marker gene NPTII and BAR is identified by pcr amplification, and IPT is accredited as the positive and commentaries on classics P that the negative plant of marker gene can be defined as rejecting marker gene _SAG12-IPT tomato plant.The result shows, has obtained the commentaries on classics P of marker-free _SAG12-IPT tomato plant.The PCR qualification result as shown in Figure 7.

Claims (2)

1, a kind of method of rejecting marker gene in the transgenic plant is characterized in that utilizing special recombination site of lambda particles phage attP and recombinase gene (INT) thereof, makes up plant conversion carrier pMFC and pMFB; Said pMFC contains INT, and said pMFB contains goal gene and selectable marker gene, and this selectable marker gene is positioned between two recognition sequences (attP) site; Obtain the transformed plant of pMFC and two kinds of carriers of pMFB respectively by Agrobacterium tumefaciens mediated or other transgenic methods; Said transformed plant is hybridized, filter out the transfer-gen plant of rejecting marker gene then from the filial generation segregating population, this plant is identified further by PCR or Southern hybrid molecule and confirms.

2, method according to claim 1, its step is:

(1) according to lambda particles phage genome sequence design primer, obtain recombinase gene and recognition sequence thereof respectively, the positive anti-primer of amplification INT is respectively: the positive anti-primer in INTF:5 '-ATGGGAAGAAGGCGAAGTC-3 ' INTB:5 '-TTATTTGATTTCAATTTTGTCCCAC-3 ' attP site is respectively: attPF:5 '-GATTGCGAGGCTTTGTGCTT-3 ' attPB:5 '-GGCAGGGAGTGGGACAAAAT-3 '

(2) make up plant conversion carrier pMFC and pMFB respectively: with SalI and SacI pMDT INT is carried out enzyme and cut, recovery INT fragment is connected with the big fragment of the pMV that XhoI, SacI enzyme are cut, names to be pMFC; With the pBI121 plasmid is underlying carrier, it is cut with the PmeI enzyme and handles with the CIAP dephosphorylation, cut pGTP with the EcoRI enzyme and obtain the attP fragment, and with T4DNA polysaccharase end-filling, thereby both are reclaimed product connect the PmeI site of fragment attP being inserted pBI121, name and be pPBIP, with HindIII the pPBIP enzyme is cut, T4 archaeal dna polymerase end-filling also uses the CIAP dephosphorylation to handle, cut pGTP with the EcoRI enzyme and obtain the attP fragment, and with T4 archaeal dna polymerase end-filling, thereby both reclaim product and connect the HindIII site that attP is inserted pPBIP, be configured to carrier pMFB, any goal gene of the replaceable one-tenth of gene GUS wherein;

(3) by the method for agriculture bacillus mediated or particle gun, utilize the carrier that makes up that plant is carried out genetic transformation, obtain transfer-gen plant, further the selfing purifying obtains the individual plant or the strain system of this gene pure;

(4) with above-mentioned two kinds of transgenosis homozygous lines or hybrid material hybridization, reject marker gene, separating the individual plant that the offspring screens does not have resistance marker;

(5) from the plant that step (4) is obtained, extract total DNA, carry out pcr amplification simultaneously with the special primer of marker gene and goal gene and detect, or utilize the label probe of marker gene and goal gene to carry out Southern hybridization and detect.

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