CN115491384A - Vector for expressing plant source herbicide-resistant mutant gene and application thereof - Google Patents

Vector for expressing plant source herbicide-resistant mutant gene and application thereof Download PDF

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CN115491384A
CN115491384A CN202110669517.XA CN202110669517A CN115491384A CN 115491384 A CN115491384 A CN 115491384A CN 202110669517 A CN202110669517 A CN 202110669517A CN 115491384 A CN115491384 A CN 115491384A
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王东芳
张意红
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Hangzhou Fangyun Biotechnology Co ltd
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Abstract

The invention discloses a vector for expressing a plant source herbicide-resistant mutant gene and application thereof, wherein the vector comprises a 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) mutant gene or an acetolactate synthase (ALS) mutant gene; the EPSPS mutant gene has a nucleotide sequence shown as SEQ ID No:1 or SEQ ID No:3 is shown in the specification; the ALS mutant gene has a nucleotide sequence shown as SEQ ID No:5 or SEQ ID No: shown at 7. The invention ensures the high-efficiency and stable expression of the target gene by utilizing the mode that the optimized regulating elements such as a promoter, a terminator, mar and the like are connected, so that the obtained transgenic plant can resist more than four times of the target herbicide with medium dosage, and the transgenic plant can meet the industrialized planting requirement and standard.

Description

Vector for expressing plant source herbicide-resistant mutant gene and application thereof
(I) technical field
The invention relates to a vector for stably and efficiently expressing a plant source herbicide-resistant mutant gene and application thereof.
(II) background of the invention
Due to rapid depletion of water resources and shortage of labor, many farmers in asian regions are shifting from transplanting rice in paddy fields to dry farming of rice. However, weed damage is a serious problem in dry farming rice systems because dry farming and aerobic soil conditions favor weed germination and growth. Weeds can cause yield losses of 39-41% (Chauhan B S et al. Field Crops Research,2013,141 (1): 9-15.). And dry crops such as corn, soybean and cotton are always infected by weeds.
The method for preventing and controlling weeds mainly comprises artificial weeding, mechanical weeding, physical weeding, chemical weeding, biological weeding and ecological weeding. Chemical weeding is the most important weeding method. The herbicide is used for preventing and controlling weeds and plays a great role in modern agricultural production. However, most of the currently used herbicides are selective herbicides for preventing damage to crops, and the herbicides generally have the problems of narrow herbicidal spectrum, high herbicidal cost and the like.
Since 1996, crops such as transgenic soybean and corn have been grown commercially in the united states and brazil, and plants can be endowed with a herbicide-tolerant trait by the transformation of herbicide-resistant genes into plants. The herbicide-resistant genes and the trait conditions thereof used in the transgenic plants popularized and applied at present are shown in table 1.
Table 1: use of herbicide tolerant genes and trait profiles in transgenic plants
Figure BDA0003118502410000011
Most of the herbicide tolerance genes in the transgenic plant materials used for commercial planting are derived from microorganisms such as bacteria. Many genes with herbicide tolerance are now found in plants, either naturally occurring or mutated. These include the mutant EPSPS (5-enolpyruvylshikimate 3-phosphate Synthase) gene and the mutant ALS (Acetolactate Synthase) gene. EPSPS is a key enzyme in the shikimate pathway for plant synthesis of chorismate, whereas chorismase is essential for plant synthesis of aromatic amino acids (L-tryptophan, L-tyrosine and L-phenylalanine) (Funke t.et al.2006, proc.natl.acad.sci.usa 103, 13010-13015.). EPSPS enzymes can be divided into two broad classes based on their inherent glyphosate sensitivity and catalytic efficiency. The first type of EPSPS (glyphosate sensitive) is present in all plants and some bacteria; the second class of EPSPS is currently found only in some bacteria, for example staphylococcus aureus, agrobacterium CP4 strain and pseudomonas PG2982 strain, which have both an intrinsic tolerance to glyphosate and a higher affinity for PEP (phosphoenolpyruvate) (Achary Vmm et al plant Biotechnology journal.2020, 18-2504-2519. A first class of EPEPS mutant genes can be mutated to confer glyphosate resistance, such as the mutated Eleusine indica EPSPS gene (Yu Q et al 2015.Plant physiol.167, 1440-1447.), the mutated maize (Zeamays) EPSPS gene (Feng P C et al 2010, pp.45-66.Hoboken, NJ. ALS is the most common enzyme in the branched-chain amino acid, leucine, isoleucine and valine biosynthetic pathways. It is known that, following mutation of a particular amino acid in ALS, the mutated gene may develop resistance to a particular herbicide or herbicides, including Sulfonylureas (SUs) including CS and BM, imidazolinones (IMs) including IQ and IP, pyrimidylcarboxyl herbicides (PCs) including BS, PS and PM (Okuzaki a et al plant Mol biol.2007, 64-219-224.
The gene function is determined by its own characteristics and is also largely influenced by the expression level. For example, the maize transgenic mutant EPSPS gene transformant GA21, which is currently the only transformant used in commercial plantations, contains 3 complete expression cassettes in its exogenous T-DNA (Feng P C et al 2010, pp.45-66.Hoboken, nj. Therefore, the expression vector for stably and efficiently expressing the plant source herbicide-resistant mutant gene is obtained through improvement and optimization, and the method is very urgent for obtaining herbicide-resistant crops by utilizing the plant source herbicide-resistant gene.
Disclosure of the invention
The invention aims to provide a vector for stably and efficiently expressing a plant source herbicide-resistant mutant gene and application thereof in preparing herbicide-resistant plant cells and herbicide-resistant plants.
The technical scheme adopted by the invention is as follows:
the invention provides a vector for stably and efficiently expressing a plant-derived herbicide-resistant mutant gene, which comprises a 5-enolpyruvylshikimate 3-phosphate Synthase (EPSPS) mutant gene or an Acetolactate Synthase (ALS) mutant gene.
Furthermore, the EPSPS mutant gene is rice EPSPS mutant gene OsEPSPS-T, and the nucleotide sequence is shown as SEQ ID No:1, and the amino acid sequence is shown as SEQ ID No:2 is shown in the specification; the EPSPS mutant gene is a maize EPSPS mutant gene ZmEPSPS-T, and the nucleotide sequence is shown as SEQ ID No:3, and the amino acid sequence is shown as SEQ ID No:4, respectively. The ALS mutant gene is a mutant gene OsALS-T1, and the nucleotide sequence is shown as SEQ ID No:5, the amino acid sequence is shown as SEQ ID No:6 is shown in the specification; the ALS mutant gene is a mutant gene OsALS-T2, and the nucleotide sequence is shown as SEQ ID No:7, and the amino acid sequence is shown as SEQ ID No: shown in fig. 8.
The plant source herbicide-resistant mutant gene is a gene derived from a plant, is relatively sensitive to herbicides such as glyphosate, sulfonylureas, imidazolinones and pyrimidylcarboxyls before mutation, but leads one or more amino acid residues to be replaced, deleted or inserted through gene mutation, thereby leading the tolerance of the mutant gene to the herbicide or the herbicides to be enhanced, and the mutated gene is the plant source herbicide-resistant mutant gene.
The plant-derived EPSPS mutant gene provided by the invention can also be a gene which is obtained by mutating other plant-derived EPSPS genes and has resistance to glyphosate, the amino acid sequence of the protein coded by the plant-derived EPSPS genes is shown in Table 2, and the plant-derived EPSPS gene provided by the invention comprises but is not limited to the gene and homologous genes thereof.
Table 2: plant source EPSPS gene coding protein
Encoding Source plant Latin name NCBI Serial number ID
1 Rice (Oryza sativa L.) with improved resistance to stress Oryza sativa XP_015643046.1
2 All-grass of Bisui Brevipedunculata Brachypodium distachyon XP_003557242.1
3 Corn (corn) Zea mays XP_008659331.2
4 (sorghum) Sorghum bicolor XP_002436424.1
5 All-grass of Bull's tendon Eleusine indica AAN63155.1
6 Millet Setaria italica XP_004964446.1
7 Herb of common Setaria Setaria viridis XP_034590364.1
8 Millet Panicum hallii XP_003557242.1
The plant-derived ALS mutant gene provided by the present invention may also be a gene having resistance to one or more herbicides such as sulfonylureas, imidazolinones, pyrimidylcarboxyls, etc. obtained by mutating other plant-derived ALS genes, and the amino acid sequence of the protein encoded by these plant-derived ALS genes is shown in table 3, and the plant-derived ALS gene provided by the present invention includes, but is not limited to, the above-mentioned genes and their homologous genes.
Table 3: plant source ALS gene coding protein
Encoding Source plant Short for sequence NCBI sequence number
1 Rice (Oryza sativa L.) with improved resistance to stress Oryza sativa XP_015626459
2 All-grass of Bisui Brevipedunculata Brachypodium distachyon XP_003575020.1
3 Corn (corn) Zea mays NP_001151761.2
4 (sorghum) Sorghum bicolor XP_021315526.1
5 All-grass of Bull's tendon Eleusine indica AOF40433.1
6 Wild two-grain wheat Triticum dicoccoides XP_037452582.1
7 Switchgrass Panicum virgatum XP_039789731.1
8 Millet Panicum hallii XP_025801953.1
9 Millet Setaria italica XP_004952560.1
10 Arabidopsis thaliana Arabidopsis thaliana NP_190425.1
11 Amaranthus hypochondriacus (lour.) Merr Amaranthus hybridus AXN57277.1
Furthermore, the vector also comprises a promoter for mediating the expression of the plant source herbicide-resistant mutant gene, wherein the promoter is a nucleotide sequence capable of mediating the transcription of the gene; the promoter includes plant ubiquitin promoter (rice UBI-2 promoter pOsUbi or maize UBI-1 promoter pZmUbi) or constitutive promoter, wherein the constitutive promoter includes actin promoter (McElroy D et al plant cell.1990,2 (2): 163-71), cassava veinmosacivirus (CsVMV) promoter, australianbanNAnaramekvrus (BSV) promoter, mirabilismosis virus (MMV) promoter, etc. Preferably rice UBI-2 promoter pOsUbi (nucleotide sequence is shown as SEQ ID No: 9) or corn UBI-1 promoter pZmUbi (nucleotide sequence is shown as SEQ ID No: 10).
Furthermore, the vector also comprises an HSP gene terminator for terminating the expression of the plant source herbicide-resistant mutant gene, wherein the terminator is a nucleotide sequence capable of terminating a gene patent. The terminator may be a terminator derived from a plant itself, or may be a terminator derived from a virus or other organism, or may be artificially synthesized. Preferably, the terminator Ter1 (nucleotide sequence is shown in SEQ ID No: 11) of the rice HSP17.9 gene and the terminator Ter2 (nucleotide sequence is shown in SEQ ID No: 12) of the Arabidopsis HSP18.2 gene are selected.
Furthermore, the vector of the invention also comprises a regulatory element for enhancing the expression quantity or stability of the plant-derived herbicide-resistant mutant gene, wherein the regulatory element refers to a nucleotide sequence which can regulate and control the gene expression, and comprises an enhancer, a matrix attachment region sequence and the like, preferably a matrix attachment region sequence (MAR), and the nucleotide sequence is SEQ ID No:13, respectively. The regulatory element is arranged at the 5 'end of the plant source herbicide-resistant mutant gene expression frame or the 3' end of the terminator.
The basic vector used to provide the vector backbone in the present invention may be a pCambia series of vectors (CAMBIA, canberra, australi a) or other vectors, preferably pCambia1300 vector. More preferably, the CaMV35S terminator in the pCambia1300 vector is replaced by the terminator Ter1 of the rice HSP17.9 gene or the terminator Ter2 of the Arabidopsis HSP18.2 gene.
The vector T-DNA comprises one of the following components: (1) SEQ ID No:1, rice EPSPS mutant gene OsEPSPS-T shown in the specification, SEQ ID No:9, and the promoter pOsUbi shown in SEQ ID No: a terminator Ter1 shown in 11; (2) SEQ ID No:1, rice EPSPS mutant gene OsEPSPS-T shown in the specification, SEQ ID No:9, and a promoter pOsUbi shown in SEQ ID No:13, MAR, SEQ ID No: a terminator Ter1 shown in 11; (3) SEQ ID No:3, maize EPSPS mutant gene ZmEPSPS-T shown in SEQ ID No:10, and the promoter pZmUbi shown in SEQ ID No: a terminator Ter2 shown as 12; (4) SEQ ID No:5, the rice ALS mutant gene OsALS-T1 shown in SEQ ID No:9, and the promoter pOsUbi shown in SEQ ID No: a terminator Ter1 shown in 11; (5) SEQ ID No:7, the rice ALS mutant gene OsALS-T2 shown in SEQ ID No:10, and the promoter pZmUbi shown in SEQ ID No: and a terminator Ter2 shown at 12.
The invention also provides an application of the vector in preparation of herbicide-tolerant plant cells, wherein the application is to use the herbicide-tolerant gene plant source EPSPS mutant gene or plant ASL mutant gene as a screening marker in plant transgenic cell culture.
The invention also provides a plant cell containing the vector T-DNA for efficiently expressing the plant source herbicide-resistant mutant gene.
The invention also provides a plant containing the vector T-DNA of the high-efficiency expression plant source herbicide-tolerant mutant gene.
The invention further provides a method for obtaining plant cells or plants stably and efficiently expressing the plant source herbicide-tolerant mutant genes by using the vector, wherein the method can be an agrobacterium-mediated transformation method, a gene gun method, a protoplast infection method or other plant genetic transformation methods, and preferably the agrobacterium-mediated transformation method.
The expression vector constructed by the invention is suitable for expression of monocotyledons or dicotyledons, wherein the monocotyledons comprise: corn, rice, etc.; dicotyledonous plants include: soybean, rape, cotton, etc.
Compared with the prior art, the invention has the following beneficial effects: the high-efficiency expression plant source herbicide-tolerant gene vector has two major breakthroughs and improvements. Firstly, the high-efficiency and stable expression of a target gene is ensured by utilizing the connection mode of optimized regulatory elements such as a promoter, a terminator, mar and the like, so that the obtained transgenic plant can resist more than four times of medium-dose target herbicide, and the transgenic plant can meet the industrial planting requirement and standard. Secondly, the genes, promoters, terminators and regulatory elements used by the vector are all derived from conventional crops, and compared with genes derived from other sources such as bacteria and animals, the safety is easier to be understood and accepted by the public, and the later safety evaluation and industrialization are facilitated.
Description of the drawings
FIG. 1: a schematic structural diagram of a T-DNA (deoxyribonucleic acid) of a plant source herbicide-tolerant mutant gene high-efficiency expression vector. pUbi represents the plant ubiquitin promoter; the plant source herbicide-resistant mutant gene represents a plant source EPSPS mutant gene or ALS mutant gene; the HSP terminator denotes a plant HSP gene terminator.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
it is within the scope of the present invention to modify or replace the steps, methods or conditions of the present invention without departing from the spirit of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 selection of terminator in expression vector of OsEPSPS-T Gene of Rice
1. Vector containing Ter1 terminator of rice HSP17.9 gene
To optimize the terminator, we compared the commonly used nos terminator (SEQ ID NO: 14) with the non-coding region of the rice heat shock protein OsHSP17.9 at the 3' end.
(1) Artificially synthesizing rice EPSPS mutant gene OsEPSPS-T, wherein the nucleotide sequence of the rice EPSPS mutant gene OsEPSPS-T is shown as SEQ ID No:1, and the amino acid sequence is shown as SEQ ID No:2, respectively.
(2) Transitional vector pCambia1300-Ter1
The CaMV35S terminator in the pCambia1300 vector is replaced by a Ter1 terminator of a rice HSP17.9 gene: a rice (Xiushui 134) genome is taken as a template, a terminator sequence Ter1 (a primer is Ter1-F:5' -TAACTCGAGGCATCGCCGGCGTGCCGCGT; ter1-R:5' -GAATTAACGCCGAATCGGCTAGAGGGGTTAAATCC) of a rice HSP17.9 gene is cloned by PCR, and an XhoI enzyme cutting site is arranged at the 5' end. Using pCambia1300 vector as template, cloning a vector skeleton sequence (primer is LBcl1-F:5'-AACCCCTCTAGCCGATTCGGCGTTAATTCAGTAC; LBcl1-R:5' -GCGGTGATCACAGGCAGCAACGCTCTGTCATC) by PCR, and naming as LBcl1, the nucleotide sequence is shown as 6319bp-6648bp in pCambia1300 vector (NCBI Accession Number: AF 234296). The terminator sequence Ter1 and the vector framework sequence LBcl1 of the rice HSP17.9 gene obtained by PCR cloning are used as templates, DNA fragments which are respectively the terminator of the rice HSP17.9 gene and the vector framework sequence LBcl1 from 5 'to 3' are obtained by PCR cloning (primers are Ter1-F:5'-TAACTCGAGGCATCGCCGGCGTGCCGCGT; LBcl1-R:5' -GCGGTGATCACAGGCAGCAACGCTCTGTCATC), and are named as fragments Ter1-LBcl1, and the 5 'end and the 3' end of each fragment are respectively provided with XhoI enzyme cutting sites and BclI enzyme cutting sites. And (3) carrying out double enzyme digestion on the pCambia1300 vector and the PCR cloning fragment Ter1-LBcl1 by using XhoI and BclI respectively, recovering the pCambia1300 vector and the Ter1-LBcl1 fragment after enzyme digestion respectively, and then cloning after connecting the fragments to obtain the transition vector pCambia1300-Ter1.
(3) T-DNA vector pCambia1300-pOsUbi-OsEPSPS-T-Ter1
The OsEPSPS-T gene (primer OE-F:5'-ATCGGATCCACCATGGCGGCGACCATGGCGTCC; OE-R:5' -GGGTCTCGAGTTATCAGTTCCTGACGAAAGTGCTTAGAAC) is cloned by PCR with artificially synthesized OsEPSPS-T as a template, a BamHI site is arranged at the 5 'end of the sequence, and an XhoI enzyme cutting site is arranged at the 3' end.
A rice (Xiushui 134) genome is used as a template, a rice UBI-2 promoter pOsUbi (a primer is Osubi-F1:5'-CCGGGTACCCTGCAGAAATGCAAATTTCATAAAACAAAC; osubi-R1:5' -CATGGTGGATCCGATCTGCAAGAAATAATCACCAAAC) is cloned by PCR, and the nucleotide sequence is shown as SEQ ID No:9, a KpnI site is arranged at the 5 'end of the sequence, and a BamHI site is arranged at the 3' end of the sequence.
Then, the transition vector pCambia1300-Ter1 is digested by KpnI and XhoI, and the digested vector is recovered; carrying out double enzyme digestion on the PCR product rice UBI-2 promoter pOsUbi by KpnI and BamHI, recovering the enzyme digested pOsUbi, carrying out double enzyme digestion on the PCR product OsEPSPS-T gene by BamHI and XhoI, and recovering the enzyme digested OsEPSPS-T gene. Then, the recovered vector and the two fragments are connected and cloned to obtain a final vector pCambia1300-pOsUbi-OsEPSPS-T-Ter1.
2. Vectors containing nos terminator
(1) Artificially synthesizing a nos terminator, wherein the nucleotide sequence is shown as SEQ ID No: as shown at 14.
The CaMV35S terminator in the pCambia1300 vector was replaced with a nos terminator: an artificially synthesized nos terminator is taken as a template, a terminator sequence nos T (a primer is nos T-F:5' -GATAACTCGAGTTCAAACATTTGGCAATAAAGTTTC; nos T-R:5' -TGAATTAACGCCGAATGATCTAGTAACATAGATGAC) is amplified through PCR, and an XhoI enzyme digestion site is arranged at the 5' end.
(2) The transition vector pCambia1300-nosT: using pCambia1300 vector as a template, cloning a vector framework sequence (primers are LBcl1-F:5'-CTATGTTACTAGATCATTCGGCGTTAATTCAGTACATT; LBcl1-R:5' -GCGGTGATCACAGGCAGCAACGCTCTGTCATC) by PCR, and naming the vector framework sequence as LBcl1, wherein the nucleotide sequence is shown in 6319bp-6648bp in pCambia1300 vector (NCBI Access Number: AF 234296). The nos terminator sequence nosT and the vector framework sequence LBcl1 obtained by PCR cloning are used as templates, DNA fragments of which the 5 'end to the 3' end are respectively the terminator sequence nosT and the vector framework sequence LBcl1 are obtained by PCR cloning (primers are nosT-F:5'-GATAACTCGAGTTCAAACATTTGGCAATAAAGTTTC; LBcl1-R:5' -GCGGTGATCACAGGCAGCAACGCTCTGTCATC), and are marked as fragment nosT-LBcl1, and the 5 'end and the 3' end of the fragment are respectively provided with XhoI and BclI enzyme cutting sites. XhoI and BclI are used for carrying out double enzyme digestion on a pCambia1300 vector and a PCR cloning fragment nosT-LBcl1 respectively, the digested pCambia1300 vector and the digested fragment nosT-LBcl1 are recovered respectively, and then the fragments are connected and cloned to obtain a transition vector pCambia1300-nosT.
(3) T-DNA vector pCambia1300-pOsUbi-OsEPSPS-T-nosT
The intermediate vector pCambia1300-nosT is digested by KpnI and XhoI, and the digested vector is recovered; carrying out double enzyme digestion on a PCR product rice UBI-2 promoter pOsUbi (synchronous step 1) by KpnI and BamHI, and recovering the enzyme digested pOsUbi; the product of PCR, osEPSPS-T gene (step 1), was digested with BamHI and XhoI, and the digested OsEPSPS-T gene was recovered. Then, the recovered vector and the two fragments are connected and cloned to obtain a final vector pCambia1300-pOsUbi-OsEPSPS-T-nosT.
3. Agrobacterium containing T-DNA vector
The obtained T-DNA vectors (pCambia 1300-pOsUbi-OsEPSPS-T-Ter1 and pCambia 1300-pOsUbi-OsEPSPS-T-nosT) are electrically shocked to transform agrobacterium EHA105, strains containing plant transformation vectors are obtained through kan screening, and the strains are stored in a refrigerator of-80 ℃ after glycerol is added for infection of transgenic crops.
4. Transformation of rice
Transgenic rice was obtained using the prior art (Lu Xiongbin, gong Zuxun, 1998, life sciences.10: 125-131; liu Fan et al, 2003, molecular plant breeding.1: 108-115). Mature and full 'Xishui-134' seeds are selected to be hulled, and callus is generated by induction and is used as a transformation material. And (3) respectively drawing the agrobacterium containing the T-DNA vectors pCambia1300-pOsUbi-OsEPSPS-T-Ter1 and pCambia1300-pOsUbi-OsEPSPS-T-nosT constructed in the step 3. Selecting a single colony and inoculating the single colony to a culture medium (the composition of the culture medium is 3g/L K) 2 HPO 4 、1g/LNaH 2 PO 4 、1g/LNH 4 Cl、0.3g/L MgSO 4 ·7H 2 O、0.15g/L KCl、0.01g/L CaCl 2 、0.0025g/L FeSO 4 ·7H 2 O, 5g/L sucrose, 20mg/L acetosyringone, solvent water, pH = 5.8), cultured at 28 ℃ to OD600 of 0.6. Putting the callus to be transformed into Agrobacterium tumefaciens bacterial liquid with OD600 of 0.6, culturing at 28 deg.C for 3 days to allow the Agrobacterium to growThe bacillus is combined on the surface of the callus, then the callus is transferred to a co-culture medium (MS +2mg/L2,4-dichlorophenoxyacetic acid (2,4-D) +30g/L glucose +30g/L sucrose +3g/L agar (sigma 7921) +20mg/L acetosyringone), and the co-culture is carried out for 2-3 days at 28 ℃. The transformed calli were rinsed with sterile water, transferred to selection medium (MS +2mg/L2,4-D +30g/L sucrose +3g/L agar (Sigma 7921) +20mg/L acetosyringone +2mM glyphosate (Sigma)), and screened for two months at 28 ℃ (once for intermediate passage). Transferring the callus with good growth activity after screening to a pre-differentiation culture medium (MS +0.1g/L inositol +5mg/L abscisic acid (ABA) +1mg/L naphthylacetic acid (NAA) +5 mg/L6-benzylaminopurine (6-BA) +20g/L sorbitol +30g/L sucrose +2.5g/L plant gel (gelrite)), culturing for 20 days at 28 ℃, then transferring the pre-differentiated callus to a differentiation culture medium, and performing differentiation and germination for 14 hours every day. After 2-3 weeks, the resistant regenerated plants are transferred to rooting medium (1/2MS +0.2mg/L NAA +20g/L sucrose +2.5g/L gelrite) for strong seedlings to root, and finally the regenerated plants are washed off and the agar is transplanted to the greenhouse. Transgenic rice plants of the vectors pCambia1300-pOsUbi-OsEPSPS-T-Ter1 and pCambia1300-pOsUbi-OsEPSPS-T-nosT were designated OEHT and OENT, respectively.
5. Analysis of glyphosate resistance of transgenic OsEPSPS-T gene rice:
transplanting the T0 generation plant of the transgenic rice plant prepared by the method in the step 4 into a greenhouse, and comparing and analyzing herbicide resistance of the transgenic rice plant and a non-transgenic receptor control plant 'Xiushui-134'. We performed different concentrations of glyphosate resistance assay on 105 transgenic lines (named OEHT) transformed with pCambia1300-pOsUbi-OsEPSPS-T-Ter1 vector and 91 transgenic lines (named OENT) transformed with pCambia1300-pOsUbi-OsEPSPS-T-nosT vector, and the resistance effect is shown in Table 4:
table 4: spraying glyphosate with different concentrations to rice plants 15 days after sowing for resistance detection
1x 2x 4x
Number of OEHT-resistant transformation events 82 42 3
Number of OENT resistant transformation events 85 80 62
* Note: the plant line with the plant height, the number of leaves and the growth potential which are not obviously different from those of the plant sprayed with the blank control after the glyphosate is sprayed for 30 days is a resistant transformation event. 1x is the dilution of the noda-41% glyphosate (monta usa) at a volume ratio of 1. OEHT represents the pCambia1300-pOsUbi-OsEPSPS-T-Ter1 transgenic plant; OENT denotes the transgenic plant pCambia1300-pOsUbi-OsEPSPS-T-nosT.
The test results show that the resistance of the transgenic rice plant obtained by the transformation vector pCambia1300-pOsUbi-OsEPSPS-T-Ter1 to glyphosate is obviously higher than that of the transgenic rice plant obtained by the transformation vector pCambia1300-pOsUbi-OsEPSPS-T-nosT on the whole. Therefore, the terminator Ter1 of the rice HSP17.9 gene is considered to be capable of mediating the high expression of a target gene in plants such as rice better than a nos terminator. Therefore, we chose to perform subsequent experiments using the terminator Ter1 of the rice HSP17.9 gene or a terminator having a similar effect thereto.
Example 2 vector construction
1. Artificially synthesizing a maize EPSPS mutant gene ZmEPSPS-T, wherein the nucleotide sequence of the maize EPSPS mutant gene ZmEPSPS-T is shown as SEQ ID No:3, and the amino acid sequence is shown as SEQ ID No:4, respectively. Artificially synthesizing a rice ALS mutant gene OsALS-T1, wherein the nucleotide sequence of the gene OsALS-T1 is shown as SEQ ID No:5, the amino acid sequence is shown as SEQ ID No: and 6. Artificially synthesizing a rice ALS mutant gene OsALS-T2, wherein the nucleotide sequence of the gene OsALS-T2 is shown as SEQ ID No:7, and the amino acid sequence is shown as SEQ ID No: shown in fig. 8.
2. Constructing a corn EPSPS mutant gene ZmEPSPS-T expression vector:
the ZmEPSPS-T gene (primer ZE-F:5'-ATCGGATCCACCATGGCGGCCATGGCGACC; ZE-R:5' -GGCGTCTCGAGTTATCAATTCTTGACGAAAGTGCTCAGC) was cloned by PCR using artificially synthesized ZmEPSPS-T as a template, and a BamHI site was placed at the 5 'end of the sequence and an XhoI cleavage site was placed at the 3' end. The maize UBI-1 promoter pZmUbi (primer ZMubi-F1:5'-CCGGGTACCTGCAGTGCAGCGTGACCCGGTC; ZMubi-ZmR: 5' -CATGGTGGATCCGATCTGCAGAAGTAACACCAAACAAC) was cloned by PCR using maize genome (B73) as template, and the nucleotide sequence was as shown in SEQ ID No:10, a KpnI site is arranged at the 5 'end of the sequence, and a BamHI site is arranged at the 3' end of the sequence. The CaMV35S terminator in the pCambia1300 vector is replaced by a terminator Ter2 of a homologous gene Arabidopsis HSP18.2 gene of the rice OsHSP17.2 gene: an arabidopsis genome (Columbia ecotype) is used as a template, a terminator Ter2 (nucleotide sequence is shown in SEQ ID No. 12) of an arabidopsis HSP18.2 gene is cloned by PCR (a primer is Ter2-F:5' -GCGGCTCGAGATATGAAGATGAAGATGAAATATTT; ter2-R:5' -CTGAATTAACGCCGAATCTTATCTTTAATCATATTCC), and an XhoI enzyme cutting site is arranged at the 5' end. Using pCambia1300 vector as template, cloning a vector skeleton sequence (primer is LBcl1-F2:5'-GAATATGATTAAAGATAAGATTCGGCGTTAATTCAGTAC; LBcl1-R:5' -GCGGTGATCACAGGCAGCAACGCTCTGTCATC) by PCR, and naming as LBcl1, the nucleotide sequence is shown as 6319bp-6648bp in pCambia1300 vector (NCBI Accession Number: AF 234296). The terminator sequence Ter2 and the vector framework sequence LBcl1 of the Arabidopsis HSP18.2 gene obtained by PCR cloning are used as templates, DNA fragments which are respectively the terminator of the Arabidopsis HSP18.2 gene and the vector framework sequence LBcl1 from 5 'to 3' and are marked as fragments Ter2-LBcl1 are obtained by PCR cloning (primers are Ter2-F:5'-GCGGCTCGAGATATGAAGATGAAGATGAAATATTT; LBcl1-R:5' -GCGGTGATCACAGGCAGCAACGCTCTGTCATC), and the 5 'end and the 3' end of the fragments are respectively provided with XhoI enzyme cutting sites and BclI enzyme cutting sites. And (3) carrying out double enzyme digestion on the pCambia1300 vector and the PCR cloning fragment Ter2-LBcl1 by using XhoI and BclI respectively, recovering the pCambia1300 vector and the Ter2-LBcl1 fragment after enzyme digestion respectively, and then cloning after connecting the fragments to obtain the transition vector pCambia1300-Ter2.
Then, the transition vector pCambia1300-Ter2 is digested by KpnI and XhoI, and the digested vector is recovered; carrying out double enzyme digestion on a PCR product corn UBI-1 promoter pZmUbi by KpnI and BamHI, and recovering the enzyme digested pZmUbi; bamHI and XhoI are used for carrying out double enzyme digestion on the ZmEPSPS-T gene of the PCR product, and the enzyme digested ZmEPSPS-T gene is recovered. Then the recovered vector and the two fragments are connected and cloned to obtain a final vector pCambia1300-pZmUbi-ZmEPSPS-T-Ter2.
3. Constructing an expression vector of rice ALS mutant gene OsALS-T1:
an artificially synthesized rice ALS mutant gene OsALS-T1 is used as a template, an OsALS-T1 gene (primers are ALS-F:5'-CAGATCGGATCCACCATGGCTACGACCGCCGC; ALS-R:5' -CATATCTCGAGTTATCAATACACAGTCCTGCCATCACC) is cloned by PCR, a BamHI site is arranged at the 5 'end of a sequence, and an XhoI enzyme cutting site is arranged at the 3' end.
In order to construct a complete OsALS-T1 gene expression vector, kpnI and XhoI are used for recovering the vector after enzyme digestion from the transition vector pCambia1300-Ter1 obtained in example 1 respectively; carrying out double enzyme digestion on the PCR product rice UBI-2 promoter pOsUbi by KpnI and BamHI, and recovering the enzyme digested pOsUbi; carrying out double enzyme digestion on the OsALS-T1 gene of the PCR product by using BamHI and XhoI, and recovering the OsALS-T1 gene after enzyme digestion. Then, the recovered vector and the two fragments are connected and cloned to obtain a final vector pCambia1300-pOsUbi-OsALS-T1-Ter1.
4. Constructing a rice ALS mutant gene OsALS-T2 expression vector:
an artificially synthesized rice ALS mutant gene OsALS-T2 is taken as a template, an OsALS-T1 gene (primer is ALS-F:5'-CAGATCGGATCCACCATGGCTACGACCGCCGC; ALS-R:5' -CATATCTCGAGTTATCAATACACAGTCCTGCCATCACC) is cloned through PCR, a BamHI site is arranged at the 5 'end of a sequence, and an XhoI enzyme cutting site is arranged at the 3' end of the sequence.
In order to construct a complete OsALS-T2 gene expression vector, kpnI and XhoI are respectively used for the transition vector pCambia1300-Ter2, and the vector after enzyme digestion is recovered; carrying out double enzyme digestion on a PCR product corn UBI-1 promoter pZmUbi by KpnI and BamHI, and recovering the enzyme digested pZmUbi; carrying out double enzyme digestion on the OsALS-T2 gene of the PCR product by using BamHI and XhoI, and recovering the OsALS-T2 gene after enzyme digestion. Then, the recovered vector and the two fragments are connected and cloned to obtain a final vector pCambia1300-pZmUbi-OsALS-T2-Ter2.
5. Expression vector construction for increasing MAR regulatory elements
A tobacco genome (Nicotiana benthamiana) is used as a template, a MAR fragment (a primer is MARF:5'-GGTAAGCTTTCGATTAAAAATCCCAATTATATTTGGTC; MARR:5' -CAGGGTACCACTATTTTCAGAAGAAGTTCCCAATAG) is cloned by PCR, and the nucleotide sequence is shown as SEQ ID No:13, a HindIII site is arranged at the 5 'end of the sequence, and a KpnI enzyme cutting site is arranged at the 3' end of the sequence. The vector pCambia1300-pOsUbi-OsEPSPS-T-Ter1 and the PCR product MAR are subjected to double enzyme digestion by restriction enzymes HindIII and KpnI respectively, the digested pCambia1300-pOsUbi-OsEPSPS-T-Ter1 vector and MAR fragment are recovered, and the final vector pCambia1300-MAR-pOsUbi-OsEPSPS-T-Ter1 is obtained by cloning after connection.
6. Transformation of Agrobacterium
The obtained T-DNA vectors (pCambia 1300-pZmUbi-ZmEPSPS-T-Ter2, pCambia1300-MAR-pOsUbi-OsEPSPS-T-Ter1, pCambia1300-pOsubi-OsALS-T1-Ter1 and pCambia 1300-pOsubi-ALS OsT 2-Ter 2) were electrically shocked to transform Agrobacterium EHA105, and strains containing plant transformation vectors were obtained by kan selection, and after glycerol addition, they were stored in a-80 ℃ refrigerator for transgenic crop infection.
Example 3 obtaining of herbicide tolerant Rice
In this example, transgenic rice plants of pCambia1300-pOsUbi-OsEPSPS-T-Ter1, pCambia1300-pZmUbi-ZmEPSPS-T-Ter2 and pCambia1300-MAR-pOsUbi-OsEPSPS-T-Ter1, which were prepared in example 2, were obtained and named OEHT, ZEHT and MOEHT, respectively, as in example 1.
To obtain ALS mutant transgenic rice, 2mM glyphosate in the selection medium in the rice transformation method of example 1 was replaced with 0.7mM bispyribac-sodium (Sigma-Aldrich, cat # 32967) using the ALS mutant as a selection marker. Transgenic rice plants of pCambia1300-pOsUbi-OsALS-T1-Ter1 and pCambia1300-pZmUbi-OsALS-T2-Ter2 prepared in example 2 were obtained by this method and named OATA and OATB, respectively.
Example 4 obtaining of herbicide tolerant soybeans
The procedure used here to obtain transgenic soybeans is from the established art (Deng et al, 1998, plant Physiology Communications 34. Healthy, full and mature soybeans of 'Tianlong No. 1' are selected, sterilized by 80% ethanol for 2 minutes, washed by sterile water and then placed in a dryer filled with chlorine (generated by the reaction of 50ml of NaClO and 2ml of concentrated HCl) for sterilization for 4-6 hours. Spreading sterilized semen glycines in B5 culture medium in clean bench, culturing at 25 deg.C for 5 days with optical density of 90-150 μmol photon/m 2 S level. When the seeds She Bianlu burst the seed coat, the aseptic bean sprouts grow. The bean sprouts with the hypocotyl removed were cut into five-five pieces in length so that both explants had cotyledons and epicotyls. The explants are cut at about 7-8 of the node of the cotyledon and epicotyl and can be used as the target tissue to be infected.
Single-clone Agrobacterium containing the vector pCambia1300-pOsUbi-OsEPSPS-T-Ter1 prepared in example 1 and pCambia1300-pZmUbi-ZmEPSPS-T-Ter2 and pCambia1300-pOsUbi-MAR-OsEPSPS-T-Ter1 prepared in example 2 were separately cultured for use. The prepared explants are immersed in the Agrobacterium culture solution for 30 minutes at 28 ℃. Then, the excess cell suspension on the infected tissue is absorbed and cleaned by absorbent paper, and then transferred to 1/10B5 co-culture medium for dark culture at 25 ℃ for 3-5 days.
The co-cultured plant tissue was washed with B5 liquid medium to remove excess Agrobacterium, and then placed in B5 solid medium for 5 days at 25 ℃ until it germinated. The induced germ tissue was transferred to B5 selection medium containing 25mg/L glyphosate and incubated at 25 ℃ with light for 4 weeks, during which the medium was changed every two weeks. Transferring the selected embryo tissue to a solid culture medium, culturing at 25 deg.C, and growing into plantlet. Subsequently, transgenic plants were transferred to 1/2B5 medium for rooting induction. Finally, the grown plantlets are washed to remove agar and planted in a greenhouse.
Example 5 obtaining of herbicide tolerant corn
The transformation technology of corn is mature. References such as Vladimir Sidorov&David Duncan(in M.Pa ul Scott(ed.),Methods in Molecular Biology:Transgenic Maize,vol:526；Yuji Ishida,Yukoh Hiei&Toshihiko Komari (2007) Agrobacterium-mediated transformation of mail. Nature Protocols 2. The basic method is as follows: collecting Hi-II corn ear 8-10 days after pollination, and collecting all immature embryos (with size of 1.0-1.5 mm). Agrobacterium (OD 660= 0.70) containing the vectors pCambia1300-pOsUbi-OsEPSPS-T-Ter1 prepared in example 1 and pCambia1300-pZmUbi-ZmEPSPS-T-Ter2 prepared in example 2 and pCambia1300-pOsUbi-MAR-OsEPSPS-T-Ter1, respectively, were co-cultured with immature embryos on co-culture medium (MS +2mg/L2,4-D +30g/L sucrose +3g/L agar (sigma 7921) +40mg/L acetosyringone) for 2-3 days (22 ℃). Transfer immature embryos onto callus induction Medium (MS +2mg/L2,4-D +30g/L sucrose +2.5g/L gelrite +5mg/L AgNO) 3 +200mg/L acetosyringone), dark culture at 28 ℃ for 10-14 days. All calli were transferred to selection medium (same as callus induction medium) with 2mM glyphosate and incubated in the dark at 28 ℃ for 2-3 weeks. All tissues were transferred to fresh 2mM glyphosate in selection medium and incubated at 28 ℃ for 2-3 weeks in the dark. Then, all viable embryonic tissues after selection were transferred to regeneration medium (MS +30g/L sucrose +0.5 mg/L6-furfurylaminopurine (kinetin) +2.5g/L gelrite +200mg/L acetosyringone), and cultured in the dark at 28 ℃ for 10-14 days, one line per dish. Transfer of embryonic tissue to freshCulturing in regeneration medium at 26 deg.C under illumination for 10-14 days. All fully developed plants are transferred to rooting medium (1/2MS +20g/L sucrose +2.5g/L gelrite +200mg/L acetosyringone), and cultured under light at 26 ℃ until roots are fully developed. Transgenic maize plants containing the T-DNA of the transformation vectors pCambia1300-pOsUbi-OsEPSPS-T-Ter1, pCambia1300-pZmUbi-ZmEPSPS-T-Ter2 and pCambia1300-pOsUbi-MAR-OsEPSPS-T-Ter1 were obtained.
Example 6 herbicide resistance identification
1. Determination of glyphosate resistance of transgenic plant EPSPS mutant genes rice OEHT, ZEHT and MOEHT
And (3) determining the glyphosate resistance of the field: example 3T 2 generation transgenic rice and conventional rice seeds of rice OEHT, ZEHT and MOEHT (xishu 134) in rice OEHT, ZEHT and MOEHT, respectively, were sprayed with tap water at a volume ratio of 1: 200. 1:100 and 1: a50-diluted 41% glyphosate isopropylamine salt (Monsanto) was applied at 40L/acre and rice growth and development and mortality were recorded after 14 days.
The glyphosate resistance of the transgenic rice was determined by spraying glyphosate in the field during 4-6 leaves, and the results are shown in table 5.
TABLE 5 glyphosate resistance test of EPSPS mutant gene-transformed rice #
Figure BDA0003118502410000091
# Spraying 40L of 1:200 of a carrier; 1:100 or 1. 1. OEHT represents a transgenic plant of a vector pCambia1300-pOsUbi-OsEPSPS-T-Ter 1; ZEHT represents the vector pCambia1300-pZmUbi-ZmEPSPS-T-Ter2 transgenic plant; MOEHT represents the vector pCambia1300-MAR-pOsUbi-OsEPSPS-T-Ter1 transgenic plant. The numbers following the transformant abbreviation indicate the transformants with different numbers. Xishui 134 is a major cultivar in the middle and downstream Yangtze river.
The results show that most transformants can tolerate higher concentrations of glyphosate. The resistance levels of OEHT-7, OEHT-15, OEHT-51, ZEHT-9, ZEHT-22, ZEHT-58, MOEHT-5, MOEHT-8, MOEHT-19, MOEHT-22 and MOEHT-35 were relatively high, with a 40L spray of 1: under the condition of 50-diluted agricultural product (41% glyphosate isopropylamine salt, monsanto), namely under the condition of 4 times of medium dose, the transgenic rice is not obviously influenced.
2. Determination of bispyribac-sodium resistance of transgenic plant ALS mutant gene rice OATA and OATB
Large Tian Naishuang nitrofen performance assay: in example 3, transgenic rice of the T2 generation of rice OATA and OATB and conventional rice seeds (Xiushui 134) were sprayed with bispyribac (Jiangsu Runze agrichemical Co., ltd.) in amounts of 0.84 g/mu, 1.68 g/mu and 2.52 g/mu at the 4-6 leaf stage 15-20 days after germination, respectively, at a dose of 30L/mu, and the growth and mortality of rice were recorded after 14 days.
Bispyribac-sodium resistance of the transgenic rice was determined by spraying bispyribac-sodium in the field during 4-6 leaves, and the results are shown in Table 6.
TABLE 6 ALS-transgenic rice bispyribac-sodium resistance performance test #
Figure BDA0003118502410000101
# Spraying bispyribac-sodium (Jiangsu Ruizu agrichemical Co., ltd.) at 0.84 g/mu, 1.68 g/mu and 2.52 g/mu. 0.84 g/mu is the recommended dosage. 30L of the fertilizer is sprayed per mu. OATA represents the vector pCambia1300-pOsUbi-OsALS-T1-Ter1 transgenic plant; OATB represents the vector pCambia1300-pZmUbi-OsALS-T2-Ter2 transgenic plant. The numbers following the transformant abbreviations indicate the differently numbered transformants. Xishui 134 is a major cultivar in the middle and downstream Yangtze river.
The results show that most transformants can tolerate higher concentrations of bispyribac-sodium. The resistance levels of OATA-2, OATA-11, OATA-35, OATB-14, OATB-27, OATB-45 and OATB-51 are higher, and the resistance levels of the genes have no obvious influence on transgenic rice under the condition that 2.52g of bispyribac (Jiangsu Runze agriculture chemical Co., ltd.) is sprayed per mu, namely under the condition that the medium dose is 4 times.
It is also noted that the above-mentioned lists merely illustrate several embodiments of the invention. The invention is not limited to the above embodiments but may be extended and expanded in many ways. All extensions that can be derived or suggested by a person of ordinary skill in the art from the present disclosure should be considered within the scope of the present invention.
Sequence listing
<110> Hangzhou aromatic rhyme Biotechnology Co., ltd
<120> vector for expressing plant source herbicide resistant mutant gene and application thereof
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1548
<212> DNA
<213> Unknown (Unknown)
<400> 1
atggcggcga ccatggcgtc caacgccgcg gctgcggcgg cggtgtccct ggaccaggcc 60
gtggcggcgt cggcggcgtt ctcgtcgcgg aagcagctgc ggctgcccgc cgcggcgcgc 120
ggggggatgc gggtgcgggt gcgggcgcgg gggcggcggg aggcggtggt ggtggcgtcc 180
gcgtcgtcgt cgtcggtggc agcgccggcg gcgaaggcgg aggagatcgt gctccagccc 240
atcagggaga tctccggggc ggttcagctg ccagggtcca agtcgctctc caaccgcatc 300
ctcctcctct ccgccctctc cgagggcaca acagtggtgg acaacttgct gaacagtgag 360
gatgttcact acatgcttga ggccctgaaa gccctcgggc tctctgtgga agcagataaa 420
gttgcaaaaa gagctgtagt cgttggctgt ggtggcaagt ttcctgttga gaaggatgcg 480
aaagaggaag tgcaactctt cttggggaac gctggaattg caatgcgatc attgacagca 540
gccgtgactg ctgctggtgg aaatgcaact tatgtgcttg atggagtgcc acgaatgagg 600
gagagaccga ttggtgactt ggttgtcggg ttgaaacaac ttggtgcgga tgtcgactgt 660
ttccttggca ctgaatgccc acctgttcgt gtcaagggaa ttggaggact tcctggtggc 720
aaggttaagc tctctggttc catcagcagt cagtacttga gtgccttgct gatggctgct 780
cctttggccc ttggggatgt ggagatcgaa atcattgaca aactaatctc cattccttac 840
gttgaaatga cattgagatt gatggagcgt tttggtgtga aggcagagca ttctgatagt 900
tgggacagat tctatattaa gggagggcag aagtacaaat ctcctggaaa tgcctatgtt 960
gaaggtgatg cctcaagcgc gagctatttc ttggctggtg ctgcaatcac tggaggcact 1020
gtgacagttc aaggttgtgg tacgaccagt ttgcagggtg atgtcaaatt tgctgaggta 1080
cttgagatga tgggagcaaa ggttacatgg actgacacca gtgtaaccgt aactggtcca 1140
ccacgtgagc cttatgggaa gaaacacctg aaagctgttg atgtcaacat gaacaaaatg 1200
cctgatgttg ccatgaccct tgccgttgtt gcactcttcg ctgatggtcc aactgctatc 1260
agagatgtgg cttcctggag agtaaaggaa accgaaagga tggttgcaat tcggaccgag 1320
ctaacaaagc tgggagcatc ggttgaagaa ggtcctgact actgcatcat caccccaccg 1380
gagaagctga acatcacggc aatcgacacc tacgatgatc acaggatggc catggccttc 1440
tccctcgctg cctgcgccga cgtgcccgtg acgatcaggg accctggttg cacccgcaag 1500
accttcccca actacttcga cgttctaagc actttcgtca ggaactga 1548
<210> 2
<211> 515
<212> PRT
<213> Unknown (Unknown)
<400> 2
Met Ala Ala Thr Met Ala Ser Asn Ala Ala Ala Ala Ala Ala Val Ser
1 5 10 15
Leu Asp Gln Ala Val Ala Ala Ser Ala Ala Phe Ser Ser Arg Lys Gln
20 25 30
Leu Arg Leu Pro Ala Ala Ala Arg Gly Gly Met Arg Val Arg Val Arg
35 40 45
Ala Arg Gly Arg Arg Glu Ala Val Val Val Ala Ser Ala Ser Ser Ser
50 55 60
Ser Val Ala Ala Pro Ala Ala Lys Ala Glu Glu Ile Val Leu Gln Pro
65 70 75 80
Ile Arg Glu Ile Ser Gly Ala Val Gln Leu Pro Gly Ser Lys Ser Leu
85 90 95
Ser Asn Arg Ile Leu Leu Leu Ser Ala Leu Ser Glu Gly Thr Thr Val
100 105 110
Val Asp Asn Leu Leu Asn Ser Glu Asp Val His Tyr Met Leu Glu Ala
115 120 125
Leu Lys Ala Leu Gly Leu Ser Val Glu Ala Asp Lys Val Ala Lys Arg
130 135 140
Ala Val Val Val Gly Cys Gly Gly Lys Phe Pro Val Glu Lys Asp Ala
145 150 155 160
Lys Glu Glu Val Gln Leu Phe Leu Gly Asn Ala Gly Ile Ala Met Arg
165 170 175
Ser Leu Thr Ala Ala Val Thr Ala Ala Gly Gly Asn Ala Thr Tyr Val
180 185 190
Leu Asp Gly Val Pro Arg Met Arg Glu Arg Pro Ile Gly Asp Leu Val
195 200 205
Val Gly Leu Lys Gln Leu Gly Ala Asp Val Asp Cys Phe Leu Gly Thr
210 215 220
Glu Cys Pro Pro Val Arg Val Lys Gly Ile Gly Gly Leu Pro Gly Gly
225 230 235 240
Lys Val Lys Leu Ser Gly Ser Ile Ser Ser Gln Tyr Leu Ser Ala Leu
245 250 255
Leu Met Ala Ala Pro Leu Ala Leu Gly Asp Val Glu Ile Glu Ile Ile
260 265 270
Asp Lys Leu Ile Ser Ile Pro Tyr Val Glu Met Thr Leu Arg Leu Met
275 280 285
Glu Arg Phe Gly Val Lys Ala Glu His Ser Asp Ser Trp Asp Arg Phe
290 295 300
Tyr Ile Lys Gly Gly Gln Lys Tyr Lys Ser Pro Gly Asn Ala Tyr Val
305 310 315 320
Glu Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile
325 330 335
Thr Gly Gly Thr Val Thr Val Gln Gly Cys Gly Thr Thr Ser Leu Gln
340 345 350
Gly Asp Val Lys Phe Ala Glu Val Leu Glu Met Met Gly Ala Lys Val
355 360 365
Thr Trp Thr Asp Thr Ser Val Thr Val Thr Gly Pro Pro Arg Glu Pro
370 375 380
Tyr Gly Lys Lys His Leu Lys Ala Val Asp Val Asn Met Asn Lys Met
385 390 395 400
Pro Asp Val Ala Met Thr Leu Ala Val Val Ala Leu Phe Ala Asp Gly
405 410 415
Pro Thr Ala Ile Arg Asp Val Ala Ser Trp Arg Val Lys Glu Thr Glu
420 425 430
Arg Met Val Ala Ile Arg Thr Glu Leu Thr Lys Leu Gly Ala Ser Val
435 440 445
Glu Glu Gly Pro Asp Tyr Cys Ile Ile Thr Pro Pro Glu Lys Leu Asn
450 455 460
Ile Thr Ala Ile Asp Thr Tyr Asp Asp His Arg Met Ala Met Ala Phe
465 470 475 480
Ser Leu Ala Ala Cys Ala Asp Val Pro Val Thr Ile Arg Asp Pro Gly
485 490 495
Cys Thr Arg Lys Thr Phe Pro Asn Tyr Phe Asp Val Leu Ser Thr Phe
500 505 510
Val Arg Asn
515
<210> 3
<211> 1518
<212> DNA
<213> Unknown (Unknown)
<400> 3
atggcggcca tggcgaccaa ggccgccgcg ggcaccgtgt cgctggacct cgccgcgccg 60
tcgcgccgcc accaccgccc gagctcggcg cgcccgcccg cccgccccgc cgtccgcggg 120
ctgcgggcgc ctgggcgccg cgtgatcgcc gcgccgccgg cggcggcagc ggcggcggcg 180
gtgcaggcgg gtgccgagga gatcgtgctg cagcccatca aggagatctc cggcaccgtc 240
aagctgccgg ggtccaagtc gctttccaac cgcatcctcc tgctcgccgc cctgtccgag 300
gggacaacag tggttgataa cctgttgaac agtgaggatg tccactacat gctcggggcc 360
ttgaggactc ttggtctctc tgtcgaagcg gacaaagctg ccaaaagagc tgtagttgtt 420
ggctgtggtg gaaagttccc agttgaggat tctaaagagg aagtgcagct cttcttgggg 480
aatgctggaa ttgcaatgcg gtcattgaca gcagctgtta ctgctgctgg tggaaatgca 540
acttacgtgc ttgatggagt accaagaatg agggagagac ccattggcga cttggttgtc 600
ggattgaagc agcttggtgc agatgttgat tgtttccttg gcactgactg cccacctgtt 660
cgtgtcaatg gaatcggagg gctacctggt ggcaaggtca agctgtctgg ctccatcagc 720
agtcagtact tgagtgcctt gctgatggct gctcctttgg ctcttgggga tgtggagatt 780
gaaatcattg ataaattaat ctccattccc tacgtcgaaa tgacattgag attgatggag 840
cgttttggtg tgaaagcaga gcattctgat agctgggaca gattctacat taagggaggt 900
caaaaataca agtcccctaa aaatgcctat gttgaaggtg atgcctcaag cgcaagctat 960
ttcttggctg gtgctgcaat tactggaggg actgtgactg tggaaggttg tggcaccacc 1020
agtttgcagg gtgatgtgaa gtttgctgag gtactggaga tgatgggagc gaaggttaca 1080
tggaccgaga ctagcgtaac tgttactggc ccaccgcggg agccatttgg gaggaaacac 1140
ctcaaggcga ttgatgtcaa catgaacaag atgcctgatg tcgccatgac tcttgctgtg 1200
gttgccctct ttgccgatgg cccgacagcc atcagagacg tggcttcctg gagagtaaag 1260
gagaccgaga ggatggttgc gatccggacg gagctaacca agctgggagc atctgttgag 1320
gaagggccgg actactgcat catcacgccg ccggagaagc tgaacgtgac ggcgatcgac 1380
acgtacgacg accacaggat ggccatggcc ttctcccttg ccgcctgtgc cgaggtcccc 1440
gtgaccatcc gggaccctgg gtgcacccgg aagaccttcc ccgactactt cgatgtgctg 1500
agcactttcg tcaagaat 1518
<210> 4
<211> 506
<212> PRT
<213> Unknown (Unknown)
<400> 4
Met Ala Ala Met Ala Thr Lys Ala Ala Ala Gly Thr Val Ser Leu Asp
1 5 10 15
Leu Ala Ala Pro Ser Arg Arg His His Arg Pro Ser Ser Ala Arg Pro
20 25 30
Pro Ala Arg Pro Ala Val Arg Gly Leu Arg Ala Pro Gly Arg Arg Val
35 40 45
Ile Ala Ala Pro Pro Ala Ala Ala Ala Ala Ala Ala Val Gln Ala Gly
50 55 60
Ala Glu Glu Ile Val Leu Gln Pro Ile Lys Glu Ile Ser Gly Thr Val
65 70 75 80
Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu Leu Ala
85 90 95
Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu Asn Ser Glu
100 105 110
Asp Val His Tyr Met Leu Gly Ala Leu Arg Thr Leu Gly Leu Ser Val
115 120 125
Glu Ala Asp Lys Ala Ala Lys Arg Ala Val Val Val Gly Cys Gly Gly
130 135 140
Lys Phe Pro Val Glu Asp Ser Lys Glu Glu Val Gln Leu Phe Leu Gly
145 150 155 160
Asn Ala Gly Ile Ala Met Arg Ser Leu Thr Ala Ala Val Thr Ala Ala
165 170 175
Gly Gly Asn Ala Thr Tyr Val Leu Asp Gly Val Pro Arg Met Arg Glu
180 185 190
Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu Gly Ala Asp
195 200 205
Val Asp Cys Phe Leu Gly Thr Asp Cys Pro Pro Val Arg Val Asn Gly
210 215 220
Ile Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly Ser Ile Ser
225 230 235 240
Ser Gln Tyr Leu Ser Ala Leu Leu Met Ala Ala Pro Leu Ala Leu Gly
245 250 255
Asp Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Ile Pro Tyr Val
260 265 270
Glu Met Thr Leu Arg Leu Met Glu Arg Phe Gly Val Lys Ala Glu His
275 280 285
Ser Asp Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln Lys Tyr Lys
290 295 300
Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser Ala Ser Tyr
305 310 315 320
Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr Val Thr Val Glu Gly
325 330 335
Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala Glu Val Leu
340 345 350
Glu Met Met Gly Ala Lys Val Thr Trp Thr Glu Thr Ser Val Thr Val
355 360 365
Thr Gly Pro Pro Arg Glu Pro Phe Gly Arg Lys His Leu Lys Ala Ile
370 375 380
Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu Ala Val
385 390 395 400
Val Ala Leu Phe Ala Asp Gly Pro Thr Ala Ile Arg Asp Val Ala Ser
405 410 415
Trp Arg Val Lys Glu Thr Glu Arg Met Val Ala Ile Arg Thr Glu Leu
420 425 430
Thr Lys Leu Gly Ala Ser Val Glu Glu Gly Pro Asp Tyr Cys Ile Ile
435 440 445
Thr Pro Pro Glu Lys Leu Asn Val Thr Ala Ile Asp Thr Tyr Asp Asp
450 455 460
His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala Glu Val Pro
465 470 475 480
Val Thr Ile Arg Asp Pro Gly Cys Thr Arg Lys Thr Phe Pro Asp Tyr
485 490 495
Phe Asp Val Leu Ser Thr Phe Val Lys Asn
500 505
<210> 5
<211> 1935
<212> DNA
<213> Unknown (Unknown)
<400> 5
atggctacga ccgccgcggc cgcggccgcc gccctgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgccc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc ccccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc cgtgccggtc 720
tgggacacct cgatgaatct accagggtac atcgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgacgaa ttgcgctggt ttgttgagct gactggtatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgacgaccc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg ccgtggataa ggctgacctg 1020
ttgcttgcgt ttggtgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcag agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag ctcgctttac agggcttgaa tgctctgcta 1200
caacagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaaa acttttggtg aagagatccc accgcaatat 1320
gccattcagg tgctggatga gctgacgaaa ggtgaggcaa tcatcgctac tggtgttggg 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagctggcat tgatccgcat tgagaacctc cctgtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggtggtgca actggaggat aggttttaca aggcgaatag ggcgcataca 1680
tacttgggca acccggaatg tgagagcgag atatatccag attttgtgac tattgctaag 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctgg agactccagg gccatacttg ttggatatca tcgtcccgca ccaggagcat 1860
gtgctgccta tgatcccaat cgggggcgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactgtgt attaa 1935
<210> 6
<211> 644
<212> PRT
<213> Unknown (Unknown)
<400> 6
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Ala Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Trp Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Gln Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Leu Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ile Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Val Tyr
<210> 7
<211> 1935
<212> DNA
<213> Unknown (Unknown)
<400> 7
atggctacga ccgccgcggc cgcggccgcc gccctgtccg ccgccgcgac ggccaagacc 60
ggccgtaaga accaccagcg acaccacgtc cttcccgccc gaggccgggt gggggcggcg 120
gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc gccggccacg 180
ccgctccggc cgtgggggcc ggccgagccc cgcaagggcg cggacatcct cgtggaggcg 240
ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc catggagatc 300
caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca cgagcagggc 360
gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt ctgcgtcgcc 420
acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct gctcgactcc 480
gtcccgatgg tcgccatcac gggccaggtc ttccgccgca tgatcggcac cgacgccttc 540
caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta ccttgtcctt 600
gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc ctcgggccgt 660
cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc cgtgccggtc 720
tgggacacct cgatgaatct accagggtac atcgcacgcc tgcccaagcc acccgcgaca 780
gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat tctctatgtc 840
ggtggtggct gctctgcatc tggtgacgaa ttgcgctggt ttgttgagct gactggtatc 900
ccagttacaa ccactctgat gggcctcggc aatttcccca gtgacgaccc gttgtccctg 960
cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg ccgtggataa ggctgacctg 1020
ttgcttgcgt ttggtgtgcg gtttgatgat cgtgtgacag ggaaaattga ggcttttgca 1080
agcagggcca agattgtgca cattgacatt gatccagcag agattggaaa gaacaagcaa 1140
ccacatgtgt caatttgcgc agatgttaag ctcgctttac agggcttgaa tgctctgcta 1200
caacagagca caacaaagac aagttctgat tttagtgcat ggcacaatga gttggaccag 1260
cagaagaggg agtttcctct ggggtacaaa acttttggtg aagagatccc accgcaatat 1320
gccattcagg tgctggatga gctgacgaaa ggtgaggcaa tcatcgctac tggtgttggg 1380
cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca gtggctgtct 1440
tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc ttctgtggct 1500
aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat gaacattcag 1560
gagctggcat tgatccgcat tgagaacctc cctgtgaagg tgatggtgtt gaacaaccaa 1620
catttgggta tggtggtgca atgggaggat aggttttaca aggcgaatag ggcgcataca 1680
tacttgggca acccggaatg tgagagcgag atatatccag attttgtgac tattgctaag 1740
gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc cgccatcaag 1800
aagatgctgg agactccagg gccatacttg ttggatatca tcgtcccgca ccaggagcat 1860
gtgctgccta tgatcccaag tgagtccgca ttcaaggaca tgatcctgga tggtgatggc 1920
aggactgtgt attaa 1935
<210> 8
<211> 644
<212> PRT
<213> Unknown (Unknown)
<400> 8
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Ala Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Phe Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Trp Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Gln Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Glu Ser Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Val Tyr
<210> 9
<211> 2517
<212> DNA
<213> Unknown (Unknown)
<400> 9
ctgcagaaat gcaaatttca taaaacaaac tactagtact gtttgttcat tggtcttatc 60
caaaacttag ccactgcaac aagttcttga cccttagcac aatcatattg tgcatgcact 120
tgtttattgc aaagaatggt gcgtagggaa cacgcatgat ttttgaattg ctggcacata 180
attttatcat tagaaactgg aatgcaacat gtaccctttg tcatggtttc tttccgagac 240
attgcactgt tttttttaat cctatcatta tcataatgcc aagaactggt caccaaccag 300
caatttgcat catggttagt tgagctgtcc ccatgtatca ataggtgcat tgtattggtc 360
cgaaatataa atgcagtgga tgcaaaccta tctcatggcc gtcaacaaaa gaaatcaaaa 420
gggaaatgca ccatcttata tctccagttt atatgaacag attggataag atcataagat 480
caagtggttt atattatttt gaggaatata acatggattc atcctaatca ctcgtctagg 540
cagtatgtgt attcatgatg gatatggtac tatactacgg agttttttct tcacaaaata 600
acctgttatt ttgacctcca accaaacacg aattatacca aaaattgggt tatttcatct 660
atagtacaac tctattataa acatgcagta aattatccta cacatatacc aaaattcaag 720
tgtaataatc ctaatacaca gacttaaaaa acaaactatt tcctttttaa gaaaaggaaa 780
accatttttt taacggaagg aaaacaaatt cgggtcaagg cggaagccag cgcgccaccc 840
cacgtcagca aatacggagg cgcggggttg acggcgtcac ccggtcctaa cggcgaccaa 900
caaaccagcc agaagaaatt acagtaaaaa aaaagtaaat tgcactttga tccacctttt 960
attacctaag tctcaatttg gatcaccctt aaacctatct tttcaatttg ggccgggttg 1020
tggtttggac taccatgaac aacttttcgt catgtctaac ttccctttca gcaaacatat 1080
gaaccatata tagaggagat cggccgtata ctagagctga tgtgtttaag gtcgttgatt 1140
gcacgagaaa aaaaaatcca aatcgcaaca atagcaaatt tatctggttc aaagtgaaaa 1200
gatatgttta aaggtagtcc aaagtaaaac ttatagataa taaaatgtgg tccaaagcgt 1260
aattcactca aaaaaaatca acgagacgtg taccaaacgg agacaaacgg catcttctcg 1320
aaatttccca accgctcgct cgcccgcctc gtcttcccgg aaaccgcggt ggtttcagcg 1380
tggcggattc tccaagcaga cggagacgtc acggcacggg actcctccca ccacccaacc 1440
gccataaata ccagccccct catctcctct cctcgcatca gctccacccc cgaaaaattt 1500
ctccccaatc tcgcgaggct ctcgtcgtcg aatcgaatcc tctcgcgtcc tcaaggtacg 1560
ctgcttctcc tctcctcgct tcgtttcgat tcgatttcgg acgggtgagg ttgttttgtt 1620
gctagatccg attggtggtt agggttgtcg atgtgattat cgtgagatgt ttaggggttg 1680
tagatctgat ggttgtgatt tgggcacggt tggttcgata ggtggaatcg tggttaggtt 1740
ttgggattgg atgttggttc tgatgattgg ggggaatttt tacggttaga tgaattgttg 1800
gatgattcga ttggggaaat cggtgtagat ctgttgggga attgtggaac tagtcatgcc 1860
tgagtgattg gtgcgatttg tagcgtgttc catcttgtag gccttgttgc gagcatgttc 1920
agatctactg ttccgctctt gattgagtta ttggtgccat gggttggtgc aaacacaggc 1980
tttaatatgt tatatctgtt ttgtgtttga tgtagatctg tagggtagtt cttcttagac 2040
atggttcaat tatgtagctt gtgcgtttcg atttgatttc atatgttcac agattagata 2100
atgatgaact cttttaatta attgtcaatg gtaaatagga agtcttgtcg ctatatctgt 2160
cataatgatc tcatgttact atctgccagt aatttatgct aagaactata ttagaatatc 2220
atgttacaat ctgtagtaat atcatgttac aatctgtagt tcatctatat aatctattgt 2280
ggtaatttct ttttactatc tgtgtgaaga ttattgccac tagttcattc tacttatttc 2340
tgaagttcag gatacgtgtg ctgttactac ctatctgaat acatgtgtga tgtgcctgtt 2400
actatctttt tgaatacatg tatgttctgt tggaatatgt ttgctgtttg atccgttgtt 2460
gtgtccttaa tcttgtgcta gttcttaccc tatctgtttg gtgattattt cttgcag 2517
<210> 10
<211> 1991
<212> DNA
<213> Unknown (Unknown)
<400> 10
tgcagtgcag cgtgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa 60
gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 120
ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 180
atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 240
tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 300
tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 360
tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 420
gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata atttagatat 480
aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 540
actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 600
gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 660
ggcacggcat ctctgtcgct gcctctggac ccctctcgag agttccgctc caccgttgga 720
cttgctccgc tgtcggcatc cagaaattgc gtggcggagc ggcagacgtg agccggcacg 780
gcaggcggcc tcctcctcct ctcacggcac ggcagctacg ggggattcct ttcccaccgc 840
tccttcgctt tcccttcctc gcccgccgta ataaatagac accccctcca caccctcttt 900
ccccaacctc gtgttgttcg gagcgcacac acacacaacc agatctcccc caaatccacc 960
cgtcggcacc tccgcttcaa ggtacgccgc tcgtcctccc cccccccccc tctctacctt 1020
ctctagatcg gcgttccggt ccatggttag ggcccggtag ttctacttct gttcatgttt 1080
gtgttagatc cgtgtttgtg ttagatccgt gctgctagcg ttcgtacacg gatgcgacct 1140
gtacgtcaga cacgttctga ttgctaactt gccagtgttt ctctttgggg aatcctggga 1200
tggctctagc cgttccgcag acgggatcga tttcatgatt ttttttgttt cgttgcatag 1260
ggtttggttt gcccttttcc tttatttcaa tatatgccgt gcacttgttt gtcgggtcat 1320
cttttcatgc ttttttttgt cttggttgtg atgatgtggt ctggttgggc ggtcgttcta 1380
gatcggagta gaattctgtt tcaaactacc tggtggattt attaattttg gatctgtatg 1440
tgtgtgccat acatattcat agttacgaat tgaagatgat ggatggaaat atcgatctag 1500
gataggtata catgttgatg cgggttttac tgatgcatat acagagatgc tttttgttcg 1560
cttggttgtg atgatgtggt gtggttgggc ggtcgttcat tcgttctaga tcggagtaga 1620
atactgtttc aaactacctg gtgtatttat taattttgga actgtatgtg tgtgtcatac 1680
atcttcatag ttacgagttt aagatggatg gaaatatcga tctaggatag gtatacatgt 1740
tgatgtgggt tttactgatg catatacatg atggcatatg cagcatctat tcatatgctc 1800
taaccttgag tacctatcta ttataataaa caagtatgtt ttataattat tttgatcttg 1860
atatacttgg atgatggcat atgcagcagc tatatgtgga tttttttagc cctgccttca 1920
tacgctattt atttgcttgg tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt 1980
tacttctgca g 1991
<210> 11
<211> 389
<212> DNA
<213> Unknown (Unknown)
<400> 11
gcatcgccgg cgtgccgcgt gcgcgacgga gaggagcacg gcggggtttt cgctttggcc 60
tggtttgtct gtcgtgaagg agcaaataaa atcgggtccg gttgagtcca gtgtgtgtcc 120
gtgtctgtct tgaacagtcg tgtgtcgcgt cgtgtcactg ggtcagtgtg ttgttccagt 180
gcgccgttca tcagccgatc agcgtctgta ccggtctttt gcaactagtt aagtgatgaa 240
tactataatc tgttaatacg tgatctgtct gatgcattct gtgctttaat ttgattgagt 300
ttgggtatta ctacctgaca agattagaaa ccatacaagc acctttcgaa tttcgtaact 360
ctcactggcg gatttaaccc ctctagccg 389
<210> 12
<211> 250
<212> DNA
<213> Unknown (Unknown)
<400> 12
atatgaagat gaagatgaaa tatttggtgt gtcaaataaa aagcttgtgt gcttaagttt 60
gtgttttttt cttggcttgt tgtgttatga atttgtggct ttttctaata ttaaatgaat 120
gtaagatctc attataatga ataaacaaat gtttctataa tccattgtga atgttttgtt 180
ggatctcttc tgcagcatat aactactgta tgtgctatgg tatggactat ggaatatgat 240
taaagataag 250
<210> 13
<211> 1168
<212> DNA
<213> Unknown (Unknown)
<400> 13
tcgattaaaa atcccaatta tatttggtct aatttagttt ggtattgagt aaaacaaatt 60
cgaaccaaac caaaatataa atatatagtt tttatatata tgcctttaag actttttata 120
gaattttctt taaaaaatat ctagaaatat ttgcgactct tctggcatgt aatatttcgt 180
taaatatgaa gtgctccatt tttattaact ttaaataatt ggttgtacga tcactttctt 240
atcaagtgtt actaaaatgc gtcaatctct ttgttcttcc atattcatat gtcaaaatct 300
atcaaaattc ttatatatct ttttcgaatt tgaagtgaaa tttcgataat ttaaaattaa 360
atagaacata tcattattta ggtatcatat tgatttttat acttaattac taaatttggt 420
taactttgaa agtgtacatc aacgaaaaat tagtcaaacg actaaaataa ataaatatca 480
tgtgttatta agaaaattct cctataagaa tattttaata gatcatatgt ttgtaaaaaa 540
aattaatttt tactaacaca tatatttact tatcaaaaat ttgacaaagt aagattaaaa 600
taatattcat ctaacaaaaa aaaaaccaga aaatgctgaa aacccggcaa aaccgaacca 660
atccaaaccg atatagttgg tttggtttga ttttgatata aaccgaacca actcggtcca 720
tttgcacccc taatcataat agctttaata tttcaagata ttattaagtt aacgttgtca 780
atatcctgga aattttgcaa aatgaatcaa gcctatatgg ctgtaatatg aatttaaaag 840
cagctcgatg tggtggtaat atgtaattta cttgattcta aaaaaatatc ccaagtatta 900
ataatttctg ctaggaagaa ggttagctac gatttacagc aaagccagaa tacaaagaac 960
cataaagtga ttgaagctcg aaatatacga aggaacaaat atttttaaaa aaatacgcaa 1020
tgacttggaa caaaagaaag tgatatattt tttgttctta aacaagcatc ccctctaaag 1080
aatggcagtt ttcctttgca tgtaactatt atgctccctt cgttacaaaa attttggact 1140
actattggga acttcttctg aaaatagt 1168
<210> 14
<211> 248
<212> DNA
<213> Unknown (Unknown)
<400> 14
ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat 60
tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac 120
gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat 180
agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt 240
actagatc 248

Claims (10)

1. A vector for expressing a plant-derived herbicide-resistant mutant gene, characterized in that the vector comprises a 5-enolpyruvylshikimate 3-phosphate synthase EPSPS mutant gene or an acetolactate synthase ALS mutant gene; the EPSPS mutant gene has a nucleotide sequence shown as SEQ ID No:1 or SEQ ID No:3 is shown in the specification; the ALS mutant gene has a nucleotide sequence shown as SEQ ID No:5 or SEQ ID No: shown at 7.
2. The vector of claim 1, wherein said vector comprises a promoter that mediates expression of a plant-derived herbicide resistant mutant gene.
3. The vector according to claim 1, wherein said promoter is a rice UBI-2 promoter, pysubi, or a maize UBI-1 promoter, pZmUbi; the nucleotide sequence of the rice UBI-2 promoter pOsUbi is shown as SEQ ID No:9, the nucleotide sequence of the corn UBI-1 promoter pZmUbi is shown as SEQ ID No: shown at 10.
4. The vector of claim 1, wherein said vector comprises an HSP gene terminator which terminates expression of a plant-derived herbicide resistant mutant gene; the terminator is a terminator Ter1 of a rice HSP17.9 gene or a terminator Ter2 of an Arabidopsis HSP18.2 gene.
5. The vector of claim 4, wherein the terminator Ter1 nucleotide sequence is as set forth in SEQ ID No:11, the nucleotide sequence of the terminator Ter2 is shown as SEQ ID No: shown at 12.
6. The vector of claim 4, wherein the basic vector of said vector is the one obtained by replacing the CaMV35S terminator in the pCambia1300 vector with the terminator Ter1 of the rice HSP17.9 gene or the terminator Ter2 of the Arabidopsis HSP18.2 gene.
7. The vector of claim 1, wherein said vector comprises regulatory elements that enhance the expression level or stability of a plant-derived herbicide resistant mutant gene; the regulatory element is a matrix attachment region sequence MAR, and the nucleotide sequence is SEQ ID No:13, respectively.
8. The vector of claim 1, wherein said vector composition comprises one of: (1) SEQ ID No:1, rice EPSPS mutant gene OsEPSPS-T shown in the specification, SEQ ID No:9, and the promoter pOsUbi shown in SEQ ID No: a terminator Ter1 shown in 11; (2) SEQ ID No:1, rice EPSPS mutant gene OsEPSPS-T shown in the specification, SEQ ID No:9, and the promoter pOsUbi shown in SEQ ID No:13, MAR, SEQ ID No: a terminator Ter1 shown in 11; (3) SEQ ID No:3, maize EPSPS mutant gene ZmEPSPS-T shown in SEQ ID No:10, and the promoter pZmUbi shown in SEQ ID No: a terminator Ter2 shown as 12; (4) SEQ ID No:5, the rice ALS mutant gene OsALS-T1 shown in SEQ ID No:9, and the promoter pOsUbi shown in SEQ ID No: a terminator Ter1 shown in 11; (5) SEQ ID No:7, the rice ALS mutant gene OsALS-T2 shown in SEQ ID No:10, and the promoter pZmUbi shown in SEQ ID No: terminator Ter2 shown at 12.
9. Use of the vector of claim 1 for expressing a plant-derived herbicide-resistant mutant gene in the preparation of herbicide-tolerant plant cells.
10. Use according to claim 9, wherein the plant comprises maize, rice, soybean, oilseed rape or cotton.
CN202110669517.XA 2021-06-17 2021-06-17 Vector for expressing plant source herbicide-resistant mutant gene and application thereof Pending CN115491384A (en)

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