CN101501196A - Modified dicamba monooxygenase enzyme and methods of its use - Google Patents

Abstract

The invention provides a modified variant of dicamba monooxygenase (DMO). The invention relates to the unexpected finding that cells expressing this DMO exhibit high levels of tolerance to the herbicide dicamba. Compositions comprising DMO-encoding nucleic acids and methods of use are provided.

Description

The dicamba monooxygenase enzyme and the using method thereof of modifying

Background of invention

The application requires the right of priority of U.S. Provisional Patent Application sequence number of submitting on June 6th, 2,006 60/811,152 and the U.S. Patent Application Serial Number of submitting on June 5th, 2,007 11/758,657, is incorporated herein by reference disclosing of they is complete.

1. Invention field

The present invention relates generally to biological technical field.More specifically, the dicamba monooxygenase enzyme that the present invention relates to modify, it can give the tolerance to the weedicide dicamba 98 in genetically modified organism.

2. Association area is described

Because by using the genetically engineered plant technology to introduce proterties such as insect-resistant and herbicide tolerant, remarkable change has taken place in the method for producing field crops such as corn, soybean and cotton in the past few decades.These change in the production cost caused the bigger productivity of per hectare, reduction, the production decision greater flexibility and efficient, sterilant and use and reduce, and for the situation of insect resistant cotton, peasant health improves.Thereby genetically modified crops are adopted in a large number and are grown in now in the world the millions of acre.Yet,, need to increase the proterties of new value for the render transgenic crop has lasting competitive power on market.

The new proterties of the quality and quantity of farm crop and garden crop has occurred and will continue speedup in the several years afterwards occurs although improve, and still needs to improve the proterties of food, feed and other product processes.For example, although there is blank spot in the current transgenic plant that can obtain herbicide-tolerant glyphosate, bromoxynil, sulphur urea and other herbicide treatment in the weeds scope of being controlled with by developing in the extra soluble processing selecting of herbicide tolerant crop.In addition, the appearance (although normally partial and variable) that tolerates the weeds of above-mentioned weedicide has caused the needs to extra or alternative weeds measure of control.

Although it is valuable having proved the transgenosis herbicide tolerant, therefore need the option of plant to avoid being overly dependent upon any single weedicide and to increase the unmanageable weeds kind of management of other weedicides of tolerance in business contexts.What especially need is for obtaining eco-friendly and developing herbicide tolerant at the effective weedicide of height aspect the control weeds.Dicamba 98 is effective and such example of eco-friendly weedicide, and it was used more than 40 year by the peasant.Dicamba 98 especially can be used for controlling annual and perennial broadleaf weed and several grassy weed (Crop Protection Reference, 1995) in corn, Chinese sorghum, millet, herbage, hay, pasture, sugarcane, asparagus, turf and the grass-seed crop.Unfortunately, dicamba 98 can injure many commercial crops and dicotyledons, as soybean, cotton, pea, potato, Sunflower Receptacle and rape, they in addition low-level this weedicide also responsive especially.However, dicamba 98 control provide in the weed growth effectively and from but important tool the agricultural.

Recently, separated the gene of coding dicamba monooxygenase enzyme (DMO) from Pseudomonas Maltophilia (Pseudomonas maltophilia), it gives the tolerance to dicamba 98 (U.S. Patent number 7,022,896).DMO participates in weedicide dicamba 98 (3,6-two chloro-neighbour-anisic acids) is converted into nontoxic 3, the 6-dichlorosalicylic acid.This gene is at U.S. Patent number 7,022, is disclosed as the tolerance that provides dicamba 98 in 896 in the plant of expressing the DMO gene.Yet the exploitation of the variant of this gene will have very big benefit.This type of variant may have the efficient of change under certain environmental conditions.By this way, can be chosen as the variant that specific environment is optimized, in described environment, this variant will be intended to be used and can demonstrate especially useful dynamic characteristic.This variant especially can demonstrate maximum efficiency under different temperature or pH condition, thereby and the plant growth condition of condition in the cell and/or expection that depends on be selected for specific crop species.

Summary of the invention

On the one hand, the invention provides isolated nucleic acid sequences, it is selected from: a) nucleotide sequence of the polypeptide of coding SEQ IDNO:1; B) comprise the nucleotide sequence of the sequence of SEQ ID NO:2; And c) coding and the polypeptide of SEQ ID NO:1 have the nucleotide sequence of the polypeptide of at least 90% sequence identity, and it is active and comprise halfcystine in the position corresponding to the amino acid/11 12 of SEQ ID NO:1 that wherein back one polypeptide has dicamba monooxygenase enzyme.In other embodiments, provide dna vector, it comprises the DMO that operability is connected in the coding nucleic acid described herein of promotor.This promotor can have function in vegetable cell.In some embodiments, the nucleotide sequence of coding dicamba monooxygenase enzyme can operability be connected in chloroplast transit peptides.

On the other hand, the invention provides the peptide sequence that at least 90% identity is arranged with SEQ ID NO:1, it is active and comprise halfcystine in the position corresponding to the amino acid/11 12 of SEQ ID NO:1 that wherein this polypeptide has dicamba monooxygenase enzyme.

More on the one hand, the invention provides nucleic acid transformed host cells or tissue with coding dicamba monooxygenase enzyme as herein described.In some embodiments, host cell can be a vegetable cell.In other embodiments, vegetable cell can be defined as dicotyledons cell or monocot plant cell.In specific embodiments, host cell is soybean, cotton, corn or rape vegetable cell.In further embodiment, provide the tissue culture that comprises transgenic cell as herein described.

More on the one hand, the invention provides nucleic acid transgenic plant transformed and its offspring with coding dicamba monooxygenase enzyme as herein described.In some embodiments, plant can be defined as dicotyledons or monocotyledons.In specific embodiments, plant is soybean, cotton, corn or rape plant.

More on the one hand, the invention provides the method that produces the dicamba 98 tolerant plants, it comprises importing transformation construct provided herein in plant.In an embodiment of this method, importing transformation construct can and carry out described one or more cell regenerationes by the one or more vegetable cells of stable conversion for the dicamba 98 tolerant plants.In another embodiment, by producing the dicamba 98 tolerant plants with the mother plant selfing or with second kind of plant hybridization, wherein this mother plant and/or second kind of plant have comprised transformation construct and the heredity of dicamba 98 tolerant plants from the transformation construct of the mother plant and/or second kind of plant.

More on the one hand, the invention provides the method for producing food or feed, comprising: a) obtain as plant of the present invention provided herein or its part; And b) prepares food or feed from described plant or its part.In one embodiment of the invention, described plant part is a seed.In certain other embodiments, described food or feed are oil, meal, protein, grain, starch or protein.In other embodiments, feed comprises forage or pasture plant, as hay.The present invention also provides producd fibers, medicine, nutritious prod (neutraceuticals) and industrial chemical, comprises biofuel and from the method for any other product of plant provided herein.

More on the one hand, the invention provides the method for control weed growth in comprising as the crop growth environment of plant of the present invention provided herein or its seed, comprise the Banvel D of crop growth environment being used the amount of effective control weed growth.In some embodiments of the present invention, Banvel D can be applied to the top of crop growth environment.In specific embodiments, the amount of the Banvel D plant that do not damage plant of the present invention or its seed and infringement and lack DMO coding nucleic acid provided by the invention has the plant of homologous genes type.

In another embodiment of the present invention, plant is provided, it comprises DMO coding nucleic acid provided by the invention and at least a other transgenes encoding sequences, for example comprises at least two kinds, three kinds, four kinds, five kinds or more kinds of this type of encoding sequence.In specific embodiments, described plant comprises the transgenosis of giving one or more extra useful proterties such as weedicide or insect/insect tolerance.For example,, except the dicamba 98 tolerance, can also provide tolerance to one or more weedicides as hereinafter describing, and other useful proterties.Therefore the present invention provides plant especially, and it comprises and the nucleic acid of extra transgenosis proterties with the encoding D MO of the present invention of the combination " stack " of any hope.

The accompanying drawing summary

Following accompanying drawing has formed the part of this specification sheets and has been comprised in order to further specify some aspect of the present invention.The detailed description of the particular that one or more these accompanying drawings of reference and this paper provide, the present invention may be better understood.

Fig. 1. be used for the sketch map of the box of genetic modification dicamba monooxygenase enzyme gene (DMOc) in order to express higher plant, wherein use FLt36 promotor from peanut chlorotic streak virus, marmor erodens (TEV leader sequence) translational enhancer sequence and from the termination subarea of pea Rubisco small ylidene gene.The DMOc gene of prepared another kind of genetic modification form contains the transit peptides coding region from pea Rubisco small ylidene gene, and it is used for the chloroplast(id) location of DMO between the coding region of TEV translational enhancer district and DMOc.

Fig. 2 .DNA, RNA and western blotting figure, the DMO gene that shows genetic modification is at T ₁For existence in the rotaring gene tobacco plant and expression.Swimming lane Q has described from multiple T to V ₁DNA, mRNA and DMO kind for the rotaring gene tobacco plant extraction.Extract from the non-transgenic tobacco plant is described in swimming lane WT, and swimming lane Ox has shown the restriction enzyme digestion product (last figure) of the DMO gene construct of cloning and about 37kDa DMO enzyme (figure below) of excessive generation in intestinal bacteria (E.coli).By in the DMO antiserum(antisera), adding the Rubisco detection of antibodies, in western blotting, detect the big subunit of about 55kDa of Rubisco, the interior mark that Rubisco loads with protein total in each swimming lane of making comparisons.As what judge, loaded the RNA of equal quantities at each swimming lane by the ethidium bromide staining of duplicate gel.Arrow is pointed out the position of DMODNA, mRNA or protein belt.

Fig. 3. handle two kinds of T with dicamba 98 with 2.2kg/ha ₁The effect of tobacco plant, a kind of DMOc gene (right side) that contains the genetic modification that lacks the chloroplast transit peptide-coding sequence wherein, a kind of DMOc gene (right several second) that lacks.Transgenic plant on the right views demonstrate the damage from the dicamba 98 processing of very little (if there is).The two strain plants on left side dicamba 98 of no use is handled and represent transgenic plant (left side) and contain the transgenic plant (left several second) of DMOc gene.

The formation that Fig. 4 .DMOw causes along with time D CSA.

Fig. 5. determine the suitableeest mensuration pH of DMOw.

Fig. 6. determine the suitableeest mensuration temperature of DMOw.

Fig. 7. determine the optimal pH of DMOc.

Fig. 8. determine the optimum temperuture of DMOc.

The temperature of Fig. 9 .DMOc and DMOw and the general introduction of pH optimum condition.

The stability kinetics of Figure 10 .DMOw.

The stability kinetics of Figure 11 .DMOc.

Figure 12. 45 minutes effect of 30 ℃ of following preincubation DMOc in 50mM TRIS pH7.5 and 100mM KPi pH 7.0.

Figure 13. with leave standstill a week and at the enzyme of 4 ℃ of preservations at TRIS damping fluid (two assay method on the lefts; Be respectively before preservation and preserve after assay method) and the KPi damping fluid (two assay methods on the right; Be respectively before preservation and preserve after assay method) in the DMOc assay method.

Figure 14. be used at the tobacco chloroplast homologous recombination and the dicamba 98 monooxygenase construct of expressing through genetic modification.

Figure 15. with illustrating of the homoplasmon state of the chloroplast gene group of the transgene tobacco of DMO gene transformation system, described DMO gene is designed in tobacco chloroplast homologous recombination and expresses.Left figure has shown the construct (as showing among Figure 14) that is used for by homologous recombination DMO being incorporated into chloroplast(id).The dna fragmentation that is amplified for the hybridization probe for preparing the digoxigenin mark has been pointed out in the bar shaped of left side targeting sequencing top.Right figure has shown southern blotting technique: swimming lane 1 contains big tick marks.Swimming lane 2 contains the DNA from the non-transgenic tobacco plant.Swimming lane 3-11 contain that first round in the presence of spectinomycin is selected and regeneration after soon (last figure) and several take turns selection and regenerate after when obtaining the apparent homoplasmon of chloroplast gene group (figure below) from the transgenic plant separated DNA.Separate the DNA that is used for the southern blotting technique analysis with non-transgenic plant and use the restriction enzyme of BamH I to digest from transgenosis, electrophoretic separation and use with " left targeting sequencing " complementary of chloroplast gene group conversion carrier and survey DNA then through trace through the dna fragmentation (that is the hybridization probe of digoxigenin mark) of mark.5.6kb the DNA band corresponding to the chloroplast DNA fragment that contains the DMO gene and 3.3kb band corresponding to the natural chloroplast(id) band of the homology of the DMO gene construct that lacks insertion.

Figure 16. the T of the dicamba monooxygenase enzyme gene of handling with the dicamba 98 of 28kg/ha level that contains the chloroplast(id) coding ₁For the homoplasmon rotaring gene tobacco plant (R that plant 1-2 and plant 3-4 independently transform from two strains ₀Plant).

Figure 17. at the non-transgenic tobacco plant with the susceptibility and the resistance that contain in the chloroplast gene group that DMO in the rotaring gene tobacco plant of DMO gene expresses and dicamba 98 is handled.Western blotting with the detection of DMO antibody: swimming lane 1 contains the DMO of purifying.Swimming lane 2 is that barren and swimming lane 3 contain the protein extract from the non-transgenic tobacco plant.Swimming lane 4 and 8 contains from the isolating protein of " false positive " tobacco plant, and described tobacco plant demonstrates antibiotics resistance during selecting with spectinomycin, but lacks complete DMO gene.Swimming lane 5-7 contains the extract of transgenic plant, and described transgenic plant are expressed the DMO that is incorporated into the DMO genes encoding in the chloroplast gene group.S=is to the plant of the dicamba 98 sensitivity of 0.56kg/ha; The plant of the dicamba 98 of the anti-5.6kg/ha of R=.As by what judge with the proteinic amount of the big subunit of Rubisco of anti-Rubisco antibody test, almost the extract of equivalent is loaded among the swimming lane 4-8, and obviously more protein from non-transgenic plant is loaded in the swimming lane 3.Arrow is pointed out the proteinic position of DMO.

Figure 18. the comparison of the part of wild-type DMO peptide sequence and the conserved regions of other iron-sulphur oxygenase, show that DMO is unique, have low identity with known enzyme, but W112 (arrow) in other iron-sulphur oxygenase be guard and be that Rieske and Non-Haem Fe (SEQ ID NOS:4-23) center on by two conserved domains.

Detailed Description Of The Invention

The invention provides dicamba monooxygenase enzyme (DMO) variant that is known as in this article DMOc, it comprises cysteine at 112 bit positions corresponding to the DMO shown in the SEQ ID NO:1. Shown that DMOc produces the high-level tolerance to herbicide dicamba in genetically modified plants when expressing. The result is wonderful, because the amino acid position that changes is high conservative in other iron-sulphur oxygenase. In 78 kinds of iron from 45 species of analyzing-sulphur oxygenase sequence, all the 52 kinds of oxygenase sequences with at least 15% homogeneity have W in the position corresponding to the amino acid/11 12 of SEQ ID NO:1, although total the highest homogeneity is only 38%. This position is also centered on (Figure 18) by two conservative functional domains. Thereby the high-caliber herbicide tolerant DMOc that produces is unexpected.

DMOc is with respect to not changing sequence (DMOw; U.S. Patent number 7,022,896) as if it is different aspect catalytic efficiency that Mi-Man parameter analysis discloses described enzyme: DMOc has higher update times and stricter Binding Capacity than the efficient of DMOw high 5 times and DMOc. In addition, DMOc brings into play function with respect to native enzyme better under lower pH condition and higher temperature. These results show the service condition based on expectation, and selection is used for the possibility of the DMO variant of specific genetically modified plants such as the plant growth condition. Therefore an aspect of of the present present invention is related at least one crop species and identifies candidate's crop growth environment, and identifies the DMO enzyme that is suitable for this environment based on the dynamics of for example DMOc and DMOw. For example, those skilled in the art can be in specific embodiments, select the DMOc coded sequence, be used for having (inplanta) pH condition in the lower plant and/or in the situation of growing environment, having the plant of higher temperature with respect to other plant species or growing environment. By mixing soil (mixing before the plantation); Spraying soil (before emerging); Be sprayed at plant top (post processing of emerging) and use Mediben, and to the tolerance level of Mediben can be during plant growth different time and difference.

Point out as top, in the genetically modified plants of expressing DMOc, obtained the tolerance to extremely high-caliber herbicide dicamba. For example usually in addition the tobacco of extremely low-level Mediben sensitivity in, produced the genetically modified plants of expressing DMOc, its tolerance 5.6kg/ha or above Mediben are processed, for example, than the high 10-20 of field rate of application that usually recommends for the control broad leaved weed doubly. When in the chloroplast gene group that the DMOc gene is inserted into tobacco plant, obtain the Mediben tolerance at least 28 kg/ha. Also produced genetically engineered soybean, tomato and arabidopsis (Arabidopsis thaliana) plant of the DMOc gene of encoding with nucleus and found that they tolerate high-caliber Mediben. For example, DMOc has produced the tolerance that 2.8kg/ha is processed to the insertion in the karyogene group of bean plant, thereby allows to use Mediben control to express the plant Tanaka's of DMOc weeds.

Thereby show that DMOc is effectively, does not need extra coded sequence, such as Pseudomonas Maltophilia DI-6 bacterial strain, ferredoxin or reductase in giving the Mediben tolerance. Modified DMO gene is stable and not obviously forfeiture genepenetrance or expression as mendelian factor in heredity. Although get stronger to a certain degree expression with chloroplast transit peptides, the genetically modified genetically modified plants of DMO that have that lack the transit peptides coded sequence also demonstrate the high-caliber rear Mediben tolerance of emerging.

A. nucleic acid and recombinant precursor

1. dicamba monooxygenase enzyme (DMO)

In one embodiment of the invention, provide DNA construct, it comprises the nucleic acid of coding dicamba monooxygenase enzyme polypeptide, and described dicamba monooxygenase enzyme polypeptide comprises cysteine at 112 bit positions corresponding to SEQ ID NO:1. Exemplary DMO coded sequence provides with SEQ ID NO:2 in this article. This sequence also comprises with respect to natural coded sequence adding GCC codon (alanine) to add Nco I restriction enzyme sites and convenient clone after the ATG initiation codon except 112 the cysteine that comprises SEQ ID NO:1. Therefore, the polypeptide among the SEQ ID NO:1 also comprises the extra Ala residue that is close to behind the Met of initiation codon coding. Transit peptide sequence is excised from plasmid with Bgl II and EcoR I, then be cloned among the BamH I and EcoR I site of pBluescript II KS+ carrier. This construct is as the template in the PCR reaction, and the primer that adds Nco I restriction site at the arbitrary end of transit peptides coded sequence is used in described PCR reaction. Allow in the ATG initiation codon site of modified DMO gene, to insert the transit peptides coded sequence with Nco I digestion PCR product.

Thereby, in one embodiment of the invention, provide the sequence of the polypeptide of coding SEQ ID NO:1, include but not limited to SEQ ID NO:2. As known in the art, the homologous sequence of these sequences and derivative can easily prepare and use. For example, can use coding and the DMOc polypeptide of SEQ ID NO:1 that at least 90% sequence homogeneity is arranged, comprise that sequence therewith is at least about 92%, 94%, 95%, 96%, 97%, 98%, 99% or the nucleic acid of the DMO polypeptide of higher homogeneity. Also can use the nucleic acid that provides with SEQ ID NO:2 that at least 90% sequence homogeneity is arranged, comprise that therewith sequence is at least about 92%, 94%, 95%, 96%, 97%, 98%, 99% or higher homogeneity and be coded in 112 nucleic acid that comprise the DMO of cysteine. In one embodiment, use GCG Wisconsin Package (Accelrys, San Diego, CA), MEGAlign (DNAStar, Inc., 1228S.Park St., Madison, Wis.53715) the sequence analysis software bag determine sequence homogeneity with default parameters. This software is by distributing similitude or homogeneity degree to mate similar sequence.

According to the disclosure, can obtain expressing the polynucleotide molecule of DMO polypeptide by technology well known in the art.Thereby can prepare the dicamba 98 of can degrading DMOs provided herein variant and measure active according to method disclosed herein.This type of sequence also can for example be identified (U.S. Patent number 5,445,962 from suitable biology (bacterium that comprises the dicamba 98 of degrading); Krueger etal., 1989; Cork and Krueger, 1991; Cork and Khalil, 1995).A kind of method of the DMO sequence of separating clone is by for example with from the biological library that makes up of originating carrying out suitable hybridization, perhaps using from the mRNA of source biology and the primer that designs based on disclosed DMO and carry out RT-PCR.Therefore the present invention comprises the nucleic acid that use is hybridized with DMO encoding sequence as herein described under stringent condition.Those skilled in the art understand can be so that the severity of condition reduces by increasing salt concn and reducing temperature.Thereby, can easily operate the method for selection that hybridization conditions and its will normally depend on the result of hope.An example of high stringent condition is 5X SSC, 50% methane amide and 42 ℃.By under this type of condition, washing,, will not be removed under these conditions with those sequences of particular target sequence hybridization as 10 minutes.Use the DMO coding nucleic acid thereby one embodiment of the invention comprise, described nucleic acid is defined in 5X SSC, under 50% methane amide and the 42 ℃ of wash conditions that continue 10 minutes down with the nucleic acid of the nucleic acid hybridization of SEQ ID NO:2.

According to technology well known in the art, also can the chemosynthesis variant with DMO polynucleotide sequence described herein.For example, can the synthetic DNA sequence in the automatization dna synthesizer by the phosphoramidite chemistry.Chemosynthesis has many advantages.Particularly, chemosynthesis is desirable, because the preferred codon of host's (dna sequence dna will be expressed therein) can be used for optimization expression.Thereby the example that uses the selection of Arabidopis thaliana codon to optimize this type of sequence that is used for expressing in dicotyledons is the DMO sequence that shows among the SEQ ID NO:3.Respectively 2,3,112 have Ala through prediction, Thr, and the polypeptide of Cys provides in SEQ ID NO:1.Owing to after next-door neighbour ATG initiator codon, add the L-Ala codon, added the Ala residue at 2 with respect to wild-type DMO to simplify vector construction (as following).

Be not to need all codons to be changed expression to be improved, but the codon that preferably seldom uses in the host to the major general changes over the preferred codon of host, for example, more in being usually used in the host and usually than the codon of the rare not preferred easier translation of codon.By will be greater than about 50%, most preferably change over the preferred codon of host at least about 80% not preferred codon, can obtain high level expression.The codon priority of many host cells is known (PCT WO 97/31115; PCT WO 97/11086; EP 646643; EP 553494; And U.S. Patent number: 5,689,052; 5,567,862; 5,567,600; 5,552,299 and 5,017,692).By can the derive codon priority of other host cells of methods known in the art.And, use chemosynthesis, the sequence that can easily change dna molecular or its encoded protein matter is with for example optimization expression (for example, eliminating the mRNA secondary structure that interference is transcribed or translated), at the unique restriction site of site adding easily, and remove proteolytic enzyme cutting site.

Can be to proteinic peptide sequence, modify and change as DMO sequence provided herein and keep enzymic activity simultaneously.Be based on below and change that proteinic amino acid is equal to generation or even the improved modified polypeptide and the discussion of corresponding codes sequence.In specific embodiments of the present invention, can change the DMO sequence by this way and be used for method of the present invention.Can realize amino acid change by the codon that changes dna sequence dna.

For example known, some amino acid can substitute in the protein structure other amino acid and and structure, do not have significantly sacrificing as the ability of mutually combining of the binding site on the substrate molecule.Because be that protein interactions ability and character define this proteinic biological function activity, so can be at protein sequence and certain, in dna encoding sequence, carry out some aminoacid sequence and substitute as the basis, and the protein that still obtains having similar quality.Thereby expection can be carried out multiple change in DMO peptide sequence as herein described and corresponding dna encoding sequence and their biological effectiveness or activity do not have significantly sacrificing.

Carrying out this type of when changing, hydrophilic index that can considered amino acid.The hydrophile amino acid number is (Kyte etal., 1982) well known in the art in the importance of protein being given in the interaction biological function.Generally acknowledge that amino acid whose hydrophilic relatively feature has contribution to the proteinic secondary structure of gained, described secondary structure has been determined the interaction of this protein and other molecules such as enzyme, substrate, acceptor, DNA, antibody, antigen or the like again.Each amino acid has been assigned with hydrophobicity index (Kyte et al., 1982) based on their hydrophobicity and charge characteristic, and these are: Isoleucine (+4.5); Xie Ansuan (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Halfcystine/Gelucystine (+2.5); Methionine(Met) (+1.9); L-Ala (+1.8); Glycine (0.4); Threonine (0.7); Serine (0.8); Tryptophane (0.9); Tyrosine (1.3); Proline(Pro) (1.6); Histidine (3.2); L-glutamic acid (3.5); Glutamine (3.5); Aspartic acid (3.5); L-asparagine (3.5); Methionin (3.9); And arginine (4.5).

Amino acid known in the art can be had other amino acid replacements of similar hydrophilic index or score and still be obtained having similar bioactive protein, promptly still obtains the protein that biological function is equal to.Carrying out this type of when changing, hydrophilic index is preferred ± 2 with interior amino acid replacement, ± 1 with interior be especially preferred, the ± 0.5th, especially preferred.Here, consider hydrophilic indexes different between natural and the amino acid that changes and will be not used for producing functional variant according to prior art by those skilled in the art, thus 112 tryptophane with halfcystine alternate DMO biologically active and this observations of plant that obtains tolerating high-level dicamba 98 be wonderful.

This area is also understood based on wetting ability can effectively carry out similar amino acid whose substituting.United States Patent (USP) 4,554, the 101 local average wetting abilities that disclose proteinic maximum (as the wetting ability control by its adjacent amino acid) are relevant with proteinic biological property.As United States Patent (USP) 4,554, describe in detail in 101, following hydrophilicity value is assigned to amino-acid residue: arginine (+3.0); Methionin (+3.0); Aspartic acid (+3.0 ± 1); L-glutamic acid (+3.0 ± 1); Serine (+0.3); L-asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (0.4); Proline(Pro) (0.5 ± 1); L-Ala (0.5); Histidine (0.5); Halfcystine (1.0); Methionine(Met) (1.3); Xie Ansuan (1.5); Leucine (1.8); Isoleucine (1.8); Tyrosine (2.3); Phenylalanine (2.5); Tryptophane (3.4).Be appreciated that amino acid can substitute another amino acid with similar hydrophilicity value and still obtain the protein that biology is equal to.In this type of changed, hydrophilicity value was preferred ± 2 with interior amino acid replacement, and ± 1 is especially preferred with interior amino acid replacement, ± 0.5 with interior amino acid replacement was even more specifically preferred.Exemplary the substituting of considering these and multiple aforementioned feature is to well known to a person skilled in the art and comprise: arginine and Methionin; L-glutamic acid and aspartic acid; Serine and Threonine; Glutamine and l-asparagine; And Xie Ansuan, leucine and Isoleucine.Once more, consider change and natural amino acid between very different hydrophilicity values and should substitute and will be not used for producing functional variant according to prior art by those skilled in the art, the activity of DMOc is wonderful.

Modification according to DMO sequence of the present invention can be instructed by considering the conserved domain in this enzyme.For example, be elucidated later herein below the DMO enzyme and contain functional domain, as the binding site (for example, seeing Figure 18) of Rieske iron-sulphur bunch and free iron.Therefore this information and this area general can be used for producing the activity that modified DMO enzyme keeps enzyme simultaneously within the scope of the present invention and (for example see Mason and Cammack, 1992 about the knowledge of proteinic functional domain and modification is combined; Jiang et al., 1996).

2. transformation construct

Being used for DMO coded polynucleotide of the present invention will be imported into cell usually as construct, and it includes the necessary expression controlling elements of efficient expression.It is (Maniatis et al., 1982 well known in the art that operability connects the method for expressing controlling elements and encoding sequence; Sambrook et al., 1989).Expression control sequenc is by any way with reference to the dna sequence dna of transcribing control.Suitable expression control sequenc and their method of use are well known in the art.Especially can use promotor, use or do not use enhanser, 5 ' non-translational region is used for protein or RNA product targeted plants organoid, and the especially transit peptides of chloroplast(id) or signal peptide and 3 ' non-translational region are as the polyadenylation site.One skilled in the art will know that multiple enhanser, promotor, intron, transit peptides, targeting signal sequence and 5 ' and 3 ' non-translational region (UTRs) can be used for designing effective plant expression vector, those as describing in the U.S. Patent Application Publication 2003/01403641.

It is well known in the art being suitable for current and promotors other purposes.The example of describing this type of promotor comprises United States Patent (USP) 6,437,217 (corn RS81 promotors), United States Patent (USP) 5,641,876 (rice actin promoters), United States Patent (USP) 6,426,446 (corn RS324 promotors), United States Patent (USP) 6,429,362 (corn PR-1 promotors), United States Patent (USP) 6,232,526 (corn A3 promotors), United States Patent (USP) 6,177,611 (composing type corn promotors), United States Patent (USP) 5,322,938,5,352,605,5,359,142 and 5,530,196 (35S promoters), United States Patent (USP) 6,433,252 (corn L3 oleosin promotors), United States Patent (USP) 6,429,357 (rice Actin muscle 2 promotors and rice Actin muscle 2 introns), United States Patent (USP) 5,837,848 (root-specific promoters), United States Patent (USP) 6,294,714 (photoinduction type promotors), United States Patent (USP) 6,140,078 (salt inducible promoters), United States Patent (USP) 6,252,138 (pathogen-inducible promoters), United States Patent (USP) 6,175,060 (phosphorus shortage inducible promoter), United States Patent (USP) 6,635,806 (γ-coixin promotor), with u.s. patent application serial number 09/757,089 (corn chloroplast(id) zymohexase promotor).Operable extra promotor is nopaline synthase (NOS) promotor (Ebert et al., 1987), octopine synthase (OCS) promotor (it carries on the tl plasmid of agrobacterium tumefaciens), cauliflower mosaic virus promoter, as cauliflower mosaic virus (CaMV) 19S promotor (Lawton et al., 1987), CaMV 35S promoter (Odell et al., 1985), radix scrophulariae mosaic virus 35 S-promotor (Walker et al., 1987), sucrose synthase promotor (Yang et al., 1990), R gene combined promoter (Chandler et al., 1989) and chlorophyll a/b binding protein gene promotor or the like.For be used for the present invention especially useful can be CaMV35S (U.S. Patent number 5,322,938; 5,352,605; 5,359,142; With 5,530,196), FMV35S (United States Patent (USP) 6,051,753; 5,378,619), PC1SV promotor (for example United States Patent (USP) 5,850,019 and SEQ ID NO:24), and AGRtu.nos (GenBank searching number V00087; Depicker et al, 1982; Bevan et al., 1983) promotor.

Also can obtain an advantage by the sequence expression of heterologous genes of using the coding transit peptides.Transit peptides is often referred to when being connected to target protein matter the peptide molecule with this protein targeting particular organization, cell, subcellular location or organoid.Example includes, but not limited to chloroplast transit peptides, nuclear targetting signal and vacuole signal.The expression that chloroplast transit peptides especially can be used for being used among the present invention the DMO enzyme is directed at chloroplast(id).Expection can promote the DMO function by endogenous reductase enzyme and the ferredoxin degraded dicamba 98 of finding in the vegetable cell.Plant chloroplast especially is rich in reductase enzyme and ferredoxin.Therefore,, can use the sequence of encoded peptide, in the oxygenase guiding chloroplast(id) of its dicamba 98 of will degrading in the preferred embodiment that is used for producing genetically modified dicamba 98 tolerant plants.Alternatively or extraly, also can be in cell expressing heterologous reductase enzyme and/or ferredoxin.

The DNA of coding chloroplast(id) targeting sequencing can preferably be placed in the sequence upstream (5 ') of encoding D MO, but also can be placed in the downstream (3 ') of encoding sequence, the perhaps upstream and downstream of encoding sequence.Especially chloroplast transit peptides (CTP) can be transform as with the proteinic N-that will be directed to plant chloroplast is terminal and merge.The localized protein of many chloroplast(id)s is expressed as precursor and is directed at chloroplast(id) by CTP from nuclear gene, and CTP is removed in input step.The example of chloroplast protein comprises ribulose-1,5-bisphosphate, 5, and the small subunit of-bisphosphate carboxylase (RbcS2), ferredoxin, ferredoxin oxide-reductase, capturing optical conjugated protein I and protein I I and Trx F.In vivo with external illustrate by use protein blend zoarium with CTP non-chloroplast protein can be directed at chloroplast(id) and CTP enough with protein targeting in chloroplast(id).For example, shown in transgenic plant, suitable chloroplast transit peptides, as Arabidopis thaliana (Arabidopsisthaliana) EPSPS CTP (Klee et al., 1987) and mixing of Petuniahybrida EPSPS CTP (della-Cioppa et al., 1986) allos EPSPS protein sequence is directed in the chloroplast(id).Other exemplary chloroplast(id) targeting sequencings comprise corn cab-m7 signal sequence (Becker etal., 1992; PCT WO 97/41228) and pea glutathione reductase signal sequence (Creissen et al., 1991; PCT WO 97/41228).In the present invention, AtRbcS4 (CTP1; United States Patent (USP) 5,728,925), AtShkG (CTP2; Klee et al., 1987), AtShkGZm (CTP2synthetic; See the SEQ ID NO of WO04009761; 14), and PsRbcS (Coruzziet al., 1984) be especially useful aspect the DMO polypeptide expression for example.

5 ' UTR as translation leader sequence performance function is in the promoter sequence of gene and the DNA genetic elements between the encoding sequence.The translation leader sequence is present among the mRNA that processes fully of translation initiation sequence upstream.The translation leader sequence can influence processing, mRNA stability or the translation efficiency of one-level transcript to mRNA.The example of translation leader sequence comprises corn and morning glory heat shock protein leader sequence (U.S. Patent number 5,362,865), plant virus envelope protein leader sequence, plant rubisco leader sequence, or the like (Turner and Foster, 1995).In the present invention, the 5 ' UTR that especially can find benefit is GmHsp (United States Patent (USP) 5,659,122), PhDnaK (United States Patent (USP) 5,362,865), AtAnt1, TEV (Carrington and Freed, 1990), and AGRtunos (GenBank searching number V00087; Bevan etal., 1983).

3 ' non-translated sequence, 3 ' transcription termination region or polyadenylation district refer to dna molecular, its be connected to the coding region of gene and be positioned at the downstream, coding region of gene and comprise provide that polyadenylation signal and can influencing is transcribed, the polynucleotide of other conditioning signals of mRNA processing or genetic expression.Polyadenylation signal is brought into play function in plant, cause 3 ' the terminal polyadenylic acid Nucleotide that adds at the mRNA precursor.The polyadenylation sequence can be from natural gene, from the various plants gene, or from the T-DNA gene.An example of 3 ' transcription termination region be nopaline synthase 3 ' district (nos3 '; Fraley et al., 1983).The purposes (Ingelbrecht et al., 1989) of different 3 ' non-translational regions has been described.From pea (Pisum sativum) RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al., 1984) and the polyadenylic acid chemoattractant molecule of AGRtu.nos (Rojiyaa et al., 1987, Genbank searching number E01312) be especially useful for the present invention.

The polynucleotide molecule ceneme of encoding D MO can be connected to second kind of polynucleotide molecule in the ceneme of the genetic elements that contains the gene that can screen/can mark mark or give desired proterties.The gene commonly used that is used to screen the transformant of supposition comprises β-glucuronidase (GUS), beta-galactosidase enzymes, luciferase and E.C. 2.3.1.28 (Jefferson, 1987; Teeri et al., 1989; Koncz etal., 1987; De Block et al., 1984), green fluorescent protein (GFP) (Chalfie et al., 1994; Haseloff et al., 1995; With PCT application WO97/41228).

Second kind of polynucleotide molecule can include but not limited to, as the gene of selective marker.Second kind or another kind of gene can provide the desirable feature relevant with phytomorph, physiology, g and D, output, nutrition enhancing, disease or pest resistance or environment or chemical resistance and can comprise genetic elements, described genetic elements comprises Herbicid resistant (United States Patent (USP) 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; 5,463,175), the output of increase (United States Patent (USP) RE38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,098; 5,716,837), insect control (United States Patent (USP) 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,468,523; 6,326,351; 6,313,378; 6,284,949; 6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695; 5,959,091; 5,942,664; 5,942,658,5,880,275; 5,763,245; 5,763,241), fungal disease resistance (United States Patent (USP) 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436; 6,316,407; 6,506,962), virus resistance (United States Patent (USP) 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; 5,304,730), nematode resistance (United States Patent (USP) 6,228,992), bacterial disease resistance (United States Patent (USP) 5,516,671), plant-growth and growth (United States Patent (USP) 6,723,897; 6,518,488), Starch Production (United States Patent (USP) 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), oil production (United States Patent (USP) 6,444,876 of change; 6,426,447; 6,380,462), high oil yield (United States Patent (USP) 6,495,739; 5,608,149; 6,483,008; 6,476,295), the fatty acid content of modification (United States Patent (USP) 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,461; 6,459,018), high protein output (United States Patent (USP) 6,380,466), fruit maturation (United States Patent (USP) 5,512,466), the nutrition of enhanced animal and human class (United States Patent (USP) 6,723,837; 6,653,530; 6,5412,59; 5,985,605; 6,171,640), biological polymer (United States Patent (USP) RE37,543; 6,228,623; 5,958,745 and U.S. Patent Publication No. US20030028917), tolerance to environmental stress (United States Patent (USP) 6,072,103), but medicinal peptide and excretory peptide (United States Patent (USP) 6,812,379; 6,774,283; 6,140,075; 6,080,560), improved processing trait (United States Patent (USP) 6,476,295), the digestibility of raising (United States Patent (USP) 6,531,648), low raffinose (United States Patent (USP) 6,166,292), industrial enzyme production (United States Patent (USP) 5,543,576), improved local flavor (United States Patent (USP) 6,011,199), nitrogen fixation (United States Patent (USP) 5,229,114), hybrid seeds (United States Patent (USP) 5,689,041), fiber production (United States Patent (USP) 6,576,818; 6,271,443; 5,981,834; 5,869,720) and biofuel production (United States Patent (USP) 5,998,700).Be appreciated that with reference to the disclosure these or other genetic elements, method and genetically modified any can be used for the present invention as those skilled in the art.

Ceneme can be used as the right margin (RB) of first kind of plasmid and the T-DNA between left margin (LB) district provides in Agrobacterium (Agrobacterium) with the second kind of plasmid that carries T-DNA transfer and integration function.The plasmid main chain DNA section that provides copy function and microbiotic to select in bacterial cell also can be provided construct, as the intestinal bacteria replication orgin, as ori322, wide host range replication orgin is as the coding region of oriV or oriRi and selective marker, as the Spec/Strp of the Tn7 aminoglycoside adenylic acid (AMP) transferring enzyme (aadA) of encoding, it gives the resistance to spectinomycin or Streptomycin sulphate, perhaps gentamicin (Gm, Gent) selectable marker gene.For Plant Transformation, the host bacteria bacterial strain is agrobacterium tumefaciens ABI, C58 or LBA4404 normally.Yet known other bacterial strains of Plant Transformation those skilled in the art can be brought into play function in the present invention.

3. the preparation of transgenic cell

Can realize transformed plant cells (for example seeing Miki et al., 1993) by any technology well known in the art with the transgenosis transfered cell.Think that the example of these class methods comprises almost any method with the DNA transfered cell.The method of having described comprises as U.S. Patent number 5,384, the electroporation of illustrating in 253; U.S. Patent number 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; With 6,403, the microparticle bombardment of illustrating in 865; As U.S. Patent number 5,635,055; 5,824,877; 5,591,616; 5,981,840; With 6,384, the agriculture bacillus mediated conversion of illustrating in 301; With U.S. Patent number 5,508, the protoplast transformation of illustrating in 184.By using such as these technology, can be according to the present invention stable conversion and selection almost any plant species cell and these cell developments are become transgenic plant.

The method of extensive utilization that expression vector is imported plant is based on the natural conversion system (for example, Horsch et al., 1985) of Agrobacterium.Agrobacterium tumefaciens and Agrobacterium rhizogenes (A.rhizogenes) are the plant-pathogenic soil bacterias, its genetic transformation plant cell.Ti of agrobacterium tumefaciens and Agrobacterium rhizogenes and Ri plasmid carry the gene (for example, Kado, 1991) of the genetic transformation of being responsible for plant respectively.The description of agrobacterium vector system and agriculture bacillus mediated gene transfer method is provided by many reference, comprises Miki et al., above, Moloney et al., 1989, and U.S. Patent number: 4,940,838 and 5,464,763.With interactional other bacteriums of natural plant, can be transferred in many different plants with mediated gene by modification as soybean nodulation Pseudomonas (Sinorhizobium), rhizobium (Rhizobium) and middle rhizobium (Mesorhizobium).The Ti-plasmids that unloads first (disarmed) by acquisition can be so that the relevant symbiotic bacterium of these plants be competent (Brothers et al, 2005) for transgenosis with suitable binary vector.

B. tissue culture and plant regeneration

Can realize the plant of institute's plant transformed cell regeneration by at first on the germination substratum, also on root media, cultivating explant subsequently for educating.Sometimes, can transfer to then on the germination substratum at cultivation explant on the callus culture base.Can and optimize multiple substratum and shift the recovery that requires to be used for Plant Transformation and transgenic plant for every kind of plant system's realization.Therefore, this type of substratum and culture condition can be modified or the component that is equal to nutrition or be used to is selected and the similar approach that reclaims transgenic event substitutes.

Nutritional medium can be prepared as liquid, but this can solidify by the material that can provide solid to support is provided in liquid.Agar is most commonly used to this purpose.Bactoagar, Hazelton agar, Gelrite and Gelgro are the solid supports of particular type, and it is suitable for the growth of vegetable cell in the tissue culture.Some cell types will be in liquid suspension or solid medium or growth and division on two kinds of substratum.

Recipient cell targets includes, but not limited to meristematic cell, callus, immature embryo and gametid [cell, as sporule pollen, spermoblast and ovum.Any cell of the transgenic plant that can educate of can being used for regenerating can be used for some embodiments.For example, can transform immature embryo, then the transgenic plant of selecting and starting callus and regenerate and to educate subsequently.The needs to long-term exploitation recipient cell culture have been avoided in the direct conversion of immature embryo.Meristematic cell (that is, can p cell division and be characterized as the undifferentiated cytology outward appearance vegetable cell of (be present in vegetative point or the tissue of plant usually, locate as the tip of a root, stem apex, lateral bud etc.)) also can be used as the recipient plant cell.Because their undifferentiated growths and can organ differentiation and totipotency, so can reclaim complete conversion plant from the meristematic cell of single conversion.

Somatocyte is polytype.Embryo generation cell is can be by embryogeny by the somatic example of regeneration induction plant.Non-embryo generation cell is those cells of not replying by this way usually.

Can use some technology of enrichment recipient cell in cell colony.For example, II type healing tissue development, then artificial selection and the brittle embryo of cultivation are organized, and cause enrichment to be used for for example recipient cell of particle conversion usually.

In some embodiments, recipient cell is selected in the growth back in cultivation.Cultured cells can be grown on solid support or with the form of liquid suspension.In arbitrary situation, can be that cell provides nutrition with the form of substratum, and the condition that controls environment.The tissue culture medium (TCM) that many types are arranged, it comprises amino acid, salt, sugar, growth regulator and VITAMIN.The most of substratum that are used for the present invention practice will have some similar components, and according to known tissue culture practice, substratum can form with components in proportions aspect different.For example, the various kinds of cell type is grown in more than one substratum usually, but will depend on growth medium and demonstrate different growth velocitys and different forms.In some substratum, cell survival does not still divide.Also often optimize the substratum composition based on selected species or cell type.

The polytype substratum that is suitable for culture plant cell had been described in the past.The example of these substratum includes but not limited to, N6 substratum and MS substratum (Murashige ﹠amp that Chu et al. (1975) describes; Skoog, 1962).In some embodiments, preferred substratum such as the N6 that use with lower in a way ammonia/nitrate ratio, thus so that being remained on, cell can continue the generation that splitted proembryo state promotes recipient cell.Also can use Woody plant culture (WPM) (Lloyd and McCown, 1981).

Keep the method for cell culture can promote their application as the recipient cell source that is used to transform.The frequency of fresh culture, the composition of substratum are selected, are transferred in the craft that is used to transfer to the cell of fresh culture, and environmental factor, include but not limited to that light quality and quantity and temperature all are to keep as the callus in recipient cell source and/or the factor in the suspension culture.Callus is useful for enrichment recipient cell in cultivation alternately between different culture condition.For example, cell can be cultivated in suspension culture, still be transferred in the solid medium with the timed interval of rule.After growth for some time on the solid medium, can select cell to be used for turning back to liquid nutrient medium by hand.Repeat this order of transferring to fresh culture and can be used for the enrichment recipient cell.Cell culture medium passed 1.9mm sieve also can be used to keep the fragility of callus or suspension culture and the transformable cell of enrichment when this type of cell type of use.

C. transgenic plant

In case selected transgenic cell, can use the transgenic plant of technology well known in the art with this cell regeneration Cheng Keyu.Subsequent analysis transforms plant to determine to exist or do not exist specific purpose nucleic acid in DNA construct.Analysis of molecules can include but not limited to, southern blotting technique (Southern, 1975) or pcr analysis, immune diagnostic method.Also can use the field assessment.Can carry out these and other known method and confirm the stability of the conversion plant that disclosed method produces.These methods are to well known to a person skilled in the art (Sambrook et al., 1989).

Thereby can produce the transgenic plant that comprise DMO encoding sequence provided herein.Particularly, important economically plant comprises that crop, tree and other plant can make that they are dicamba 98 tolerances tolerance or that have increase with DNA construct conversion of the present invention.Currently think that thereby the plant of tolerance growth hormone sample weedicide can be transformed to increase their tolerances to this weedicide.Some limiting examples that can be used for plant of the present invention comprise clover, barley, Kidney bean, beet, green Cauliflower, wild cabbage, Radix Dauci Sativae, rape, Cauliflower, celery, Chinese cabbage, corn, cotton, cucumber, eggplant, leek, lettuce, muskmelon, oat, onion, pea, pepper, peanut, potato, summer squash, radish, rice, sweet corn, Chinese sorghum, soybean, spinach, pumpkin, sugar beet, Sunflower Receptacle, tomato, watermelon and wheat.

Contain genetically modified transgenic plant in case prepared, can this transgenosis importing be planted the compatible any plant of rerum natura with first kind by hybridization, and not need directly to transform second kind of plant.Therefore, as used herein, term " offspring " refers to the offspring of any generation of mother plant prepared in accordance with the present invention, and wherein the offspring comprises selected DNA construct prepared in accordance with the present invention." transgenic plant " can be the transgenic plant of any generation.As disclosed herein, be defined as following technology so that the transgenosis or the allelic plant lines that have one or more interpolations with respect to initial plant lines to be provided with plant " hybridization ": its by with initial strain with comprise the hybridization of transgenosis of the present invention or allelic donor plant lines and cause particular sequence to be imported in the plant lines.In order to realize this purpose, can for example carry out following step: (a) seed of plantation first kind (initial strain) and second kind of (comprising desirable transgenosis or allelic donor plant lines) mother plant; (b) seed growth with described second kind and second kind mother plant is to have colored plant; (c) will pollinate with pollen from the flower of first kind of mother plant from second kind of mother plant; (d) gather in the crops the seed that on first kind of plant that has the flower of being fertilized, produces.

Thereby the present invention provides the transgenic plant that comprise DMO coding nucleic acid provided herein tissue.This tissue can be directly transform or hereditary nucleic acid from progenitor cell with the DMO coding nucleic acid.Tissue provided by the invention particularly including but be not limited to cell, embryo, immature embryo, meristematic cell, prematurity male flower fringe, sporule, pollen, leaf, flower pesticide, root, the tip of a root, flower and seed.Thereby the present invention provides any this type of tissue that comprises nucleic acid as herein described, comprises any plant part.Seed especially is used for commerce or food applications in the form with grain, and is used for planting and the extra crop that grows is useful especially.

Embodiment

Comprise that the following examples are used to illustrate embodiment of the present invention.It will be appreciated by those skilled in the art that the technology that in practice of the present invention, plays a role well that on behalf of the inventor, disclosed technology find among the following embodiment.Yet those skilled in the art will understand according to the disclosure and can carry out many changes in disclosed particular and still obtain similar or similar result and do not deviate from notion of the present invention, spirit and scope.More specifically, it is evident that some relative material of chemistry or physiology can replace material as herein described and realize same or analogous result.Think conspicuous all these type of similar replacement schemes of those skilled in the art and being modified within spirit of the present invention, scope and the notion of claims definition.

Embodiment 1

The vector construction that is used for the DMO gene of genetic modification

Produce DMOc variant encoding sequence from the DMOw template by pcr amplification at first.In this amplification, from the coding region of plasmid pPLH1 amplification DMOw, described plasmid contains the DMOw gene as Pseudomonas Maltophilia bacterial strain DI-6, the 3.5kbp Xho I/Sst I fragment of DNA.For DNA cloning, use 5 ' primer and 3 ' primer, 5 ' primer inserts the codon of the L-Ala that is close to after Nco I restriction site and the ATG initiator codon near 5 ' end of PCR product, 3 ' primer is at 3 ' the terminal Xba I restriction site (step details provides hereinafter) that produces of PCR product.Identify the change of 112W by nucleic acid sequencing subsequently to 112C.

In order to produce plant conversion carrier, with 5 ' and 3 ' terminal NcoI and the Xba I site that adds the coding region respectively the DMOc gene is inserted into pRTL2 carrier (Carrington and Freed, 1990) in, thereby the coding region is fused to marmor erodens (TEV leader sequence) the translational enhancer element of carrier.Use specially designed PCR primer to import 5 ' Nco I site and after the ATG initiator codon, add GCC codon (L-Ala) and at 3 ' the terminal Xba of generation I restriction site of coding region.In order to allow that DMOc is delivered to chloroplast(id), will place the DMOC upstream of coding region to allow to be directed at chloroplast(id) from the chloroplast transit peptide-coding region (Coruzzi et al., 1983) of pea Rubisco subunit gene.The transit peptides encoding sequence that carries on Bgl II and the EcoR I fragment is cloned among the BamH I and EcoR I site of pBluescript II KS+ carrier.This construct is as the template in the PCR reaction, and its 3 ' end and 5 ' end at transit peptide sequence all inserts Nco I site.With the product cloning of amplification make in the Nco I site of pRLT2 carrier transit peptide sequence be located immediately at the DMO gene coding region the upstream and with meet its reading frame.The box of being made up of TEV leader sequence, transit peptides district and DMODNA encoding sequence from the excision of pRTL2 carrier with Xho I and Xba I also is cloned into pKLP36 carrier (U.S.5,850,019 with it; Fig. 5), use identical restriction enzyme sites to be used to connect this box and PC1SV promotor and PsRbcS2-E9 polyadenylic acid sequence.New carrier is marked as pKLP36-TEV-TP-DMOc (being also referred to as pKLP36-DMOc), and be deposited in American type culture collection (ATCC) on February 2nd, 2006,10801 University Boulevard, Manassas, Va.20110-2209USA, and distribute ATCC preserving number PTA-7357.

The pKLP36-DMOc carrier is used for transformation of tobacco, Arabidopis thaliana and tomato plants.Transform for soybean, the DMOc box is cut out as EcoR I/Acc I section and is cloned among the pPZP101 (Hajdukiewicz et al., 1994) of EcoR I/Acc I digestion from pKLP36-TEV-TP-DMOc be used to obtain the right side and left margin.Cut this carrier (pPZP101+DMOc box) and the DMOc box be cloned into binary vector pPTN200 (seeing below) with ScaI then---derivative (the Hajdukiewicz et al. of pPZP201,1994) in, it contains flank selects the regeneration transformant for the bar box and the permission on a left side and right T-DNA border in the presence of weedicide Basta.Two new T-DNA binary vectors are called pPTN348 and are used for the soybean conversion.By at first will be from pGPTV-bar (Becker et al., 1992) no promotor-bar element is cloned into pPZP201 as the PstI/BamHI section and (is seen Hajdukiewicz et al., 1994) preparation pPTN200 carrier in, and with gained plasmid called after pPTN193.Will from the no terminator of pE7113-GUS (seeing Mitsuhara et al., 1996) be cloned into be in no promotor-bar element among the pPTN193 the downstream to obtain the bar box.

Obtain restriction enzyme and other enzymes from Fermentas or Invitrogen.Obtain DIG-11-dUTP (the alkali mark) from Roche, CSPD (ready-to-use), DIG III molecular weight marker, anti-digoxigenin-AP (Fab fragment) and closed reagent.Obtain prehybridization solution ULTRAhyb from Ambion.Obtain DIG-RNA molecular weight standard I from Roche.Obtain the antibody (donkey) and Hybond ECL (nitrocellulose) film of anti-rabbit igg, peroxidase-connection from AmershamBiosciences.Carry out DNA, RNA, western blotting, recombinant DNA technology and other molecular biology methods with standard technique (Ausubel et al., 1995).

Embodiment 2

Generation of transgenic plant and analysis

Tobacco, tomato, soybean and Arabidopis thaliana are used for the DMOc gene of transgene expression genetic modification and confirm dicamba 98 tolerance the plant of expressing this gene.DMOc encoding sequence among the binary vector pKLP36 is contained among the soil Agrobacterium bacterial strain C58C1 that unloads first Ti-plasmids pMP90 (Koncz and Schell, 1986) by triparental mating (Ditta 1980) importing.The leaf dish scheme of describing with Horsch etal. (Horsch 1985) is used for tobacco (cv Xanthi) and tomato (cv Rutgers) conversion with the transconjugant of gained.By flower immersion technology (Clough and Bent, 1998) arabidopsis thaliana transformation.Carry out the conversion of soybean varieties Thorne and NE-3001 by the agriculture bacillus mediated conversion system of cotyledonary node (Zhang et al., 1999).

The DMOc gene has produced the T in several independent sources to the genomic agriculture bacillus mediated transgenosis of tobacco plant ₁For plant.Existence and expression with the DMOc gene of DNA, RNA and western blot analysis test plants.Although all transgenic plant (swimming lane 1-6) in this analysis of Fig. 2 explanation all contain the identical dna fragmentation of DMO gene (swimming lane 8) with the clone after restriction enzyme digestion, mRNA transcript and DMO protein level are significantly different between transformant.For example, but the plant described in swimming lane 5 of extract demonstrates high-caliber relatively DMOmRNA extremely low-level enzyme.On the contrary, the DMO mRNA of level and the high expression level coupling of DMO much at one in the extract that in swimming lane 3, shows.Yet, show by this method to obtain having the incident of strongly expressed all the time.

The track sprayer with flat fan 8002E nozzle solvent and the commercial grade dicamba 98 (Clarity that use pressurized air, motor to drive; BASF) plant in the spraying greenhouse, this atomizer is sprayed with 1.87mph.Additive comprises: the 28% urea nitrate ammonium of 1.25%v/v and the nonionogenic tenside of 1.0%v/v.Use the solution that contains the different concns dicamba 98 with 182L/ha (40 gallons every acre).With the Clarity weedicide with 2.8kg/ha (2.51b/ac) spraying soybean field plant.

Tobacco plant as most of dicotyledonss, is handled quite responsive to dicamba 98.This is by more untreated and illustrate with the non-transgenic tobacco plant that the increment dicamba 98 is handled.Detect weedicide infringement symptom behind the horizontal spraying dicamba 98 with 0.017kg/ha easily.The normally used level of weeds control in agricultural is used: symptom is quite serious under 0.28kg/ha and the 0.56kg/ha.

Caused symptom seldom (if existence) with 5.6kg/ha (higher 10 to 20 times) at the rotaring gene tobacco plant that the aftertreatment of emerging contains DMOc, and non-transgenic plant suffers grievous injury than normal rate of application.By the injury that can duplicate the following square leaf of transgenic plant with the solvent solution that contains tensio-active agent (as the dicamba 98 application carrier) spraying plant.Handle the leaf that produces behind the transgenic plant with dicamba 98 and do not demonstrate visible infringement sign.The transgenic Fructus Lycopersici esculenti plant of carrying the DMOc gene of genetic modification does not demonstrate infringement equally yet when spraying with high-level dicamba 98, in this particular case, at first use 0.56kg/ha, handles with 5.6kg/ha subsequently.The Arabidopis thaliana of expressing the DMOc gene also shows the strong tolerance that dicamba 98 is handled.In this research, the dicamba 98 concentration of use provides the dosage of 1.12kg/ha.It is unexpected that discovery be to observe the aftertreatment of emerging that also tolerates the dicamba 98 of mean concns with the tobacco plant of the DMOc gene transformation that lacks the transit peptides coding region, and described tolerance only is lower than the tolerance of the plant that has the DMOc gene with transit peptides coding region slightly.In this research, compared with the 2.2kg/ha dicamba 98 two strain T ₁The processing of tobacco plant, the DMOc that lacks chloroplast transit peptides is carried in a strain, and another strain lacks the DMOc gene fully because heredity separates.Back one plant is handled the complete sensitivity of the injury that causes and dies from this processing (Fig. 3) dicamba 98.The transgenic plant of carrying the DMOc gene that lacks transit peptides tolerate the processing of 2.2.kg/ha dicamba 98 fully.In the rotaring gene tobacco plant genetics research of DMOc gene genetic also show this proterties in most plants with the heredity of normal Mendelian's mode and about the initial expression level of herbicide tolerant maintenance.

In soybean, produced above 50 R ₀The genetically engineered soybean incident is also collected T ₁, T ₂And T ₃For seed.Because used agrobacterium tumefaciens binary vector system, have marker gene and the transgenic plant unmarked DMOc of containing gene so reclaimed.In arbitrary situation, most genetically engineered soybean product tie up to and demonstrate the strong tolerance that 2.8kg/ha and 5.6kg/ha dicamba 98 are handled under the greenhouse experiment and demonstrate strong tolerance to 2.8kg/ha (the highest testing level) dicamba 98 in the field test in 2 years.Genetically engineered soybean that carries the DMOc gene that these results suggest are relevant with the broadleaf weeds of efficient control wide region and the wide safety limit of other crops.

High-level dicamba 98 tolerance shows that can use dicamba 98 soybean Tanaka comes the competition of strongly inhibited and broadleaf weeds and do not have crop damage in having the genetically engineered soybean plant of DMO gene.In addition, use genetically modified herbicide tolerant crop, dicamba 98 tolerance crop can be the important supplement that current weeds control is selected.Promptly, they are valuable properties in the appearance of existing herbicide tolerant weeds of control and the extra herbicide tolerant weeds of inhibition, and described extra herbicide tolerant weeds can finally threaten life-time service and the value of current weedicide and herbicide tolerant crop.

Embodiment 3

The comparison of crossing expression, purifying and enzymatic character of DMOw and DMOc

A. clone and cross expression

Wild-type (DMOw) and variant (DMOc) DMO encoding sequence are cloned into carrier pET28b (Novagen from plasmid pMON95900DMO (DMOw) and pMON58499DMO (DMOc), .San Diego, CA) in and be transformed into the e. coli bl21 cell (Novagen, San Diego, CA) in.Cell grows into 600nm under 37 ℃ in 1 liter of LB nutrient solution absorbancy is 0.4 to 0.6.By adding 50 μ M Fe (NH ₄) SO ₄, 100 μ M Na ₂S and 1mM sec.-propyl-β-sulfo-galactopyranoside (IPTG) also is transformed into 15 ℃ and induced protein with cell and expresses.15 ℃ after 48-72 hour, with centrifugal 20 minutes harvested cells of 10000xg.Down preserve standby at-20 ℃ cell.

DMOw is different with the protein expression output of DMOc in intestinal bacteria.Arrive the 150mg true protein although DMOw output is every liter of LB substratum about 100, the output of DMOc is low 10 times, or every liter about 10 is arrived the 15mg true protein.This is a beyong contemplation, because intestinal bacteria do not have rare codon and tryptophane that a codon is only arranged for halfcystine, but shows in two kinds of situations no matter output how, can allos produce protein in intestinal bacteria.Proteinic amount is low in both cases in the inclusion body, and prompting protein mainly is retained in the solvable fraction.

See reorganization DMOc from the His mark of expressing among the reorganization DMOw albumen of the His mark of Pseudomonas Maltophilia (Pseudomonas maltophilia) bacterial strain DI-6 and the coli strain BL21 by the Ni-NTA column chromatography purification to homogeneous.Cell suspension is destroyed in lysis buffer (100mM NaPi pH 8.0,300mM NaCl and 10mM imidazoles) and by supersound process.With 55000xg eccentric cell lysate 1 hour.With the supernatant liquor Ni-NTA post of packing into, with lavation buffer solution (100mM NaPi pH 8.0,300mM NaCl and 20mM imidazoles) washing pillar to remove the non-special protein that is attached to resin.Protein with elution buffer (100mM NaPipH8.0,300mM NaCl and 250mM imidazoles) wash-out His mark.For the DMOw purifying, progressively gradient enough obtains 95% pure enzyme, and needs the linear gradient of 20 to 250mM concentration imidazoles to realize the purity of par for DMOc.From the enzyme of pillar wash-out is about 95% purity, estimated to the western blotting of enzyme after separating as size fractionation on the SDS-polyacrylamide gel electrophoresis.Move to that the single main bar carrying means of about 40kDa (37.3kDa DMO enzyme adds the 3kDAHis mark) is excessive to have produced correct protein.

The assay method of B.DMOc and DMOw and stability kinetics

Use rabbit igg as the standard test protein concn by the Bradford assay method.By the SDS-PAGE isolated protein and use Coomassie blue stain.Measure the DMO activity by forming DCSA, (St.Louis is MO) by HPLC (Waters Corporation, Milford, MA) separation DCSA for Supelco, Sigma-Aldrich with Discovery C18 post.The retention time of DCSA is 8 minutes, and the retention time of dicamba 98 is 9.5 minutes.For dynamics research, after separating from reaction mixture on the HPLC post, detect DCSA and quantitative by the fluorescent emission under 420nm (excitation wavelength 310nm).The setting concentration of DCSA (12 and 24 μ M) is as quantitative criterion.

Use the storage liquid (100,200,400,800,1000,2000,5000 and 10000 μ M) of dicamba 98,0.1M KPi pH 7.2,0.1M FeSO ₄, 0.1M NADH and 1M MgCl ₂Assay method carried out 20 minutes and passed through adding 40 μ l H at 30 ℃ ₂SO ₄The cancellation reaction.In order to measure activity, DMO and ferredoxin and reductase enzyme coupling from the excessive purifying of Pseudomonas Maltophilia bacterial strain DI-6.

Because the active assay method of DMO is discontinuous assay method, so importantly be established as the time that this assay method of significant kinetic parameter must be moved that obtains.Thereby this assay method must be moved under starting condition, because the amount of the DCSA that produces is linear (Fig. 4) for time of the assay method of just moving.This assay method of results suggest can be moved 20 to 30 minutes and still keep linear.Fig. 5 has shown that the optimal pH of the assay method of carrying out is 7.2 and has found that optimum temperuture is about 37 ℃ (Fig. 6) in the presence of 0.1M Kpi damping fluid.

C. dynamics data analysis

By data substitution Nonlinear Steady equation (equation 1) is determined Mi-Man parameter (equation 1).With Sigma plot 8.0 (Jandel Scientific) analytical data.

V _o=Vmax*[S]/(Km+[S]) equation 1

The also optimal pH of DMOw of Ce Dinging and DMOc and temperature.For the enzyme of two kinds of forms, optimal pH is measured 20 minutes at 30 ℃, and optimum temperuture mensuration was also measured under pH7.2 20 minutes.The result summarizes in Fig. 7-9 and discusses hereinafter.

The result shows that the kinetic property of DMOw and DMOc is different.For example, Mi-Man parameter of calculating for DMOw and DMOc is: for DMOw, and Km=49 ± 7 μ M and Vmax=633 ± 24 nmoles/minute/mg, for DMOc, Km=20.5 ± 5 μ M and Vmax=676 ± 37 nmoles/minute/mg.These results in Figure 10 and 11, show and table 1 below in the general introduction.In addition, two extra analyses carrying out for DMOw and DMOc have produced similar result (table 2 and 3).

Can see that aspect catalytic efficiency, DMOw has different character with the DMOc enzyme: by this analysis, DMOc is the enzyme than five times of DMOw.The pH spectrum of DMOc is different with DMOw's.At first, as if DMOc compares the buffering system (TRIS vs.KPi) responsive (Fig. 9,12 and 13) to using with DMOw.Secondly, compare (this moment, the activity of DMOc reduced along with the rising of pH unit) with TRIS, when measuring in the KPi damping fluid, DMOc demonstrates stable activity in the pH of wide region.The TEMPERATURE SPECTROSCOPY of the DMOc of incubation is similar in KPi or TRIS damping fluid.

Observe the TEMPERATURE SPECTROSCOPY between the enzyme of these two kinds of forms, DMOw 37 ℃ with better function, and DMOc (Fig. 9) with better function under to a certain degree lower temperature.Fig. 9 shows the low optimum temperuture of DMOc, and it can be used for early stage transgenic plant in vegetative period.

The stability kinetics parameter of table 1.DMOw and DMOc

Enzyme	Km(M)	Vmax(U/mg)	kcat(s -1)	Kcat/Km?(M -1s -1)
					DMOw	49±7×10 -6	633±24×10 -3	36.63	7.47×10 5
DMOc	20±5×10 -6	676±37×10 -3	70.41	35.21×10 5

The Mi of table 2.DMOw-Man parameter general introduction

Research number	Rsqr	Vmax (nmole/minute/mg)	Km(μM)
				1.	0.983	633±24	49±7
2.	0.988	583±18	46±5
				3.	0.987	590±19	46±5.5

The Mi of table 3.DMOc-Man parameter general introduction

Research number	Rsqr	Vmax (nmole/minute/mg)	Km(μM)
				1.	0.933	713±43	21±6
2.	0.948	676±37	20±5

Embodiment 4

The bioinformatic analysis of the conservative region of DMO

Carry out bioinformatic analysis with the peptide sequence of relatively DMO and other iron-sulphur oxygenase and identify conserved regions.At first, based on 70% DMO sequence coverage on the e value cutoff value of 1e-08 and the sequence alignment, select 78 sequences to be used for analyzing.The further analysis of these 78 sequences discloses and have two structural domains having identified in other research, comprises Rieske and non--haem Fe structural domain (Herman et al., 2005).In these 78 sequences, 68 contain two structural domains, and 10 only have a structural domain.68 kinds of molecules with these two structural domains are used to further motif analysis.

This 68 kinds of molecules comparison in different identity levels with two structural domains has disclosed new WXWX motif.Although some sequences do not contain this motif, phylogenetic analysis shows that the molecule of this motif not falls into some clade of genealogical tree, and it does not belong to identical group with the molecule with this motif.Therefore those sequences of this motif do not remove from initial data set, stay 52 kinds of residue sequences being compared again and are used for further analysis.

Again Bi Dui 52 sequences show two W residues conservative property on every side of form below containing: WX ₁WX ₂(W is Trp to G, and G is the Gly residue, X ₁Be non-polar residue, X ₂Be any amino acid).Second W in this situation is corresponding to 112 of SEQ ID NO:1.Reported WX recently ₁WX ₂The WXG of G motif, and have the protein relevant with the emiocytosis system (Desvaux et al., 2005) of WXG motif.

Tryptophane (W) is the residue with remarkable different sizes with halfcystine (C).W is big residue, and C is relatively little residue.Because W and C are polare Aminosaerens, their more total common features are supplied with as proton.Because the W residue is encoded by TGC and TGT by TGG coding and Cys, thus some conversion in the 3rd password (G-〉C or G-〉T) can produce the missense mutation from W to C or from C to W.This type of conversion in nature evaluation and biological function and active the change by those sudden changes (for example see the BRCAl gene in hereditary breast cancer and the ovarian cancer (Xiaoman and Jinghe, 1999); Lipoprotein lipid enzyme mutant (Hoffmann et al., 2000) in Hageman factor defective (Wada et al., 2003) and the I type hyperlipoproteinemia).

Therefore, although showing DMO, the result of front is unique and has low identity that W112 guards in other relevant iron-sulphur oxygenases with known enzyme.In addition, 112 by two conservative functional domains around (Figure 18).In addition, W influences biological activity usually to the C conversion.Following discovery is especially wonderful: DMOc has produced than wild-type DMOw enzyme to have the functional enzyme of more excellent kinetic parameter and high-level tolerance to dicamba 98 is provided when expressing in transgenic plant.

Embodiment 5

The DMO of chloroplast(id) coding has produced high-level dicamba 98 tolerance

In order to determine the whether only possibility of performance function and research restricted passage pollen floating " gene diffusion " in chloroplast(id) of DMO, based on the pFMDV1 carrier (for example, Svab et al., 1990) produced construct to allow the DMOc gene and be incorporated in the chloroplast gene group of tobacco by homologous recombination and to use the antibiotics resistance to select to separate transformant (Figure 14).In this construct, the DMOc gene coding region is by the psbA chloroplast gene promoters driven that contains complete psbA5 ' UTR sequence.The preliminary southern blotting technique analysis (Figure 15 A) of antibiotics resistance transgenic plant shows and has DMOc transgenosis (5.6kb band) and integrated alternate natural gene district (3.3kb band) (that is, chloroplast(id) is heterogeneous for natural gene and DMOc transgenosis) by the homology of DMOc gene in the chloroplast gene group.Containing repeated regeneration on the antibiotic substratum and selecting transgenic plant to cause the chloroplast(id) of obvious homogeneity, but it has the DMOc gene fragment does not have replaced natural gene district (Figure 15 B).

To transform the T of body from the chloroplast(id) in two independent sources ₁, T ₂And T ₃Test the tolerance of handling with the various dose dicamba 98 for the offspring.All all demonstrate high-level tolerance.In fact, when spraying with the dicamba 98 of 28kg/ha (25lb/ac) ratio, chloroplast gene group transformant does not demonstrate obvious damage (being different from down " the only solvent infringement " of square leaf) (Figure 16) and when with 112 and the high dicamba 98 of 224kg/ha when using the processing plant, only observes temporary transient infringement.Under these high levels, initial infringement is mainly caused by the solvent tensio-active agent that is used for sending dicamba 98 and other components; Demonstrate approaching normal phenotype to normal phenotype from the tissue of impaired summit growth, behind initial growth retardation, do not demonstrate the ability that growth velocity descends and keeps producing the seed of common number and quality.

The result is consistent with following possibility: the reductive ferredoxin may be that dicamba 98 is oxidized to the required DMO electron donor of DCSA in the tobacco chloroplast.Direct test as this possibility, from Pseudomonas Maltophilia DI-6 bacterial strain purifying or from intestinal bacteria the DMO of excessive generation and purifying existence and not in the presence of, checked that the spinach ferredoxin of purifying is supported the ability (table 4) that dicamba 98 transforms to DCSA.The result shows fully can be external for DMO supplies with electronics, as occurring measured by dicamba 98 degraded or DCSA from the ferredoxin that is reduced of spinach or fusobacterium.

It is 3 as electronics source catalysis dicamba 98 vitro conversion that the dicamba monooxygenase enzyme of table 4A-B. purifying can utilize reductive chloroplast(id) ferredoxin or reductive clostridium ferredoxin, the 6-dichlorosalicylic acid.

The degraded of table 4A-dicamba 98

Reaction type	The degraded of dicamba 98 (%)
		(Ferr+Red) DI-6+NADH	0
(Oxy+Ferr+Red) DI-6+NADH	86
		(Oxy) DI-6+(Ferr) Spinach+(Ferr:Oxidored) Spinach+NADPH	83
(Oxy) DI-6+(Ferr:Oxidored) Spinach+NADPH	ND
		(Oxy) DI-6+(Ferr) Clostridium+(Ferr:Oxidored) Spinach+No?NADPH	ND
(Oxy) DI-6+(Ferr) Clostridium+(Ferr:Oxidored) Spinach+NADPH	82
		(Ferr) Clostridium+(Ferr:Oxidored) Spinach+NADPH	ND

The formation of table 4B-DCSA

Reaction type	The formation of DCSA (%)
		(Ferr+Red) DI-6+NADH	ND
(Oxy+Ferr+Red) DI-6+NADH	100
		(Oxy) DI-6+(Ferr) Spinach+(Ferr:Oxidored) Spinach+NADPH	95
(Oxy) DI-6+(Ferr:Oxidored) Spinach+NADPH	2.5
		(Oxy) DI-6+(Ferr) Spinach+(Ferr:Oxidored) Spinach+No?NADPH	1.2
(Oxy) DI-6+(Ferr) Clostridium+(Ferr:Oxidored) Spinach+NADPH	90
		(Ferr) Clostridium+(Ferr:Oxidored) Spinach+NADPH	1.5

ND does not determine

Although the result among Fig. 2 shows that the DMO level that is produced is variable and sometimes DMOc level and dicamba 98 tolerance level are not closely related, the result shows the high-level tolerance that can as one man obtain dicamba 98.Shown in transformant from examining localized DMOc gene and producing DMOc from the localized DMOc gene of chloroplast(id).In the nuclear transformant, constitute extra high-caliber total DMOc with respect to gross protein, and the amount of DMOc not significantly different and is lower than the nuclear transformant sometimes in the chloroplast(id) conversion body.The estimation of relative activity shows that the DMOc of the higher percent that produces more has activity and thinks than synthetic DMOc in the tenuigenin and is transferred in the chloroplast(id) in the cell-free extract of leaf texture's sample in chloroplast(id).

In all plants of analyzing,, realized the dicamba 98 tolerance without ferredoxin or reductase gene cotransformation.The result shows that described plant contains one or more molecules, described molecule can be with required transfer transport to DMO allowing dicamba 98 to 3, the conversion of 6-dichlorosalicylic acid (DCSA).Use transit peptide sequence, DMO is the potential ferredoxin that is reduced that can make full use of in the chloroplast(id) that utilizes to the target of the initial guiding of chloroplast(id).In this respect, be important to note that the plant that obtains tolerating the aftertreatment of emerging of dicamba 98 with the DMOc gene construct transformation of tobacco plant that lacks the chloroplast(id) peptide-coding sequence unexpectedly.Yet minority T uses by oneself ₁Result for the limited test of plant shows that the tolerance level that obtains with these transgenic plant is on average a shade below the tolerance level that obtains with the tobacco plant that produces the DMOc that contains transit peptides.These observationss have caused about being the interesting problem of the molecule of DMO productivity supplies electrons in the transgenic plant.The homogeneity chloroplast(id) demonstration that produces DMO from the DMOc gene that is incorporated into the chloroplast gene group inherently can all point out the chloroplast(id) ferredoxin to interact to allow transfer transport with the DMO productivity with this fact (table 4) of reductive spinach chloroplast ferredoxin performance function external to this fact of resistance (Figure 16) of high horizontal dicamba 98 and the DMO of purifying.Yet it is unknown that the electronics source of the DMO that produces from the nuclear gene that lacks the chloroplast transit peptide-coding sequence remains.It is external to suppose that ferredoxin is not positioned at plant leaf green, must think that a kind of cytoplasm protein of the unknown may provide the possibility of stable electronics supply for DMO.Alternatively, DMO self may contain available chloroplast transit peptides, and it allows enough DMO to enter in the chloroplast(id) to provide protection to prevent that dicamba 98 from entering in the cell after dicamba 98 is handled.

*?*?*?*?*?*

All compositions that this paper is open and claimed and/or method can be carried out without undo experimentation and implement according to the disclosure.Although described the compositions and methods of the invention according to a preferred embodiment, those skilled in the art it is evident that the scheme of change can be applicable in the order of the step of described composition and/or method and method disclosed herein or step and do not deviate from notion of the present invention, spirit and scope.More particularly, it is evident that chemistry some material relevant with physiology can replace material described herein and will realize same or analogous result.Think that conspicuous all these type of the similar alternative and modifications of those skilled in the art are all within spirit of the present invention, scope and notion that claims limit.

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The reference of listing below is incorporated herein by reference, replenishes, explain, provide background or instruct the degree of method used herein, technology and/or composition until them.

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Sequence table

<110>CLEMENTE.THOMAS?E.

DUMITRU，RAZVAN

FENG，PAUL?C.C.

FLASINSKI，STAN

WEEKS，DONALD?P.

＜120〉dicamba monooxygenase enzyme of Xiu Shiing and using method thereof

<130>MONS：093WO

＜140〉the unknown

<141>2007-06-06

<150>11/758,657

<151>2007-06-05

<150>60/811,152

<151>2006-06-06

<160>24

<170>PatentIn?Ver.2.1

<210>1

<211>340

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>1

<210>2

<211>1023

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic primer

<400>2

<210>3

<211>1023

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic primer

<400>3

<210>4

<211>120

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>4

<210>5

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>5

<210>6

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>6

<210>7

<211>59

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>7

<210>8

<211>57

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>8

<210>9

<211>57

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>9

<210>10

<211>57

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>10

<210>11

<211>57

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>11

<210>12

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>12

<210>13

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>13

<210>14

<211>60

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>14

<210>15

<211>57

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>15

<210>16

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>16

<210>17

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>17

<210>18

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>18

<210>19

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>19

<210>20

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>20

<210>21

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>21

<210>22

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>22

<210>23

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic peptide

<400>23

<210>24

<211>433

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic primer

<400>24

Claims (24)

1. isolated nucleic acid molecule, it is selected from:

A) nucleic acid molecule of the polypeptide of coding SEQ ID NO:1;

B) comprise the nucleic acid molecule of the sequence of SEQ ID NO:2; With

C) nucleic acid molecule, its coding and the polypeptide of SEQ ID NO:1 have the polypeptide of at least 90% sequence identity, and it is active and comprise halfcystine on the position corresponding to the amino acid/11 12 of SEQ IDNO:1 that wherein back one polypeptide has dicamba monooxygenase enzyme.

2. the isolated nucleic acid molecule of claim 1, the wherein dicamba monooxygenase enzyme of this nucleic acid molecule encoding plasmid pKLP36-TEV-TP-DMOc (ATCC preserving number PTA-7357) coding.

3.DNA construct, it comprises the nucleic acid molecule that operability is connected in the claim 1 of promotor.

4. the construct of claim 3, wherein this promotor has function in vegetable cell.

5. the construct of claim 3, wherein said isolated nucleic acid molecule operability is connected in chloroplast transit peptides.

6. peptide sequence, it comprises the aminoacid sequence that at least 90% identity is arranged with SEQ ID NO:1, and it is active and comprise halfcystine on the position corresponding to the amino acid/11 12 of SEQ ID NO:1 that wherein this polypeptide has dicamba monooxygenase enzyme.

7. use the nucleic acid molecule plant transformed cell of claim 1.

8. the cell of claim 7, wherein said vegetable cell is the dicotyledons cell.

9. the cell of claim 7, wherein said vegetable cell is a monocot plant cell.

10. the cell of claim 7, wherein said dicotyledons cell is soybean, cotton, corn or Semen Brassicae campestris vegetable cell.

11. comprise the plant tissue cultures of the cell of claim 7.

12. nucleic acid molecule transgenic plant transformed with claim 1.

13. the transgenic plant of claim 12, wherein said plant is a dicotyledons.

14. the transgenic plant of claim 12, wherein said plant is a monocotyledons.

15. the transgenic plant of claim 12, wherein said plant are soybean, cotton, corn or Semen Brassicae campestris plant.

16. produce the method for dicamba 98 tolerant plants, it comprises the construct that imports claim 3 in plant.

17. the method for claim 16, it comprises the construct that becomes the dicamba 98 tolerant plants by the initial vegetable cell of stable conversion and with described cell regeneration and import claim 3 in described plant.

18. the method for claim 16, wherein by producing the dicamba 98 tolerant plants with the mother plant selfing or with second kind of plant hybridization, the wherein said mother plant and/or second kind of plant have comprised transformation construct and the heredity of described dicamba 98 tolerant plants from the transformation construct of the described mother plant and/or second kind of plant.

19. the method for control weed growth in the crop growth environment of the plant that comprises claim 12 or its seed, it comprises the Banvel D of described crop growth environment being used the amount of effective control weed growth.

20. the method for claim 19 is wherein at the applied on top Banvel D of described crop growth environment.

The plant that lacks the nucleic acid of claim 1 21. the method for claim 19, the described plant of the amount without prejudice the right requirement 12 of wherein said Banvel D or the plant of its seed and infringement and claim 12 have the homologous genes type.

22. produce the method for food, feed or Industrial products, it comprises:

A) plant or its part of acquisition claim 12; With

B) prepare food, feed or Industrial products from described plant or its part.

23. the method for claim 22, wherein said food or feed are oil, meal, grain, starch, flour or protein.

24. the method for claim 22, wherein said Industrial products are biofuel, fiber, industrial chemical, medicine or nutritious prod.

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