CN101003815A - Method for destroying gramineous transgenic plant selectively - Google Patents

Abstract

This invention discloses a method for selectively diminishing transgenic gramineous plants. The method comprises: (1) constructing a vector containing one or more target gene expression frames and the DNA fragment coding antisense RNA or RNAi in the same T-DNA fragment of a plasmid or in one fragment that can be transferred into plant genome; (2) transferring the vector into Gramineous plants to obtain transgenic Gramineous plants; (3) spraying herbicides such as bentazon and sulfonylurea onto the transgenic gramineous plants. Transgenic Gramineous plants can be killed by this method, and non-transgenic plants can survive. The method is simple and efficient in controlling and identifying transgenic gramineous plants.

Description

The method of destroying gramineous transgenic plant optionally

Technical field

The invention belongs to plant genetic engineering field; Specifically, the present invention relates to a kind of method of optionally eliminating transgenic graminaceous plant.

Background technology

Transgenic crop is planted in the world many countries large-scale popularization at present.For example in the North America large-scale popularization, transgene cotton is also planted in China in a large number for transgenic pest-resistant, antiweed corn.Further, utilize transgenic crop as bio-reactor production pharmaceutical protein, industrial enzymes etc. also just broad research and exploitation (Larrick and Thomas, Current Opinion in Biotechnology, 2001,12:411-418).One of important process that transgenic crop is used is their propagation of control, prevents that they are blended among the non-transgenic farm crop; Particularly prevent as bio-reactor production pharmaceutical protein, industrial enzyme and other short-terms or long-term edible may have the transformed variety of undesirable action to sneak into to be used for grain and hay varieties is very important humans and animals.Therefore invent a kind of easy method control transgenic crop and have great value.

Ben Dasong and sulfonylurea herbicide are the agriculture weedicides of using always.They have the good careless ability of killing to many width leaf weeds.But gramineae farm crop is owing to have the toxenzyme of separating of these weedicides, therefore to these weedicides have good resistance (Pan et al., Plant Molecular Biology, 2006,61:933-943).If so suppressed the expression of separating toxenzyme of Ben Dasong and sulfonylurea herbicide when expressing target gene in transgenic gramineae farm crop, these transgenic plant just can optionally be eliminated by Ben Dasong or sulfonylurea herbicide in case of necessity.

Summary of the invention

The technical problem to be solved in the present invention provides a kind of method of optionally eliminating transgenic graminaceous plant, uses this kind method can control and discern genetically modified grass simply, effectively.

In order to solve the problems of the technologies described above, the invention provides the method that a kind of Ben Dasong of utilization or sulfonylurea herbicide are optionally eliminated transgenic graminaceous plant, carry out following steps successively:

1), with one or more target gene expression cassette and the sense-rna of the expression of separating toxenzyme that suppresses Ben Dasong or sulfonylurea herbicide or the dna segment of RNAi, be structured in the same T-DNA segment on the same plasmid or be structured in the same segment that imports Plant Genome, form carrier;

2), the dna segment in the above-mentioned carrier is imported in the gramineae farm crop by transgenic method, form genetically modified gramineae farm crop;

3), select for use Ben Dasong or sulfonylurea herbicide that above-mentioned genetically modified gramineae farm crop is carried out conventional sprinkling to handle, transgenic graminaceous plant then is killed, and can survive be the non-transgenic grass.

Improvement as the inventive method: target gene is anti insect gene, anti-herbicide gene, pharmaceutical protein plasmagene or industrial enzyme gene; This anti-herbicide gene can be selected Antiglyphosate gene for use.

Further improvement as the inventive method: transgenic method is particle gun method or agriculture bacillus mediated method.

Further improvement as the inventive method: the sense-rna of the expression of separating toxenzyme of inhibition Ben Dasong or sulfonylurea herbicide or the dna segment of RNAi design according to SEQ ID NO:1 in paddy rice; And in corn, design according to SEQ ID NO:2.

Design concept of the present invention is as follows: express in transgenic graminaceous plant in the target gene, by the expression of the detoxification genes of Ben Dasong and sulfonylurea herbicide in the method inhibition gramineae farm crop that utilizes sense-rna or RNAi, thereby the render transgenic farm crop are to Ben Dasong and sulfonylurea herbicide sensitivity.And in the present invention, the detoxifying gene of target gene expression cassette and Ben Dasong, sulfonylurea herbicide suppresses expression cassette and is connected same DNA and imports on the segment, thereby reaches the inhibition expressing gene close linkage of the detoxifying gene of target gene and Ben Dasong, sulfonylurea herbicide in transgenic crop.Particularly above-mentioned target gene comprises a kind of Antiglyphosate gene, thereby makes the transgenic crop of acquisition have high resistance to glyphosate herbicidal and to Ben Dasong and sulfonylurea herbicide sensitivity.The transgenic gramineae farm crop that utilizes method of the present invention and obtain just can optionally be killed by Ben Dasong and sulfonylurea herbicide.Therefore in case of necessity can by use Ben Dasong commonly used and sulfonylurea herbicide prevent transgenic gramineae farm crop propagation, eliminate the transgenic gramineae farm crop of sneaking in the non-transgenic gramineae farm crop.

The gramineae farm crop of render transgenic of the present invention is to Ben Dasong and sulfonylurea herbicide sensitivity, and this to herbicide sensitive proterties and goal gene close linkage.And this chain be to realize by the structure that inserts DNA: i.e. the present invention suppresses expression cassette with the detoxification genes of target gene expression cassette and Ben Dasong and sulfonylurea and is connected on the same carrier.When utilizing Agrobacterium-mediated Transformation, then be in being connected same T-DNA segment.When using " particle gun method ", then the same DNA on same carrier inserts in the segment.

Target gene among the present invention can be anti-herbicide gene, anti insect gene, pharmaceutical protein plasmagene, industrial enzyme gene or other valuable genes; Can be one of them, two or a plurality of.The detoxification genes of Ben Dasong and sulfonylurea suppresses expression cassette and is made up of two parts.One is promotor, it can be corn ubiquitin (Ubiqutin) promotor (Christensen and Quail, TransgenicRes., 1996,5:213-8), perhaps paddy rice ubiquitin promoter, perhaps paddy rice Actin promotor, or CaMV35S promotor, or other promoters active in gramineae farm crop.Another part and promoter function link is the dna profiling of sense-rna that produces the detoxifying gene of Ben Dasong and sulfonylurea, perhaps produces the dna profiling of RNAi.Mechanism and the technology of utilizing sense-rna and RNAi suppress to express have had complete description, be the technology that had (Nature ReviewsGenetics 2006,7,334-335).The dna profiling that produces sense-rna can be a full-length gene, perhaps only is one of them or several segment.The detoxifying gene dna sequence dna is SEQ IDNO:1 in paddy rice, is SEQ ID NO:2 in corn.Producing RNA interferential sequence (RNAi) is prior art, can design according to SEQ ID NO:2 in corn according to SEQ ID NO:1 design in paddy rice.

The carrier that has the detoxification genes inhibition expression cassette of target gene expression cassette and Ben Dasong and sulfonylurea simultaneously can be by the stable gramineae farm crop that imports of transgenic method.This method comprises particle gun method and agriculture bacillus mediated method, and these two kinds of methods all are sophisticated known method (Volume 17 for Plant, Cell andEnvironment, and Issue 4).

A kind of transgenic plant can be by pollen diffusion or directly by the place of seed dispersal beyond the plan plantation.The present invention produces susceptibility by the render transgenic plant to Ben Dasong and sulfonylurea and optionally kills, thereby has guaranteed that goal gene can Be Controlled.In the non-transgenic kind, as long as use Ben Dasong and sulfonylurea herbicide controlling weeds just can eliminate the transformed variety that to sneak into simultaneously.

The present invention also provides a kind of and utilizes different weedicides can just select and bear the technology of selecting to transgenic plant.A kind of Antiglyphosate gene is structured on the same plasmid with the dna segment of the detoxification genes that suppresses Ben Dasong and sulfonylurea.Like this, to the resistance of glyphosate and chain at the transgenic gramineae farm crop camber to the susceptibility of Ben Dasong and sulfonylurea.The transgenic plant that this method obtains can utilize glyphosate to screen and control of weeds, also can utilize the control of Ben Dasong and sulfonylurea herbicide simultaneously and eliminate transgenic gramineae farm crop.

The present invention also provides a kind of plasmid: comprise the expression cassette of Antiglyphosate gene and the inhibition expression cassette of control Ben Dasong and the expression of sulfonylurea detoxifying gene simultaneously.

The present invention also provides a kind of gramineae farm crop cell that comprises above-mentioned plasmid.

The present invention also provides a kind of method that plant is transformed: comprise and utilize above-mentioned vegetable cell, more corresponding transfer-gen plant is cultivated in its differentiation.The plant that is obtained can be expressed goal gene, but has obviously reduced the ability of anti-Ben Dasong and sulfonylurea herbicide simultaneously significantly.

Therefore the method for optionally eliminating transgenic graminaceous plant of the present invention relates to a kind of biotechnology, this kind method can be killed by the Ben Dasong and the sulfonylurea herbicide of normal all concentration on render transgenic crop-selective ground, therefore can prevent the transgenic gramineae farm crop diffusion effectively or sneak into the non-transgenic gramineae farm crop.

Description of drawings

Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.

Fig. 1 is the synoptic diagram of rice conversion T-DNA carrier pCAMB1300Rice-GlyR-450i. of the present invention;

Fig. 2 is the synoptic diagram that corn of the present invention transforms T-DNA carrier pCAMB1300Corn-GlyR-450i-AMY.

Embodiment

The structure of embodiment 1, Agrobacterium-mediated Transformation T-DNA carrier:

1), the structure of rice conversion T-DNA carrier pCAMB1300Rice-GlyR-450i:

In Fig. 1: S35 represents the CaMV35S promotor; The 450i representative produces the dna profiling of RNAi; Ubi represents corn corn ubiquitin (Ubiqutin) promotor; The Antiglyphosate gene representative is a kind of to the insensitive enol pyruvic acid shikimic acid of glyphosate-3-phosphate synthase (EPSPS); LB and RB represent the insertion border of T-DNA respectively.

Paddy rice Ben Dasong and sulfonylurea herbicide detoxifying gene segment are to obtain from the method amplification of rice genome by PCR.A segment is R450FR, 207bp, and the PCR primer is respectively 450F, 5 ' CTCGAGCAG TGC ACC AGA GTC ACA GAA ACA CAT CAC AC and 450R, 5 ' AGA CTC CT TCT TGA CGA GGT GGA GGT GT.Another segment is R450FR2,327bp, and the PCR primer is respectively 450F, 5 ' CTCGAGCAG TGC ACCAGA GTC ACA GAA ACA CAT CAC AC and 450R2,5 ' AGA TCTCGG TGAAGC ACT CCC TGG CGC AC.These two segments are cloned into respectively in the T carrier (worker is given birth in Shanghai), obtain T-vector-R450FR1 and T-vector-R450FR1 respectively.

The T-DNA carrier pCAMB1300-GlyR (based on pCAMB1300) that will contain Antiglyphosate gene carries out enzyme with Xho1 and cuts, excision moisture resistance mycin (hyg+) gene, and dephosphorylation.Simultaneously with the R450FR among carrier T-vector-R450FR1 and the T-vector-R450FR1, the R450FR2 fragment is all cut with Xho1 and Bg12 enzyme, acquire 2 and insert fragment (being respectively 207bp and 327bp), then, with the pCAMB1300-GlyR of excision moisture resistance mycin, R450FR and R450FR2,3 fragments connect, in intestinal bacteria, clone, obtain pCAMB 1300Rice-GlyR-450i.Like this, this T-DNA carrier contains Antiglyphosate gene and CaMV35S promotor control inhibition Ben Dasong that can produce RNAi and the detoxify gene of expression of enzymes of sulfonylurea herbicide down.The dna sequence dna of CaMV35S promotor control is R450FR and the R450FR2 that has connected, and serial number is SEQ ID NO:3.

2), corn transforms the structure of T-DNA carrier pCAMB1300Corn-GlyR-450i-AMY:

In Fig. 2: the 450i representative produces the maize dna template of RNAi; Ubi represents corn ubiquitin (Ubiqutin) promotor; The Antiglyphosate gene representative is a kind of to the insensitive enol pyruvic acid shikimic acid of glyphosate-3-phosphate synthase (EPSPS); LB and RB represent the insertion border of T-DNA respectively; Gt1 represents the rice paddy seed specific expression promoter.The synthetic alpha-amylase gene (aminoacid sequence is gb:A24549) that on behalf of a kind of codon, amylase optimize.

Two segments of Ben Dasong and sulfonylurea herbicide detoxifying gene are that the method amplification by PCR obtains from the corn gene group in the corn.Segment T-ZM450-1,259bp is long, and the PCR primer is respectively ZmSac731F 5 ' TCGAC GAGCTCG TGC CGT ACA TCG G, and ZmXho1R, 5 ' AGCG CTCGAG TT TAG AGC AGT GAT CAC AGT GTC AG.Segment T-R450-2,147bp is long, and the PCR primer is respectively ZmBg12-80F 5 ' GCTT AGATCTCGTAC ATC GGC ACG GCC AAC CGC T and Zm880R 5 ' AGCG CTCGAGCCAGCCTCCGCCGCTCCCCGT.These two PCR products are cloned into respectively in the T carrier (worker is given birth in Shanghai).By common molecular biology method these two segments are connected segment of acquisition, its dna sequence dna is SEQ ID NO:4.Then, with SEQ ID NO:4 and functional connection of corn ubiquitin promoter (Ubi), obtain corn Ben Dasong and sulfonylurea herbicide detoxifying gene and suppress expression cassette (Ubi-SEQ ID NO:4).This utilizes the detoxifying gene inhibition expression cassette of RNAi method further to be cloned among the T-DNA carrier pCAMB1300-GlyR (based on pCAMB1300) that contains Antiglyphosate gene, is contained the T-DNA plasmid vector (pCAMB1300Corn-GlyR-450i) that detoxifying gene suppresses expression cassette and anti-Antiglyphosate gene expression cassette simultaneously.Utilizing Hind3 to carry out enzyme pCAMB1300Corn-GlyR-450i cuts, obtain the carrier segments of 15.8kb, CIAP dephosphorylation then, further with the α-Dian Fenmei expression cassette (alpha-amylase gene of rice starter gt1 control, the fragment of about 3kb) is cloned into the T-DNA plasmid vector, obtain T-DNA carrier pCAMB1300Corn-GlyR-450i-AMY, be used for the conversion of corn.Therefore, the T-DNA of pCAMB1300Corn-GlyR-450i-AMY comprises gene, an Antiglyphosate gene and an amylase gene that suppresses Ben Dasong and the expression of sulfonylurea herbicide detoxifying gene.

The acquisition of embodiment 2, transgenic paddy rice:

The preparation method of transgenic plant is to adopt prior art (the refined Gong ancestral of Lu Xiong an ancient egg-shaped, holed wind instrument 1998 life science 10:125-131; Liu Fan etc., 2003 Molecular Plant Breeding 1:108-115)." elegant water 110 " seed of choosing mature and plump shells, and induces to produce callus as converting material.Get the Agrobacterium that contains order T-DNA carrier pCAMB1300Rice-GlyR-450i and draw plate, choose single colony inoculation preparation conversion and use Agrobacterium.Callus to be transformed is put into the agrobacterium liquid (containing Syringylethanone) of proper concn, allow Agrobacterium be attached to the callus surface, then callus is transferred in the common substratum, cultivated altogether 2～3 days.With the callus after the aseptic water washing conversion, transfer on the screening culture medium that contains the 2mM glyphosate screening and culturing two months (middle subculture once).After screening, the callus that growth vigor is good is transferred on the pre-differentiation substratum and was cultivated about 20 days, will break up good callus then in advance and move on to division culture medium, and illumination in 14 hours differentiation is germinateed.2-3 transfers to strengthening seedling and rooting on the root media to the resistance regeneration plant after week, at last regeneration plant flush away agar is transplanted in the greenhouse, as expert evidence.

The insertion of T-DNA and expression of gene thereof can detect by PCR and western engram analysis.Genomic dna can extract from transformation tissue culture plant and non-transforming gramineous farm crop according to existing method.

The acquisition of embodiment 3, transgenic corns

Get 8-10 days the Hi-2 mealie in pollination back.Collect all immature embryos (size is 1.0-1.5mm).The Agrobacterium and the immature embryo that will contain T-DNA carrier pCAMB1300Corn-GlyR-450i-AMY are cultivated common cultivation 2-3 days (22 ℃) altogether.Shift immature embryo (M3, the Timentin that contains 200mg/L kills Agrobacterium) to the callus of induce substratum, 28 ℃ of dark cultivations 10-14 days.All callus are forwarded (M4) on the screening culture medium that has the 2mM glyphosate to 28 ℃ of dark cultivations 2-3 week.

Shift on all screening culture medium that is organized into fresh glyphosate (M4) 28 ℃ of dark cultivations 2-3 week.Then, shift the embryonal connective tissue (M5) to regeneration culture medium that survives after all screenings, 28 ℃ of dark cultivations 10-14 days, strain system of every ware.Shift embryonal connective tissue (M5) to fresh regeneration culture medium, 26 ℃ illumination cultivation 10-14 days.Shift all full-grown plants (M6) to root media, 26 ℃ of illumination cultivation are complete up to root development.

Embodiment 4, to the just selection of transgenic gramineae farm crop and negative the selection

Reuse water rice plants individual plant cultivation in the culture solution in the greenhouse that the conversion pCAMB1300Rice-GlyR-450i reuse water rice plants that obtains and not have transforms.These regeneration plants are divided into 3 processing, and each processing comprises conversion and do not have each 30 strain of transformation tissue culture plant.Handle 1: spray 30mM glyphosate; Handle 2: spray 500mg/L Ben Dasong; Handle 3: water spray.Handle the surviving rate of back 10 days record plant.Result such as following table 1.

Table 1

	Glyphosate	Ben Dasong	Water
				PCAMB1300Rice-GlyR-450i transforms	100% survives	0% survives	100% survives
Do not transform	0% survives	100% survives	100% survives

Embodiment 5: Ben Dasong is to the kill capability of the maize calli of conversion

To survive maize calli through 20 of the screening and culturing on the screening culture medium of glyphosate of agroinfection 40 days, survive maize calli with 20 without unscreened 20 of agroinfection, in the screening culture medium and the 5mg/L Ben Dasong culture medium culturing of 3mM glyphosate, observe the callus Growth situation after 10 days respectively.The obvious resistance glyphosate of callus (100% callus is obviously grown) that pCAMB1300Corn-GlyR-450i-Amy transforms, but 75% death on Ben Dasong (5mg/L) substratum.On the contrary, the callus that transforms without agroinfection is resistance glyphosate not obviously, but anti-Ben Dasong.Concrete outcome sees Table 2.

Simultaneously, the callus that takes a morsel in the 20mM of pH5.5 Tris-HCl solution, is measured amylase activity.Obviously have amylase activity through the callus that transforms, illustrate to transform successfully.

Table 2

The M4 substratum	Glyphosate (2mM)	Ben Dasong (5mg/L)	Amylase activity
				pCAMB1300Corn-G lyR-450i-Amy	100% survives	25% survives	Have
Do not transform	0% survives	100% survives	Do not have

At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.

Sequence table

SEQ ID NO：1

1 CAGTGCACCA GAGTCACAGA AACACATCAC ACATTCGTGA GCTCAGCTTA GCCATGGATA

61 ACGCCTACAT TATTGCCATT CTCTCTGTAG CTATCCTCTT CTTGCTCCAC TACTACCTCC

121 TCGGCCGCGG CAATGGCGGG GCGGCGCGGC TGCCGCCGGG TCCACCGGCC GTCCCGATCC

181 TGGGACACCT CCACCTCGTC AAGAAGCCGA TGCACGCCAC CATGTCCCGC CTCGCCGAGC

241 GGTACGGGCC GGTGTTCTCG CTGCGCCTCG GGTCGCGGCG CGCCGTGGTG GTGTCGTCGC

301 CGGGGTGCGC CAGGGAGTGC TTCACCGAGC ACGACGTGAC CTTCGCGAAC CGGCCCAGGT

361 TCGAGTCGCA GCTGCTGGTC TCGTTCAACG GCGCCGCGCT CGCCACGGCG AGCTACGGCG

421 CGCACTGGCG CAACCTCCGC CGGATCGTCG CCGTGCAGCT GCTCTCCGCG CACCGCGTCG

481 GCCTCATGTC GGGGCTCATC GCCGGCGAGG TCCGCGCCAT GGTGCGGAGG ATGTACCGCG

541 CCGCGGCCGC GTCCCCCGCC GGCGCCGCGC GCATCCAGCT GAAGCGGAGG CTGTTCGAGG

601 TCTCCCTCAG CGTGCTCATG GAGACCATCG CCCACACCAA GGCGACCCGC CCCGAGACGG

661 ACCCGGACAC CGACATGTCC GTGGAAGCCC AGGAGTTTAA GCAGGTCGTC GACGAGATCA

721 TCCCGCACAT CGGCGCGGCC AACCTGTGGG ACTACTTGCC GGCGCTCCGG TGGTTCGACG

781 TGTTCGGCGT CAGGAGGAAG ATCCTCGCCG CTGTAAGCCG GAGGGACGCG TTCCTTCGCC

841 GCCTGATCGA CGCGGAGCGG CGGAGGCTGG ACGACGGCGA CGAGGGCGAG AAGAAGAGCA

901 TGATCGCCGT GCTGCTCACT CTGCAGAAGA CAGAGCCGGA GGTGTACACC GATAACATGA

961 TCACAGCTCT AACGGCGAAC TTGTTCGGAG CAGGAACAGA GACAACCTCG ACGACATCAG

1021 AATGGGCGAT GTCGCTACTG CTGAACCACC CCGACACACT CAAGAAAGCG CAAGCCGAGA

1081 TCGACGCATC CGTCGGCAAC TCTCGCCTGA TCACCGCCGA CGACGTGACT CGCCTCGGCT

1141 ACCTCCAGTG CATCGTCAGG GAGACGCTCC GCCTGTACCC CGCCGCGCCG ATGCTCCTCC

1201 CGCACGAGTC CTCCGCCGAC TGCAAGGTCG GCGGCTACAA CATCCCGCGC GGGTCGATGT

1261 TGCTCATCAA CGCGTACGCC ATCCACCGTG ACCCGGCGGT GTGGGAGGAG CCGGAGAAGT

1321 TCATGCCGGA GAGGTTCGAG GACGGCGGGT GCGACGGCAA TCTCTTGATG CCGTTCGGGA

1381 TGGGGAGGCG GAGGTGCCCC GGCGAGACGC TGGCGCTGCG CACAGTGGGG TTGGTGCTGG

1441 GCACGCTGAT CCAGTGCTTC GACTGGGAGA GGGTCGACGG CGTGGAGGTC GACATGACTG

1501 AAGGTGGCGG GCTCACCATC CCCAAGGTCG TGCCGTTGGA GGCCATGTGC AGGCCGCGCG

1561 ACGCCATGGG TGGTGTTCTT CGCGAGCTCG TCTGAATA

SEQ ID NO：2

1 ATGGATAAGG CCTACATCGC CGCCCTCTCC GCCGCCGCCC TCTTCTTGCT CCACTACCTC

61 CTGGGCCGCC GGGCCGGCGG CGAGGGCAAG GCCAAGGCCA AGGGCTCGCG GCGGCGGCTC

121 CCGCCGAGCC CTCCGGCGAT CCCGTTCCTG GGCCACCTCC ACCTCGTCAA GGCCCCGTTC

181 CACGGGGCGC TGGCCCGCCT CGCGGCGCGC CACGGCCCGG TGTTCTCCAT GCGCCTGGGG

241 ACCCGGCGCG CCGTGGTCGT GTCGTCGCCG GACTGCGCCA GGGAGTGCTT CACGGAGCAC

301 GACGTGAACT TCGCGAACCG GCCGCTGTTC CCGTCGATGC GGCTGGCGTC CTTCGACGGC

361 GCCATGCTCT CCGTGTCCAG CTACGGCCCG TACTGGCGCA ACCTGCGCCG CGTCGCCGCC

421 GTGCAGCTCC TCTCCGCGCA CCGCGTCGGG TGCATGGCCC CCGCCATCGA AGCGCAGGTG

481 CGCGCCATGG TGCGGAGGAT GGACCGCGCC GCCGCGGCCG GCGGCGGCGG CGTCGCGCGC

541 GTCCAGCTCA AGCGGCGGCT GTTCGAGCTC TCCCTCAGCG TGCTCATGGA GACCATCGCG

601 CACACCAAGA CGTCCCGCGC CGAGGCCGAC GCCGACTCGG ACATGTCGAC CGAGGCCCAC

661 GAGTTCAAGC AGATCGTCGA CGAGCTCGTG CCGTACATCG GCACGGCCAA CCGCTGGGAC

721 TACCTGCCGG TGCTGCGCTG GTTCGACGTG TTCGGCGTGA GGAACAAGAT CCTCGACGCC

781 GTGGGCAGAA GGGACGCGTT CCTGGGGCGG CTCATCGACG GGGAGCGGCG GAGGCTGGAC

841 GCTGGCGACG AGAGCGAAAG TAAGAGCATG ATTGCGGTGC TGCTCACTCT GCAGAAGTCC

901 GAGCCAGAGG TCTACACTGA CACTGTGATC ACTGCTCTAA AGAACCTATT CGGCGCCGGA

961 ACGGAGACCA CGTCCACCAC GACGGAATGG GCCATGTCAC TGCTGCTGAA CCACCGGGAG

1021 GCGCTCAAGA AGGCGCAGGC CGAGATCGAC GCGGCGGTGG GCACCTCCCG CCTGGTGACC

1081 GCGGACGACG TGCCCCACCT CACCTACCTG CAGTGCATCG TCGACGAGAC GCTGCGCCTG

1141 CACCCGGCCG CGCCGCTGCT GCTGCCGCAC GAGTCCGCCG CGGACTGCAC GGTCGGCGGC

1201 TACGACGTGC CGCGCGGCAC GATGCTGCTG GTCAACGTGC ACGCGGTCCA CAGGGACCCC

1261 GCGGTGTGGG AGGACCCGGA CAGGTTCGTG CCGGAGCGGT TCGAGGGCGC CGGCGGCAAG

1321 GCCGAGGGGC GCCTGCTGAT GCCGTTCGGG ATGGGGCGGC GCAAGTGCCC CGGGGAGACG

1381 CTCGCGCTGC GGACCGTCGG GCTGGTGCTC GCCACGCTGC TCCAGTGCTT CGACTGGGAC

1441 ACGGTTGATG GAGCTCAGGT TGACATGAAG GCTAGCGGCG GGCTGACCAT GCCCCGGGCC

1501 GTCCCGTTGG AGGCCATGTG CAGGCCGCGT ACAGCTATGC GTGGTGTTCT TAAGAGGCTC

1561 TGAAAACCTC ATGGATCGAA TTGCTGGCAT CGTCTGAAGG GTGTATGACG TAGCTTCCGA

SEQ ID NO：3

1 GCAGTGCACC AGAGTCACAG AAACACATCA CACATTCGTG AGCTCAGCTT AGCCATGGAT

61 AACGCCTACA TTATTGCCAT TCTCTCTGTA GCTATCCTCT TCTTGCTCCA CTACTACCTC

121 CTCGGCCGCG GCAATGGCGG GGCGGCGCGG CTGCCGCCGG GTCCACCGGC CGTCCCGATC

181 CTGGGACACC TCCACCTCGT CAAGAAGCCG ATGCACGCCA CCATGTCCCG CCTCGCCGAG

241 CGGTACGGGC CGGTGTTCTC GCTGCGCCTC GGGTCGCGGC GCGCCGTGGT GGTGTCGTCG

301 CCGGGGTGCG CCAGGGAGTG CTTCACCGAG ATCTGCTTCT TGACGAGGTG GAGGTGTCCC

361 AGGATCGGGA CGGCCGGTGG ACCCGGCGGC AGCCGCGCCG CCCCGCCATT GCCGCGGCCG

421 AGGAGGTAGT AGTGGAGCAA GAAGAGGATA GCTACAGAGA GAATGGCAAT AATGTAGGCG

481 TTATCCATGG CTAAGCTGAG CTCACGAATG TGTGATGTGT TTCTGTGACT CCTGGTGCAC

541 TGC

SEQ ID NO：4

1 TCGAGGGGGG GCCCGGTACC CACTGGATTT TGGTTTTAGG AATTAGAAAT TTTATTGATA

61 GAAGTATTTT ACAAATACAA ATACATACTA AGTTCTGCAC AAAGTGGAGT AGTCAGTCAT

121 CGATCAGGAA CCAGACACCA GACTTTTATT CATACAGTGA AGTGAAGTGA AGTGCAGTGC

181 AGTGAGTTGC TGGTTTTTGT ACAACAGATC TTGAGCTCGT GCCGTACATC GGCACGGCCA

241 ACCGCTGGGA CTACCTGCCG GTGCTGCGCT GGTTCGACGT GTTCGGCGTG AGGAACAAGA

301 TCCTCGACGC CGTGGGCAGA AGGGACGCGT TCCTGAGGCG GCTCATCGAC GGGGAGCGGC

361 GGAGGCTGGA CGCTGGCGAC GACAGCGAAA GTAAGAGCAT GATTGCGGTG CTGCTCACTC

421 TGCAGAAGTC CGAGCCAGAG GTCTACACTG ACACTGTGAT CACTGCTCTA AACTCGAGCC

481 AGCCTCCGCC GCTCCCCGTC GATGAGCCGC CCCAGGAACG CGTCCCTTCT GCCCACGGCG

541 TCGAGGATCT TGTTCCTCAC GCCGAACACG TCGAACCAGC GCAGCACCGG CAGGTAGTCC

601 CAGCGGTTGG CCGTGCCGAT GTACGAGATC CTCTAGAGTC

Claims (6)

1, a kind of method of optionally eliminating transgenic graminaceous plant is characterized in that carrying out successively following steps:

1), with one or more target gene expression cassette and the sense-rna of the expression of separating toxenzyme that suppresses Ben Dasong or sulfonylurea herbicide or the dna segment of RNAi, be structured in the same T-DNA segment on the same plasmid or be structured in the same segment that imports Plant Genome, form carrier;

2), the dna segment in the above-mentioned carrier is imported in the gramineae farm crop by transgenic method, form genetically modified gramineae farm crop;

3), select for use Ben Dasong or sulfonylurea herbicide that above-mentioned genetically modified gramineae farm crop is carried out conventional sprinkling to handle, transgenic graminaceous plant then is killed, and can survive be the non-transgenic grass.

2, the method for optionally eliminating transgenic graminaceous plant according to claim 1 is characterized in that: described target gene is anti insect gene, anti-herbicide gene, medical protein plasmagene or industrial use protein gene.

3, the method for optionally eliminating transgenic graminaceous plant according to claim 2 is characterized in that: described transgenic method is particle gun method or agriculture bacillus mediated method.

4, the method for optionally eliminating transgenic graminaceous plant according to claim 3 is characterized in that: the sense-rna of the expression of separating toxenzyme of described inhibition Ben Dasong or sulfonylurea herbicide or the dna segment of RNAi design according to SEQ ID NO:1 in paddy rice.

5, the method for optionally eliminating transgenic graminaceous plant according to claim 3 is characterized in that: the sense-rna of the expression of separating toxenzyme of described inhibition Ben Dasong or sulfonylurea herbicide or the dna segment of RNAi design according to SEQ ID NO:2 in corn.

6, according to claim 4 or the 5 described methods of optionally eliminating transgenic graminaceous plant, it is characterized in that: described anti-herbicide gene is an Antiglyphosate gene.

Images