CN1298850C - Method capable of auto rejecting marking gene in trans gene plant - Google Patents

Abstract

The present invention belongs to the field of plant genetic engineering. A plant conversion vector pAMF is constructed by using lambda bacteriophage attP specific recombination sites and a recombinase gene INT thereof; the plant conversion vector pAMF comprises the recombinase gene INT, a purpose gene and a selective mark gene, wherein the INT is driven by a decasone specific inducible promoter, the INT and the selective mark gene are jointly positioned between the two attP sites, and the purpose gene is positioned at the external side of the attP sites. A conversion plant or callus of the vector is obtained by agricultural bacillus conversion or other methods, the expression of the INT is induced by decasone to specifically cut a mark gene and the decasone between the two attP sites after the conversion plant is purified in a selfing way, a plant without the mark gene us selected from a separation generation of the conversion plant after selfing, or the callus is induced by the decasone to cause the expression of the INT to specifically cut the decasone and the mark gene between the two sites; the callus cut with the mark gene does not have mark gene resistance, non-resistance callus is selected to be induced into seedlings regarded as transgenic plants without the mark gene, and the transgenic plants without the mark gene are confirmed finally by PCR or Southern molecular detection.

Description

A kind of method of can the render transgenic plant independently rejecting marker gene

Technical field

The invention discloses a kind of can the render transgenic plant in the autonomous method of rejecting marker gene, belong to plant genetic engineering field.Separating clone, the related vector that the present invention is specifically related to genes involved and structural element makes up, plant genetic transforms and methods such as the field screening of marker-free transfer-gen plant and Molecular Identification.Purpose mainly is to create a kind of method of new high efficiency, rejects the entrained marker gene of transgenic plant body, obtains only to contain the transfer-gen plant of goal gene, improves the biological safety of transgene agricultural product.

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 corresponding enzymic synthesis in plant, consume the cell resource, and the Biosafety problem of marker gene also 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, the method that obtains the marker-free transgenic plant can reduce avoidance strategy and reject strategy two big classes, the former comprises marker-free conversion method, physiological metabolism gene Selection method, goal gene carrier and marker gene carrier cotransformation method, and the latter comprises marker gene excision method.The marker-free conversion method needs directly the non-selection regrowth that obtains to be detected evaluation by pcr amplification, and the later stage selects workload big, the expense height, and limitation is big.The physiological metabolism genes involved that physiological metabolism gene Selection method need change over to influences plant individual and grows, and also should not adopt.Comparatively practical is back two kinds of methods, promptly two carrier cotransformation methods and marker gene excision method (being the special recombination method in site) etc.

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, 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, two kinds of transformed plant hybridization that obtain, recombinase gene is expressed the special excision that realizes marker gene.The five class site-specific recombination systems of microorganism have at present mainly been found to derive from, i.e. Gin-gix recombinase system of the R/RS system of the Cre-loxP system of bacteriophage P1, yeast plasmid FLP-FRT system, Zygosaccharomyces rouxii, Mu phage and lambda particles phage attB-P system.

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 marker gene in the Cre-loxP specific site recombination system excision transgenic plant tobacco, (Gleave succeeds on the plants such as Arabidopis thaliana, A.p, Mitra, D.S.and Morris, B.A.M., 1999, Selectable marker-free transgenic plants without sexual crossing:transient expression of Crerecombinase 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).But there is report to point out, the excision of introducing transient expression Cre recombinase is not thorough in the Cre-loxP system, efficient is very low, also need a tissue culture regeneration process of taking turns again, so relatively poor (Zuo J of its feasibility, Niu Q.W., 2001, Chemical-regulated, site-specific DNA excision intransgenic plants.Nature Biotech.19:157-161).

Yeast 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, Functionalexpression of the yeast FLP/FRT site-specific recombination system in Nicotiana tabacum.Mol.Gen.Genet.242:653-657).And the Gin-gix recombinase system of the R/RS system of Zygosaccharomyces rouxii and Mu phage then with Cre-loxP and FLP-FRT system similarity, but yet there are no the report of application in plant.

Above method not only efficient is low, and all needs by the separate chosen process of recombinase excision with the offspring, and the cycle is longer, and workload is big.Utilize lambda particles phage attB-P system not only can realize the autonomous rejecting of marker gene, and the cycle is short, is convenient to artificial control, can also be applied to asexually propagated plant.The tract in attB site is lacked (21bp), core sequence 7bp, and the attP site is made of the 352bp distinguished sequence, and is the same with the attB site, and core sequence also is 7bp.λ-att recombinase gene (INT), but specific recognition attB and att-P site and carry out the special excision of sequence between the site.

At present, utilize the att specific site to carry out applying for a patent of vector construction and gene clone and only have one, be that the applicant rejects innovation and creation, send bright name elsewhere and be called " a kind of method of position-point recombination counter-cloning genes and utilization thereof ", application number: 01130855.9, send elsewhere bright different with the present invention.Utilize the att specific site independently to recombinate to realize the literature search to a piece of the rejecting of marker gene, but very low (the Zubko E of its efficient, ScuttC, Meyer P., 2000, Intrachromosomal recombination between attP regions as a tool to remove selectable markergenes from tobacco transgenes.Nature Biotech.18:442-445), do not apply for a patent.The research of introducing INT recombinase identification att site excision marker gene up to now yet there are no relevant report and patent application on plant.

Summary of the invention

The objective of the invention is to utilize the special recombination site of lambda particles phage attP with and recombinase gene INT, make up special plant conversion carrier and transform plant, the render transgenic plant is the rejecting marker gene of carrying itself independently, obtain only to contain the transfer-gen plant of goal gene, help improving the biological safety of transgene agricultural product.

The present invention passes through following scheme implementation:

A kind of method of can the render transgenic plant independently rejecting marker gene, utilize special recombination site of lambda particles phage recognition sequence (attP) and recombinase gene (INT) thereof, make up plant conversion carrier pAMF, obtain the transformed plant or the callus of this carrier by agrobacterium tumefaciens or other plant genetic transforming method.The transformed plant selfing is selected to obtain to isozygoty, and after dexamethason is induced, separates the plant of selecting to reject marker gene the offspring from its selfing; The callus that obtains can be induced by dexamethason, chooses non-resistance callus of induce and becomes seedling to obtain the marker-free transfer-gen plant; Further confirm by PCR or Southern hybrid molecule authentication method.

The method of can the render transgenic plant independently rejecting marker gene is according to the following step:

(1) according to lambda particles phage genome sequence design primer: obtain recombinase gene and recognition sequence thereof (attP) 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 ', and attPB:5 '-GGCAGGGAGTGGGACAAAAT-3 ':

(2) make up plant conversion carrier pAMF: with the pBI121 plasmid is underlying carrier, it is cut with the PmeI enzyme and with the processing of CIAP dephosphorylation, cuts pGTP with the EcoRI enzyme and obtain the attP fragment, and with T4DNA polysaccharase end-filling.Both reclaim to name after product connects and are pPBIP.With SdaI the pPBIP enzyme is cut, T4DNA polysaccharase end-filling is also handled with the CIAP dephosphorylation, and cut pGTP with the EcoRI enzyme and obtain the attP fragment, and with T4DNA polysaccharase end-filling.Both reclaim and promptly are built into carrier pMFB after product connects;

Plasmid pTA7002 is carried out the SpeI enzyme cut the back, carry out second enzyme with XhoI and cut, cut glue and reclaim with T4DNA polysaccharase end-filling; Plasmid pMDTINT cuts the back with the SacI enzyme and mends flat end with the T4DNA polysaccharase, carries out second enzyme with SalI and cuts, and cuts glue and reclaims small segment; Both reclaim product and connect called after pTAINT.

PTAINT is carried out behind SdaI and the StuI double digestion T4DNA polysaccharase mend flat terminally, cut glue and reclaim small segment; PMFB is cut back T4DNA polysaccharase with the HindIII enzyme mend flat end, cut glue and reclaim; Thereby both reclaim product and connect marker gene and recombinase gene are together built up between the locus specificity recombinase recognition sequence, and wherein recombinase gene is by the special abduction delivering of dexamethason, called after pAMF, any goal gene of the replaceable one-tenth of GUS wherein;

(3) genetic transformation of plant and purifying:

By particle gun or agriculture bacillus mediated method, utilize the plant conversion carrier that makes up to transform, obtain transfer-gen plant, by screening microbiotic or screening weedicide spraying method, further the selfing purifying is selected again, obtains the individual plant or the strain system of this gene pure;

(4) rejecting of marker gene:

After utilizing the dexamethason evoked callus, choose non-resistance callus induction Cheng Miao, be the transfer-gen plant of rejecting marker gene; Or, the transfer-gen plant that isozygotys that obtains induces by being sprayed dexamethason, seedling to its self progeny sprays corresponding selection microbiotic (for example in one of them embodiment tomato of the present invention then, the microbiotic that is adopted is the 100mg/L kantlex), selection is the plant of rejecting marker gene to the plant of this antibiotic sensitive.

(5) Molecular Identification:

According to goal gene and marker gene design special primer or synthetic specific probe, simultaneously the plant that obtains is carried out pcr amplification or Southern hybridization detects, marker gene is negative and plant that goal gene is positive is the transfer-gen plant of rejecting marker gene.

More detailed operation steps is as described in following:

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

(U.S. biotechnology information center gene number of registration: NC_001416) design primer obtains recombinase gene (INT) and recognition sequence (attP) thereof respectively according to the lambda particles phage genome sequence.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 '

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/LdNTPs, 0.5 μ M/L forward and reverse primer, 0.25 μ L lambda particles phage lysate, 0.5UTqDNA polysaccharase.The reaction cycle parameter of amplification INT 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), and reclaiming product, the latter is connected with pGEM -T Easy carrier (Promega company), 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 pMDTINT and the pGTP and is respectively INT and attP site.Its sequence is as follows respectively:

INT sequence (1071bp):

ATGGGAAGAAGGCGAAGTCATGAGCGCCGGGATTTACCCCCTAACCTTTATATAAGAAACAATGGATATTACTGCTACAGGGACCCAAGGA

CGGGTAAAGAGTTTGGATTAGGCAGAGACAGGCGAATCGCTAACACTGAAGCTATACAGGCCAACATTGAGTTATTTTCAGGACACAAACA

CAAGCCTCTGACAGCGAGAATCAACAGTGATAATTCCGTTACGTTACATTCATGGCTTGATCGCTACGAAAAAATCCTGGCCAGCAGAGGA

ATCAAGCAGAAGACACTCATAAATTACATGAGCAAAATTAAAGCAATAAGGAGGGGTCTGCCTGATGCTCCACTTGAAGACATCACCACAA

AAGAAATTGCGGCAATGCTCAATGGATACATAGACGAGGGCAAGGCGGCGTCAGCCAAGTTAATCAGATCAACACTGAGCGATGCATTCCG

AGAGGCAATAGCTGAAGGCCATATAACAACAAACCATGTCGCTGCCACTCGCGCAGCAAAATCAGAGGTAAGGAGATCAAGACTTACGGCT

GACGAATACCTGAAAATTTATCAAGCAGCAGAATCATCACCATGTTGGCTCAGACTTGCAATGGAACTGGCTGTTGTTACCGGGCAACGAG

TTGGTGATTTATGCGAAATGAAGTGGTCTGATATCGTAGATGGATATCTTTATGTCGAGCAAAGCAAAACAGGCGTAAAAATTGCCATCCC

AACAGCATTGCATATTGATGCTCTCGGAATATCAATGAAGGAAACACTTGATAAATGCAAAGAGATTCTTGGCGGAGAAACCATAATTGCA

TCTACTCGTCGCGAACCGCTTTCATCCGGCACAGTATCAAGGTATTTTATGCGCGCACGAAAAGCATCAGGTCTTTCCTTCGAAGGGGATC

CGCCTACCTTTCACGAGTTGCGCAGTTTGTCTGCAAGACTCTATGAGAAGCAGATAAGCGATAAGTTTGCTCAACATCTTCTCGGGCATAA

GTCGGACACCATGGCATCACAGTATCGTGATGACAGAGGCAGGGAGTGGGACAAAATTGAAATCAAATAA。

2. the attP sequence is (353bp):

GATTGCGAGGCTTTGTGCTTCTCTGGAGTGCGACAGGTTTGATGACAAAAAATTAGCGCAAGAAGACAAAAATCACCTTGCGCTAATGCTC

TGTTACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCA

AAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTTTTATACTAAGTTGGCATTATAAAAAAGCATTGCTT

ATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTGATTTCAATTTTGTCCCACTCCCTGCC。

2. construction of expression vector:

Make up plant conversion carrier pAMF.The T-DNA district of pAMF contains lambda particles phage recombinase gene (INT), selectable marker gene and goal gene, INT and marker gene are between two lambda particles phage locus specificity recombination site attP forward tumor-necrosis factor glycoproteinss, and goal gene is positioned at the two attP outside.Wherein recombinase gene (INT) drives (as Fig. 2) by dexamethason evoked promoter pDEX.

(1) goal gene is contained (among the figure for GUS in the T-DNA district of pMFB, the any goal gene of replaceable one-tenth) and selectable marker gene (can be any selectable marker gene), 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 pBI121 plasmid (U.S.'s biotechnology information center registers number: AF485783) be underlying carrier, it is cut with the PmeI enzyme and the processing of CLAP 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 SdaI the pPBIP enzyme is cut, T4DNA polysaccharase end-filling is also handled with the CIAP dephosphorylation, and cut pGTP with the EcoRI enzyme and obtain the attP fragment, and with T4DNA polysaccharase end-filling.Thereby both reclaim product and connect the SdaI site that attP is inserted pPBIP, are built into carrier pMFB.

(2) plasmid pTA7002 carries out using T4DNA polysaccharase end-filling after the SpeI enzyme is cut, and carries out second enzyme with XhoI and cuts, and cuts glue and reclaims.Plasmid pMDTINT cuts the back with the SacI enzyme and mends flat end with the T4DNA polysaccharase, carries out second enzyme with SalI and cuts, and cuts glue and reclaims small segment.Thereby both reclaim product and connect the inducible promoter downstream that INT is built up to pTA7002, called after pTAINT.

(3) pTAINT is carried out behind SdaI and the StuI double digestion T4DNA polysaccharase and mend flat terminally, cut glue and reclaim small segment.PMFB is cut back T4DNA polysaccharase with the HindIII enzyme mend flat end, cut glue and reclaim.Connection together builds up to marker gene and recombinase gene between the locus specificity recombinase recognition sequence attP thereby both reclaim product, wherein recombinase gene (INT) is by the special abduction delivering of dexamethason, called after pAMF, any goal gene of the replaceable one-tenth of GUS wherein.

3. the genetic transformation of plant:

By agriculture bacillus mediated or other transgenic methods pAMF is changed over to plant, obtain transformed calli or transfer-gen plant, the transfer-gen plant purifying obtains the transfer-gen plant or the strain system of isozygotying.

The step that agriculture bacillus mediated genetic transformation obtains transgenic Fructus Lycopersici esculenti is as follows: 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), transferred to the selection regeneration culture medium, treated that regeneration bud grows to about 1cm, is transferred to root media.Acclimatization and transplants after taking root.

The transfer-gen plant purification step is as follows: for the pAMF plants transformed, results first-generation seed is T1 generation.Because T1 is for separating, utilize and select the microbiotic spraying method that the T1 of transfer-gen plant is screened for plant, plant with this antibiotics resistance is pressed individual plant results seed from T1 for resistant plant for containing genetically modified isozygotying and heterozygous plant, i.e. T2 generation.Continue to utilize the microbiotic 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.

4. the rejecting of marker gene:

If the callus that transforms, can utilize dexamethason to induce, the INT recombinase is expressed, identification attP site, recombinase itself and marker gene between special excision two sites, the callus of excision marker gene does not have the corresponding resistance of marker gene, chooses non-resistance callus of induce Cheng Miao, is the transfer-gen plant of rejecting marker gene.

If obtained the transfer-gen plant that isozygotys, then induce plant or seed by spraying dexamethason, the INT recombinase is expressed, identification attP site, recombinase itself and marker gene between special excision two sites.The plant of excision marker gene does not have the corresponding resistance of marker gene, can choose in selecting to induce the self progeny of plant.Promptly by spraying certain density corresponding selection microbiotic (as kantlex or weedicide) during the seedling, hickie appears in plant usually that do not possess resistance, and these plant promptly are the plant of rejecting marker gene.

5. Molecular Identification

According to goal gene and marker gene design special primer or synthetic specific probe, simultaneously the plant that obtains is carried out pcr amplification or Southern hybridization detects, marker gene is negative and transfer-gen plant that plant that goal gene is positive can be defined as rejecting marker gene.Concrete steps can be referring to shown in Figure 1.

Description of drawings

Fig. 1. be schema of the present invention;

Fig. 2. the plant conversion carrier pAMF that can independently reject marker gene of the present invention, the replaceable one-tenth goal gene of GUS;

Fig. 3. the plant conversion carrier pAMF1 that can independently reject marker gene NPTII of the present invention, goal gene is GNA;

Fig. 4. the plant conversion carrier pAMF2 that can independently reject marker gene NPTII of the present invention, goal gene is CHITINASE;

Fig. 5. the embodiment of the invention 1 Molecular Identification result, 1--does not reject the homozygous plants of marker gene among the figure, and 2--rejects the marker gene plant; 380bp fragment: GNA fragment, 740bp fragment: marker gene NPTII fragment.

Fig. 6. the embodiment of the invention 2 Molecular Identification results, 1,2:: do not reject the homozygous plants of marker gene, 3: reject the marker gene plant; 1180bp fragment: p35S-CHITINASE mosaic gene fragment, 740bp fragment: marker gene NPTII fragment, 400bp fragment: INT fragment.

Embodiment

Example 1: the acquisition of unmarked anti-aphid transgenosis (GNA) rape plant

The Homoptera aphid crop of not only biting is stopped up pore, and even more serious is because wound causes pathogenic agent to infect, to cause the crop multiple diseases.According to statistics, aphid is the transmitting carrier of more than 100 kind of different virus disease.The GNA gene (Galanthus nivalid Agglutinin, GNA) source and plant snowdrop, the single-minded opposing Homoptera insect of its expression product, harmless to people and higher animal.This gene is driven by paddy rice phloem specific expression promoter at present, has transferred in the crops such as paddy rice, tomato, has good anti-aphid and viral diseases effect.The rape step that obtains marker-free commentaries on classics GNA is as follows:

1., carrier construction:

Utilize carrier pAMF of the present invention to transform, paddy rice phloem specific promoter RSs1 and GNA are built into mosaic gene, replace goal gene and promotor thereof among the pAMF, selectable marker gene NPTII and INT are between two attP sites, and INT is driven by the dexamethason inducible promoter.This carrier called after pAMF1.

2., rape genetic transformation

Utilizing Agrobacterium tumefaciens mediated genetic transformation method, T-DNA section in the pAMF1 carrier is imported rape, is to select microbiotic with the kantlex, obtains transfer-gen plant;

3, the purifying of transfer-gen plant

According to the gene isolation law of Meng Deer, utilize selectable marker gene to be selection markers, by kantlex spraying method (50mg/L), select the individual plant and the strain system of isozygotying in generation at T1;

4, the rejecting of marker gene

The individual plant that isozygotys is sprayed the dexamethason abduction delivering, excision NPTII and INT itself, this individual plant carries out selfing, by the kantlex of foliage-spray 50mg/L, selects not have for kantlex the plant of resistance among the offspring;

5, Molecular Identification

Choose the plant that kantlex is not had resistance, extract DNA, increase with NPTII, INT and GNA special primer, GNA is accredited as the positive and NPTII and INT are accredited as the commentaries on classics GNA rape plant that negative plant is marker-free.PCR qualification result Fig. 5.

Example 2: marker-free resistance to fungal disease transgenosis (chitinase gene, CHITINASE) seed selection of tomato material

The chitinase gene coded product is to be a kind of lytic enzyme chitinase of substrate with chitin (chitin) for chitinase EC.3.2.11.14, different classifications can be divided into, acid chitinase and alkaline chitinase two big classes can be divided into generally.Chitin is the moiety of many pathogenic fungi hyphal cell walls, thereby the chitinase one side directly vertical chitin of degradative fungi mycelia suppresses fungi growth, on the other hand then by discharging a large amount of oligosaccharides behind the degrade chitin, oligosaccharides produces disease resistance response as the exciton inducing plant, thereby effectively keeps out the attack of pathogenic fungi.Therefore, plant chitinase is significant for preventing and treating controlling fungal diseases of crop.

1., carrier construction:

Utilize carrier pAMF of the present invention to transform, the goal gene among the CHITINASE gene replacement pAMF, selectable marker gene NPTII and INT are between two attP sites, and INT is driven by the dexamethason inducible promoter.This carrier called after pAMF2.

2., tomato genetic transformation:

Utilizing Agrobacterium tumefaciens mediated genetic transformation method, T-DNA section in the pAMF2 carrier is imported tomato, is to select microbiotic with the kantlex, obtains transfer-gen plant;

3, the purifying of transfer-gen plant:

According to the gene isolation law of Meng Deer, utilize selectable marker gene to be selection markers, by kantlex spraying method (100mg/L), select the individual plant and the strain system of isozygotying in generation at T1;

4, the rejecting of marker gene:

The individual plant that isozygotys is sprayed the dexamethason abduction delivering, excision NPTII and INT itself, this individual plant carries out selfing, selects not have for kantlex the plant of resistance among the offspring;

5, Molecular Identification:

Extraction does not have the plant DNA of resistance to kantlex, increases with NPTII, INT and CHITINASE special primer, and CHITINASE is accredited as the positive and NPTII and INT are accredited as the commentaries on classics CHITINASE tomato plant that negative plant is marker-free.PCR qualification result such as Fig. 6.

Claims (1)

1, a kind of method of can the render transgenic plant independently rejecting marker gene, it is characterized in that, utilize special recombination site attP of lambda particles phage and recombinase gene INT thereof, prepare plant conversion carrier pAMF, obtain the transformed plant or the callus of this carrier by agrobacterium tumefaciens or other plant genetic transforming method, make the transformed plant selfing select to obtain to isozygoty, after dexamethason is induced, separate the plant of selecting to reject marker gene the offspring from its selfing; The callus that obtains is induced by dexamethason, chooses non-resistance callus of induce and becomes seedling to obtain the marker-free transfer-gen plant; Further confirm by PCR or Southern hybrid molecule authentication method.

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