CN101636498A - Chloroplast transit peptides for efficient targeting of DMO and uses thereof - Google Patents

Abstract

The invention provides for identification and use of certain chloroplast transit peptides for efficient processing and localization of dicamba monooxygenase (DMO) enzyme in transgenic plants. Methods for producing dicamba tolerant plants, methods for controlling weed growth, and methods for producing food, feed, and other products are also provided, as well as seed that confers tolerance to dicamba when it is applied pre- or post-emergence.

Description

The chloroplast transit peptides and uses thereof that is used for the efficient targeting of DMO

Background of invention

The application requires in the right of priority of the U.S. Patent application 11/758,659 of the U.S. Provisional Patent Application 60/891,675 of submission on February 26th, 2007 and submission on June 5th, 2007, and its disclosure of the complete introducing of this paper as a reference.

Invention field

The present invention relates to plant biotechnology field.In particular, evaluation and the purposes that the present invention relates to allow effectively processing and locate the chloroplast transit peptides of the dicamba monooxygenase enzyme in the plant.

Description of Related Art

It is nontoxic 3 that DMO (dicamba monooxygenase enzyme) catalysis weedicide dicamba 98 (3,6-two chloro-o-anisic acids) in plant is degraded to, and the 6-dichlorosalicylic acid (3,6-DCSA), thus the conferring herbicide tolerance.Protein in the middle of the activity of DMO need be used for electronics is shifted to dicamba 98 from NADH two kinds: reductase enzyme and ferredoxin (United States Patent (USP) 7,022,896; People such as Herman, 2005).Yet the dicamba 98 tolerance in the transgenic plant obtains proof by transforming with DMO separately, shows that the reductase enzyme of plant endogenous property and ferredoxin can replace in transfer transport.The plant ferredoxin that participates in transfer transport is arranged in plastid.Therefore, for efficient performance that obtains DMO and the dicamba 98 tolerance that therefore obtains raising, need be with the DMO target to chloroplast(id).

In many cases, this target can be realized by the terminal existence of extending of the N-that is known as chloroplast transit peptides (CTP) or plastid transit peptides.If polypeptide expressed will be in plant plastid (as chloroplast(id)) compartmentation, the karyomit(e) transgenosis of bacterial origin must have the sequence of the coding CTP sequence that merges mutually with the sequence of coding polypeptide expressed.Therefore, allogenic polypeptide being navigated in the chloroplast(id) normally polynucleotide sequence by means of the CTP sequence of will encoding is operably connected to and finishes on the 5 ' zone of polynucleotide of encoding exogenous polypeptide.CTP is removed by procedure of processing in transfer process in plastid.But working (machining) efficiency may be influenced by the aminoacid sequence of CTP and near the sequence the peptide aminoterminal.

People such as Weeks (United States Patent (USP) 7,022,896) described corn cab-m7 signal sequence (referring to, people such as Becker, 1992 and PCT WO 97/41228; GenBank registration number X53398) and pea glutathione reductase signal sequence (people such as Creissen, 1992 and PCT WO97/41228) with the DMO target to the potential use aspect the plant plastid, but do not provide about processing or the target efficiency in data.Pea rubisco small subunit (RbcS) CTP that contains 27 aminoacid sequences that comprise pea rubisco (Rubisco) small subunit encoding sequence, also be used for the DMO target to chloroplast(id) (for example, U.S. Provisional Application 60/811,152).Yet, in western blotting (Westernblot) analytic process, find, this pea RbcS CTP produce a correct processing the DMO protein band (～38kDa), but also produce one with the corresponding bigger band of 27 amino acid of DMO and RbcS coding region (～41kDa).Extra amino acid may influence the activity of DMO unfriendly.In addition, because the incomplete processing of DMO, extra protein causes the obstacle of supervision aspect in the transgenic product, in order to make the purpose of product in government organs' registration, also need increase the expense of equipment registration thus in the extra effort of input aspect the product sign.Therefore, need to identify the CTP that produces the DMO of correct processing effectively, thereby the advantage that DMO completely is active and be easy to characterize product is provided.

Summary of the invention

An aspect of of the present present invention relates to a kind of recombinant DNA molecules, it comprises the nucleotide sequence of the coding chloroplast transit peptides that lists of nucleotides sequence that is operably connected to the coding dicamba monooxygenase enzyme, wherein, this nucleotide sequence coded chloroplast transit peptides that comprises the sequence that is selected from SEQ ID NOs:1-11.In certain embodiments, recombinant DNA molecules comprises the nucleotide sequence that is selected from SEQ ID NOs:12-22.In certain embodiments, recombinant DNA molecules comprises that coding is selected from the nucleotide sequence of SEQ ID NOs:24,26,28,30,32,34,36,38 and 40 dicamba monooxygenase enzyme.The DNA construct that comprises the dna molecular that is operably connected with the promotor that has function in vegetable cell also is an aspect of of the present present invention.

On the other hand, the present invention includes with dna molecular plant transformed cell, described dna molecular comprises the nucleotide sequence of the coding chloroplast transit peptides that lists of nucleotides sequence that is operably connected to the coding dicamba monooxygenase enzyme, and wherein, the chloroplast transit peptide sequence is selected from SEQ ID NOs:1-11.In certain embodiments, recombinant DNA molecules comprises the nucleotide sequence that is selected from SEQ ID NOs:12-22.In certain embodiments, dna molecular comprises that coding is selected from the nucleotide sequence of SEQ IDNOs:24,26,28,30,32,34,36,38 and 40 dicamba monooxygenase enzyme, wherein, dna molecular is operably connected on the promotor that has function in vegetable cell.In specific embodiment, dna molecular comprises and is selected from SEQ ID NOs:23,25,27,29,31,33,35,37 and 39 nucleotide sequence.

In certain embodiments, vegetable cell is the dicotyledons cell.In other embodiments, vegetable cell is a monocot plant cell.In specific embodiment, vegetable cell is soybean, cotton, corn or Semen Brassicae campestris vegetable cell.The present invention also relates to comprise the plant tissue cultures of such cell, relate to the transgenic seed and the transgenic plant that comprise such cell.In certain embodiments, transgenic seed or plant are dicotyledonous seed or plant.In other embodiments, transgenic seed or plant are unifacial leaf seed or plant.Transgenic seed or plant can be soybean, cotton, corn or Semen Brassicae campestris seed or plant.

The invention further relates to the method for the plant that produces the tolerance dicamba 98, comprise: in recombinant DNA molecules introduced plant cell, and by its aftergrowth, described dna molecular comprises the nucleotide sequence of the coding chloroplast transit peptides that lists of nucleotides sequence that is operably connected to the coding dicamba monooxygenase enzyme, wherein, the sequence of coding chloroplast transit peptides is selected from SEQ ID NOs:12-22.In certain embodiments, recombinant DNA molecules comprises the nucleotide sequence that coding is selected from SEQ ID NOs:24,26,28,30,32,34,36,38 and 40 dicamba monooxygenase enzyme.Dna molecular can be operatively attached on the promotor that has function in vegetable cell.This method may further include by stock plant and it self or the hybridization of second plant being produced the plant of tolerance dicamba 98, wherein the stock plant and/or second plant comprise described DNA construct, and the tolerance dicamba 98 plant from the described stock plant and/or the second plant heredity this DNA construct.

A kind of method of in vegetable cell, expressing dicamba monooxygenase enzyme be of the present invention further aspect, this method comprises that the CTP that will select is operably connected on the sequence of coding dicamba monooxygenase enzyme.

On the other hand, the present invention relates to a kind of method of in crop growth environment, controlling weed growth, comprising: plant such plant or its seed, and use the Banvel D of the amount of effective control weed growth to crop growth environment.Banvel D can be used from last direction crop growth environment, and the amount of Banvel D is not damaged described plant or its seed thus, but but damages the plant or the seed of identical shortage construct with this plant or its seed cdna type.

Further aspect of the present invention relates to the method for producing food, feed or Industrial products, comprising:

A) obtain plant, this plant comprises nucleotide sequence and the nucleotide sequence of coding dicamba monooxygenase enzyme or the nucleotide sequence that is coded in the promotor that has function in the vegetable cell on its part that is operably connected to the coding chloroplast transit peptides according to 5 ' to 3 ' direction; B) prepare food, feed, fiber or Industrial products from described plant or its part.

In some embodiment of this method, food or feed are cereal, meal, oil, starch, flour or protein.In other embodiment of this method, Industrial products are biofuel, fiber, industrial chemical, medicine or nutritious prod.

Using the seed that dicamba 98 provides the tolerance dicamba 98 of DNA protection, that comprise the coding chloroplast transit peptides on the DNA that is operably connected to the coding dicamba monooxygenase enzyme at (pre emergence) before being unearthed, is further aspect of the present invention.In certain embodiments, the seed of tolerance dicamba 98 comprises the nucleotide sequence of the chloroplast transit peptides of encoding, and for example is selected from the nucleotide sequence of SEQID NOs:12-22.The seed of tolerance dicamba 98 may further include the nucleotide sequence that coding is selected from SEQ ID NOs:24,26,28,30,32,34,36,38 and 40 dicamba monooxygenase enzyme.

Another aspect of the present invention relates to the method that improves monocotyledonous upright ability (standability), comprise: a) obtain and cultivate the plant that produces by with stock plant and it self or the hybridization of second plant, wherein this stock plant and/or second plant plant that comprises described DNA construct and tolerate dicamba 98 from the described stock plant and/or the second plant heredity this DNA construct; B) handle plant with dicamba 98.In certain embodiments, plant is a maize plant.In other embodiment, can measure the parameter relevant of shape, quantity, length and/or the structure, lodging per-cent and the output that comprise stilit root with upright ability.

The accompanying drawing summary

Fig. 1: the CTP-DMO construct is in the correct processing of DMO and application in the dicamba 98 tolerance is provided.

Detailed Description Of The Invention

According to the present invention, provide to be used for more effectively expressing and to composition and the method for chloroplast transit dicamba monooxygenase enzyme (DMO) polypeptide at plant cell. Therefore the compositions and methods of the invention are strengthening plant and cell to useful aspect the tolerance of herbicide dicamba. Especially by with chloroplast transit peptides (CTP) with the DMO target to chloroplaset, can realize that the DMO that improves expresses and to the tolerance of Mediben.

Yet surprisingly, the inventor has been found that some CTP and DMO combination can not bring into play function well. For example, number of C TP can not cause enough protein expressions. This comprises the incorrect expression of protein, and the generation and the incomplete activity in vivo that change the protein of size. This can cause incomplete herbicide tolerant and make registration examine complicated. The invention provides CTP, when this CTP and DMO unite when using, beyond thought benefit is provided, includes but not limited to: the desirable level of the protein expression of the level that is transported to chloroplaset of raising, the herbicide tolerant that in the genetically modified plants of expressing DMO, strengthens, correct size and suitable posttranslational modification. Providing the example of the CTP of beyond thought benefit after making up with DMO is transit peptides CTP2, comprises the nucleic acid of SEQ ID NO:4 or 5, and comprises the sequence of coding SEQ ID NOs:15 and 16. In other embodiments, use pea (Pisum sativum) rubisco small subunit CTP coded sequence, for example by SEQ ID NO:2 representative or coding SEQ ID NO:13. Therefore, comprise that the DNA construct that is operably connected to the DMO coded sequence on CTP2 and/or the pea rubisco small subunit CTP transit peptides coded sequence consists of one aspect of the present invention, also consist of one aspect of the present invention by the protein of its coding.

The dicamba monooxygenase enzyme of Pseudomonas Maltophilia (Pseudomonas maltophilia) bacterial strain DI6 (people such as Herman, 2005; United States Patent (USP) discloses 20030115626; GenBank registers AY786443, and the sequence that this paper introduces its encoding D MO is as a reference) detoxifcation of catalysis herbicide dicamba. DMO is nontoxic 3 for Mediben is detoxified into, and the 6-dichlorosalicylic acid (3, the part in three compositions system 6-DCSA), and need as mentioned above reductase and ferredoxin function to come metastatic electron. Since participate in endogenous plant iron oxygen that electronics shifts also protein localization in plastid, for the effective active that obtains DMO and for example Mediben tolerance in dicotyledon or the Mediben tolerance that for example strengthens in monocotyledon, preferred DMO target is to plastid (for example chloroplaset).

Test chloroplast transit peptides (CTP) is with the efficient of DMO target to plastid and the DMO processing. The plastid location of the DMO relevant with these CTP and processing never or are partly arrived and are not waited fully. Discovery only has number of C TP to allow DMO is processed into correct size fully. Therefore, based on its protein or nucleotide sequence, it is to be difficult to expectation and beat all that any given CTP provides the ability of complete and effective DMO processing.

In addition, also in arabidopsis, find in the situation that does not have suitable CTP, almost do not have or do not have the expression of DMO and almost do not have or do not have the Mediben tolerance relevant. This shows chloroplast targeted detoxifcation for Mediben and then is important for tolerance. The CTP that allows effective processing DMO is being useful aspect the chloroplaset for example with DMO target to the plastid of crops, and therefore following advantage is provided: DMO is active and to the higher tolerance of Mediben completely, and is easy to characterize product and reduces cost of registering.

The chimeric dna molecule that comprises the DNA of the coding chloroplast transit peptides on the DNA that is operably connected to the coding dicamba monooxygenase enzyme can be by molecular biology method preparation well known to those skilled in the art (for example, the people such as Sambrook, 1989). The invention provides the CTP that is operably connected on the known encoding D MO dna molecular of (comprising those that list in the table 1), be used for improving the expression of plant DMO.

Any gene and its product that come to encode in the comfortable nucleus can with polypeptide target to the chloroplast transit peptides to chloroplaset, can carry out the detection of DMO effective expression. Can separate or synthetic chloroplast transit peptide sequence. In order in dicotyledon, monocotyledon or the two, to express, nucleotide sequence that can Optimized Coding Based CTP. By each being operably connected on the DMO coded sequence transit peptides that test is following: the CTPs that PsRbcS-derives (SEQ ID NO:1 and 2: pea rubisco small subunit CTP; The people such as Coruzzi, 1984); AtRbcS CTP (SEQ ID NO:3: arabidopsis rubisco small subunit 1A CTP; CTP1; United States Patent (USP) 5,728,925); AtShkG CTP (SEQ ID NO:4: arabidopsis 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS); CTP2; The people such as Klee, 1987); (SEQ ID NO:5:CTP2 synthesizes AtShkGZm CTP; For the monocotyledon expression is carried out codon optimized; The SEQ ID NO:14 of WO04009761); PhShkG CTP (SEQ ID NO:6: petunia (Petunia hybrida) EPSPS; CTP4; For the monocotyledon expression is carried out codon optimized; The people such as Gasser, 1988); (SEQ ID NO:7: wheat (Triticum aestivum) is in conjunction with the starch synthase CTP of particle synthetic for TaWaxy CTP, for the corn expression is carried out codon optimized: the people such as Clark, 1991): OsWaxy CTP (SEQ ID NO:8: paddy rice (Oryza sativa) starch synthase CTP; Okagaki, 1992); NtRbcS CTP (SEQ ID NO:9: tobacco (Nicotiana tabacum) ribulose 1,5-diphosphonic acid carboxylase small subunit chloroplast transit peptides; Mazur waits the people, and 1985); ZmAS CTP (SEQ ID NO:10: corn (Zea mays) o-amino benzoyl acid synthase α 2 subunit gene CTP; The people such as Gardiner, 2004); With RgAS CTP (SEQ ID NO:11: rue (Ruta graveolens) o-amino benzoyl acid synthase CTP; Bohlmann waits the people, and 1995). The nucleotide sequence of coding SEQ ID NO:1-SEQ ID NO:11 provides in SEQ ID NO:12-SEQ ID NO:22 respectively.

Other transit peptides that comes in handy comprises corn cab-m7 burst (people such as Becker, 1992; PCT WO 97/41228) and pea (Pisum sativum) glutathione reductase burst (people such as Creissen, 1995; PCT WO 97/41228). CTP (described amino acid is the part of this gene coding region or merges with it) with the additional amino acid that comes from gene coding region, for example (it comprises transit peptides to AtRbcS CTP, 24 amino acid of ripe rubisco albumen, then be last 6 amino acid whose repetitions of transit peptides), can be used to produce DMO. ZmAS CTP7 also comprises extra 18 amino acid that come from gene coding region. Other CTP also can be used for producing DMO, they have the extra amino acid (for example 27 amino acid) (described amino acid is the part of this gene coding region) that comes from gene coding region, for example and then PsRbcS CTP is the amino acid (for example 3 amino acid) of introducing by cloning process. Have the CTP of the amino acid (for example 21 amino acid) of less coding total length CTP, for example RgAs CTP also can be used to produce DMO. Preferably, use the nucleotide sequence of coding total length CTP. Can comprise that one or more nucleotides adds or disappearance, so that the clone of CTP. These interpolations or disappearance can cause the modification of one or more coded amino acid in front or the back of other Expression element and code area, for example at Restriction Enzyme recognition site place or be positioned near it.

In one embodiment, the nucleotide sequence of chloroplast transit peptides the present invention relates to encode, among this chloroplast transit peptides and the SEQ ID NOs:1-11 any one or a plurality of peptide sequence have 70% homogeneity at least, comprise with these sequences having about at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher sequence homogeneity, comprise 100% homogeneity. In specific embodiment, an identical chloroplast transit peptides among described nucleic acid sequence encoding and the SEQ ID NOs:1-11. In another embodiment, the nucleotide sequence of coding CTP and any one among the SEQ ID NOs:12-22 or a plurality of nucleotide sequence have at least 70% sequence homogeneity, comprise with these sequences in one or more have about 75 %, 80%, 85%, 90%, 95%, 97%, 98%, 99% or larger sequence homogeneity at least, comprise 100% homogeneity. Determining and can carrying out as known in the art of the polypeptide of these sequences and arbitrarily other sequence as herein described or polynucleotides comparison and homogeneity for example, used MEGAlign (DNAStar, Inc., 1228S.Park St., Madison, WI), adopt default parameter. Such software mates similar sequence by determining the degree of similitude or homogeneity.

By using the presequence can be with the DMO target to other organelle mitochondria for example, be present in ferredoxin oxidation-reduction system in this organelle with utilization. Perhaps, can with the DMO target to chloroplaset and mitochondria, to utilize two ferredoxin oxidation-reduction systems, make work more effective by two targeting peptides. Such element is known to those skilled in the art. For example, the mitochondria presequence has description people such as Silva Filho in (1996). The nucleotide sequence of the two targeting peptides sequences of coding can from encode following known can be out identified to the nucleotide sequence of the protein of chloroplaset and mitochondria by target: the Zn-MP (people such as Moberg, 2003), glutathione reductase (people such as Rudhe, 2002; The people such as Creissen, 1995) and Histidyl-tRNA synthetase (people such as Akashi, 1998). For example, as shown in table 1, in the sequence of coding SEQ ID NOs:24,26,28,30,32,34,36,38,40 polypeptide, discovery can be used for the example of the DMO coded sequence of this respect.

DMO and DMO variant that table 1. uses.

DMO/ or variant	Protein s EQ ID	??DNA??SEQ??ID	Protein length	The amino acid of 2 places prediction in the position	The amino acid of 3 places prediction in the position	Amino acid in the prediction of post-11.2 place	Codon uses
								??DMO-Cat(A)	??24	??23	??340	??Ala	??Thr	??Cys	Dicotyledons
??DMO-Cat(L)	??26	??25	??340	??Leu	??Thr	??Cys	Dicotyledons
								??DMO-Wat(L)	??28	??27	??340	??Leu	??Thr	??Trp	Dicotyledons
??DMO-Cnat(A)	??30	??29	??340	??Ala	??Thr	??Cys	Bacterium
								??DMO-Wat(A)	??32	??31	??340	??Ala	??Thr	??Trp	Dicotyledons
??DMO-Wnat(T)	??34	??33	??339	??Thr	??Phe	Trp (at 111 places)	Bacterium
								??DMO-Cnat(L)	??36	??35	??340	??Leu	??Thr	??Cys	Bacterium
??DMO-Wmc(L)	??38	??37	??340	??Leu	??Thr	??Trp	Monocotyledons
								??DMO-Wmc(A)	??40	??39	??340	??Ala	??Thr	??Trp	Monocotyledons

In certain embodiments, the sequence of any one polypeptide has at least 70% identity among the nucleic acid of coding dicamba monooxygenase enzyme and the coding SEQ IDNOs:24,26,28,30,32,34,36,38 and 40, comprises with these sequences having about at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% and the sequence identity of Geng Gao.In certain embodiments, any one nucleotide sequence among nucleic acid and the SEQ ID NOs:23,25,27,29,31,33,35,37 or 39 has at least 70% sequence identity, comprise with these sequences in one have about at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% and bigger sequence identity.In further embodiment, dicamba monooxygenase enzyme can be the variant of any such sequence and/or can be the synthetic DMO molecule of through engineering approaches, for example, the U.S. Provisional Application 60/884 that is called " DMO Methods AndCompositions " as the name of submitting on January 12nd, 2007, describe in 854, this paper introduces its complete disclosure as a reference especially.

The variant of DMO with ability of degrading plant growth hormone sample weedicide, and glyphosate or other herbicide tolerant gene can easily prepare according to standard method, and measure its activity.Also can be by technology known in the art, for example, from suitable organism, identify these sequences, described organism comprises for example can degrade the vitamin H sample weedicide of dicamba 98 or bacterium (United States Patent (USP) 5,445,962 of other weedicide; Cork and Krueger, 1991; Cork and Khalil, 1995).The method of a kind of DMO of separation or other sequence is to carry out nucleic acid hybridization by the library that for example makes up with origin source organism, perhaps uses from the mRNA of source organism with based on the primer of disclosed desaturase and carries out RT-PCR.Therefore the present invention is included in the application of the nucleic acid of hybridizing with DMO encoding sequence as herein described under the stringent condition.Those skilled in the art understands by increasing salt concn and reducing temperature can provide severity lower condition.Therefore, hybridization conditions can easily be controlled, and generally is the method for selecting according to expected result.An example of high stringency is 5X SSC, 50% methane amide and 42 ℃.By washing under such condition for example 10 minutes, can remove those under these conditions not with the sequence of particular target sequence hybridization.

Variant also can chemosynthesis, for example, according to technology well-known in the art, utilizes known DMO polynucleotide sequence synthetic.For example, dna sequence dna can be synthetic on automatic dna synthesizer by phosphoramidite chemical method (phosphoroamidite).Chemosynthesis has many advantages.Particularly, chemosynthesis is desirable, because the codon of host institute preference that will the expressible dna sequence can be used for optimization expression.Be not that all codons all need to change and obtain improved expression, but preferably to the major general those codons of in the host, seldom using change into the codon of host's preference.About 50% by change surpassing, most preferably about at least 80% codon becomes the codon of host's preference, can obtain high-caliber expression.The codon preference of many host cells is that known (for example, PCT WO 97/31115; PCT WO 97/11086; EP 646643; EP 553494; And United States Patent (USP): 5,689,052; 5,567,862; 5,567,600; 5,552,299 and 5,017,692).Can pass through methods known in the art, derive the codon preference of other host cell.In addition, utilize chemosynthesis, can easily change sequence or its encoded protein matter of dna molecular, for example be used for optimization expression (for example, eliminate disturbing the mRNA secondary structure of transcribing or translating), add unique restriction site and deletion proteolytic enzyme restriction enzyme site in site easily.

When keeping as required or changing enzymic activity, can be to proteinic peptide sequence, as DMO sequence provided herein, modify and change.The exemplary method that produces the DMO sequence is provided in the U.S. Provisional Application of submitting on January 12nd, 2,007 60/884,854.Be based on below and change proteinic amino acid and produce suitable or even the discussion of improved, modified polypeptides and corresponding coding sequence.Known, for example, some amino acid can be replaced by other amino acid in protein structure, but can significantly sacrificing and substrate molecule on the ability that mutually combines such as the structure of binding site.Because protein interactions ability and character define proteinic biological function activity, so can be in protein sequence, can certainly be in its dna encoding sequence, carry out some aminoacid sequence displacement, and still obtain protein with similar quality.Therefore imagination: can carry out various changes to DMO peptide sequence described herein or other herbicide tolerant polypeptide and corresponding D NA encoding sequence, and can obviously not weaken their biological availability or activity.

When carrying out such change, need the hydrophilic index of considered amino acid.Generally understand the hydrophile amino acid number in this area in the importance (people such as Kyte, 1982) of giving aspect the protein interaction biological function.Generally believe that amino acid whose relative water-wet behavior has contribution to the proteinic secondary structure that produces, and proteinic secondary structure defines protein and for example interaction of other molecules such as enzyme, substrate, acceptor, DNA, antibody, antigen.Based on their hydrophobicity and charge characteristic, every seed amino acid has been assigned with hydrophilic index (people such as Kyte, 1982), 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).

Known in the art, amino acid can be replaced by other amino acid with similar hydrophilic index or score, and still produces the protein with similar biologic activity,, still obtains the suitable protein of biological function that is.When carrying out such transformation, the preferred amino-acid substitution of its hydrophilic index within ± 2, the especially preferred amino-acid substitution within ± 1, even the more preferably amino-acid substitution within ± 0.5.

Also understand in this area on hydrophilic basis and can effectively carry out similar amino acid whose displacement.United States Patent (USP) 4,554,101 propose, and the proteinic maximum local average wetting ability that is subjected to contiguous amino acid pro water-based control is relevant with proteinic biological property.As United States Patent (USP) 4,554,101 is described, and 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 to be understood that: amino acid can be had the amino acid replacement of similar hydrophilicity value by another kind, and still obtains the suitable protein of biology.In such change, the preferred amino-acid substitution of its hydrophilicity value within ± 2, the especially preferred amino-acid substitution within ± 1, even the more preferably amino-acid substitution within ± 0.5.The exemplary displacement that these and various above-mentioned features are taken into account is well-known to those having ordinary skill 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.

The DNA construct that comprises the CTP sequence that is operably connected on the DMO sequence can be by being operably connected on the promotor that has function in plant and is expressed in the test macros such as vegetable cell such as protoplastis, instantaneous or stable conversion.The example of describing such promotor comprises United States Patent (USP) 6,437,217 (corn RS81 promotors), United States Patent (USP) 5,641,876 (rice actin promotors; OsAct1), 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 2 promotors and rice actin 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 promoter), 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) 6,388,170 (for example, PClSV promotors), the PClSV promotor of SEQ ID NO:41, United States Patent (USP) 6,635,806 (γ-Job's tears prolamine (coixin) promotor) and United States Patent (USP) 7,151,204 (corn chloroplast(id) zymohexase promotor).Operable other promotor is nopaline synthase (NOS) promotor (people such as Ebert, 1987), octopine synthase (OCS) promotor (it carries in the plasmid of the tumor inducing of Agrobacterium tumefaciens (Agrobacterium tumefaciens)), cauliflower mosaic virus promoter is cauliflower mosaic virus (CaMV) 19S promotor (people such as Lawton for example, 1987), CaMV 35S promoter (people such as Odell, 1985), radix scrophulariae mosaic virus 35 S-promotor (people such as Walker, 1987), sucrose synthase promotor (people such as Yang, 1990), R gene complex promotor (people such as Chandler, 1989) and chlorophyll a/b binding protein gene promotor etc.In the present invention, the CaMV35S (e35S that has enhancer sequence; United States Patent (USP) 5,322,938; 5,352,605; 5,359,142 and 5,530,196), FMV35S (United States Patent (USP) 6,051,753; 5,378,619), peanut chlorisis striped cauliflower mosaic virus (PClSV; United States Patent (USP) 5,850,019), At.Act 7 (registration number #U27811), At.ANT1 (U.S. Patent application 20060236420), FMV.35S-EF1a (U.S. Patent application 20050022261), eIF4A10 (registration number #X79008) and AGRtu.nos (GenBank registration number V00087; People such as Depicker, 1982; People such as Bevan, 1983), paddy rice kytoplasm triosephosphate isomerase (OsTPI; United States Patent (USP) 7,132,528) and rice actin 15 gene (OsAct15; U.S. Patent application 2006-0162010) promotor may be particularly useful.

5 ' the UTR that works as the translation leader sequence is in the promoter sequence of gene and the DNA genetic elements between the encoding sequence, can be included between promotor and the CTP-DMO sequence.The translation leader sequence is present among the mRNA that processes fully of translation initiating sequence upstream.The translation leader sequence can influence processing, mRNA stability or the translation efficiency of primary transcript to mRNA.The example of translation leader sequence comprises corn and petunia heat shock protein(HSP) leader sequence (United States Patent (USP) 5,362,865), plant viral coat protein leader sequence, plant rubisco leader sequence, 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 registration number V00087; People such as Bevan, 1983) etc. (Turner and Foster, 1995).In the present invention, may 5 ' especially useful UTR be GmHsp (United States Patent (USP) 5,659,122), PhDnaK (United States Patent (USP) 5,362,865), AtAnt1, TEV (Carrington and Freed, 1990), OsAct1 (United States Patent (USP) 5641876), OsTPI (United States Patent (USP) 7,132,528), OsAct15 (U.S. discloses 20060162010) and AGRtunos (GenBank registration number V00087; People such as Bevan, 1983).

3 ' non-translated sequence, 3 ' transcription termination region or the polyadenylation region meaning are the dna moleculars that is connected to and is positioned at structure polynucleotide molecule downstream, and comprise provide can influence transcribe, mRNA processing or the polyadenylation signal of genetic expression and the polynucleotide of other adjustment signal.Polyadenylation signal is brought into play function in plant, to cause 3 ' the terminal polyadenylic acid that increases to the mRNA precursor.The polyadenylation sequence can derive from natural gene, various plants gene or T-DNA gene.These sequences can be contained in the downstream of CTP-DMO sequence.The example of 3 ' transcription termination region is nopaline synthase 3 ' district (no 3 '; People such as Fraley, 1983).For example understand application people such as (, 1989) Ingelbrecht of 3 ' different non-translational regions.From pea RbcS2 gene (Ps.RbcS2-E9; People such as Coruzzi, 1984), AGRtu.nos (Genbank registration number E01312), E6 (registration number #U30508) and TaHsp17 (wheat low molecular weight heat shock protein gene; Registration number #X13431) polyadenylic acid chemoattractant molecule is particularly advantageous for the present invention.

Except above-mentioned Expression element,, between promotor and 3 ' UTR, may need intron in order especially in monocotyledons, to express the coding region." intron " refers to polynucleotide molecule, and its intervening sequence that can copy from the genome of gene separates or identifies, and generally can be defined in the zone that is sheared in the translation premessenger RNA course of processing.Perhaps, intron can synthesize generation.The inferior element that intron itself can comprise the gene transcription that is operably connected of influence is cis element or enhanser zone for example." plant introne " is natural or non-natural intron of performance function in vegetable cell.Plant introne can be regulated one or more expression of gene that are operably connected as controlling element.Polynucleotide molecule sequence in the transformation construct can comprise intron.With respect to the polynucleotide sequence that can transcribe, intron can be allogenic.The example of intron comprises maize actin intron (United States Patent (USP) 5641876), corn HSP70 intron (ZmHSP70; United States Patent (USP) 5,859,347; United States Patent (USP) 5,424,412) and paddy rice TPI intron (OsTPI; United States Patent (USP) 7,132,528), and be useful in the embodiment of this invention.

Can and transform method known to the technician in field by plant tissue culture, as the test macro of the unifacial leaf of protoplastis or instantaneous or stable conversion or dicotyledons cell in test the correct processing whether the CTP-DMO construct provides DMO.According to the present invention, can use as known in the art any with transgenic constructs import plant technology (referring to, for example, people such as Miki, 1993).It is believed that in fact suitable methods for plant transformation comprises that any can be with the method in the DNA transfered cell, as United States Patent (USP) 5,384,253 described electroporations; United States Patent (USP) 5,015,580,5,550,318,5,538,880,6,160,208,6,399,861 and 6,403,865 described microprojectile bombardment methods; United States Patent (USP) 5,635,055,5,824,877,5,591,616,5,981,840 and 6,384,301 described agrobacterium-mediated conversion methods; With United States Patent (USP) 5,508, the 184 protoplast transformation methods of describing.By using the technology as these methods, the cell of in fact any plant species can stably transform, and these cells can develop into transgenic plant.United States Patent (USP) 5,846,797,5,159,135,5,004,863 and 6,624,344 disclose useful especially technology in cotton transforms.For example, at United States Patent (USP) 5,750, the technology of conversion Btassica (Brassica) plant is disclosed especially in 871; For example, people such as Zhang, 1999, disclose the technology of soybean transformation among United States Patent (USP) 6,384,301 and the US 7,002,058.The technology of maize transformation is disclosed in WO9506722.Some non-limitative examples that can be used for plant of the present invention comprise clover, barley, beans, beet, asparagus broccoli, Caulis et Folium Brassicae capitatae, Radix Dauci Sativae, rape, Cauliflower, celery, Chinese cabbage, corn, cotton, cucumber, dried beans, eggplant, fennel, French bean, cucurbit, leek, lettuce, muskmelon, oat, okra, onion, pea, pepper, pumpkin, peanut, potato, pumpkin, radish, paddy rice, Chinese sorghum, soybean, spinach, pumpkin, sweet corn, beet, Sunflower Receptacle, tomato, watermelon and wheat.

After realization imported the receptor cell with foreign DNA, next step of generation transgenic plant related generally to identify the cell transformed that is used for further cultivation and plant regeneration.In order to improve the ability of identifying transformant, may need to use and to select the marker gene that maybe can screen with conversion carrier according to the present invention's preparation.In this case, generally next may the cell transformed group by cellular exposure is detected in one or more selective agents, perhaps at the character screening cell of the marker gene of needs.

The cell of surviving after being exposed to selective agent, perhaps the positive cell of scoring in shaker test can continue to cultivate on the substratum of supporting plant regeneration.Any suitable plant tissue culture media, for example, MS or N6 substratum (Murashige and Skoog, 1962; People such as Chu, 1975), can change by other material that comprises as growth regulator.Can keep tissue containing on the basic medium of growth regulator, begin plant regeneration work up to obtaining enough tissues, perhaps carry out repeatedly several artificial selection of taking turns, up to the suitable regeneration of the form of tissue, typically at least two weeks, transfer to then on the substratum that helps seedling formation.Regularly shift culture, form up to enough seedling are taken place.In case seedling forms, just they are transferred on the substratum that helps root formation.In case form enough roots, just plant can be transferred to further growth and maturation in the soil.

In order to confirm foreign DNA or " transgenosis " existence in aftergrowth, can carry out multiple test.Such test comprises that for example, " molecular biology " test is as southern blotting technique method and RNA blotting and PCR ^TM" biological chemistry " test, as detecting the existence of protein, for example, by immunological method (ELISA and Western blotting) or by the enzyme function; The plant part test is as leaf or root test; And, by analyzing the phenotype of whole aftergrowth.

In case transgenosis is imported in the plant, just can this gene be imported in any plant compatible with this first vegetalitas, and not need directly to transform second kind of plant by hybridization.Therefore, term used herein " offspring " refers to any generation offspring according to the stock plant of the present invention's preparation, and wherein, this offspring comprises the DNA construct according to the selection of the present invention's preparation.Therefore, " transgenic plant " can be any generation.As disclosed herein, " hybridization " kind of plant to be providing transgenosis or the allelic department of botany that one or more interpolation is arranged with respect to the original plant cording, thereby is defined as by will initially being and comprising transgenosis of the present invention or the hybridization of allelic donor department of botany and cause specific sequence to be imported into technology in the department of botany.In order to realize this point, for example, can carry out following steps: the seed of (a) plantation first (initial system) and second (transgenosis or the allelic donor department of botany that comprise needs) stock plant; (b) with the plant of the cultivating seeds Cheng Youhua of first and second stock plants; (c) with the pollen of second mother plant the flower of first mother plant is pollinated; (d) results have the seed that produces on the mother plant of the flower of being fertilized.

Can stable testing plant transformed tissue and plant whether provide the dicamba 98 tolerance by the proteinic correct processing of DMO.In crop plants, provide the dicamba 98 tolerance can be used for designing the method for the weed growth in the control growing environment, comprising: Banvel D from the amount of effective control weed growth to crop growth environment that use.Banvel D is used from last direction crop growth environment with certain amount, the amount of described Banvel D is not damaged crop plants or the seed that transforms with the CTP-DMO construct, and but damage has the crop plants that same genotype still lacks the CTP-DMO construct.

Based on disclosed content, the preparation that is used for herbicidal composition of the present invention is conspicuous for a person skilled in the art.Commercially available composition so typically also comprises as components such as tensio-active agent, solid or liquid vehicle, solvent and tackiness agents except activeconstituents.The example that can be used for the tensio-active agent used to plant comprises an alkali metal salt of following material, alkaline earth salt or ammonium salt: aromatic sulphonic acid class, for example, lignosulfonic acid, sulfocarbolic acid, naphthene sulfonic acid and dibutyl naphthene sulfonic acid, and lipid acid, aromatic yl sulphonate, alkyl oxide, lauryl ether, aliphatic alcohol sulfate and Fatty Alcohol(C12-C14 and C12-C18) glycol ethers vitriol, the condenses of sulfonated naphthalene and derivative thereof and formaldehyde, the condenses of naphthalene or naphthene sulfonic acid and phenol and formaldehyde, the condenses of phenol or sulfocarbolic acid and formaldehyde, the condenses of phenol and formaldehyde and S-WAT, polyoxyethylene octyl phenyl ether, the iso-octyl of ethoxylation-, octyl group-or nonylphenol, tributyl phenyl polyglycol ether, alkyl aryl polyether alcohol, different tridecyl alcohol, the Viscotrol C of ethoxylation, the triaryl phenol of ethoxylation, the salt of the triaryl phenol ethoxylate of phosphorylation, lauryl alcohol polyglycol ether acetic ester, sorbitol ester, xylogen-sulfite waste lye or methylcellulose gum, or their mixture.The common example that tensio-active agent is used is about 0.25 weight %-1.0 weight %, is about 0.25 weight %-0.5 weight % more commonly usedly.

The composition of using to plant can be solid or liquid.When using solids composition, may need to comprise one or more solid support materials and active compound.The example of carrier comprises mineral soil, as silica, silica gel, silicate, talcum, kaolin, attaclay (attaclay), Wingdale (limestone), chalk (chalk), loess (loess), clay, rhombspar (dolomite), diatomite (diatomaceous earth), calcium sulfate, sal epsom, magnesium oxide, the ground synthetic materials, fertilizer, as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiocarbamide and urea, the product of plant origin, as the cereal meal, the bark meal, wood powder and nutshell meal, cellulose powder, attapulgite (attapulgites), montmorillonite (montmorillonites), mica (mica), vermiculite (vermiculites), synthetic silicon-dioxide and synthetic Calucium Silicate powder, or their mixture.

For liquor, can comprise water miscible compound or salt, as sodium sulfate, vitriolate of tartar, sodium-chlor, Repone K, sodium-acetate, monoammonium sulfate, ammonium chloride, ammonium acetate, ammonium formiate, ammonium oxalate, volatile salt, bicarbonate of ammonia, ammonium thiosulfate, bisphosphate hydrogen ammonium, primary ammonium phosphate, sodium ammonium biphosphate, ammonium thiocyanate, Ammonium sulfamate or ammonium carbamate.

The exemplary composition of in the herbicidal composition other comprises: tackiness agent, as polyvinyl acetate (PVA), carboxymethyl cellulose, starch, V-Pyrol RC/vinyl acetate copolymer and the polyvinyl acetate (PVA) of polyvinylpyrrolidone, polyvinyl alcohol, partial hydrolysis or their mixture; Lubricant is as Magnesium Stearate, sodium stearate, talcum or polyoxyethylene glycol or their mixture; Foam preventer is as silicone emulsion, long-chain alcohol, phosphoric acid ester, acetylenediol, lipid acid or organofluorine compound; And sequestrant, as: the salt of ethylenediamine tetraacetic acid (EDTA) (EDTA), the salt of three nitrilotriacetic acid(NTA) or the salt of polyphosphoric acid or their mixture.

Can use from about 2.5g/ha to about 10, the dicamba 98 of 080g/ha, comprise about 2.5g/ha-about 5,040g/ha, about 5g/ha-about 2,020g/ha, the about about 820g/h of 10g/a-and approximately 50g/ha-is about 1,000g/ha, approximately about 800g/ha of 100g/ha-and the about about 800g/ha of 250g/ha-.

The CTP-DMO construct can be connected to comprise and be used for can screening/can mark/selectable mark and/or give one or more polynucleotide molecule of genetic elements of the proterties of another kind of needs.Be commonly used to screen the gene of inferring cell transformed and comprise β-glucuronidase (GUS), beta-galactosidase enzymes, luciferase and E.C. 2.3.1.28 (Jefferson, 1987; People such as Teeri, 1989; People such as Koncz 1987; People such as De Block, 1984), green fluorescent protein (GFP) (people such as Chalfie, 1994; Haseloff and Amos, 1995; With PCT application WO97/41228).For example, at Miki and McHugh, selected marker's unrestricted example has been described in 2004.

The nucleic acid molecule of giving the another kind of proterties that needs comprises, but be not limited to: the gene that the characteristic relevant with phytomorph, physiology, g and D, output, nutritional fortification, disease resistance or insect-resistance or environment or chemical resistance that needs is provided, can also comprise genetic elements, comprise: 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), output (United States Patent (USP) RE38,446 of Zeng Jiaing; 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; 6,593,293; 6,555,655; 6,538,109; 6,537,756; 6,521,442; 6,501,009; 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; 6,063,756; 6,063,597; 6,023,013; 5,959,091; 5,942,664; 5,942,658,5,880,275; 5,763,245; 5,763,241), mycosis 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), bacteriosis resistance (United States Patent (USP) 5,516,671), growth and development of plants (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), production (United States Patent (USP) 6,444,876 of the oil of Gai Bianing; 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), fatty acid content (United States Patent (USP) 6,828,475 of Gai Bianing; 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), the animal and human's of high protein output (United States Patent (USP) 6,380,466), fruit maturation (United States Patent (USP) 5,512,466), reinforcement nutrition (United States Patent (USP) 6,723,837; 6,653,530; 6,541,259; 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 US20030028917), environmental stress tolerance (United States Patent (USP) 6,072,103) but, medicine 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 Ti Gaoing (United States Patent (USP) 6,531,648), low-level raffinose (United States Patent (USP) 6,166,292), industrial enzyme production (United States Patent (USP) 5,543,576), improved fragrance (United States Patent (USP) 6,011,199), fixed nitrogen (United States Patent (USP) 5,229,114), cenospecies production (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).In these or other genetic elements, method and the transgenosis any can be used for the present invention, is that those skilled in the art is understandable based on present disclosure.

In addition, one or more polynucleotide molecule that is connected on the CTP-DMO construct suppresses the RNA molecule of (for example pass through antisense, inhibitory RNA (RNAi) or be total to the mechanism that suppresses mediation) by the target that coding causes native gene to be expressed, and can realize plant characteristic above-mentioned or phenotype.RNA also can be the RNA molecule (being ribozyme) of catalytic, and design is used for cutting the endogenous mRNA product that needs.Therefore, any coding polynucleotide molecule of influencing the transcribe rna molecule of interested phenotype or morphological change can be used to put into practice the present invention.

The present invention also discloses the method for producing food, feed or Industrial products, comprise the plant that comprises the CTP-DMO construct or the part of this kind of plant, and prepare food, feed, fiber or Industrial products from plant or its part, wherein, food or feed are cereal, meal, oil, starch, flour or protein, and Industrial products are biofuel, fiber, industrial chemical, medicine or nutritious prod.

Another aspect of the present invention relates to the method for improving monocotyledonous upright ability, comprise: a) obtain and plantation by plant with stock plant and it self or second plant hybridization generation, wherein the stock plant and/or second plant comprise described DNA construct, and the tolerance dicamba 98 plant from the described stock plant and/or the second plant heredity this DNA construct; And b) handles this plant with dicamba 98.Can measure the parameter relevant of quantity, shape, length or the structure, lodging per-cent and the output that comprise stilit root with upright ability.In certain embodiments, plant is a maize plant.

Embodiment

Following these embodiment are used for illustrating embodiment of the present invention.It should be appreciated by those skilled in the art: disclosed technology has been represented inventor's technology that find, that play a role well in the following examples in practice of the present invention.Yet those skilled in the art's disclosure according to the present invention should be appreciated that, can carry out many changes to disclosed specific embodiment, still obtains identical or similar result, and does not depart from notion of the present invention, spirit and scope.More particularly, obviously some chemistry reagent relevant with physiology can replace described reagent herein, and will obtain same or analogous result.All these significantly similarly replace for those skilled in the art and revise, and are considered to be within spirit of the present invention, scope and the notion of appending claims qualification.

Embodiment 1

The preparation of the CTP-DMO construct that is used to transform

According to the DNA construct shown in standard method (for example people such as Sambrook, 1989) the preparation table 2, this DNA construct comprises the CTP that is operably connected with DMO gene or its variant between plant promoter and polyadenylation signal sequence.As described below, corn protoplastis system or in the Arabidopis thaliana of stable conversion or soybean plants these constructs of test.

Embodiment 2

The analysis of the CTP-DMO construct in the corn protoplastis

From 12 days etiolated seedlings (from LH200x LH5 hybridization) preparation leaf of Semen Maydis mesophyll protoplast.The middle portion (approximately 6cm is long) of second leaf is become the bar of 0.5mm with blade cuts, and in vacuum filtration after 30 minutes, in flask with containing 2% (w/v) cellulase RS, 0.3% (w/v) macerozyme R10 (all from Karlan Research Products Corp, SantaRosa, CA), 0.6M N.F,USP MANNITOL, 10mM MES (pH 5.7) and 1mM CaCl ₂Enzyme solution be no more than 2 hours 23 ℃ of digestion.Pass through to discharge in 5 minutes from filtering protoplastis with hand moving flask with the leaf texture that digests, and by the separation of 60-μ m nylon net filter.Protoplastis is by collecting in centrifugal 2 minutes with 150g, and is with the cold mannitol solution washing of 0.6M once centrifugal, with 2x 10 ⁶/ mL resuspending is in cold 0.6M N.F,USP MANNITOL.Use polyoxyethylene glycol (PEG) then, transform protoplastis with 12.5 μ g DNA, and at room temperature hatched 16-20 hour.

Protoplastis is stored in-80 ℃, up to analyzing by Western blotting (western blot).Protoplastis is melting on ice, and the 2xLaemmli sample buffer/dyestuff (BioRad) that contains 5.0% β-ME of 1-3 times of volume is added to protoplastis.Aliquots containig with protoplastis protein sample is heated to about 100 ℃ of maintenances 5 minutes then, and application of sample is to ready-formed Tris-HCL 10% polyacrylamide gel.Electrophoresis is about 35 minutes under the constant current of about 80-100Amps.Then protein nitrocellulose filter from gel electrotransfer to 0.2 micron under the 100V constant voltage was carried out 1-3 hour.Film at room temperature sealed 1 hour with 5% among the TBST (w/v) milk powder or seals under 4 ℃ and spend the night.The goat that is used among the TBST dilution in 1: 200 is anti--DMO antibody detection membrane 1 hour.Use TBS flushing 3 times, each 5 minutes, to remove excessive antibody.Be used among the TBST that contains 0.5% (w/v) milk powder with 1: 7, (MO) detection membrane is 1 hour for Sigma, St.Louis for the anti-goat IgG of peroxidase link coupled rabbit of 500 dilution proportion.With TBST flushing 3 times, each 5 minutes, to remove excessive superoxide enzyme conjugates.Except those particularly point out, all programs comprise that sealing and all other hatch, and at room temperature carry out.Use ECL detection system (Amersham Biosciences, Piscataway NJ) show immunocompetent band, and at Kodak BioMax ^TMExpose on the MS film.Suitably the existence of the immunocompetence band of size shows correct processing and location (table 1) of DMO.Therefore, for example, use to be operably connected to DMO and to go up and be transformed into CTP4 in the corn protoplastis produces 38kDa behind western blot analysis immunocompetence band.

Embodiment 3

The test of various CTP-DMO constructs in Arabidopis thaliana

According to the method for Clough and Bent (1998) exploitation, the environmental plant of arabidopsis thaliana transformation Colombia.The plant that the seed that obtains by this method is inoculated into the multiple concentration dicamba 98 that contains 0.5,1.0 to 2.0 or 4.0 mg/litre is selected on the substratum.These plates were hatched 48 hours at 4 ℃, transfer to then and be set at 23.5 ℃, have in 16 hours photoperiodic Percival incubators.The seed that transforms with the CTP-DMO construct grows into plant containing on the substratum of dicamba 98, and grows primary leaf and time leave, and unconverted seed and negative segregant (segregants) are dead or do not grow primary leaf and time leave.By

It is that 3 ' UTR male transgenic plant are used for further analysis that the PCR test detects.

3-5 the shot hole of leaf from the transgenic arabidopsis plant is used for western blot analysis.In Harbil paint jolting device, carried out proteins extraction 3 minutes with 500-1000 μ l PSBT and 4 granulated glass spherees.Under 4 ℃ with 3000rpm rotated sample 3 minutes.In the aliquots containig of supernatant liquor, add isopyknic 2x Laemmli sample buffer/dyestuff (cat.No.161-0737BioRad) that contains 5.0% β-ME.The same among all the other steps of western blot analysis and the embodiment 2.The existence of the immunocompetence band of correct size shows correct processing and location (table 2) of DMO.For example, as shown in table 2, after with pMON73749 or pMON73725 arabidopsis thaliana transformation in the comparison of visible band, use lacks the DMO that produces the correct processing that is positioned chloroplast(id) from the RbcSnoc-CTP of 27 amino acid whose encoding sequences of pea rubisco, uses the RbcS CTP that comprises 27 amino acid whose encoding sequences then to produce 2 immunocompetence bands.

Embodiment 4

The test of CTP-DMO construct in soybean

Use standard program to transform (for example, people such as Zhang, 1999 by agrobacterium-mediated soybean; US 7,002, and 058) acquisition genetically engineered soybean (for example, cvs.Thorne, NE3001 and A3525) plant.3-5 the shot hole of leaf from the genetically engineered soybean plant is used for western blot analysis.In Harbil paint jolting device, carried out proteins extraction 3 minutes with 500-1000 μ l PSBT and 4 granulated glass spherees.Under 4 ℃ with 3000rpm rotated sample 3 minutes.In the aliquots containig of supernatant liquor, add isopyknic 2x Laemmli sample buffer/dyestuff (BioRad)/5.0% β-ME.The same among all the other steps of western blot analysis and the embodiment 2.The existence of the immunocompetence band of correct size shows correct processing and location (table 2) of DMO.

The soybean plants that transforms with construct (its coding be connected to other 24 amino acid of being attached with the rubisco coding region and owing to the DMO that introduces on 3 amino acid whose pea rubisco transit peptides that the Restriction Enzyme recognition site adds), when the dicamba 98 processing of using 0.5lb in the last stage of being unearthed is then used the processing of 2lb dicamba 98 in back (V6) stage of being unearthed, show the damage rate of 17-20%.Its soybean plants with the construct conversion that only is connected to the DMO on the pea rubisco transit peptides with coding is compared, and the latter shows about 12% damage rate.These results show and use the transit peptides do not have additional amino acid to cause the active generation of single DMO (rather than polypeptide of a plurality of part or different processing) and to the higher tolerance of dicamba 98.The generation of the enzyme of single form also causes being easy to characterize product and reducing cost of registering.

Embodiment 5

The High-efficient Production of DMO and higher dicamba 98 tolerance need CTP

With as the environmental plant of embodiment 3 described several constructs (Fig. 1) arabidopsis thaliana transformation Colombia.Select the seed that transforms comprising from the plant tissue culture media of the dicamba 98 of the various concentration of 0.5,1.0 to 2.0 mg/litre.The seed that transforms with the CTP-DMO construct grows into plant containing on the substratum of dicamba 98, and grows primary leaf and time leave, and unconverted seed and negative segregant are dead or do not grow primary leaf and time leave.Growth and detection are used for further analysis for the transgenic plant of DMO gene masculine.

As shown in Figure 1, almost do not have with the construct plant transformed that does not contain CTP or do not show tolerance dicamba 98.When the dicamba 98 processing of using 0.5lb/a in the last stage of being unearthed is then used the dicamba 98 processing of 2lb/a in back (V6) stage of being unearthed, with coding be not connected to soybean plants that the DNA construct of the DMO on the CTP transforms do not show be unearthed before tolerance, and with DMO wherein be connected to construct plant transformed on the CTP show be unearthed before and be unearthed after to the tolerance of dicamba 98.

Embodiment 6

The generation of the rotaring gene corn plant of tolerance dicamba 98

In order to test the DMO gene in the purposes that provides to monocotyledons aspect the dicamba 98 tolerance, in gene expression in plants element such as promotor (for example, PClSV, e35S, OsAct1, OsTPI, OsAct15) and intron is (for example, OsAct1, OsAct15, OsTPI, ZmHSP70) control under, produced rotaring gene corn plant, it (for example comprises the DMO gene, SEQ ID NOS:29,33,35,37,39), has or do not have transit peptides (for example, TaWaxy, CTP1, synthetic, the CTP4 of CTP2).This Expression element comprises from first intron of rice actin 1 gene and flank UTR exon sequence, also comprises the 12nt of exons 1 and holds the 7nt that comprises exon 23 ' at 5 ' end, also has 3 ' UTR (for example, TaHsp17).

Rotaring gene corn plant produces by U.S. Patent application 20040244075 described methods basically.In the revision test of single position, estimate the dicamba 98 tolerance of transgenic corns incident with single copy.Use is from each 6 incidents in 6 constructs.Experimental design is as follows: row/project: 1; Handle: at the 0.5lb/a in V3 stage dicamba 98, then the 1lb/a in V8 stage dicamba 98 (

BASF, Raleigh, NC); Repeat: 2; Line-spacing: 30 inches; Ground block length: minimum 20 feet; Plant density: about 30 strains/17.5 feet; Passage (alley): 2.5 feet.The uniform of application of whole plot is to obtain acceptable farm crop on the agronomy.In order to control corn rootworm, use 1000 feet 5 ounces of soil insecticides of every row in the period of cultivating, as

3G (Syngenta Crop Protection, Greensboro, NC, USA).If observe the invasion of black cutworm (black cutworm), use with the amount of every acre of 4-8 ounce 3.2EC (FMC Corporation, Philadelphia, PA).In addition, use the spraying insecticide plan to control all lepidoptera pests on the ground, comprise European corn borer, corn ear worm and autumn mythimna separata.Per 3 weeks use with the amount of every acre of 4-8 ounce

3.2EC with the control lepidoptera pest; Approximately use 4 times.Pre-emergence application as Xtra 5.6L (Monsanto, St.Louis, MO) and Degree

(weedicide MO) does not have weeds to keep the plot for Monsanto, St.Louis.In whole experiment, occur if in untreated inspection, observe weeds, then by manual weeding or post-emergence application usefulness PERMIT (Monsanto, St.Louis, MO) or

(Bayer, Research Triangle Park, NC) control weeds.

When handling when then handling with the 1lb/a dicamba 98 with the 0.5lb/a dicamba 98, damage the dicamba 98 tolerance of testing with comprising the corn inbred lines that the genetically modified DNA construct of DMO transforms by measuring stilit root in the V8 stage in the V3 stage.By counting the quantity of comparing the plant of " atypia " form that shows that stilit root merges among the row with the representative configuration of " finger sample " structure, the damage of visual valuation stilit root.As shown in table 4, the maize plant that the DNA construct (pMON73699, pMON73704) of the DMO that is not connected with CTP with encoding transforms shows the stilit root damage of higher level, that is, and and the low-level protection after dicamba 98 is handled.The DNA construct (pMON73716, pMON73700, pMON73715, pMON73703) that coding is connected to the DMO on the CTP shows that the stilit root of lower level damages, that is, dicamba 98 is handled the protection of back higher level.

The stilit root damage per-cent that the transgenic corns that table 4. transforms with the DNA construct of carrying DMO demonstrates when testing the dicamba 98 tolerance.

Inbreeding/construct	Details	The stilit root damage
			??01CSI6	Inbred lines to the dicamba 98 sensitivity	??95.4
??LH244	Inbred lines to the dicamba 98 tolerance	??93.8
			??pMON73699	??PC1SV/I-OsAct1/DMO-Wmc/TaHsp17	??93.2
??pMON73704	??e35S/I-OsAct1/DMO-Wmc/TaHsp17	??91.3
			??pMON73716	??PC1SV/I-OsAct1/TaWaxy/DMO-Wmc/TaHsp17	??78.8
??pMON73700	??PC1SV/I-OsAct1/CTP1/DMO-Wmc/TaHsp17	??74.4
			??pMON73715	??PC1SV/I-OsAct1/CTP2syn/DMO-Wmc/TaHsp17	??68.2
??pMON73703	??e35S/I-OsAct1/CTP1/DMO-Wmc/TaHsp17	??68.8

From these researchs (in addition, for example, embodiment 3,4 and 8) of carrying out different plant species as seen, chloroplast transit peptides can be used for the efficient target of DMO and DMO is active produces fully, causes the tolerance higher to dicamba 98.In addition, the expression of CTP-DMO provides the preceding tolerance of being unearthed of dicamba 98 in corn.

Embodiment 7

Effectively DMO expresses unitary structure

Some genetic elements can influence the effective expression of gene, as coding region, the 3 ' UTR of promotor, chloroplast transit peptide sequence, intron, 5 ' UTR, gene.Yet, do not know which combined running is best.Need effective DMO expression unit or construct to produce seed and the plant of improved product as the tolerance dicamba 98.By each a kind of several DMO expression unit that makes up among the various promotors that are operably connected, CTP, DMO variant and the 3 ' UTR, to obtain to be used for the effective DMO expression unit of product development.These constructs are transformed in the soybean (for example, U.S.6,384,301, U.S.7,002,058 or people such as Zhang, 1999) by methods known in the art.Obtain transgenic seed, and detect the unearthed preceding and tolerance of back of being unearthed Banvel D.Table 5 has shown before seed and plant are being unearthed with 0.5lb/ acre dicamba 98 to be handled then in back V6 stage of being unearthed during with the dicamba 98 processing of 2lb/ acre, the damage per-cent (lower damage means higher tolerance) that is caused by dicamba 98.The seed that transforms with the DNA construct pMON68939 that does not carry CTP and pMON73723 can not tolerate the pre-emergence application of dicamba 98, shows in order to obtain tolerance before being unearthed of dicamba 98, need be with the DMO target to chloroplast(id).With pMON68939 and pMON73723 (not containing CTP) plant transformed, after the V3 latter stage is handled with the amount of 1lb/a with dicamba 98, show 55% and 57% the damage rate be similar to wild-type soybean (60%) respectively, and with the very low damage rate of pMON68938 (containing CTP) plant transformed demonstration.These results show that obtaining needs CTP to the preceding and unearthed back of being unearthed of dicamba 98 tolerance in soybean.

The damage per-cent that table 5. is expressed the unit conversion and shown with the soybean plants of back with the dicamba 98 processing that be unearthed before being unearthed with specificity DMO.

Express the unit	The pMON title	The % damage
			??PC1SV/CTP2syn/DMO-Wat(A)/nos	??73724	??9
??e35S/CTP1/DMO-Wat(L)/nos	??68938	??12
			??PC1SV/RbcSnoc/DMO-Wat(A)/nos	??73725	??12
??PC1SV/RbcSnoc/DMO-Wat(L)/nos	??73728	??12
			??PCSV/CTP1/DMO-Wat(A)/nos	??73729	??13
??PC1SV/CTP2syn/DMO-Wat(L)/nos	??73727	??13
			??ANT1/CTP1/DMO-Wat(L)/nos	??68945	??14
??PC1SV/RbcSnoc/DMO-Wat(A)/nos	??73730	??15
			??PC1SV/RbcS-CTP/DMO-Cnat(A)/nos	??68934	??17

??Act7/CTP1/DMO-Wat(L)/nos	??68942	??17
			??FMV.35S-EF1a/CTP1/DMO-Wat(L)/nos	??68940	??17
??PClSV/RbcS-CTP/DMO-Cnat(A)/E9	??84254	??20
			??FMV/CTP1/DMO-Wat(L)/nos	??68941	??29
??eIF4A10/CTP1/DMO-Wat(L)/nos	??68943	??60
			??e35S/CTP1/DMO-Cat(A)/nos	??68937	??62
??e35S/CTP1/DMO-Cnat(L)/nos	??68946	??73
			??e35S/DMO-Wat(A)/nos	??68939	100 (preceding)
??PC1SV/DMO-Wat(A)/nos	??73723	100 (preceding)

Embodiment 8

The generation of the transgenic cotton flowering plant of tolerance dicamba 98

In order to test the DMO gene, produce the transgenic cotton flowering plant in the purposes that provides to cotton aspect the dicamba 98 tolerance.Produced several DNA construct, they are carried at gene expression in plants element such as promotor (for example, PClSV, FMV or e35S) and 3 ' UTR (for example, E6; Registration number #U30508) control under (for example have transit peptides, PsRbcS CTP, CTP1, CTP2) the DMO coding region (for example, and as described below being transformed in the cotton (upland cotton (Gossypium hirsutum)) SEQ ID NO:23,25,27,29,31,35).Used substratum is listed in table 6.

The seedling isolated growth of cotton cv Coker130, and downcut the plumular axis part, and be inoculated in the liquid suspension of the Agrobacterium tumefaciens of carrying DNA construct, trace drying (blot dried), and cultivated altogether 2 days.Then the plumular axis explant of inoculation is transferred to glucose and selected 4 weeks in the substratum, sucrose is selected 1 week in the substratum, and glucose is selected to cultivate for 4 weeks again in the substratum, comes evoked callus.Hatch culture under (light/dark) circulation and 28 ℃ 16/8.Then the kalamycin resistance callus is transferred in the UMO substratum, and under 28-30 ℃, dark condition, cultivated and induce living embryo callus 16-24 week.Results are given birth to the embryo callus from these callus then, and under 28-30 ℃, dark condition, on the TRP+ substratum, keep and reach 4-16 week.Results are come the little embryo of spontaneous embryo callus, and on the SHSU substratum, under the 28-30 ℃ of temperature and 6/8 (light/dark) cycling condition germination down.To seem that then normal plantlet transfers in the soil to obtain sophisticated vegetable lamb.The transgenosis character of transformant confirms by DNA detection.

Table 6. is used for the composition of the various substratum of cotton conversion.

The V4-5 growth phase with the amount of 561g ae/ha (0.5lb/a) with dicamba 98 (

BASF, Raleigh, NC) handle the vegetable lamb of the conversion that comprises such DNA construct as the aftertreatment of being unearthed, each DNA construct comprises the different combination of DMO coding region and transit peptides, promotor and 3 ' UTR, discovery has tolerance, and unconverted vegetable lamb shows the damage rate of 79%-86%.The transgenic plant that select to show surpass 95% tolerance (equaling to be less than 5% damage) are used for further research.Also the aftertreatment of being unearthed has tolerance to transgenic plant to subsequently dicamba 98.For example, the V3-4 stage with the dicamba 98 processing subsequent of 0.5lb/ acre V5 stage or more late stage with 1 or the plant of the dicamba 98 processing of 2lb/ acre still dicamba 98 is tolerated.This embodiment shows that the DMO gene can provide the dicamba 98 tolerance to the cotton of each growth phase, thereby can use dicamba 98 to obtain effective weeds control in each stage.

Embodiment 9

Improve the method for the upright ability of corn

Some monocotyledons as corn produces stilit root, and stilit root is grown from the node more than the face of land, and helps to support plant and absorb moisture and nutrition in reproductive phase from topsoil.If plant runs into strong wind or makes when the subterraneous root system becomes more weak that healthy stilit root system becomes important because root nematode infections or underground water lack.In order to control broadleaf weeds, allow use as dicamba 98 and 2 synthetic herbicide of 4-D as the monocotyledons of corn.For the unearthed back weeds control of corn, dicamba 98 is the 5th weedicide of extensively using.Although being used to control the optimal amount of broadleaf weeds is 280-560 gram/hectare (g/h) or 0.25-0.5lb/ acre, but the average usage quantity in the corn is 168g/h or 0.15lb/ acre, because under higher amount of application and some envrionment conditions as hot day, dicamba 98 can damage corn.In addition, several corn hybridization bodies, as DKC61-42, DKC64-77, DKC63-46, DKC66-21 and DKC61-44, and the inbreeding body, as 01CSI6,16IUL2,70LDL5 and 90LCL6, dicamba 98 is used sensitivity.Susceptibility shows in many ways, as the formation that onion peels off (onion leafing), fringe deformity, plant height reduction or abnormal stilit root occurs, for example formation of the root of fusion or distortion.The polyoma that becomes stilit root saves, and trends towards becoming to grow together, and does not grow in soil and support plant.This may cause the upright ability of more weak corn crop, higher easy lodging and final production loss.Some contain the weedicide product of dicamba 98, for example

BANVEL, MARKSMAN, DISTINCT, NORTHSTAR and CELEBRITY PLUS can cause these effects.The raising corn also is useful near the corn field that the farm crop kind (as soybean, cotton) of tolerance dicamba 98 is planted to the tolerance of dicamba 98 in protection, and wherein allows higher dicamba 98 amount of application.

Present embodiment provides a kind of method of improving corn and other monocotyledonous upright ability, is included in to introduce the DMO gene in the corn and handle corn with dicamba 98.In one embodiment, the DMO gene is expressed under the control of constitutive promoter, and described constitutive promoter also can be expressed DMO in node region and/or stilit root.In another embodiment, the DMO gene is expressed under the control of a kind of chimeric composing type and node/stilit root specificity promoter.In another embodiment, the DMO gene is expressed under the control of root-specific promoter such as RCc3 or its variant (the SEQ ID NOs:1-6 that for example, finds in US20060101541).The expression of DMO in stilit root do not cause stilit root not or less damage is arranged, and causes the higher upright ability of corn, less lodging, thereby causes higher output.

The R1 of 3 single copy incidents of the maize plant that the various DMO constructs (listing in table 7) of using by oneself transform or F1 seed are 4.0 " germinate in the pallet.Health plant is transplanted to about 10 " the jar in.Germinate and growth medium comprise Redi-earthTM (Scotts-SierraHorticultural Products Co., Marysville, Ohio).The glass fibre that jar is placed on 60 inches of 35 inches x waters on the capillary pad in the water tray, is used for carrying out the Asia pouring at test period, thereby keeps soil moisture content best for plant-growth.With Osmocote (14-14-14 slowly-releasing; Scotts-Sierra Horticultural Products Co., Marysville Ohio) applies fertilizer to jar with the amount of 100gm/cu.ft., to keep plant-growth during greenhouse test.Plant 29 ℃/21 ℃ the daytime/night temperature, 25%-75% the greenhouse of relative humidity in grow, with the simulation growth conditions in late spring in warm season.The additional light of about 600 μ E is provided for minimum 14 hours periodicity of illumination as required.

Use has the flat fan of the atmospheric Teejet 9501E nozzle that is set at minimum 24psi (165kpa), and (Spraying Systems Co, Wheaton IL), uses dicamba 98 with track sprayer.Nozzle remains on than high about 16 inches position, plant top and sprays.Sprayed volume is 10 gallon/acre or 93 liters/hectare.When plant reaches the V4-5 leaf during stage, begin to use.

By handling with the dicamba 98 of 2lb/ acre or 4lb/ acre in the V4-5 stage, and in the stilit root damage (0% of processing assessment in back 24 days plant; Do not have visible injury, to 100%, plant is dead fully) and lodging (inclined degree), detect with comprising the stilit root that DMO expresses the plant of the corn inbred lines that unitary DNA construct transforms and damage and the lodging situation.

As shown in table 7, with the contrast inbred lines that is not transformed with only express unit (pMON73746) plant transformed and compare, express the maize plant that unitary DNA construct transforms and show do not have or almost do not have stilit root damage and lodging with containing DMO with selected marker.Present embodiment shows that the plant that comprises DMO can be used to provide the upright ability of raising when handling with dicamba 98.

Table 7. is when handling with dicamba 98, and the maize plant that transforms with various DMO constructs shows do not have or almost do not have stilit root damage and lodging.

Reference

The document of below listing is incorporated herein by reference, with method, technology and/or the composition that replenishes, explains, provides background or instruction this paper to adopt.

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People such as Miki, In:Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson (volume), CRC Press, 67-88,1993.

People such as Moberg, Plant J.36:616-628,2003.

Murashige and Skoog, Physiol Plant15:473-497,1962.

People such as Odell, Nature, 313:810-812,1985.

Okagaki，Plant?Mol.Biol.，19：513-516，1992.

PCT applies for WO 9506722.

PCT applies for WO 97/11086

PCT applies for WO 97/31115

PCT applies for WO 97/41228

People such as Rudhe, J.Molec.Biol.324:577-585,2002.

People such as Sambrook, In:Molecular cloning:a laboratory manual, the 2nd edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

People such as Silva Filho, Plant Molecular Biology 30:769-780,1996,

People such as Teeri, EMBO J., 8 (2): 343-350,1989.

Turner and Foster, Molecular Biotech., 3:225,1995.

People such as Walker, Proc.Natl.Acad.Sci.USA, 84:6624,1987.

Yang and Russell, Proc.Natl.Acad.Sci.USA, 87:4144-4148,1990.

People such as Zhang, Plant Cell Tissue Organ Cult.56:37-46,1999.

Sequence table

＜110〉Monsanto Technology LLC

Feng，Paul?C.C.

Malven，Marianne

Stanislaw?Flasinski

＜120〉be used for the chloroplast transit peptides and uses thereof of the efficient targeting of DMO

<130>MONS：107WO

＜140〉the unknown

<141>2007-06-06

<150>11/758,659

<151>2007-06-05

<150>60/891,675

<151>2007-02-26

<160>41

<170>3.3

<210>1

<211>84

<212>PRT

＜213〉pea

<400>1

Met?Ala?Ser?Met?Ile?Ser?Ser?Ser?Ala?Val?Thr?Thr?Val?Ser?Arg?Ala

1???????????????5???????????????????10??????????????????15

Ser?Arg?Gly?Gln?Ser?Ala?Ala?Met?Ala?Pro?Phe?Gly?Gly?Leu?Lys?Ser

20??????????????????25??????????????????30

Met?Thr?Gly?Phe?Pro?Val?Arg?Lys?Val?Asn?Thr?Asp?Ile?Thr?Ser?Ile

35??????????????????40??????????????????45

Thr?Ser?Asn?Gly?Gly?Arg?Val?Lys?Cys?Met?Gln?Val?Trp?Pro?Pro?Ile

50??????????????????55??????????????????60

Gly?Lys?Lys?Lys?Phe?Glu?Thr?Leu?Ser?Tyr?Leu?Pro?Pro?Leu?Thr?Arg

65??????????????????70??????????????????75??????????????????80

Asp?Ser?Arg?Ala

<210>2

<211>57

<212>PRT

＜213〉pea

<400>2

Met?Ala?Ser?Met?Ile?Ser?Ser?Ser?Ala?Val?Thr?Thr?Val?Ser?Arg?Ala

1???????????????5???????????????????10??????????????????15

Ser?Arg?Gly?Gln?Ser?Ala?Ala?Met?Ala?Pro?Phe?Gly?Gly?Leu?Lys?Ser

20??????????????????25??????????????????30

Met?Thr?Gly?Phe?Pro?Val?Arg?Lys?Val?Asn?Thr?Asp?Ile?Thr?Ser?Ile

35??????????????????40??????????????????45

Thr?Ser?Asn?Gly?Gly?Arg?Val?Lys?Cys

50??????????????????55

<210>3

<211>85

<212>PRT

＜213〉Arabidopis thaliana

<400>3

Met?Ala?Ser?Ser?Met?Leu?Ser?Ser?Ala?Thr?Met?Val?Ala?Ser?Pro?Ala

1???????????????5???????????????????10??????????????????15

Gln?Ala?Thr?Met?Val?Ala?Pro?Phe?Asn?Gly?Leu?Lys?Ser?Ser?Ala?Ala

20??????????????????25??????????????????30

Phe?Pro?Ala?Thr?Arg?Lys?Ala?Asn?Asn?Asp?Ile?Thr?Ser?Ile?Thr?Ser

35??????????????????40??????????????????45

Asn?Gly?Gly?Arg?Val?Asn?Cys?Met?Gln?Val?Trp?Pro?Pro?Ile?Glu?Lys

50??????????????????55??????????????????60

Lys?Lys?Phe?Glu?Thr?Leu?Ser?Tyr?Leu?Pro?Asp?Leu?Thr?Asp?Ser?Gly

65??????????????????70??????????????????75??????????????????80

Gly?Arg?Val?Asn?Cys

85

<210>4

<211>76

<212>PRT

＜213〉Arabidopis thaliana

<400>4

Met?Ala?Gln?Val?Ser?Arg?Ile?Cys?Asn?Gly?Val?Gln?Asn?Pro?Ser?Leu

1???????????????5???????????????????10??????????????????15

Ile?Ser?Asn?Leu?Ser?Lys?Ser?Ser?Gln?Arg?Lys?Ser?Pro?Leu?Ser?Val

20??????????????????25??????????????????30

Ser?Leu?Lys?Thr?Gln?Gln?His?Pro?Arg?Ala?Tyr?Pro?Ile?Ser?Ser?Ser

35??????????????????40??????????????????45

Trp?Gly?Leu?Lys?Lys?Ser?Gly?Met?Thr?Leu?Ile?Gly?Ser?Glu?Leu?Arg

50??????????????????55??????????????????60

Pro?Leu?Lys?Val?Met?Ser?Ser?Val?Ser?Thr?Ala?Cys

65??????????????????70??????????????????75

<210>5

<211>76

<212>PRT

＜213〉Arabidopis thaliana

<400>5

Met?Ala?Gln?Val?Ser?Arg?Ile?Cys?Asn?Gly?Val?Gln?Asn?Pro?Ser?Leu

1???????????????5???????????????????10??????????????????15

Ile?Ser?Asn?Leu?Ser?Lys?Ser?Ser?Gln?Arg?Lys?Ser?Pro?Leu?Ser?Val

20??????????????????25??????????????????30

Ser?Leu?Lys?Thr?Gln?Gln?His?Pro?Arg?Ala?Tyr?Pro?Ile?Ser?Ser?Ser

35??????????????????40??????????????????45

Trp?Gly?Leu?Lys?Lys?Ser?Gly?Met?Thr?Leu?Ile?Gly?Ser?Glu?Leu?Arg

50??????????????????55??????????????????60

Pro?Leu?Lys?Val?Met?Ser?Ser?Val?Ser?Thr?Ala?Cys

65??????????????????70??????????????????75

<210>6

<211>72

<212>PRT

＜213〉petunia

<400>6

Met?Ala?Gln?Ile?Asn?Asn?Met?Ala?Gln?Gly?Ile?Gln?Thr?Leu?Asn?Pro

1???????????????5???????????????????10??????????????????15

Asn?Ser?Asn?Phe?His?Lys?Pro?Gln?Val?Pro?Lys?Ser?Ser?Ser?Phe?Leu

20??????????????????25??????????????????30

Val?Phe?Gly?Ser?Lys?Lys?Leu?Lys?Asn?Ser?Ala?Asn?Ser?Met?Leu?Val

35??????????????????40??????????????????45

Leu?Lys?Lys?Asp?Ser?Ile?Phe?Met?Gln?Lys?Phe?Cys?Ser?Phe?Arg?Ile

50??????????????????55??????????????????60

Ser?Ala?Ser?Val?Ala?Thr?Ala?Cys

65??????????????????70

<210>7

<211>69

<212>PRT

＜213〉wheat

<400>7

Met?Ala?Ala?Leu?Val?Thr?Ser?Gln?Leu?Ala?Thr?Ser?Gly?Thr?Val?Leu

1???????????????5???????????????????10??????????????????15

Ser?Val?Thr?Asp?Arg?Phe?Arg?Arg?Pro?Gly?Phe?Gln?Gly?Leu?Arg?Pro

20??????????????????25??????????????????30

Arg?Asn?Pro?Ala?Asp?Ala?Ala?Leu?Gly?Met?Arg?Thr?Val?Gly?Ala?Ser

35??????????????????40??????????????????45

Ala?Ala?Pro?Lys?Gln?Ser?Arg?Lys?Pro?His?Arg?Phe?Asp?Arg?Arg?Cys

50??????????????????55??????????????????60

Leu?Ser?Met?Val?Val

65

<210>8

<211>77

<212>PRT

＜213〉paddy rice

<400>8

Met?Ala?Ala?Leu?Thr?Thr?Ser?Gln?Leu?Ala?Thr?Ser?Ala?Thr?Gly?Phe

1???????????????5???????????????????10??????????????????15

Gly?Ile?Ala?Asp?Arg?Ser?Ala?Pro?Ser?Ser?Leu?Leu?Arg?His?Gly?Phe

20??????????????????25??????????????????30

Gln?Gly?Leu?Lys?Pro?Arg?Ser?Pro?Ala?Gly?Gly?Asp?Ala?Thr?Ser?Leu

35??????????????????40??????????????????45

Ser?Val?Thr?Thr?Ser?Ala?Arg?Ala?Thr?Pro?Lys?Gln?Gln?Arg?Ser?Val

50??????????????????55??????????????????60

Gln?Arg?Gly?Ser?Arg?Arg?Phe?Pro?Ser?Val?Val?Val?Cys

65??????????????????70??????????????????75

<210>9

<211>57

<212>PRT

＜213〉tobacco

<400>9

Met?Ala?Ser?Ser?Val?Leu?Ser?Ser?Ala?Ala?Val?Ala?Thr?Arg?Ser?Asn

1???????????????5???????????????????10??????????????????15

Val?Ala?Gln?Ala?Asn?Met?Val?Ala?Pro?Phe?Thr?Gly?Leu?Lys?Ser?Ala

20??????????????????25??????????????????30

Ala?Ser?Phe?Pro?Val?Ser?Arg?Lys?Gln?Asn?Leu?Asp?Ile?Thr?Ser?Ile

35??????????????????40??????????????????45

Ala?Ser?Asn?Gly?Gly?Arg?Val?Gln?Cys

50??????????????????55

<210>10

<211>65

<212>PRT

＜213〉corn

<400>10

Met?Glu?Ser?Leu?Ala?Ala?Thr?Ser?Val?Phe?Ala?Pro?Ser?Arg?Val?Ala

1???????????????5???????????????????10??????????????????15

Val?Pro?Ala?Ala?Arg?Ala?Leu?Val?Arg?Ala?Gly?Thr?Val?Val?Pro?Thr

20??????????????????25??????????????????30

Arg?Arg?Thr?Ser?Ser?Arg?Ser?Gly?Thr?Ser?Gly?Val?Lys?Cys?Ser?Ala

35??????????????????40??????????????????45

Ala?Val?Thr?Pro?Gln?Ala?Ser?Pro?Val?Ile?Ser?Arg?Ser?Ala?Ala?Ala

50??????????????????55??????????????????60

Ala

65

<210>11

<211>72

<212>PRT

＜213〉rue

<400>11

Met?Gly?Ala?Ala?Ala?Thr?Ser?Met?Gln?Ser?Leu?Lys?Phe?Ser?Asn?Arg

1???????????????5???????????????????10??????????????????15

Leu?Val?Pro?Pro?Ser?Arg?Arg?Leu?Ser?Pro?Val?Pro?Asn?Asn?Val?Thr

20??????????????????25??????????????????30

Cys?Asn?Asn?Leu?Pro?Lys?Ser?Ala?Ala?Pro?Val?Arg?Thr?Val?Lys?Cys

35??????????????????40??????????????????45

Cys?Ala?Ser?Ser?Trp?Asn?Ser?Thr?Ile?Asn?Gly?Ala?Ala?Ala?Thr?Thr

50??????????????????55??????????????????60

Asn?Gly?Ala?Ser?Ala?Ala?Ser?Ser

65??????????????????70

<210>12

<211>252

<212>DNA

＜213〉pea

<400>12

atggcttcta?tgatatcctc?ttccgctgtg?acaacagtca?gccgtgcctc?tagggggcaa????60

tccgccgcaa?tggctccatt?cggcggcctc?aaatccatga?ctggattccc?agtgaggaag????120

gtcaacactg?acattacttc?cattacaagc?aatggtggaa?gagtaaagtg?catgcaggtg????180

tggcctccaa?ttggaaagaa?gaagtttgag?actctttcct?atttgccacc?attgacgaga????240

gattcccggg?cc????????????????????????????????????????????????????????252

<210>13

<211>171

<212>DNA

＜213〉pea

<400>13

atggcttcta?tgatatcctc?ttccgctgtg?acaacagtca?gccgtgcctc?tagggggcaa????60

tccgccgcaa?tggctccatt?cggcggcctc?aaatccatga?ctggattccc?agtgaggaag????120

gtcaacactg?acattacttc?cattacaagc?aatggtggaa?gagtaaagtg?c?????????????171

<210>14

<211>255

<212>DNA

＜213〉Arabidopis thaliana

<400>14

atggcttcct?ctatgctctc?ttccgctact?atggttgcct?ctccggctca?ggccactatg????60

gtcgctcctt?tcaacggact?taagtcctcc?gctgccttcc?cagccacccg?caaggctaac????120

aacgacatta?cttccatcac?aagcaacggc?ggaagagtta?actgtatgca?ggtgtggcct????180

ccgattgaaa?agaagaagtt?tgagactctc?tcttaccttc?ctgaccttac?cgattccggt????240

ggtcgcgtca?actgc?????????????????????????????????????????????????????255

<210>15

<211>228

<212>DNA

＜213〉Arabidopis thaliana

<400>15

atggcgcaag?ttagcagaat?ctgcaatggt?gtgcagaacc?catctcttat?ctccaatctc????60

tcgaaatcca?gtcaacgcaa?atctccctta?tcggtttctc?tgaagacgca?gcagcatcca????120

cgagcttatc?cgatttcgtc?gtcgtgggga?ttgaagaaga?gtgggatgac?gttaattggc????180

tctgagcttc?gtcctcttaa?ggtcatgtct?tctgtttcca?cggcgtgc?????????????????228

<210>16

<211>228

<212>DNA

＜213〉artificial sequence

<400>16

atggcgcaag?ttagcagaat?ctgcaatggt?gtgcagaacc?catctcttat?ctccaatctc????60

tcgaaatcca?gtcaacgcaa?atctccctta?tcggtttctc?tgaagacgca?gcagcatcca????120

cgagcttatc?cgatttcgtc?gtcgtgggga?ttgaagaaga?gtgggatgac?gttaattggc????180

tctgagcttc?gtcctcttaa?ggtcatgtct?tctgtttcca?cggcgtgc?????????????????228

<210>17

<211>216

<212>DNA

＜213〉artificial sequence

<400>17

atggcccaga?tcaacaacat?ggcccagggc?atccagaccc?tgaaccctaa?ctctaacttc????60

cacaagccgc?aagtgcccaa?gtctagctcc?ttcctcgtgt?tcggctccaa?gaagctcaag????120

aatagcgcca?attccatgct?ggtcctgaag?aaagactcga?tcttcatgca?gaagttctgc????180

tcctttcgca?tcagtgcttc?ggttgcgact?gcctgc??????????????????????????????216

<210>18

<211>207

<212>DNA

＜213〉artificial sequence

<400>18

atggcggcac?tggtgacctc?ccagctcgcg?acaagcggca?ccgtcctgtc?ggtgacggac????60

cgcttccggc?gtcccggctt?ccagggactg?aggccacgga?acccagccga?tgccgctctc????120

gggatgagga?cggtgggcgc?gtccgcggct?cccaagcaga?gcaggaagcc?acaccgtttc????180

gaccgccggt?gcttgagcat?ggtcgtc????????????????????????????????????????207

<210>19

<211>231

<212>DNA

＜213〉paddy rice

<400>19

atggcggctc?tcaccacgtc?ccagctcgcc?acctcggcca?ccggcttcgg?catcgccgac????60

aggtcggcgc?cgtcgtcgct?gctccgccac?gggttccagg?gcctcaagcc?ccgcagcccc????120

gccggcggcg?acgcgacgtc?gctcagcgtg?acgaccagcg?cgcgcgcgac?gcccaagcag????180

cagcggtcgg?tgcagcgtgg?cagccggagg?ttcccctccg?tcgtcgtgtg?c?????????????231

<210>20

<211>171

<212>DNA

＜213〉tobacco

<400>20

atggcttcct?cagttctttc?ctctgcagca?gttgccaccc?gcagcaatgt?tgctcaagct????60

aacatggttg?cacctttcac?tggccttaag?tcagctgcct?cattccctgt?ttcaaggaag????120

caaaaccttg?acatcacttc?cattgccagc?aacggcggaa?gagtgcaatg?c?????????????171

<210>21

<211>195

<212>DNA

＜213〉corn

<400>21

atggaatccc?tagccgccac?ctccgtgttc?gcgccctccc?gcgtcgccgt?cccggcggcg????60

cgggccctgg?ttagggcggg?gacggtggta?ccaaccaggc?ggacgagcag?ccggagcgga????120

accagcgggg?tgaaatgctc?tgctgccgtg?acgccgcagg?cgagcccagt?gattagcagg????180

agcgctgcgg?cggcc?????????????????????????????????????????????????????195

<210>22

<211>216

<212>DNA

＜213〉rue

<400>22

atgggtgcag?cggcaacgtc?gatgcaatcc?cttaaattct?ccaaccgtct?ggtcccaccc????60

agtcgccgtc?tgtctccggt?tccgaacaat?gtcacctgca?ataacctccc?caagtctgca????120

gctcccgtcc?ggacagtcaa?atgctgcgct?tcttcctgga?acagtaccat?caacggcgcg????180

gccgccacga?ccaacggtgc?gtccgccgcc?agtagc??????????????????????????????216

<210>23

<211>1023

<212>DNA

＜213〉artificial sequence

<400>23

atggccactt?tcgttagaaa?cgcttggtac?gttgctgcac?ttcctgagga?gttgagcgag????60

aagcctctag?gaagaactat?cctcgatact?ccactagctc?tctatcgtca?acctgacgga????120

gttgtcgctg?ccctgcttga?tatttgtccg?catcgcttcg?ctccgttgag?tgacggtatt????180

ctagtcaacg?gacatctcca?gtgtccatat?cacggtctgg?aatttgacgg?aggtggccag????240

tgtgtccaca?acccgcacgg?caacggagcc?cgccctgctt?ctctgaacgt?gcgatcattc????300

cctgtcgtgg?aaagagacgc?attgatctgg?atctgccctg?gagatccagc?actcgcagat????360

cccggtgcta?tccctgactt?tgggtgtcgt?gttgatccag?cttaccgtac?tgtcggaggt????420

tacggtcacg?tggactgcaa?ctacaagctc?cttgtggata?acctcatgga?tcttggacac????480

gctcagtacg?tgcaccgcgc?taacgcccaa?acagacgcct?tcgatagact?tgagcgtgag????540

gtgatcgttg?gcgacggcga?gatccaggcg?ctcatgaaga?tccctggtgg?cacaccctca????600

gttctcatgg?ctaagttctt?gcgtggtgct?aacacaccag?ttgacgcctg?gaacgacatc????660

cggtggaata?aggtgtcggc?tatgctgaac?ttcatcgcgg?tcgcgccgga?agggacgccg????720

aaggagcagt?caatccactc?ccgaggaacc?catatcctta?ctcctgagac?cgaggcaagc????780

tgccattact?tcttcggtag?ttcccgcaac?ttcggtatag?acgatccaga?gatggacggt????840

gttctcagga?gctggcaagc?tcaagccctg?gtgaaggagg?acaaagtggt?cgttgaagct????900

atcgaaaggc?ggagggctta?cgtcgaagcg?aacgggatca?gacccgccat?gttgtcctgc????960

gacgaggcag?ccgtcagggt?atccagggag?attgagaagc?tcgaacaact?agaagcggcg????1020

tga??????????????????????????????????????????????????????????????????1023

<210>24

<211>340

<212>PRT

＜213〉artificial sequence

<400>24

Met?Ala?Thr?Phe?Val?Arg?Asn?Ala?Trp?Tyr?Val?Ala?Ala?Leu?Pro?Glu

1???????????????5???????????????????10??????????????????15

Glu?Leu?Ser?Glu?Lys?Pro?Leu?Gly?Arg?Thr?Ile?Leu?Asp?Thr?Pro?Leu

20??????????????????25??????????????????30

Ala?Leu?Tyr?Arg?Gln?Pro?Asp?Gly?Val?Val?Ala?Ala?Leu?Leu?Asp?Ile

35??????????????????40??????????????????45

Cys?Pro?His?Arg?Phe?Ala?Pro?Leu?Ser?Asp?Gly?Ile?Leu?Val?Asn?Gly

50??????????????????55??????????????????60

His?Leu?Gln?Cys?Pro?Tyr?His?Gly?Leu?Glu?Phe?Asp?Gly?Gly?Gly?Gln

65??????????????????70??????????????????75??????????????????80

Cys?Val?His?Asn?Pro?His?Gly?Asn?Gly?Ala?Arg?Pro?Ala?Ser?Leu?Asn

85??????????????????90??????????????????95

Val?Arg?Ser?Phe?Pro?Val?Val?Glu?Arg?Asp?Ala?Leu?Ile?Trp?Ile?Cys

100?????????????????105?????????????????110

Pro?Gly?Asp?Pro?Ala?Leu?Ala?Asp?Pro?Gly?Ala?Ile?Pro?Asp?Phe?Gly

115?????????????????120?????????????????125

Cys?Arg?Val?Asp?Pro?Ala?Tyr?Arg?Thr?Val?Gly?Gly?Tyr?Gly?His?Val

130?????????????????135?????????????????140

Asp?Cys?Asn?Tyr?Lys?Leu?Leu?Val?Asp?Asn?Leu?Met?Asp?Leu?Gly?His

145?????????????????150?????????????????155?????????????????160

Ala?Gln?Tyr?Val?His?Arg?Ala?Asn?Ala?Gln?Thr?Asp?Ala?Phe?Asp?Arg

165?????????????????170?????????????????175

Leu?Glu?Arg?Glu?Val?Ile?Val?Gly?Asp?Gly?Glu?Ile?Gln?Ala?Leu?Met

180?????????????????185?????????????????190

Lys?Ile?Pro?Gly?Gly?Thr?Pro?Ser?Val?Leu?Met?Ala?Lys?Phe?Leu?Arg

195?????????????????200?????????????????205

Gly?Ala?Asn?Thr?Pro?Val?Asp?Ala?Trp?Asn?Asp?Ile?Arg?Trp?Asn?Lys

210?????????????????215?????????????????220

Val?Ser?Ala?Met?Leu?Asn?Phe?Ile?Ala?Val?Ala?Pro?Glu?Gly?Thr?Pro

225?????????????????230?????????????????235?????????????????240

Lys?Glu?Gln?Ser?Ile?His?Ser?Arg?Gly?Thr?His?Ile?Leu?Thr?Pro?Glu

245?????????????????250?????????????????255

Thr?Glu?Ala?Ser?Cys?His?Tyr?Phe?Phe?Gly?Ser?Ser?Arg?Asn?Phe?Gly

260?????????????????265?????????????????270

Ile?Asp?Asp?Pro?Glu?Met?Asp?Gly?Val?Leu?Arg?Ser?Trp?Gln?Ala?Gln

275?????????????????280?????????????????285

Ala?Leu?Val?Lys?Glu?Asp?Lys?Val?Val?Val?Glu?Ala?Ile?Glu?Arg?Arg

290?????????????????295?????????????????300

Arg?Ala?Tyr?Val?Glu?Ala?Asn?Gly?Ile?Arg?Pro?Ala?Met?Leu?Ser?Cys

305?????????????????310?????????????????315?????????????????320

Asp?Glu?Ala?Ala?Val?Arg?Val?Ser?Arg?Glu?Ile?Glu?Lys?Leu?Glu?Gln

325?????????????????330?????????????????335

Leu?Glu?Ala?Ala

340

<210>25

<211>1023

<212>DNA

＜213〉artificial sequence

<220>

<400>25

atgctcactt?tcgttagaaa?cgcttggtac?gttgctgcac?ttcctgagga?gttgagcgag????60

aagcctctag?gaagaactat?cctcgatact?ccactagctc?tctatcgtca?acctgacgga????120

gttgtcgctg?ccctgcttga?tatttgtccg?catcgcttcg?ctccgttgag?tgacggtatt????180

ctagtcaacg?gacatctcca?gtgtccatat?cacggtctgg?aatttgacgg?aggtggccag????240

tgtgtccaca?acccgcacgg?caacggagcc?cgccctgctt?ctctgaacgt?gcgatcattc????300

cctgtcgtgg?aaagagacgc?attgatctgg?atctgccctg?gagatccagc?actcgcagat????360

cccggtgcta?tccctgactt?tgggtgtcgt?gttgatccag?cttaccgtac?tgtcggaggt????420

tacggtcacg?tggactgcaa?ctacaagctc?cttgtggata?acctcatgga?tcttggacac????480

gctcagtacg?tgcaccgcgc?taacgcccaa?acagacgcct?tcgatagact?tgagcgtgag????540

gtgatcgttg?gcgacggcga?gatccaggcg?ctcatgaaga?tccctggtgg?cacaccctca????600

gttctcatgg?ctaagttctt?gcgtggtgct?aacacaccag?ttgacgcctg?gaacgacatc????660

cggtggaata?aggtgtcggc?tatgctgaac?ttcatcgcgg?tcgcgccgga?agggacgccg????720

aaggagcagt?caatccactc?ccgaggaacc?catatcctta?ctcctgagac?cgaggcaagc????780

tgccattact?tcttcggtag?ttcccgcaac?ttcggtatag?acgatccaga?gatggacggt????840

gttctcagga?gctggcaagc?tcaagccctg?gtgaaggagg?acaaagtggt?cgttgaagct????900

atcgaaaggc?ggagggctta?cgtcgaagcg?aacgggatca?gacccgccat?gttgtcctgc????960

gacgaggcag?ccgtcagggt?atccagggag?attgagaagc?tcgaacaact?agaagcggcg???1020

tga?????????????????????????????????????????????????????????????????1023

<210>26

<211>340

<212>PRT

＜213〉artificial sequence

<220>

<400>26

Met?Leu?Thr?Phe?Val?Arg?Asn?Ala?Trp?Tyr?Val?Ala?Ala?Leu?Pro?Glu

1???????????????5???????????????????10??????????????????15

Glu?Leu?Ser?Glu?Lys?Pro?Leu?Gly?Arg?Thr?Ile?Leu?Asp?Thr?Pro?Leu

20??????????????????25??????????????????30

Ala?Leu?Tyr?Arg?Gln?Pro?Asp?Gly?Val?Val?Ala?Ala?Leu?Leu?Asp?Ile

35??????????????????40??????????????????45

Cys?Pro?His?Arg?Phe?Ala?Pro?Leu?Ser?Asp?Gly?Ile?Leu?Val?Asn?Gly

50??????????????????55??????????????????60

His?Leu?Gln?Cys?Pro?Tyr?His?Gly?Leu?Glu?Phe?Asp?Gly?Gly?Gly?Gln

65??????????????????70??????????????????75??????????????????80

Cys?Val?His?Asn?Pro?His?Gly?Asn?Gly?Ala?Arg?Pro?Ala?Ser?Leu?Asn

85??????????????????90??????????????????95

Val?Arg?Ser?Phe?Pro?Val?Val?Glu?Arg?Asp?Ala?Leu?Ile?Trp?Ile?Cys

100?????????????????105?????????????????110

Pro?Gly?Asp?Pro?Ala?Leu?Ala?Asp?Pro?Gly?Ala?Ile?Pro?Asp?Phe?Gly

115?????????????????120?????????????????125

Cys?Arg?Val?Asp?Pro?Ala?Tyr?Arg?Thr?Val?Gly?Gly?Tyr?Gly?His?Val

130?????????????????135?????????????????140

Asp?Cys?Asn?Tyr?Lys?Leu?Leu?Val?Asp?Asn?Leu?Met?Asp?Leu?Gly?His

145?????????????????150?????????????????155?????????????????160

Ala?Gln?Tyr?Val?His?Arg?Ala?Asn?Ala?Gln?Thr?Asp?Ala?Phe?Asp?Arg

165?????????????????170?????????????????175

Leu?Glu?Arg?Glu?Val?Ile?Val?Gly?Asp?Gly?Glu?Ile?Gln?Ala?Leu?Met

180?????????????????185?????????????????190

Lys?Ile?Pro?Gly?Gly?Thr?Pro?Ser?Val?Leu?Met?Ala?Lys?Phe?Leu?Arg

195?????????????????200?????????????????205

Gly?Ala?Asn?Thr?Pro?Val?Asp?Ala?Trp?Asn?Asp?Ile?Arg?Trp?Asn?Lys

210?????????????????215?????????????????220

Val?Ser?Ala?Met?Leu?Asn?Phe?Ile?Ala?Val?Ala?Pro?Glu?Gly?Thr?Pro

225?????????????????230?????????????????235?????????????????240

Lys?Glu?Gln?Ser?Ile?His?Ser?Arg?Gly?Thr?His?Ile?Leu?Thr?Pro?Glu

245?????????????????250?????????????????255

Thr?Glu?Ala?Ser?Cys?His?Tyr?Phe?Phe?Gly?Ser?Ser?Arg?Asn?Phe?Gly

260?????????????????265?????????????????270

Ile?Asp?Asp?Pro?Glu?Met?Asp?Gly?Val?Leu?Arg?Ser?Trp?Gln?Ala?Gln

275?????????????????280?????????????????285

Ala?Leu?Val?Lys?Glu?Asp?Lys?Val?Val?Val?Glu?Ala?Ile?Glu?Arg?Arg

290?????????????????295?????????????????300

Arg?Ala?Tyr?Val?Glu?Ala?Asn?Gly?Ile?Arg?Pro?Ala?Met?Leu?Ser?Cys

305?????????????????310?????????????????315?????????????????320

Asp?Glu?Ala?Ala?Val?Arg?Val?Ser?Arg?Glu?Ile?Glu?Lys?Leu?Glu?Gln

325?????????????????330?????????????????335

Leu?Glu?Ala?Ala

340

<210>27

<211>1023

<212>DNA

＜213〉artificial sequence

<220>

<400>27

atgctcactt?tcgttagaaa?cgcttggtac?gttgctgcac?ttcctgagga?gttgagcgag????60

aagcctctag?gaagaactat?cctcgatact?ccactagctc?tctatcgtca?acctgacgga????120

gttgtcgctg?ccctgcttga?tatttgtccg?catcgcttcg?ctccgttgag?tgacggtatt????180

ctagtcaacg?gacatctcca?gtgtccatat?cacggtctgg?aatttgacgg?aggtggccag????240

tgtgtccaca?acccgcacgg?caacggagcc?cgccctgctt?ctctgaacgt?gcgatcattc????300

cctgtcgtgg?aaagagacgc?attgatctgg?atctggcctg?gagatccagc?actcgcagat????360

cccggtgcta?tccctgactt?tgggtgtcgt?gttgatccag?cttaccgtac?tgtcggaggt????420

tacggtcacg?tggactgcaa?ctacaagctc?cttgtggata?acctcatgga?tcttggacac????480

gctcagtacg?tgcaccgcgc?taacgcccaa?acagacgcct?tcgatagact?tgagcgtgag????540

gtgatcgttg?gcgacggcga?gatccaggcg?ctcatgaaga?tccctggtgg?cacaccctca????600

gttctcatgg?ctaagttctt?gcgtggtgct?aacacaccag?ttgacgcctg?gaacgacatc????660

cggtggaata?aggtgtcggc?tatgctgaac?ttcatcgcgg?tcgcgccgga?agggacgccg????720

aaggagcagt?caatccactc?ccgaggaacc?catatcctta?ctcctgagac?cgaggcaagc????780

tgccattact?tcttcggtag?ttcccgcaac?ttcggtatag?acgatccaga?gatggacggt????840

gttctcagga?gctggcaagc?tcaagccctg?gtgaaggagg?acaaagtggt?cgttgaagct????900

atcgaaaggc?ggagggctta?cgtcgaagcg?aacgggatca?gacccgccat?gttgtcctgc????960

gacgaggcag?ccgtcagggt?atccagggag?attgagaagc?tcgaacaact?agaagcggcg????1020

tga??????????????????????????????????????????????????????????????????1023

<210>28

<211>340

<212>PRT

＜213〉artificial sequence

<220>

<400>28

Met?Leu?Thr?Phe?Val?Arg?Asn?Ala?Trp?Tyr?Val?Ala?Ala?Leu?Pro?Glu

1???????????????5???????????????????10??????????????????15

Glu?Leu?Ser?Glu?Lys?Pro?Leu?Gly?Arg?Thr?Ile?Leu?Asp?Thr?Pro?Leu

20??????????????????25??????????????????30

Ala?Leu?Tyr?Arg?Gln?Pro?Asp?Gly?Val?Val?Ala?Ala?Leu?Leu?Asp?Ile

35??????????????????40??????????????????45

Cys?Pro?His?Arg?Phe?Ala?Pro?Leu?Ser?Asp?Gly?Ile?Leu?Val?Asn?Gly

50??????????????????55??????????????????60

His?Leu?Gln?Cys?Pro?Tyr?His?Gly?Leu?Glu?Phe?Asp?Gly?Gly?Gly?Gln

65??????????????????70??????????????????75??????????????????80

Cys?Val?His?Asn?Pro?His?Gly?Asn?Gly?Ala?Arg?Pro?Ala?Ser?Leu?Asn

85??????????????????90??????????????????95

Val?Arg?Ser?Phe?Pro?Val?Val?Glu?Arg?Asp?Ala?Leu?Ile?Trp?Ile?Trp

100?????????????????105?????????????????110

Pro?Gly?Asp?Pro?Ala?Leu?Ala?Asp?Pro?Gly?Ala?Ile?Pro?Asp?Phe?Gly

115?????????????????120?????????????????125

Cys?Arg?Val?Asp?Pro?Ala?Tyr?Arg?Thr?Val?Gly?Gly?Tyr?Gly?His?Val

130?????????????????135?????????????????140

Asp?Cys?Asn?Tyr?Lys?Leu?Leu?Val?Asp?Asn?Leu?Met?Asp?Leu?Gly?His

145?????????????????150?????????????????155?????????????????160

Ala?Gln?Tyr?Val?His?Arg?Ala?Asn?Ala?Gln?Thr?Asp?Ala?Phe?Asp?Arg

165?????????????????170?????????????????175

Leu?Glu?Arg?Glu?Val?Ile?Val?Gly?Asp?Gly?Glu?Ile?Gln?Ala?Leu?Met

180?????????????????185?????????????????190

Lys?Ile?Pro?Gly?Gly?Thr?Pro?Ser?Val?Leu?Met?Ala?Lys?Phe?Leu?Arg

Gly?Ala?Asn?Thr?Pro?Val?Asp?Ala?Trp?Asn?Asp?Ile?Arg?Trp?Asn?Lys

210?????????????????215?????????????????220

Val?Ser?Ala?Met?Leu?Asn?Phe?Ile?Ala?Val?Ala?Pro?Glu?Gly?Thr?Pro

225?????????????????230?????????????????235?????????????????240

Lys?Glu?Gln?Ser?Ile?His?Ser?Arg?Gly?Thr?His?Ile?Leu?Thr?Pro?Glu

245?????????????????250?????????????????255

Thr?Glu?Ala?Ser?Cys?His?Tyr?Phe?Phe?Gly?Ser?Ser?Arg?Asn?Phe?Gly

260?????????????????265?????????????????270

Ile?Asp?Asp?Pro?Glu?Met?Asp?Gly?Val?Leu?Arg?Ser?Trp?Gln?Ala?Gln

275?????????????????280?????????????????285

Ala?Leu?Val?Lys?Glu?Asp?Lys?Val?Val?Val?Glu?Ala?Ile?Glu?Arg?Arg

290?????????????????295?????????????????300

Arg?Ala?Tyr?Val?Glu?Ala?Asn?Gly?Ile?Arg?Pro?Ala?Met?Leu?Ser?Cys

305?????????????????310?????????????????315?????????????????320

Asp?Glu?Ala?Ala?Val?Arg?Val?Ser?Arg?Glu?Ile?Glu?Lys?Leu?Glu?Gln

325?????????????????330?????????????????335

Leu?Glu?Ala?Ala

340

<210>29

<211>1023

<212>DNA

＜213〉artificial sequence

<220>

<400>29

atggccacct?tcgtccgcaa?tgcctggtat?gtggcggcgc?tgcccgagga?actgtccgaa????60

aagccgctcg?gccggacgat?tctcgacaca?ccgctcgcgc?tctaccgcca?gcccgacggt????120

gtggtcgcgg?cgctgctcga?catctgtccg?caccgcttcg?cgccgctgag?cgacggcatc????180

ctcgtcaacg?gccatctcca?atgcccctat?cacgggctgg?aattcgatgg?cggcgggcag????240

tgcgtccata?acccgcacgg?caatggcgcc?cgcccggctt?cgctcaacgt?ccgctccttc????300

ccggtggtgg?agcgcgacgc?gctgatctgg?atctgtcccg?gcgatccggc?gctggccgat????360

cctggggcga?tccccgactt?cggctgccgc?gtcgatcccg?cctatcggac?cgtcggcggc????420

tatgggcatg?tcgactgcaa?ctacaagctg?ctggtcgaca?acctgatgga?cctcggccac????480

gcccaatatg?tccatcgcgc?caacgcccag?accgacgcct?tcgaccggct?ggagcgcgag????540

gtgatcgtcg?gcgacggtga?gatacaggcg?ctgatgaaga?ttcccggcgg?cacgccgagc????600

gtgctgatgg?ccaagttcct?gcgcggcgcc?aatacccccg?tcgacgcttg?gaacgacatc????660

cgctggaaca?aggtgagcgc?gatgctcaac?ttcatcgcgg?tggcgccgga?aggcaccccg????720

aaggagcaga?gcatccactc?gcgcggtacc?catatcctga?cccccgagac?ggaggcgagc????780

tgccattatt?tcttcggctc?ctcgcgcaat?ttcggcatcg?acgatccgga?gatggacggc????840

gtgctgcgca?gctggcaggc?tcaggcgctg?gtcaaggagg?acaaggtcgt?cgtcgaggcg????900

atcgagcgcc?gccgcgccta?tgtcgaggcg?aatggcatcc?gcccggcgat?gctgtcgtgc????960

gacgaagccg?cagtccgtgt?cagccgcgag?atcgagaagc?ttgagcagct?cgaagccgcc????1020

tga??????????????????????????????????????????????????????????????????1023

<210>30

<211>340

<212>PRT

＜213〉artificial sequence

<220>

<400>30

Met?Ala?Thr?Phe?Val?Arg?Asn?Ala?Trp?Tyr?Val?Ala?Ala?Leu?Pro?Glu

1???????????????5???????????????????10??????????????????15

Glu?Leu?Ser?Glu?Lys?Pro?Leu?Gly?Arg?Thr?Ile?Leu?Asp?Thr?Pro?Leu

20??????????????????25??????????????????30

Ala?Leu?Tyr?Arg?Gln?Pro?Asp?Gly?Val?Val?Ala?Ala?Leu?Leu?Asp?Ile

35??????????????????40??????????????????45

Cys?Pro?His?Arg?Phe?Ala?Pro?Leu?Ser?Asp?Gly?Ile?Leu?Val?Asn?Gly

50??????????????????55??????????????????60

His?Leu?Gln?Cys?Pro?Tyr?His?Gly?Leu?Glu?Phe?Asp?Gly?Gly?Gly?Gln

65??????????????????70??????????????????75??????????????????80

Cys?Val?His?Asn?Pro?His?Gly?Asn?Gly?Ala?Arg?Pro?Ala?Ser?Leu?Asn

85??????????????????90??????????????????95

Val?Arg?Ser?Phe?Pro?Val?Val?Glu?Arg?Asp?Ala?Leu?Ile?Trp?Ile?Cys

100?????????????????105?????????????????110

Pro?Gly?Asp?Pro?Ala?Leu?Ala?Asp?Pro?Gly?Ala?Ile?Pro?Asp?Phe?Gly

115?????????????????120?????????????????125

Cys?Arg?Val?Asp?Pro?Ala?Tyr?Arg?Thr?Val?Gly?Gly?Tyr?Gly?His?Val

130?????????????????135?????????????????140

Asp?Cys?Asn?Tyr?Lys?Leu?Leu?Val?Asp?Asn?Leu?Met?Asp?Leu?Gly?His

145?????????????????150?????????????????155?????????????????160

Ala?Gln?Tyr?Val?His?Arg?Ala?Asn?Ala?Gln?Thr?Asp?Ala?Phe?Asp?Arg

165?????????????????170?????????????????175

Leu?Glu?Arg?Glu?Val?Ile?Val?Gly?Asp?Gly?Glu?Ile?Gln?Ala?Leu?Met

180?????????????????185?????????????????190

Lys?Ile?Pro?Gly?Gly?Thr?Pro?Ser?Val?Leu?Met?Ala?Lys?Phe?Leu?Arg

195?????????????????200?????????????????205

Gly?Ala?Asn?Thr?Pro?Val?Asp?Ala?Trp?Asn?Asp?Ile?Arg?Trp?Asn?Lys

210?????????????????215?????????????????220

Val?Ser?Ala?Met?Leu?Asn?Phe?Ile?Ala?Val?Ala?Pro?Glu?Gly?Thr?Pro

225?????????????????230?????????????????235?????????????????240

Lys?Glu?Gln?Ser?Ile?His?Ser?Arg?Gly?Thr?His?Ile?Leu?Thr?Pro?Glu

245?????????????????250?????????????????255

Thr?Glu?Ala?Ser?Cys?His?Tyr?Phe?Phe?Gly?Ser?Ser?Arg?Asn?Phe?Gly

260?????????????????265?????????????????270

Ile?Asp?Asp?Pro?Glu?Met?Asp?Gly?Val?Leu?Arg?Ser?Trp?Gln?Ala?Gln

275?????????????????280?????????????????285

Ala?Leu?Val?Lys?Glu?Asp?Lys?Val?Val?Val?Glu?Ala?Ile?Glu?Arg?Arg

290?????????????????295?????????????????300

Arg?Ala?Tyr?Val?Glu?Ala?Asn?Gly?Ile?Arg?Pro?Ala?Met?Leu?Ser?Cys

305?????????????????310?????????????????315?????????????????320

Asp?Glu?Ala?Ala?Val?Arg?Val?Ser?Arg?Glu?Ile?Glu?Lys?Leu?Glu?Gln

325?????????????????330?????????????????335

Leu?Glu?Ala?Ala

340

<210>31

<211>1023

<212>DNA

＜213〉artificial sequence

<220>

<400>31

atggccactt?tcgttagaaa?cgcttggtac?gttgctgcac?ttcctgagga?gttgagcgag????60

aagcctctag?gaagaactat?cctcgatact?ccactagctc?tctatcgtca?acctgacgga????120

gttgtcgctg?ccctgcttga?tatttgtccg?catcgcttcg?ctccgttgag?tgacggtatt????180

ctagtcaacg?gacatctcca?gtgtccatat?cacggtctgg?aatttgacgg?aggtggccag????240

tgtgtccaca?acccgcacgg?caacggagcc?cgccctgctt?ctctgaacgt?gcgatcattc????300

cctgtcgtgg?aaagagacgc?attgatctgg?atctggcctg?gagatccagc?actcgcagat????360

cccggtgcta?tccctgactt?tgggtgtcgt?gttgatccag?cttaccgtac?tgtcggaggt????420

tacggtcacg?tggactgcaa?ctacaagctc?cttgtggata?acctcatgga?tcttggacac????480

gctcagtacg?tgcaccgcgc?taacgcccaa?acagacgcct?tcgatagact?tgagcgtgag????540

gtgatcgttg?gcgacggcga?gatccaggcg?ctcatgaaga?tccctggtgg?cacaccctca????600

gttctcatgg?ctaagttctt?gcgtggtgct?aacacaccag?ttgacgcctg?gaacgacatc????660

cggtggaata?aggtgtcggc?tatgctgaac?ttcatcgcgg?tcgcgccgga?agggacgccg????720

aaggagcagt?caatccactc?ccgaggaacc?catatcctta?ctcctgagac?cgaggcaagc????780

tgccattact?tcttcggtag?ttcccgcaac?ttcggtatag?acgatccaga?gatggacggt????840

gttctcagga?gctggcaagc?tcaagccctg?gtgaaggagg?acaaagtggt?cgttgaagct????900

atcgaaaggc?ggagggctta?cgtcgaagcg?aacgggatca?gacccgccat?gttgtcctgc????960

gacgaggcag?ccgtcagggt?atccagggag?attgagaagc?tcgaacaact?agaagcggcg????1020

tga??????????????????????????????????????????????????????????????????1023

<210>32

<211>340

<212>PRT

＜213〉artificial sequence

<220>

<400>32

Met?Ala?Thr?Phe?Val?Arg?Asn?Ala?Trp?Tyr?Val?Ala?Ala?Leu?Pro?Glu

1???????????????5???????????????????10??????????????????15

Glu?Leu?Ser?Glu?Lys?Pro?Leu?Gly?Arg?Thr?Ile?Leu?Asp?Thr?Pro?Leu

20??????????????????25??????????????????30

Ala?Leu?Tyr?Arg?Gln?Pro?Asp?Gly?Val?Val?Ala?Ala?Leu?Leu?Asp?Ile

35??????????????????40??????????????????45

Cys?Pro?His?Arg?Phe?Ala?Pro?Leu?Ser?Asp?Gly?Ile?Leu?Val?Asn?Gly

50??????????????????55??????????????????60

His?Leu?Gln?Cys?Pro?Tyr?His?Gly?Leu?Glu?Phe?Asp?Gly?Gly?Gly?Gln

65??????????????????70??????????????????75??????????????????80

Cys?Val?His?Asn?Pro?His?Gly?Asn?Gly?Ala?Arg?Pro?Ala?Ser?Leu?Asn

85??????????????????90??????????????????95

Val?Arg?Ser?Phe?Pro?Val?Val?Glu?Arg?Asp?Ala?Leu?Ile?Trp?Ile?Trp

100?????????????????105?????????????????110

Pro?Gly?Asp?Pro?Ala?Leu?Ala?Asp?Pro?Gly?Ala?Ile?Pro?Asp?Phe?Gly

115?????????????????120?????????????????125

Cys?Arg?Val?Asp?Pro?Ala?Tyr?Arg?Thr?Val?Gly?Gly?Tyr?Gly?His?Val

130?????????????????135?????????????????140

Asp?Cys?Asn?Tyr?Lys?Leu?Leu?Val?Asp?Asn?Leu?Met?Asp?Leu?Gly?His

145?????????????????150?????????????????155?????????????????160

Ala?Gln?Tyr?Val?His?Arg?Ala?Asn?Ala?Gln?Thr?Asp?Ala?Phe?Asp?Arg

165?????????????????170?????????????????175

Leu?Glu?Arg?Glu?Val?Ile?Val?Gly?Asp?Gly?Glu?Ile?Gln?Ala?Leu?Met

180?????????????????185?????????????????190

Lys?Ile?Pro?Gly?Gly?Thr?Pro?Ser?Val?Leu?Met?Ala?Lys?Phe?Leu?Arg

195?????????????????200?????????????????205

Gly?Ala?Asn?Thr?Pro?Val?Asp?Ala?Trp?Asn?Asp?Ile?Arg?Trp?Asn?Lys

210?????????????????215?????????????????220

Val?Ser?Ala?Met?Leu?Asn?Phe?Ile?Ala?Val?Ala?Pro?Glu?Gly?Thr?Pro

225?????????????????230?????????????????235?????????????????240

Lys?Glu?Gln?Ser?Ile?His?Ser?Arg?Gly?Thr?His?Ile?Leu?Thr?Pro?Glu

245?????????????????250?????????????????255

Thr?Glu?Ala?Ser?Cys?His?Tyr?Phe?Phe?Gly?Ser?Ser?Arg?Asn?Phe?Gly

260?????????????????265?????????????????270

Ile?Asp?Asp?Pro?Glu?Met?Asp?Gly?Val?Leu?Arg?Ser?Trp?Gln?Ala?Gln

275?????????????????280?????????????????285

Ala?Leu?Val?Lys?Glu?Asp?Lys?Val?Val?Val?Glu?Ala?Ile?Glu?Arg?Arg

290?????????????????295?????????????????300

Arg?Ala?Tyr?Val?Glu?Ala?Asn?Gly?Ile?Arg?Pro?Ala?Met?Leu?Ser?Cys

305?????????????????310?????????????????315?????????????????320

Asp?Glu?Ala?Ala?Val?Arg?Val?Ser?Arg?Glu?Ile?Glu?Lys?Leu?Glu?Gln

325?????????????????330?????????????????335

Leu?Glu?Ala?Ala

340

<210>33

<211>1020

<212>DNA

＜213〉Pseudomonas Maltophilia

<400>33

atgaccttcg?tccgcaatgc?ctggtatgtg?gcggcgctgc?ccgaggaact?gtccgaaaag????60

ccgctcggcc?ggacgattct?cgacacaccg?ctcgcgctct?accgccagcc?cgacggtgtg????120

gtcgcggcgc?tgctcgacat?ctgtccgcac?cgcttcgcgc?cgctgagcga?cggcatcctc????180

gtcaacggcc?atctccaatg?cccctatcac?gggctggaat?tcgatggcgg?cgggcagtgc????240

gtccataacc?cgcacggcaa?tggcgcccgc?ccggcttcgc?tcaacgtccg?ctccttcccg????300

gtggtggagc?gcgacgcgct?gatctggatc?tggcccggcg?atccggcgct?ggccgatcct????360

ggggcgatcc?ccgacttcgg?ctgccgcgtc?gatcccgcct?atcggaccgt?cggcggctat????420

gggcatgtcg?actgcaacta?caagctgctg?gtcgacaacc?tgatggacct?cggccacgcc????480

caatatgtcc?atcgcgccaa?cgcccagacc?gacgccttcg?accggctgga?gcgcgaggtg????540

atcgtcggcg?acggtgagat?acaggcgctg?atgaagattc?ccggcggcac?gccgagcgtg????600

ctgatggcca?agttcctgcg?cggcgccaat?acccccgtcg?acgcttggaa?cgacatccgc????660

tggaacaagg?tgagcgcgat?gctcaacttc?atcgcggtgg?cgccggaagg?caccccgaag????720

gagcagagca?tccactcgcg?cggtacccat?atcctgaccc?ccgagacgga?ggcgagctgc????780

cattatttct?tcggctcctc?gcgcaatttc?ggcatcgacg?atccggagat?ggacggcgtg????840

ctgcgcagct?ggcaggctca?ggcgctggtc?aaggaggaca?aggtcgtcgt?cgaggcgatc????900

gagcgccgcc?gcgcctatgt?cgaggcgaat?ggcatccgcc?cggcgatgct?gtcgtgcgac????960

gaagccgcag?tccgtgtcag?ccgcgagatc?gagaagcttg?agcagctcga?agccgcctga????1020

<210>34

<211>339

<212>PRT

＜213〉Pseudomonas Maltophilia

<400>34

Met?Thr?Phe?Val?Arg?Asn?Ala?Trp?Tyr?Val?Ala?Ala?Leu?Pro?Glu?Glu

1???????????????5???????????????????10??????????????????15

Leu?Ser?Glu?Lys?Pro?Leu?Gly?Arg?Thr?Ile?Leu?Asp?Thr?Pro?Leu?Ala

20??????????????????25??????????????????30

Leu?Tyr?Arg?Gln?Pro?Asp?Gly?Val?Val?Ala?Ala?Leu?Leu?Asp?Ile?Cys

35??????????????????40??????????????????45

Pro?His?Arg?Phe?Ala?Pro?Leu?Ser?Asp?Gly?Ile?Leu?Val?Asn?Gly?His

50??????????????????55??????????????????60

Leu?Gln?Cys?Pro?Tyr?His?Gly?Leu?Glu?Phe?Asp?Gly?Gly?Gly?Gln?Cys

65??????????????????70??????????????????75??????????????????80

Val?His?Asn?Pro?His?Gly?Asn?Gly?Ala?Arg?Pro?Ala?Ser?Leu?Asn?Val

85??????????????????90??????????????????95

Arg?Ser?Phe?Pro?Val?Val?Glu?Arg?Asp?Ala?Leu?Ile?Trp?Ile?Trp?Pro

100?????????????????105?????????????????110

Gly?Asp?Pro?Ala?Leu?Ala?Asp?Pro?Gly?Ala?Ile?Pro?Asp?Phe?Gly?Cys

115?????????????????120?????????????????125

Arg?Val?Asp?Pro?Ala?Tyr?Arg?Thr?Val?Gly?Gly?Tyr?Gly?His?Val?Asp

130?????????????????135?????????????????140

Cys?Asn?Tyr?Lys?Leu?Leu?Val?Asp?Asn?Leu?Met?Asp?Leu?Gly?His?Ala

145?????????????????150?????????????????155?????????????????160

Gln?Tyr?Val?His?Arg?Ala?Asn?Ala?Gln?Thr?Asp?Ala?Phe?Asp?Arg?Leu

165?????????????????170?????????????????175

Glu?Arg?Glu?Val?Ile?Val?Gly?Asp?Gly?Glu?Ile?Gln?Ala?Leu?Met?Lys

180?????????????????185?????????????????190

Ile?Pro?Gly?Gly?Thr?Pro?Ser?Val?Leu?Met?Ala?Lys?Phe?Leu?Arg?Gly

195?????????????????200?????????????????205

Ala?Asn?Thr?Pro?Val?Asp?Ala?Trp?Asn?Asp?Ile?Arg?Trp?Asn?Lys?Val

210?????????????????215?????????????????220

Ser?Ala?Met?Leu?Asn?Phe?Ile?Ala?Val?Ala?Pro?Glu?Gly?Thr?Pro?Lys

2252?????????????????30?????????????????235?????????????????240

Glu?Gln?Ser?Ile?His?Ser?Arg?Gly?Thr?His?Ile?Leu?Thr?Pro?Glu?Thr

245?????????????????250?????????????????255

Glu?Ala?Ser?Cys?His?Tyr?Phe?Phe?Gly?Ser?Ser?Arg?Asn?Phe?Gly?Ile

260?????????????????265?????????????????270

Asp?Asp?Pro?Glu?Met?Asp?Gly?Val?Leu?Arg?Ser?Trp?Gln?Ala?Gln?Ala

275?????????????????280?????????????????285

Leu?Val?Lys?Glu?Asp?Lys?Val?Val?Val?Glu?Ala?Ile?Glu?Arg?Arg?Arg

290?????????????????295?????????????????300

Ala?Tyr?Val?Glu?Ala?Asn?Gly?Ile?Arg?Pro?Ala?Met?Leu?Ser?Cys?Asp

305?????????????????310?????????????????315?????????????????320

Glu?Ala?Ala?Val?Arg?Val?Ser?Arg?Glu?Ile?Glu?Lys?Leu?Glu?Gln?Leu

325?????????????????330?????????????????335

Glu?Ala?Ala

<210>35

<21l>1023

<212>DNA

＜213〉artificial sequence

<400>35

atgctcacct?tcgtccgcaa?tgcctggtat?gtggcggcgc?tgcccgagga?actgtccgaa????60

aagccgctcg?gccggacgat?tctcgacaca?ccgctcgcgc?tctaccgcca?gcccgacggt????120

gtggtcgcgg?cgctgctcga?catctgtccg?caccgcttcg?cgccgctgag?cgacggcatc????180

ctcgtcaacg?gccatctcca?atgcccctat?cacgggctgg?aattcgatgg?cggcgggcag????240

tgcgtccata?acccgcacgg?caatggcgcc?cgcccggctt?cgctcaacgt?ccgctccttc????300

ccggtggtgg?agcgcgacgc?gctgatctgg?atctgtcccg?gcgatccggc?gctggccgat????360

cctggggcga?tccccgactt?cggctgccgc?gtcgatcccg?cctatcggac?cgtcggcggc????420

tatgggcatg?tcgactgcaa?ctacaagctg?ctggtcgaca?acctgatgga?cctcggccac????480

gcccaatatg?tccatcgcgc?caacgcccag?accgacgcct?tcgaccggct?ggagcgcgag????540

gtgatcgtcg?gcgacggtga?gatacaggcg?ctgatgaaga?ttcccggcgg?cacgccgagc????600

gtgctgatgg?ccaagttcct?gcgcggcgcc?aatacccccg?tcgacgcttg?gaacgacatc????660

cgctggaaca?aggtgagcgc?gatgctcaac?ttcatcgcgg?tggcgccgga?aggcaccccg????720

aaggagcaga?gcatccactc?gcgcggtacc?catatcctga?cccccgagac?ggaggcgagc????780

tgccattatt?tcttcggctc?ctcgcgcaat?ttcggcatcg?acgatccgga?gatggacggc????840

gtgctgcgca?gctggcaggc?tcaggcgctg?gtcaaggagg?acaaggtcgt?cgtcgaggcg????900

atcgagcgcc?gccgcgccta?tgtcgaggcg?aatggcatcc?gcccggcgat?gctgtcgtgc????960

gacgaagccg?cagtccgtgt?cagccgcgag?atcgagaagc?ttgagcagct?cgaagccgcc????1020

tga??????????????????????????????????????????????????????????????????1023

<210>36

<21l>340

<212>PRT

＜213〉artificial sequence

<400>36

Met?Leu?Thr?Phe?Val?Arg?Asn?Ala?Trp?Tyr?Val?Ala?Ala?Leu?Pro?Glu

1???????????????5???????????????????10??????????????????15

Glu?Leu?Ser?Glu?Lys?Pro?Leu?Gly?Arg?Thr?Ile?Leu?Asp?Thr?Pro?Leu

20??????????????????25??????????????????30

Ala?Leu?Tyr?Arg?Gln?Pro?Asp?Gly?Val?Val?Ala?Ala?Leu?Leu?Asp?Ile

35??????????????????40??????????????????45

Cys?Pro?His?Arg?Phe?Ala?Pro?Leu?Ser?Asp?Gly?Ile?Leu?Val?Asn?Gly

50??????????????????55??????????????????60

His?Leu?Gln?Cys?Pro?Tyr?His?Gly?Leu?Glu?Phe?Asp?Gly?Gly?Gly?Gln

65??????????????????70??????????????????75??????????????????80

Cys?Val?His?Asn?Pro?His?Gly?Asn?Gly?Ala?Arg?Pro?Ala?Ser?Leu?Asn

85??????????????????90??????????????????95

Val?Arg?Ser?Phe?Pro?Val?Val?Glu?Arg?Asp?Ala?Leu?Ile?Trp?Ile?Cys

100?????????????????105?????????????????110

Pro?Gly?Asp?Pro?Ala?Leu?Ala?Asp?Pro?Gly?Ala?Ile?Pro?Asp?Phe?Gly

115?????????????????120?????????????????125

Cys?Arg?Val?Asp?Pro?Ala?Tyr?Arg?Thr?Val?Gly?Gly?Tyr?Gly?His?Val

130?????????????????135?????????????????140

Asp?Cys?Asn?Tyr?Lys?Leu?Leu?Val?Asp?Asn?Leu?Met?Asp?Leu?Gly?His

145?????????????????150?????????????????155?????????????????160

Ala?Gln?Tyr?Val?His?Arg?Ala?Asn?Ala?Gln?Thr?Asp?Ala?Phe?Asp?Arg

165?????????????????170?????????????????175

Leu?Glu?Arg?Glu?Val?Ile?Val?Gly?Asp?Gly?Glu?Ile?Gln?Ala?Leu?Met

180?????????????????185?????????????????190

Lys?Ile?Pro?Gly?Gly?Thr?Pro?Ser?Val?Leu?Met?Ala?Lys?Phe?Leu?Arg

195?????????????????200?????????????????205

Gly?Ala?Asn?Thr?Pro?Val?Asp?Ala?Trp?Asn?Asp?Ile?Arg?Trp?Asn?Lys

210?????????????????215?????????????????220

Val?Ser?Ala?Met?Leu?Asn?Phe?Ile?Ala?Val?Ala?Pro?Glu?Gly?Thr?Pro

225?????????????????230?????????????????235?????????????????240

Lys?Glu?Gln?Ser?Ile?His?Ser?Arg?Gly?Thr?His?Ile?Leu?Thr?Pro?Glu

245?????????????????250?????????????????255

Thr?Glu?Ala?Ser?Cys?His?Tyr?Phe?Phe?Gly?Ser?Ser?Arg?Asn?Phe?Gly

260?????????????????265?????????????????270

Ile?Asp?Asp?Pro?Glu?Met?Asp?Gly?Val?Leu?Arg?Ser?Trp?Gln?Ala?Gln

275?????????????????280?????????????????285

Ala?Leu?Val?Lys?Glu?Asp?Lys?Val?Val?Val?Glu?Ala?Ile?Glu?Arg?Arg

290?????????????????295?????????????????300

Arg?Ala?Tyr?Val?Glu?Ala?Asn?Gly?Ile?Arg?Pro?Ala?Met?Leu?Ser?Cys

305?????????????????310?????????????????315?????????????????320

Asp?Glu?Ala?Ala?Val?Arg?Val?Ser?Arg?Glu?Ile?Glu?Lys?Leu?Glu?Gln

325?????????????????330?????????????????335

Leu?Glu?Ala?Ala

340

<210>37

<211>1023

<212>DNA

＜213〉artificial sequence

<400>37

atgctcacct?tcgttaggaa?cgcctggtac?gtcgccgctc?tccctgagga?gctgagcgag????60

aagcccttgg?gtcgcaccat?cctagacact?ccgttagccc?tttaccgcca?gcctgacggc????120

gtagtggcgg?ccctgcttga?catctgcccg?cataggttcg?ctccgctcag?cgacggcatc????180

ctcgtcaacg?ggcatcttca?gtgcccgtac?cacgggctgg?aatttgacgg?cggtgggcag????240

tgtgtccaca?acccgcacgg?caacggcgca?cggccagctt?ccctcaacgt?taggtcgttc????300

cctgttgtcg?agcgcgacgc?actgatctgg?atctggcctg?gcgacccagc?tctggccgat????360

ccaggagcca?ttcccgactt?cggttgccgc?gtggacccag?cctatcggac?ggtcggcggt????420

tacgggcacg?tcgattgtaa?ctataagctc?cttgtggaca?accttatgga?tttgggccac????480

gctcagtacg?tgcaccgggc?taacgctcag?actgacgcct?ttgaccgtct?cgaaagggag????540

gtcatcgtcg?gcgacggaga?gattcaggcg?ctgatgaaga?tccctggagg?cacgccctct????600

gtgctcatgg?cgaagtttct?cagaggcgcg?aacacgcccg?tggacgcctg?gaacgacatc????660

cgctggaata?aggtctccgc?gatgctgaac?ttcatcgccg?ttgcgcccga?gggcacaccc????720

aaagagcagt?caatccacag?cagagggacc?catattctta?caccggaaac?cgaggctagt????780

tgccactact?tcttcggctc?gtcacggaat?ttcgggatag?acgatccgga?gatggacggt????840

gttcttcgat?cttggcaagc?gcaagctctc?gtcaaggaag?ataaggtggt?cgtggaggct????900

atcgagcgta?ggcgcgccta?cgttgaggcg?aacggtatta?ggcccgcgat?gctgtcctgc????960

gacgaggccg?cagttagagt?gtcgcgcgag?atagaaaagc?tggagcagct?agaggccgcc????1020

tga??????????????????????????????????????????????????????????????????1023

<210>38

<211>340

<212>PRT

＜213〉artificial sequence

<400>38

Met?Leu?Thr?Phe?Val?Arg?Asn?Ala?Trp?Tyr?Val?Ala?Ala?Leu?Pro?Glu

1???????????????5???????????????????10??????????????????15

Glu?Leu?Ser?Glu?Lys?Pro?Leu?Gly?Arg?Thr?Ile?Leu?Asp?Thr?Pro?Leu

20??????????????????25??????????????????30

Ala?Leu?Tyr?Arg?Gln?Pro?Asp?Gly?Val?Val?Ala?Ala?Leu?Leu?Asp?Ile

35??????????????????40??????????????????45

Cys?Pro?His?Arg?Phe?Ala?Pro?Leu?Ser?Asp?Gly?Ile?Leu?Val?Asn?Gly

50??????????????????55??????????????????60

His?Leu?Gln?Cys?Pro?Tyr?His?Gly?Leu?Glu?Phe?Asp?Gly?Gly?Gly?Gln

65??????????????????70??????????????????75??????????????????80

Cys?Val?His?Asn?Pro?His?Gly?Asn?Gly?Ala?Arg?Pro?Ala?Ser?Leu?Asn

85??????????????????90??????????????????95

Val?Arg?Ser?Phe?Pro?Val?Val?Glu?Arg?Asp?Ala?Leu?Ile?Trp?Ile?Trp

100?????????????????105?????????????????110

Pro?Gly?Asp?Pro?Ala?Leu?Ala?Asp?Pro?Gly?Ala?Ile?Pro?Asp?Phe?Gly

115?????????????????120?????????????????125

Cys?Arg?Val?Asp?Pro?Ala?Tyr?Arg?Thr?Val?Gly?Gly?Tyr?Gly?His?Val

130?????????????????135?????????????????140

Asp?Cys?Asn?Tyr?Lys?Leu?Leu?Val?Asp?Asn?Leu?Met?Asp?Leu?Gly?His

145?????????????????150?????????????????155?????????????????160

Ala?Gln?Tyr?Val?His?Arg?Ala?Asn?Ala?Gln?Thr?Asp?Ala?Phe?Asp?Arg

165?????????????????170?????????????????175

Leu?Glu?Arg?Glu?Val?Ile?Val?Gly?Asp?Gly?Glu?Ile?Gln?Ala?Leu?Met

180?????????????????185?????????????????190

Lys?Ile?Pro?Gly?Gly?Thr?Pro?Ser?Val?Leu?Met?Ala?Lys?Phe?Leu?Arg

195?????????????????200?????????????????205

Gly?Ala?Asn?Thr?Pro?Val?Asp?Ala?Trp?Asn?Asp?Ile?Arg?Trp?Asn?Lys

210?????????????????215?????????????????220

Val?Ser?Ala?Met?Leu?Asn?Phe?Ile?Ala?Val?Ala?Pro?Glu?Gly?Thr?Pro

225?????????????????230?????????????????235?????????????????240

Lys?Glu?Gln?Ser?Ile?His?Ser?Arg?Gly?Thr?His?Ile?Leu?Thr?Pro?Glu

245?????????????????250?????????????????255

Thr?Glu?Ala?Ser?Cys?His?Tyr?Phe?Phe?Gly?Ser?Ser?Arg?Asn?Phe?Gly

260?????????????????265?????????????????270

Ile?Asp?Asp?Pro?Glu?Met?Asp?Gly?Val?Leu?Arg?Ser?Trp?Gln?Ala?Gln

275?????????????????280?????????????????285

Ala?Leu?Val?Lys?Glu?Asp?Lys?Val?Val?Val?Glu?Ala?Ile?Glu?Arg?Arg

290?????????????????295?????????????????300

Arg?Ala?Tyr?Val?Glu?Ala?Asn?Gly?Ile?Arg?Pro?Ala?Met?Leu?Ser?Cys

305?????????????????310?????????????????315?????????????????320

Asp?Glu?Ala?Ala?Val?Arg?Val?Ser?Arg?Glu?Ile?Glu?Lys?Leu?Glu?Gln

325?????????????????330?????????????????335

Leu?Glu?Ala?Ala

340

<210>39

<211>1023

<212>DNA

＜213〉artificial sequence

<400>39

atggccacct?tcgttaggaa?cgcctggtac?gtcgccgctc?tccctgagga?gctgagcgag????60

aagcccttgg?gtcgcaccat?cctagacact?ccgttagccc?tttaccgcca?gcctgacggc????120

gtagtggcgg?ccctgcttga?catctgcccg?cataggttcg?ctccgctcag?cgacggcatc????180

ctcgtcaacg?ggcatcttca?gtgcccgtac?cacgggctgg?aatttgacgg?cggtgggcag????240

tgtgtccaca?acccgcacgg?caacggcgca?cggccagctt?ccctcaacgt?taggtcgttc????300

cctgttgtcg?agcgcgacgc?actgatctgg?atctggcctg?gcgacccagc?tctggccgat????360

ccaggagcca?ttcccgactt?cggttgccgc?gtggacccag?cctatcggac?ggtcggcggt????420

tacgggcacg?tcgattgtaa?ctataagctc?cttgtggaca?accttatgga?tttgggccac????480

gctcagtacg?tgcaccgggc?taacgctcag?actgacgcct?ttgaccgtct?cgaaagggag????540

gtcatcgtcg?gcgacggaga?gattcaggcg?ctgatgaaga?tccctggagg?cacgccctct????600

gtgctcatgg?cgaagtttct?cagaggcgcg?aacacgcccg?tggacgcctg?gaacgacatc????660

cgctggaata?aggtctccgc?gatgctgaac?ttcatcgccg?ttgcgcccga?gggcacaccc????720

aaagagcagt?caatccacag?cagagggacc?catattctta?caccggaaac?cgaggctagt????780

tgccactact?tcttcggctc?gtcacggaat?ttcgggatag?acgatccgga?gatggacggt????840

gttcttcgat?cttggcaagc?gcaagctctc?gtcaaggaag?ataaggtggt?cgtggaggct????900

atcgagcgta?ggcgcgccta?cgttgaggcg?aacggtatta?ggcccgcgat?gctgtcctgc????960

gacgaggccg?cagttagagt?gtcgcgcgag?atagaaaagc?tggagcagct?agaggccgcc????1020

tga??????????????????????????????????????????????????????????????????1023

<210>40

<211>340

<212>PRT

＜213〉artificial sequence

<400>40

Met?Ala?Thr?Phe?Val?Arg?Asn?Ala?Trp?Tyr?Val?Ala?Ala?Leu?Pro?Glu

1???????????????5???????????????????10??????????????????15

Glu?Leu?Ser?Glu?Lys?Pro?Leu?Gly?Arg?Thr?Ile?Leu?Asp?Thr?Pro?Leu

20??????????????????25??????????????????30

Ala?Leu?Tyr?Arg?Gln?Pro?Asp?Gly?Val?Val?Ala?Ala?Leu?Leu?Asp?Ile

35??????????????????40??????????????????45

Cys?Pro?His?Arg?Phe?Ala?Pro?Leu?Ser?Asp?Gly?Ile?Leu?Val?Asn?Gly

50??????????????????55??????????????????60

His?Leu?Gln?Cys?Pro?Tyr?His?Gly?Leu?Glu?Phe?Asp?Gly?Gly?Gly?Gln

65??????????????????70??????????????????75??????????????????80

Cys?Val?His?Asn?Pro?His?Gly?Asn?Gly?Ala?Arg?Pro?Ala?Ser?Leu?Asn

85??????????????????90??????????????????95

Val?Arg?Ser?Phe?Pro?Val?Val?Glu?Arg?Asp?Ala?Leu?Ile?Trp?Ile?Trp

100?????????????????105?????????????????110

Pro?Gly?Asp?Pro?Ala?Leu?Ala?Asp?Pro?Gly?Ala?Ile?Pro?Asp?Phe?Gly

115?????????????????120?????????????????125

Cys?Arg?Val?Asp?Pro?Ala?Tyr?Arg?Thr?Val?Gly?Gly?Tyr?Gly?His?Val

130?????????????????135?????????????????140

Asp?Cys?Asn?Tyr?Lys?Leu?Leu?Val?Asp?Asn?Leu?Met?Asp?Leu?Gly?His

145?????????????????150?????????????????155?????????????????160

Ala?Gln?Tyr?Val?His?Arg?Ala?Asn?Ala?Gln?Thr?Asp?Ala?Phe?Asp?Arg

165?????????????????170?????????????????175

Leu?Glu?Arg?Glu?Val?Ile?Val?Gly?Asp?Gly?Glu?Ile?Gln?Ala?Leu?Met

180?????????????????185?????????????????190

Lys?Ile?Pro?Gly?Gly?Thr?Pro?Ser?Val?Leu?Met?Ala?Lys?Phe?Leu?Arg

195?????????????????200?????????????????205

Gly?Ala?Asn?Thr?Pro?Val?Asp?Ala?Trp?Asn?Asp?Ile?Arg?Trp?Asn?Lys

210?????????????????215?????????????????220

Val?Ser?Ala?Met?Leu?Asn?Phe?Ile?Ala?Val?Ala?Pro?Glu?Gly?Thr?Pro

225?????????????????230?????????????????235?????????????????240

Lys?Glu?Gln?Ser?Ile?His?Ser?Arg?Gly?Thr?His?Ile?Leu?Thr?Pro?Glu

245?????????????????250?????????????????255

Thr?Glu?Ala?Ser?Cys?His?Tyr?Phe?Phe?Gly?Ser?Ser?Arg?Asn?Phe?Gly

260?????????????????265?????????????????270

Ile?Asp?Asp?Pro?Glu?Met?Asp?Gly?Val?Leu?Arg?Ser?Trp?Gln?Ala?Gln

275?????????????????280?????????????????285

Ala?Leu?Val?Lys?Glu?Asp?Lys?Val?Val?Val?Glu?Ala?Ile?Glu?Arg?Arg

290?????????????????295?????????????????300

Arg?Ala?Tyr?Val?Glu?Ala?Asn?Gly?Ile?Arg?Pro?Ala?Met?Leu?Ser?Cys

305?????????????????310?????????????????315?????????????????320

Asp?Glu?Ala?Ala?Val?Arg?Val?Ser?Arg?Glu?Ile?Glu?Lys?Leu?Glu?Gln

325?????????????????330?????????????????335

Leu?Glu?Ala?Ala

340

<210>41

<211>433

<212>DNA

＜213〉artificial sequence

<220>

<400>41

agatcttgag?ccaatcaaag?aggagtgatg?tagacctaaa?gcaataatgg?agccatgacg????60

taagggctta?cgcccatacg?aaataattaa?aggctgatgt?gacctgtcgg?tctctcagaa????120

cctttacttt?ttatgtttgg?cgtgtatttt?taaatttcca?cggcaatgac?gatgtgaccc????180

aacgagatct?tgagccaatc?aaagaggagt?gatgtagacc?taaagcaata?atggagccat????240

gacgtaaggg?cttacgccca?tacgaaataa?ttaaaggctg?atgtgacctg?tcggtctctc????300

agaaccttta?ctttttatat?ttggcgtgta?tttttaaatt?tccacggcaa?tgacgatgtg????360

acctgtgcat?ccgctttgcc?tataaataag?ttttagtttg?tattgatcga?cacggtcgag????420

aagacacggc?cat???????????????????????????????????????????????????????433

Claims (39)

1. recombinant DNA molecules, it comprises the dna sequence dna of the coding chloroplast transit peptides on the dna sequence dna that is operably connected to the coding dicamba monooxygenase enzyme, wherein, the dna sequence encoding of coding chloroplast transit peptides is selected from the sequence of SEQ ID NOs:1-11.

2. recombinant DNA molecules as claimed in claim 1, wherein, the dna sequence dna of described coding chloroplast transit peptides comprises the sequence that is selected from SEQ ID NOs:12-22.

3. recombinant DNA molecules, it comprises the dna sequence dna of the coding chloroplast transit peptides on the dna sequence dna that is operably connected to the coding dicamba monooxygenase enzyme, wherein, the dna sequence encoding of coding chloroplast transit peptides is selected from the sequence of Arabidopis thaliana 5-enol pyruvoyl shikimic acid-3-phosphate synthase CTP2 chloroplast transit peptide sequence and pea rubisco small subunit chloroplast transit peptide sequence.

4. recombinant DNA molecules as claimed in claim 1, it comprises the dna sequence dna of the coding chloroplast transit peptides on the dna sequence dna that is operably connected to the coding dicamba monooxygenase enzyme, wherein, the dna sequence encoding of coding chloroplast transit peptides is selected from the sequence of SEQ ID NOs:2-7.

5. recombinant DNA molecules as claimed in claim 1, it comprises the dna sequence dna of the coding chloroplast transit peptides on the dna sequence dna that is operably connected to the coding dicamba monooxygenase enzyme, wherein, the dna sequence encoding of coding chloroplast transit peptides is selected from the sequence of SEQ ID NO:2, SEQ IDNO:4 and SEQ ID NO:5.

6. recombinant DNA molecules as claimed in claim 1, it comprises the dna sequence dna of the coding chloroplast transit peptides on the dna sequence dna that is operably connected to the coding dicamba monooxygenase enzyme, wherein, the dna sequence dna of coding chloroplast transit peptides comprises the sequence that is selected from SEQ ID NOs:13-18.

7. recombinant DNA molecules as claimed in claim 1, it comprises the dna sequence dna of the coding chloroplast transit peptides on the dna sequence dna that is operably connected to the coding dicamba monooxygenase enzyme, wherein, the dna sequence dna of coding chloroplast transit peptides comprises and is selected from SEQ ID NOs:13,15 and 16 sequence.

8. recombinant DNA molecules as claimed in claim 1, wherein, the dna sequence encoding of described coding dicamba monooxygenase enzyme is selected from SEQ ID NOs:24,26,28,30,32,34,36,38 and 40 polypeptide.

9. recombinant DNA molecules as claimed in claim 8, wherein, described dna sequence dna is selected from SEQ ID NOs:23,25,27,29,31,33,35,37 and 39.

10. DNA construct, it comprises the dna molecular as claimed in claim 1 that is operably connected with promotor.

11. DNA construct as claimed in claim 5, wherein, described promotor is selected from FMV35S promotor, At.ANT1 promotor, FMV.35S-EF1a promotor, eIF4A10 promotor, AGRtu.nos promotor, paddy rice kytoplasm triosephosphate isomerase (OsTPI) promotor, rice actin 15 genes (OsAct15) promotor and the molten protein promoter of γ-coixol.

12. construct as claimed in claim 10, wherein, described promotor has function in vegetable cell.

13. with the described DNA construct plant transformed of claim 10 cell.

14. cell as claimed in claim 13, wherein, described vegetable cell is the dicotyledons cell.

15. cell as claimed in claim 13, wherein, described vegetable cell is a monocot plant cell.

16. cell as claimed in claim 13, wherein, described vegetable cell is soybean, cotton, corn or Semen Brassicae campestris vegetable cell.

17. a plant tissue cultures, it comprises the described cell of claim 13.

18. plant tissue cultures as claimed in claim 17, it comprises the dicotyledons cell.

19. plant tissue cultures as claimed in claim 17, it comprises monocot plant cell.

20. plant tissue cultures as claimed in claim 17, it comprises soybean, cotton, corn or Semen Brassicae campestris vegetable cell.

21. with the described DNA construct transgenic plant transformed of claim 10.

22. transgenic plant as claimed in claim 21, wherein, described plant is a dicotyledons.

23. transgenic plant as claimed in claim 21, wherein, described plant is a monocotyledons.

24. transgenic plant as claimed in claim 21, wherein, described plant is soybean, cotton, corn or Semen Brassicae campestris plant.

25. a method that produces the plant of tolerance dicamba 98 comprises in the described construct introduced plant of claim 10 cell, and is comprised the plant of the described construct of claim 10 by its regeneration.

26. method as claimed in claim 25, further comprise by stock plant and it self or the hybridization of second plant being produced the plant of tolerance dicamba 98, wherein, the described stock plant and/or second plant comprise described DNA construct, and the tolerance dicamba 98 plant from the described stock plant and/or the second plant heredity described DNA construct.

27. a method of expressing dicamba monooxygenase enzyme in vegetable cell comprises that the CTP that will select is operably connected on the sequence of coding dicamba monooxygenase enzyme.

28. the method for a control weed growth in crop growth environment comprises the described plant of claim 20 or its seed, and uses the Banvel D of the amount of effective control weed growth to crop growth environment.

29. method as claimed in claim 28, wherein, described Banvel D is used from last direction crop growth environment.

30. method as claimed in claim 28, wherein, the amount of described Banvel D is not damaged the described plant of claim 21 or its seed, but the but identical plant that lacks the described construct of claim 10 of damage with the genotype of the described plant of claim 20.

31. a method of producing food, feed or Industrial products comprises:

A) obtain the described plant of claim 21 or its part; With

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

32. method as claimed in claim 31, wherein, described food or feed are cereal, meal, oil, starch, flour or protein.

33. method as claimed in claim 31, wherein, described Industrial products are biofuel, fiber, industrial chemical, medicine or nutritious prod.

34. one kind provides the seed of the tolerance dicamba 98 of protection at the pre-emergence application dicamba 98, it comprises the DNA of the coding chloroplast transit peptides on the DNA that is operably connected to the coding dicamba monooxygenase enzyme.

35. the seed of tolerance dicamba 98 as claimed in claim 34, wherein, described dna encoding is selected from the chloroplast transit peptides of SEQ ID NOs:1-11.

36. the seed of tolerance dicamba 98 as claimed in claim 35, wherein, the DNA of described coding chloroplast transit peptides comprises the sequence that is selected from SEQ ID NOs:12-22.

37. the seed of tolerance dicamba 98 as claimed in claim 34, wherein, described dna encoding comprises the dicamba monooxygenase enzyme that is selected from SEQ ID NOs:24,26,28,30,32,34,36,38 and 40 sequence.

38. a method that improves monocotyledonous upright ability comprises: a) cultivate the plant or the seed that produce by the described method of claim 26; And b) handles this plant or seed with dicamba 98.

39. method as claimed in claim 38 further comprises: c) measurement is selected from the parameter relevant with upright ability of quantity, shape, length or structure, lodging per-cent and the output of stilit root.

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