CN109182369B

CN109182369B - Multi-herbicide-resistant expression vector, transformant and application thereof

Info

Publication number: CN109182369B
Application number: CN201810875895.1A
Authority: CN
Inventors: 肖珍珍; 沈志成
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-08-03
Filing date: 2018-08-03
Publication date: 2021-01-12
Anticipated expiration: 2038-08-03
Also published as: CN109182369A

Abstract

The invention relates to a multi-herbicide-resistant expression vector, a transformant and application thereof. The carrier contains glyphosate resistance gene G10 and multi-resistance herbicide gene ZJ 3-1. The Escherichia coli is transformed by the plant polygene expression vector to obtain an Escherichia coli transformant containing the plasmid; agrobacterium transformants containing the plasmid can be obtained by transforming agrobacterium. The agrobacterium transformant can be used for transforming plants to obtain transgenic plants resistant to various herbicides.

Description

Multi-herbicide-resistant expression vector, transformant and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a high-efficiency plant polygene expression vector, a transformant containing the vector and application of the vector.

Technical Field

Glyphosate is a broad-spectrum biocidal herbicide, is convenient to use and safe to the environment, and has the action target of chloroplast 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which can condense shikimate-3-phosphate and phosphoenolpyruvate to generate 5-enolpyruvylshikimate-3-phosphate and inorganic phosphate, and can competitively bind with the phosphoenolpyruvate, a substrate of the EPSPS, to inhibit the shikimate synthesis pathway, resulting in the synthesis of essential amino acids such as aromatic amino acids, such as tryptophan, tyrosine, phenylalanine, etc. The glyphosate has the excellent characteristics of wide weed control spectrum, quick plant absorption, easy degradation in soil, safety to people, livestock and natural environment and the like, and is the herbicide type with the largest use amount at present.

In the last decade, due to the extensive spread of transgenic glyphosate resistant crops (e.g., "Roundup Ready" corn, soybean, "GA 21" corn from Syngenta, etc.) and the ever decreasing price of glyphosate, the use of glyphosate has reached an unprecedented level. However, within only a decade of commercial application of glyphosate, weeds are resistant to glyphosate (Green and Owen, 2011). At present, more and more weeds also develop glyphosate resistance, and the high efficiency and the service life of glyphosate are seriously threatened.

Cytochrome P450(Cytochrome P450) is a multifunctional heme oxidase system and widely exists in animals, plants and microorganisms. The compound is named because the compound can be combined with CO, and the formed compound has a characteristic absorption peak at 450 nm. P450 is composed of various isoenzymes, can catalyze the mono-oxygenation activity of various hydrophobic substances, enhances the water solubility of heterologous substances, and enables the heterologous substances to be easier to be discharged out of a body. Cytochrome P450 not only catalyzes the biosynthesis of some endogenous substances with important physiological functions, such as hormones, terpenes, fatty acids and the like, but also participates in the metabolism of many exogenous substances, such as drugs, herbicides, pesticides and the like, so that the toxicity of the substances is reduced. There are a large number of cytochrome P450 enzyme lines in higher plants, for example over 300 cytochrome P450 genes in the arabidopsis genome. Compared with the animal cytochrome P450 enzyme system, the plant cytochrome P450 enzyme system has higher herbicide metabolism capability and stricter substrate specificity. Frear et al first discovered that mixed function oxidase in cotton seed microsomes can metabolize meturon (monuron), and then a great deal of research shows that the plant cytochrome P450 enzyme system is involved in the metabolism and detoxification of various herbicides. For example, CYP73A1, CYP76B1, CYP81B1 from Jerusalem artichoke (Helianthus tuberosus), CYP81B2 and CYP71A11 from tobacco, and CYP71A10 from soybean are all involved in the detoxification of some sulfonylurea herbicides, such as chlortoluron (chlorotoluron) and linuron (linuron). In 2006, Pan and the like clone from rice to obtain a cytochrome P450 gene CYP81A6, and the transgenic plant is transferred into arabidopsis thaliana and tobacco to find that the resistance of the transgenic plant to bentazon and sulfonylurea herbicides is obviously improved, and the development of herbicide-resistant transgenic crops by using the cytochrome P450 gene has wider prospects along with further research.

The large-area planting of the glyphosate-resistant crops greatly changes a weed control management system, so that the weed control is simpler and more convenient. However, with the continuous reuse of glyphosate, the original efficiency of glyphosate is greatly reduced, many weeds have resistance to glyphosate, and a glyphosate-based weed management system is in an endangered state. Therefore, the novel herbicide resistance gene is excavated and combined with the existing resistance gene to culture the second generation of multi-herbicide-resistant transgenic crops, and the method has important significance for controlling the generation of weed resistance, prolonging the service life of glyphosate and realizing the sustainable development of a crop production system.

Disclosure of Invention

The invention aims to provide a novel high-efficiency plant polygene expression vector, a transformant containing the vector and application thereof in order to cultivate more ideal multi-herbicide-resistant crops. The engineering bacteria carrying the carrier is used for transforming plants, and the transgenic plants with high resistance to various herbicides can be obtained through screening.

The technical scheme adopted by the invention is as follows:

the invention provides a multi-herbicide-resistant expression vector, which simultaneously comprises an expression frame for expressing a glyphosate-resistant gene G10 and an expression frame for expressing a herbicide degradation gene ZJ 3-1.

Furthermore, the multi-herbicide-resistant vector of the invention is preferably formed by inserting an expression frame of a glyphosate-resistant gene G10 into an XhoI site of a basic vector pCAMBIA1300 and inserting an expression frame of a multi-herbicide-resistant gene ZJ3-1 into an MCS site.

Furthermore, the nucleotide sequence of the glyphosate resistant gene G10 is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2.

Furthermore, the nucleotide sequence of the herbicide-resistant gene ZJ3-1 is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4.

Furthermore, the expression cassette of the glyphosate-resistant gene G10 comprises: the promoter CaMV35S of the transcription regulation region shown by SEQ ID NO. 5, the 5 ' untranslated region (TEV 5 ' UTR) shown by SEQ ID NO. 6, the Arabidopsis thaliana transduction peptide sequence CTP shown by SEQ ID NO. 7, the glyphosate resistant gene G10 shown by SEQ ID NO. 1 and the CaMV35S terminator at the 3 ' end shown by SEQ ID NO. 8 are arranged in sequence from the 5 ' end to the 3 ' end.

Further, the expression cassette of the herbicide-resistant gene ZJ3-1 comprises: the promoter CSV of the transcription regulation region shown in SEQ ID NO. 9, the coding sequence of the multi-herbicide-resistant gene ZJ3-1 shown in SEQ ID NO. 3 and the NOS terminator sequence of the 3 ' end shown in SEQ ID NO. 10 are arranged in sequence from the 5 ' end to the 3 ' end.

The invention also provides a transformant containing the multi-herbicide-resistant expression vector.

The invention relates to application of a multi-resistance herbicide expression vector in transforming plant cells, wherein the plants are rice, soybean, corn, rape, cotton and arabidopsis thaliana.

The plant polygene expression vector can be formed by inserting a glyphosate resistance gene G10 expression frame into an XhoI site of a basic vector pCAMBIA1300 and inserting a polyherbicide resistance gene ZJ3-1 expression frame into an MCS site. The basic vector pCAMBIA1300 contains left and right border sequences (LB and RB) of T-DNA in Ti plasmid, the fragment contains hygromycin resistance gene expression frame with XhoI enzyme cutting sites at two ends, the hygromycin resistance gene expression frame is replaced by a glyphosate resistant gene G10 expression frame (SEQ ID NO:11) which is artificially synthesized and is preferred according to dicotyledon codon, meanwhile, the basic vector contains Multiple Cloning Sites (MCS), and a multiple herbicide resistant gene ZJ3-1 expression frame (SEQ ID NO:12) is inserted at the MCS sites. The obtained plant polygene expression vector is named as p1300-CSV/ZJ3-1-35s/G10, the carried glyphosate-resistant gene is G10, the polyherbicide-resistant gene is ZJ3-1, and the genes are positioned in the left and right border sequences LB and RB regions of the T-DNA of the vector.

Coli TG1 was transformed with the plant multi-expression vector of the present invention to obtain E.coli transformants containing the plasmid vector.

Agrobacterium transformants containing the plasmid can be obtained by transforming Agrobacterium LBA4404 with the plant polygene expression vector. The agrobacterium transformant can be used for transforming plants to obtain transgenic plants simultaneously resistant to herbicides such as glyphosate, flazasulfuron, mesotrione, dicamba and the like.

The agrobacterium transformant containing the plasmid vector obtained by transforming agrobacterium LBA4404 by the plant polygene expression vector is used for transforming soybean, corn, rice, rape, cotton or arabidopsis thaliana, and glyphosate is used as a screening marker for screening, so that soybean, corn, rice, rape, cotton or arabidopsis thaliana plants which simultaneously resist herbicides such as glyphosate, flazasulfuron, mesotrione, dicamba and the like can be obtained.

Compared with the prior art, the invention has the following beneficial effects:

the invention combines the glyphosate-resistant gene and the ZJP450 gene for use, and the obtained transgenic plant can weed by using the glyphosate, can use the herbicides which can be degraded by the ZJP450, such as the herbicides of flazasulfuron, mesotrione, dicamba and the like, and can also weed by using the mixed herbicides. The plant obtained by transformation can provide new resources for culturing new generation of transgenic herbicide-resistant crops, avoids the occurrence of weed resistance caused by large-scale long-time repeated use of the same herbicide, and has important significance for prolonging the service life of glyphosate and realizing the sustainable development of modern crop production systems.

Drawings

FIG. 1 is a schematic diagram of a p1300-CSV/ZJ3-1-35s/G10 plant polyvalent gene expression plasmid containing G10 gene and ZJ3-1 gene.

Detailed Description

EXAMPLE 1 construction of plant multivalent expression vectors

The plasmid pCAMBIA1300(NCBI gene bank Access: AF234296.1) is cut by XhoI according to the enzyme digestion reaction and electrophoresis system shown in Table 1 to remove hygromycin resistance gene, and FastAP is added to dephosphorize the enzyme digestion product, and the vector fragment is recovered after agarose gel electrophoresis: 1300-35S (X, AP).

The TEV 5' untranslated region (SEQ ID NO:6) and an Arabidopsis thaliana transduction peptide sequence CTP (SEQ ID NO:7) are functionally spliced together, namely TEV-CTP-G10(SEQ ID NO:11), a TEV-CTP-G10(SEQ ID NO:11) is artificially synthesized, the synthesized gene is cloned to a pUC57 vector, and the obtained vector is named pUC 57-G10.

TEV-CTP-G10 was excised from pUC57-G10 using XhoI, ligated to 1300-35S (XhoI, AP), the ligation product transformed E.coli strain TG1, clones were selected and correctly oriented clones identified as p 1300-35S/G10.

The functional splicing of ZJ3-1(SEQ ID NO:3) and the NOS terminator (SEQ ID NO:10) gave ZJ3-1-NOSter (SEQ ID NO:12), SEQ ID NO:12 was artificially synthesized and cloned into pUC57, and the resulting vector was designated as pUC57-ZJ 3-1-NOSter. ZJ3-1-NOSter was digested simultaneously with (BamHI, Kpn I) from pUC57-ZJ3-1-NOSter vector according to the double digestion reaction and electrophoresis conditions shown in Table 1, and the fragment ZJ3-1-NOSter (BamHI, Kpn I) was recovered by agarose gel electrophoresis.

The CSV promoter (SEQ ID NO:9) was synthesized and cloned into the pUC57 vector, designated pUC 57-CSV. The synthetic CSV promoter sequence was double-digested with (Hind III, BamHI) from pUC57 vector according to the double-digestion reaction and electrophoresis conditions shown in Table 1, and the fragment CSV (Hind III, BamHI) was recovered by agarose gel electrophoresis.

The plasmid p1300-35s/G10 was digested simultaneously with HindIII and KpnI according to the double digestion reaction and electrophoresis conditions shown in Table 1, and the vector fragment p1300-35s/G10(HindIII and KpnI) was recovered after agarose gel electrophoresis.

The final vector p1300-CSV-ZJ3-1-35S/G10 (see FIG. 1) was obtained by ligating the fragment ZJ3-1-NOSter (BamHI, Kpn I), CSV (Hind III, BamHI) and the vector fragment p1300-35S/G10(Hind III, Kpn I) with T4 ligase (see Table 3 for ligation reaction).

TABLE 1 digestion and electrophoresis System

After incubation at 37 ℃ for 1-1.5 hours, 1.0% agarose gel electrophoresis was performed using 1 XTAE (0.04mol/L Tris-acetate, 0.001mol/L EDTA) electrophoresis buffer, and EB (ethidium bromide) was added to the agarose gel electrophoresis to a final concentration of 0.5. mu.g/ml, followed by electrophoresis at a voltage of 3-5V/cm.

TABLE 2 two-segment ligation reaction System

Ligation was performed overnight at 16 ℃.

TABLE 3 three-fragment ligation reaction System

Ligation was performed overnight at 16 ℃.

Example 2 obtaining of E.coli transformant

E, transformation of escherichia coli: adding 10ul of the ligation product (multivalent plasmid vector p1300-CSV/ZJ3-1-35s/G10) in example 1 into an escherichia coli TG1 competent cell, gently mixing uniformly, and standing on ice for about 30 min; ② the heating is carried out for 90s at 42 ℃, and then the mixture is rapidly placed on ice for cooling for 2 min; ③ 1ml of antibiotic-free sterilized LB liquid Medium (10g of peptone, 5g of yeast extract, 10g of NaCl, 1L H)₂O), placing the mixture into an incubator at 37 ℃ for about 1 hour; fourthly, centrifuging for 3min at 4000rpm, discarding the supernatant, and uniformly mixing the residual LB culture solution at the bottom of the centrifugal tube and the escherichia coli by using a gun head; evenly coating the bacterial liquid on an LB solid culture medium (containing 50 mug/L kanamycin) containing corresponding antibiotics, firstly placing the bacterial liquid in an incubator at 37 ℃ from the front side upwards for 1h until the bacterial liquid is completely absorbed, and then carrying out inverted culture for about 8-10h until white bacterial colonies appear.

And (3) selecting a single colony, shaking the bacteria overnight, extracting plasmids by adopting an Axygene Miniprep plasmid Miniprep kit, selecting positive plasmids after PCR and enzyme digestion detection, and storing corresponding colonies, namely transformants.

Example 3 transformation of Agrobacterium tumefaciens LBA4404

Electric shock transformation of agrobacterium tumefaciens: taking the multivalent plasmid vector p1300-CSV/ZJ3-1-35S/G10, 1ul in the example 2, adding the multivalent plasmid vector into the competent cells of the agrobacterium tumefaciens LBA4404, uniformly mixing, and adding into an electric shock cup (Eppendorf); secondly, the electric shock cup is put into a sample groove of the electric shock instrument according to the electric shock cup electrodesSelecting proper voltage electric shock (1.8mm 1800V, 2.0mm 2500V) from the size, and converting the electric shock; ③ to the shocked Agrobacterium cells, 1ml of YEP liquid medium (10g peptone, 10g yeast extract, 5g NaCl, 1L H) without antibiotics was added₂O), mixing evenly, transferring the mixture into a sterilized 1.5ml centrifuge tube, and culturing for about 3 hours at the temperature of 28 ℃; fourthly, the bacterial liquid is centrifuged for 3min at 4000rpm, and the supernatant is discarded. The residual YEP liquid medium and the precipitated cells were mixed well and spread evenly on YEP solid medium plates (10g peptone, 10g yeast extract, 5g NaCl, 15g agar, 1L H) containing 12mg/L tetracycline and 50mg/L kanamycin₂O), placing the mixture in an incubator at 28 ℃ for 1h with the front side facing upwards until the bacteria liquid is completely absorbed, and then carrying out inverted culture for about 36-48h until bacterial colonies appear.

A single colony is picked (which is confirmed to contain a multivalent plasmid vector p1300-CSV/ZJ3-1-35s/G10 by enzyme digestion and PCR detection), and the obtained agrobacterium strain is named as C3G.

Example 4 acquisition of transgenic plants

The arabidopsis transformation adopts an agrobacterium-mediated inflorescence dipping method: drawing lines on a YEP solid culture medium containing 50mg/L kanamycin and 12mg/L tetracycline for agrobacterium tumefaciens C3G with a T-DNA plasmid p1300-CSV/ZJ3-1-35S/G10, and carrying out dark culture at 28 ℃ for 2 d; selecting a single clone to 2-3ml of YEP liquid culture medium containing corresponding antibiotics, and carrying out dark culture at 28 ℃ and 220rpm for 24-27 h; ③ according to the volume ratio of 1: transferring the bacterial liquid to 250ml of YEP culture solution containing corresponding antibiotics according to a proportion of 100, carrying out dark culture at 28 ℃ and 220rpm for about 13-16h until OD600 reaches 1.5-2.0, and collecting the bacteria at 4 ℃, 5000rpm and 5 min; fourthly, the supernatant is poured off, the cells are resuspended in the transformation fluid, and OD is adjusted₆₀₀About 0.8. Preparation of transformation liquid: 2.21g of MS powder, 5g of cane sugar, Silwet L-771 ml and H₂O1L. Transforming Arabidopsis thaliana plant. The plant is watered thoroughly one day before transformation, inflorescences are immersed in transformation liquid for 20s, the plant is wrapped by a preservative film to preserve moisture, and then the plant is directly placed into an incubator with proper temperature for dark culture for 18-24h, watered and normally cultured, and the transformation is carried out once again after one week.

Example 5 herbicide resistance assay

The arabidopsis thaliana T0 generation seeds obtained by transformation in example 4 were germinated and harvested to obtain T1 generation seeds, which were sprayed with 1: the glyphosate resistance of transgenic plants was initially determined by 200 dilutions of agroda (41% aqueous glyphosate). Subsequently, the resistant plants were sprayed with different kinds of herbicides, respectively: mesotrione 200mg/L (10% suspending agent, piondard), dicamba 300mg/L (48% aqueous solution, sublimed bayer), and flazasulfuron 300mg/L (25% water dispersible granule, japaneth).

The results showed that 18 out of 20 transgenic arabidopsis lines had excellent resistance to glyphosate, and the controls sprayed with glyphosate all died. Of 18 well glyphosate resistant Arabidopsis lines: the 5 strains have good resistance to mesotrione, dicamba and flazasulfuron, and can tolerate 200mg/L of mesotrione, 300mg/L of dicamba and 300mg/L of flazasulfuron without influencing the growth; 10 strains show mild symptoms to mesotrione, dicamba and flazasulfuron, and can be recovered after a short period of time; there were 3 lines that were substantially non-resistant to mesotrione, dicamba, flazasulfuron and the non-transgenic controls to which the corresponding herbicide was applied.

The determination result shows that the resistance of the transgenic arabidopsis thaliana to glyphosate, mesotrione, dicamba and flazasulfuron is generally improved.

Example 7 resistance analysis of transgenic Arabidopsis thaliana to Mixed herbicides

The 18 transgenic arabidopsis thaliana with glyphosate resistance obtained in example 6 were sprayed with mixed herbicide (glyphosate diluted 200 times to the agricultural standard, flazasulfuron 100mg/L, dicamba 100mg/L) at the 3-4 leaf stage, and after 14 days, 5 strains were found to have no obvious phytotoxicity, 10 strains had mild phytotoxicity, 3 strains had serious phytotoxicity, and none of the transgenic controls were dead.

The determination result shows that the transgenic arabidopsis thaliana grows well generally under the condition of mixing and spraying different herbicides.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

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Ser Phe Asn Gly Ala Ala Leu Ser Thr Ala Ser Tyr Gly Pro Tyr Trp

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Leu Leu Pro His Leu Gly Thr Ala Asn Leu Trp Asp Tyr Met Pro Val

225 230 235 240

Leu Arg Trp Phe Asp Val Phe Gly Val Arg Lys Lys Ile Val Ser Ala

245 250 255

Val Arg Arg Arg Asp Ala Phe Leu Arg His Leu Val Asp Ala Glu Arg

260 265 270

Thr Arg Leu Asp Asp Gly Asn Asp Ala Gly Glu Lys Lys Ser Ile Ile

275 280 285

Ala Met Leu Leu Thr Leu Gln Lys Ser Glu Pro Asp Val Tyr Ser Asp

290 295 300

Thr Met Ile Met Ala Leu Cys Gly Asn Leu Phe Gly Ala Gly Thr Glu

305 310 315 320

Thr Thr Ser Thr Thr Thr Glu Trp Ala Met Ser Leu Leu Leu Asn His

325 330 335

Pro Glu Lys Leu Arg Lys Ala Gln Ala Glu Ile Asp Ala Val Val Gly

340 345 350

Thr Ser Arg Leu Leu Thr Ala Asp Asp Met Pro Arg Leu Thr Tyr Leu

355 360 365

Arg Cys Ile Ile Asp Glu Thr Met Arg Leu Tyr Pro Ala Ala Pro Leu

370 375 380

Leu Leu Pro His Glu Ser Ser Thr His Cys Lys Val Gly Gly Tyr Asp

385 390 395 400

Val Pro Ala Gly Thr Met Leu Leu Val Asn Val Tyr Ala Ile His Arg

405 410 415

Asp Pro Ala Val Trp Asp Gly Pro Thr Glu Phe Val Pro Glu Arg Phe

420 425 430

Glu Asp Gly Lys Ala Glu Gly Arg Leu Leu Met Pro Phe Gly Met Gly

435 440 445

Arg Arg Lys Cys Pro Gly Glu Thr Leu Ala Leu Arg Thr Ile Gly Leu

450 455 460

Val Leu Gly Thr Leu Ile Gln Cys Phe Asp Trp Asp Arg Val Asp Gly

465 470 475 480

Leu Glu Val Asp Met Thr Glu Ser Gly Gly Leu Thr Ile Pro Arg Ala

485 490 495

Val Pro Leu Glu Ala Met Cys Arg Pro Arg Ala Thr Met Arg Glu Val

500 505 510

Leu Gln Glu Leu

515

<210> 5

<211> 799

<212> DNA

<213> Unknown (Unknown)

<400> 5

ctagagcagc ttgccaacat ggtggagcac gacactctcg tctactccaa gaatatcaaa 60

gatacagtct cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga 120

aacctcctcg gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag 180

gaaggtggca cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc 240

tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa 300

gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg ataacatggt ggagcacgac 360

actctcgtct actccaagaa tatcaaagat acagtctcag aagaccaaag ggctattgag 420

acttttcaac aaagggtaat atcgggaaac ctcctcggat tccattgccc agctatctgt 480

cacttcatca aaaggacagt agaaaaggaa ggtggcacct acaaatgcca tcattgcgat 540

aaaggaaagg ctatcgttca agatgcctct gccgacagtg gtcccaaaga tggaccccca 600

cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat 660

tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac 720

cttcctctat ataaggaagt tcatttcatt tggagaggac acgctgaaat caccagtctc 780

tctctacaaa tctatctct 799

<210> 6

<211> 129

<212> DNA

<213> Unknown (Unknown)

<400> 6

tcaacacaac atatacaaaa caaacgaatc tcaagcaatc aagcattcta cttctattgc 60

agcaatttaa atcatttctt ttaaagcaaa agcaattttc tgaaaatttt caccatttac 120

gaacgatag 129

<210> 7

<211> 231

<212> DNA

<213> Unknown (Unknown)

<400> 7

atggctcaag ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60

tctaaatcca gtcaaaggaa atctccctta tcggtttctc tgaagactca gcagcatcca 120

cgagcttatc caatttcttc atcttgggga ttgaagaaga gtgggatgac tttaattggc 180

tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcggagaa g 231

<210> 8

<211> 185

<212> DNA

<213> Unknown (Unknown)

<400> 8

tttctccata ataatgtgtg agtagttccc agataaggga attagggttc ctatagggtt 60

tcgctcatgt gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt 120

ctatcaataa aatttctaat tcctaaaacc aaaatccagt actaaaatcc agatcccccg 180

aatta 185

<210> 9

<211> 700

<212> DNA

<213> Unknown (Unknown)

<400> 9

aagcttccag aaggtaatta tccaagatgt agcatcaaga atccaatgtt tacgggaaaa 60

actatggaag tattatgtga actcagcaag aagcagatca atatgcggca catatgcaac 120

ctatgttcaa aaatgaagaa tgtacagata caagatccta tactgccaga atacgaagaa 180

gaatacgtag aaattgaaaa agaagaacca ggcgaagaaa agaatcttga agacgtaagc 240

actgacgaca acaatgaaaa gaagaagata aggtcggtga ttgtgaaaga gacatagagg 300

acacatgtaa ggtggaaaat gtaagggcgg aaagtaacct tatcacaaag gaatcttatc 360

ccccactact tatcctttta tatttttccg tgtcactagt gaagacgtaa gcactgacga 420

caacaatgaa aagaagaaga taaggtcggt gattgtgaaa gagacataga ggacacatgt 480

aaggtggaaa atgtaagggc ggaaagtaac cttatcacaa aggaatctta tcccccacta 540

cttatccttt tatatttttc cgtgtcattt ttgcccttga gttttcctat ataaggaacc 600

aagttcggca tttgtgaaaa caagaaaaaa tttggtgtaa gctattttct ttgaagtact 660

gaggatacaa cttcagagaa atttgtaagt ttgtggatcc 700

<210> 10

<211> 266

<212> DNA

<213> Unknown (Unknown)

<400> 10

gtcgacgagc tcttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg 60

tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat 120

gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat 180

ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt 240

gtcatctatg ttactagatc ggtacc 266

<210> 11

<211> 1700

<212> DNA

<213> Unknown (Unknown)

<400> 11

ctcgagtcaa cacaacatat acaaaacaaa cgaatctcaa gcaatcaagc attctacttc 60

tattgcagca atttaaatca tttcttttaa agcaaaagca attttctgaa aattttcacc 120

atttacgaac gatagccatg gctcaagtta gcagaatctg caatggtgtg cagaacccat 180

ctcttatctc caatctctct aaatccagtc aaaggaaatc tcccttatcg gtttctctga 240

agactcagca gcatccacga gcttatccaa tttcttcatc ttggggattg aagaagagtg 300

ggatgacttt aattggctct gagcttcgtc ctcttaaggt catgtcttct gtttccacgg 360

cggagaaggg atccgacgct cttccagcta ccttcgacgt tatcgtgcat ccagctagag 420

aactcagagg tgaacttaga gcacagccat ccaagaacta caccactaga tacctcctcg 480

ccgctgctct cgctgagggt gaaaccagag ttgttggtgt ggctacctct gaggatgccg 540

aagctatgct cagatgcctc agagattggg gtgctggtgt tgagcttgtt ggtgatgacg 600

ccgtgatcag aggtttcggt gctagaccac aggctggtgt tacccttaac ccaggtaacg 660

ctgctgcggt ggccagactc cttatgggtg ttgctgctct cacctctggt acaactttcg 720

ttaccgatta ccctgattcc cttggtaaga gacctcaggg tgaccttctt gaagccctcg 780

aaagacttgg tgcttgggtg tcctccaacg atggtagact ccctatctcc gtttccggtc 840

cagttagagg tggtacagtg gaggtttccg ccgaaagatc ctcccagtac gcttccgccc 900

ttatgttcct cggtcctctt cttcctgacg gactcgaact tagactcacc ggtgatatca 960

agtcccacgc tcctcttaga cagacacttg acaccctctc tgatttcggt gttagagcta 1020

ctgcctccga tgaccttaga agaatctcca tccctggtgg tcagaagtac agaccaggta 1080

gagtgctcgt tcctggtgat taccctggtt ccgctgctat ccttaccgcc gctgctcttc 1140

tcccaggtga ggttagactt tctaacctta gagaacacga cctccagggt gagaaggaag 1200

ctgtgaacgt tcttagagag atgggtgctg atatcgttag agaaggtgat acccttaccg 1260

tgagaggtgg tagacctctc cacgctgtta ctagagatgg tgattccttc accgacgccg 1320

tgcaagctct taccgctgct gctgccttcg ctgagggtga taccacctgg gaaaacgttg 1380

ctactcttag actcaaggaa tgcgatagaa tctctgacac cagagctgag cttgaaagac 1440

ttggtcttag agcaagagag accgccgatt ctctctccgt tactggttct gctcaccttg 1500

ctggtggtat caccgctgat ggtcacggtg accacagaat gatcatgctt ctcacccttc 1560

ttggtctcag agcagatgct ccacttagaa tcaccggtgc acaccacatc agaaagtcct 1620

accctcagtt cttcgctcac cttgaagctc ttggtgctag attcgaatac gctgaggcta 1680

ccgcctaata ggagctcgag 1700

<210> 12

<211> 1827

<212> DNA

<213> Unknown (Unknown)

<400> 12

ggatccaaca atggataagg cttatgttgc tattctttct ttcgctttct tcttcgttct 60

tcattatctt attggaagag gaaacggaac ttggaaggct ggaaagggaa gacaacttcc 120

accatctcca ccagctcttc cacttattgg acatcttcat cttgttaaga ctccattcca 180

tgctgctctt gctagacttg ctgcttgcag aggaccagtt ttctctctta gaatgggatc 240

tagaccagct gttgtggttt cttctccaga ttgcgctaga gagtgcttca ctgatcatga 300

tgttgctttc gctaacagac cacttttccc aactcttcaa cttgtttctt tcaacggagc 360

tgctctttct actgcttctt atggaccata ttggagagat cttagaagag ttgcttctgt 420

tcatctcctt tctgctcata gagttaactg catggctgga actatttctg ctgaggttag 480

agctatggtt agaagaatgt ctagagctgc tgctgctgct ccaggaggag ctgctagaat 540

tgagcttaag agaagacttt tcgaggtttc tctttctgtt cttatggaga ctattgctag 600

aactaagact tctagaactg aggctgatga tgatactgat atgtctccag aggctagaga 660

gttcaagcaa attgttgatg agcttcttcc acatcttgga actgctaacc tttgggatta 720

tatgccagtt cttagatggt tcgatgtttt cggagttaga aagaagattg tttctgctgt 780

tagaaggaga gatgctttcc ttagacatct tgttgatgct gagagaacta gacttgatga 840

tggaaacgat gctggagaga agaagtctat tattgctatg cttctcactc ttcaaaagtc 900

tgagccagat gtttattctg atactatgat tatggctctt tgcggaaacc ttttcggagc 960

tggaactgag actacttcta ctacaactga gtgggctatg tctctccttc ttaaccatcc 1020

agagaagctc agaaaggctc aagctgagat tgatgctgtt gttggaactt ctagacttct 1080

tactgctgat gatatgccaa gacttactta tcttagatgc attattgatg agactatgag 1140

actttatcca gctgctccac ttctccttcc acatgagtct tctactcatt gcaaggttgg 1200

aggatatgat gttccagctg gaactatgct tcttgttaac gtttatgcta ttcatagaga 1260

tccagctgtt tgggatggac caactgagtt cgttccagag agattcgagg atggaaaggc 1320

tgagggaaga cttcttatgc cattcggaat gggaagaaga aagtgcccag gagagactct 1380

tgctcttaga actattggac ttgttcttgg aactcttatt caatgcttcg attgggatag 1440

agttgatgga cttgaggttg atatgactga gtctggagga cttactattc caagagctgt 1500

tccacttgag gctatgtgca gaccaagagc tactatgaga gaggttcttc aagagcttta 1560

agtcgacgag ctcttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620

gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680

tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740

tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800

tgtcatctat gttactagat cggtacc 1827

Claims

1. A multi-herbicide-resistant expression vector is characterized in that the vector is formed by inserting an glyphosate-resistant gene G10 expression frame into an XhoI site of a basic vector pCAMBIA1300 and inserting a multi-herbicide-resistant gene ZJ3-1 expression frame into an MCS site; the expression cassette of the glyphosate-resistant gene G10 comprises: the promoter CaMV35S of the transcription regulation region shown by SEQ ID NO. 5, the 5 'untranslated region shown by SEQ ID NO. 6, the Arabidopsis thaliana transduction peptide sequence CTP shown by SEQ ID NO. 7, the glyphosate resistant gene G10 shown by SEQ ID NO. 1 and the CaMV35S terminator at the 3' end shown by SEQ ID NO. 8 are arranged in sequence from the 5 'end to the 3' end; the expression cassette of the herbicide-resistant gene ZJ3-1 comprises: the promoter CSV of the transcription regulation region shown in SEQ ID NO. 9, the coding sequence of the multi-herbicide-resistant gene ZJ3-1 shown in SEQ ID NO. 3 and the NOS terminator sequence of the 3 ' end shown in SEQ ID NO. 10 are arranged in sequence from the 5 ' end to the 3 ' end.

2. A transformant containing the multi-herbicide-resistant expression vector of claim 1.

3. Use of the multi-herbicide-resistant expression vector of claim 1 for transforming plant cells.

4. Use according to claim 3, characterized in that the plant is soybean, maize, rice, rape, cotton, Arabidopsis thaliana.