CN114854787B

CN114854787B - Plant recombinant expression vector, construction method and application thereof

Info

Publication number: CN114854787B
Application number: CN202210396439.5A
Authority: CN
Inventors: 邱丽娟; 郭勇; 金龙国; 郭兵福
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2022-04-15
Filing date: 2022-04-15
Publication date: 2023-08-18
Anticipated expiration: 2042-04-15
Also published as: CN114854787A

Abstract

The invention provides a plant recombinant expression vector, a construction method and application thereof, and belongs to the technical field of genetic engineering. The plant recombinant expression vector pGR contains a herbicide-resistant gene expression frame, an insect-resistant gene expression frame and a stress-resistant gene expression frame. The herbicide-resistant gene expression frame comprises a CaMV35S promoter, a g2m-epsps gene with optimized codons and a CaMVPolyA terminator, the insect-resistant gene expression frame comprises a CaMV35S promoter, a cry1C gene with optimized codons and a NOS terminator, and the stress-resistant gene expression frame comprises a CaMV35S promoter, a soybean GmNFYB1 gene and a NOS terminator. The expression vector can be used for obtaining the herbicide-resistant, insect-resistant and stress-resistant composite transgenic plant, and has good application prospect in cultivating composite plant varieties.

Description

Plant recombinant expression vector, construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a plant recombinant expression vector, a construction method and application thereof.

Background

Grass damage, insect damage and abiotic stress are important factors influencing crop production, especially rural labor force transfer and large area per unit area of main areas in northeast China, the field management level is reduced, chemical weeding is mainly relied on in the aspect of weed control, and mechanical and manual weeding are assisted. If rainwater is concentrated or chemical weeding fails during seedling field management, the yield is reduced by more than 10% generally, and the yield is seriously abandoned. And the use of the traditional herbicide causes the problems of common weed damage, soil environment deterioration, serious phytotoxicity to crops and the like. The occurrence and presence of pests directly affect the yield and quality of crops, and it is often necessary to apply large amounts of pesticides to control the pests. If the pesticide spraying prevention and control are not performed, the yield reduction caused by insect damage reaches more than 10 percent, and the serious year yield reduction can reach more than 50 percent. In addition, drought and salt and alkali are also one of the main factors affecting crop production.

The transgenic crop with herbicide resistance, insect resistance and stress tolerance is cultivated by using the genetic engineering technology, and a better solution is provided for effectively preventing and controlling weeds and insect pests and coping with drought, saline-alkali and other stress. The glyphosate-tolerant herbicide characteristic enables the transgenic crops to be planted with high-efficiency herbicides with short residual effect period and wide herbicide control spectrum, and avoids the harm to the next crop, thereby realizing reasonable rotation. The cultivation and planting of the insect-resistant crops bring great economic and environmental benefits, and the use amount of the insecticide is greatly reduced. The transgenic stress-resistant variety is cultivated, so that the yield of soybean on drought and salinized soil can be increased, the idle lands can be effectively utilized, and a new way is opened for solving the problems of more population and less cultivated land in China.

The continuous emergence of the transgenic crops with the composite traits is the trend and the characteristics of the development of the transgenic industrialization, is also the trend of the future development of the transgenic crops, and meets the diversified demands of growers and consumers. The planting area of the transgenic crops with the composite insect resistance property in 2018 is increased by 4 percent, and the transgenic crops occupy 42 percent of the global planting area. The method for cultivating transgenic crops with complex characters is mainly 3 methods, namely, transforming the existing transgenic crops, connecting a plurality of genes to the same vector, transferring the genes into a receptor, and polymerizing the existing transgenic characters by using a conventional hybridization breeding technology. The transformation of existing transgenic crops and the polymerization of different shapes by using conventional crossbreeding technology have the problems of time consumption, complexity, low transformation efficiency, different screening marker genes and the like. The method of connecting multiple genes to the same vector and simultaneously carrying out genetic transformation is a relatively rapid method, and transgenic plants with multiple characters can be obtained simultaneously, but because of more gene expression frames and longer vector sequences, precise design is needed in the process of constructing the vector.

Disclosure of Invention

Therefore, the invention aims to provide a plant recombinant expression vector, a construction method and application thereof, herbicide-resistant, insect-resistant and stress-resistant genes are constructed into the same expression vector, and the plant is genetically transformed by using the expression vector, so that herbicide-resistant, insect-resistant and stress-resistant composite property transgenic plants can be cultivated, thereby providing a rapid method for developing and applying composite property transgenic crops, and having good application prospect

The invention provides a plant recombinant expression vector pGR which comprises a herbicide-resistant gene expression frame, an insect-resistant gene expression frame and a stress-resistant gene expression frame.

Preferably, the herbicide-resistant gene expression frame sequentially comprises a CaMV35S promoter, a glyphosate-resistant gene with optimized codons and a CaMV PolyA terminator;

the grass resistance Ganji is due to g2m-epsps;

the herbicide resistant gene expression frame is one of the following DNA fragments:

A. a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 1;

B. a DNA fragment complementary to SEQ ID NO. 1;

C. and (3) replacing, deleting or inserting one or more bases on the basis of the DNA fragment shown in SEQ ID NO. 1.

Preferably, the insect-resistant gene expression frame sequentially comprises a CaMV35S promoter, an insect-resistant gene with optimized codons and an NOS terminator; the insect-resistant genes comprise cry1C genes;

the nucleotide sequence of the insect-resistant gene expression frame is one of the following DNA fragments:

1) A DNA fragment with a nucleotide sequence shown as SEQ ID NO. 2;

2) A DNA fragment complementary to SEQ ID NO. 2;

3) And (3) replacing, deleting or inserting one or more bases on the basis of the DNA fragment shown in SEQ ID NO. 2.

Preferably, the stress-tolerant gene expression cassette sequentially comprises a CaMV35S promoter, a soybean stress-tolerant gene and an NOS terminator; the stress-tolerant genes comprise GmNFYB1 genes;

the stress-tolerant gene expression frame is one of the following DNA fragments:

a DNA fragment with the nucleotide sequence shown in SEQ ID NO. 3;

II DNA fragment complementary to SEQ ID NO. 3;

III substitution, deletion or insertion of one or several bases on the basis of the DNA fragment shown in SEQ ID NO. 3.

Preferably, the multiple cloning site of the herbicide-resistant gene expression cassette is XhoI;

the multiple cloning site of the insect-resistant gene expression frame is KpnI/HindIII;

the multiple cloning site of the stress-resistant gene expression frame is BglII/BstEII;

the backbone vector of the recombinant expression vector pGR includes pCAMBIA3301.

Preferably, the nucleotide sequence of pGR20 is shown as SEQ ID NO. 4.

The invention provides a construction method of the plant recombinant expression vector pGR, which comprises the following steps:

the GmNFYB1 gene fragment containing BglII and BstEII enzyme cutting sites and the commercial vector pCAMBIA3301 are respectively connected after double enzyme cutting by BglII and BstEII, so as to construct a p3301-NF vector;

the insect-resistant gene cry1C and a commercial vector pBI121 are respectively cut by BamHI and SacI and then connected to construct a pBI121-crylC vector;

the pBI121-crylC vector is used as a template, a specific primer with KpnI and HindIII enzyme cutting sites at two ends is utilized to amplify and obtain a fragment of an insect-resistant gene expression frame, and the fragment is connected with a p3301-NF vector which is cut by KpnI and HindIII enzyme, so as to construct a p3301-NF-cry1C plant expression vector;

the g2m-epsps fragment containing the XhoI enzyme cutting site synthesized by the sequence and the p3301-NF-cry1C vector are connected after being cut by the XhoI enzyme, so as to form a plant recombinant expression vector pGR containing the herbicide-resistant gene expression frame.

The invention provides application of the plant recombinant expression vector pGR or the plant recombinant expression vector pGR obtained by the construction method in cultivation of transgenic plants.

Preferably, the transgenic plant is a herbicide-resistant, insect-resistant and stress-resistant composite trait transgenic plant.

The invention provides a cultivation method of herbicide-resistant, insect-resistant and stress-resistant transgenic plants, which comprises the following steps: the plant recombinant expression vector pGR20 is introduced into a receptor plant to obtain a transgenic plant.

The invention provides a plant recombinant expression vector pGR which comprises a herbicide-resistant gene expression frame, an insect-resistant gene expression frame and a stress-resistant gene expression frame. The herbicide-resistant, insect-resistant and stress-resistant genes are constructed into the same expression vector, and the plant is subjected to genetic transformation by using the expression vector, so that the herbicide-resistant, insect-resistant and stress-resistant composite trait transgenic plant can be cultivated, a rapid method is provided for development and application of composite trait transgenic crops, and the method has good application prospects.

Drawings

FIG. 1 is a schematic diagram of a plant expression vector pGR provided by the invention;

FIG. 2 is T ₂ PCR detection of g2m-epsps, cry1C and GmNFYB1 genes in the transgenic soybean; m: DNA marker, lane 1: no template control, lane 2: a plasmid positive control; lane 3: a non-transgenic receptor control; lanes 4-13: transgenic soybean of different strains;

FIG. 3 is an identification of glyphosate tolerant herbicide characteristics of transgenic soybeans;

FIG. 4 is an identification of insect resistance of transgenic soybeans;

FIG. 5 is an identification of drought tolerance of transgenic soybeans.

Detailed Description

In the present invention, the herbicide-resistant gene expression cassette preferably comprises a CaMV35S promoter, a glyphosate-resistant gene with optimized codons, and a CaMV PolyA terminator in that order. The glyphosate-tolerant gene is preferably g2m-epsps. The glyphosate-resistant gene expression frame is one of the following DNA fragments:

A. a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 1;

B. a DNA fragment complementary to SEQ ID NO. 1;

The glyphosate-resistant gene expression frame can efficiently express the g2m-epsps gene, so that a genetic transformation system shows stronger herbicide-resistant property.

In the present invention, the insect-resistant gene expression cassette preferably comprises a CaMV35S promoter, an insect-resistant gene optimizing codons, and an NOS terminator in this order. The insect-resistant gene preferably includes cry1C gene. The nucleotide sequence of the insect-resistant gene expression frame is one of the following DNA fragments: 1) A DNA fragment with a nucleotide sequence shown as SEQ ID NO. 2;

2) A DNA fragment complementary to SEQ ID NO. 2;

The insect-resistant gene expression frame can efficiently express cry1C genes, so that a genetic transformation system shows stronger insect-resistant characteristics.

In the present invention, the stress-tolerant gene expression cassette preferably comprises a CaMV35S promoter, a soybean gene and an NOS terminator in this order. The stress-tolerant gene preferably comprises the GmNFYB1 gene. The stress-tolerant gene expression frame is one of the following DNA fragments: a DNA fragment with the nucleotide sequence shown in SEQ ID NO. 3;

II DNA fragment complementary to SEQ ID NO. 3;

The stress-tolerant gene expression frame can efficiently express GmNFYB1 genes, so that a genetic transformation system shows stronger stress-tolerant characteristics. The stress tolerance is preferably drought tolerance.

In the present invention, the multiple cloning site of the glyphosate-tolerant gene expression cassette is preferably XhoI. The multiple cloning site of the insect-resistant gene expression frame is preferably KpnI/HindIII; the multiple cloning site of the stress-tolerant gene expression cassette is preferably BglII/BstEII. The backbone vector of the recombinant expression vector pGR includes pCAMBIA3301. The nucleotide sequence of pGR is preferably shown in SEQ ID NO. 4.

the insect-resistant gene cry1C containing BamHI and SacI double enzyme cutting sites and a commercial vector pBI121 are respectively connected after being cut by BamHI and SacI double enzyme, so as to construct a pBI121-crylC vector;

the pBI121-crylC vector is used as a template, a specific primer with KpnI and HindIII enzyme cutting sites at two ends is utilized to amplify and obtain a fragment of an insect-resistant gene expression frame, and the fragment is connected with a p3301-NF vector after KpnI and HindIII enzyme cutting to construct a p3301-NF-cry1C plant expression vector;

In the invention, the GmNFYB1 gene fragment containing BglII and BstEII enzyme cutting sites is preferably obtained by amplifying a primer pair shown in SEQ ID NO. 11 and SEQ ID NO. 12, and the nucleotide sequence of the obtained fragment is shown in SEQ ID NO. 13. The amplification system is preferably 10 XBuffer 3. Mu. L, dNTP 3. Mu. L, mgCl as follows ₂ 3. Mu.L of each of the upstream and downstream primers, 0.5. Mu.L of KOD Plus Neo, 1. Mu.L of the template and the balance ddH ₂ O. The procedure for amplification is preferably as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 58℃for 30s, elongation at 68℃for 1min,34 cycles; extending at 72℃for 5min. The reaction system of the double digestion is preferably 10 mu L of PCR recovery product, 10 XBuffer 2 mu L, bglII 1 mu L, bstEII 1 mu L and the rest ddH ₂ O. The reaction conditions for the double cleavage are preferably 37℃overnight. The said connectionThe grafting is preferably performed with T4 ligase. The conditions for the ligation are preferably 16℃for 4 hours. After the ligation, verification of the recombinant vector is preferably performed. The method of the present invention is not particularly limited, and the method of the present invention may be performed by any method known in the art, such as culturing in transformed E.coli, extracting plasmids, and performing colony PCR amplification or sequencing. The source of the commercial vector pCAMBIA3301 is not particularly limited in the present invention, and the commercial vector pCAMBIA3301 known in the art may be used. The commercial vector pCAMBIA3301 in the examples of the present invention was purchased from Cambia corporation.

In the invention, the insect-resistant gene cry1C containing BamHI and SacI double enzyme cutting sites is preferably obtained by amplifying a primer pair shown as SEQ ID NO. 14 and SEQ ID NO. 15, and the nucleotide sequence of the obtained fragment is shown as SEQ ID NO. 16. The amplification system is preferably 10 XBuffer 3. Mu. L, dNTP 3. Mu. L, mgCl as follows ₂ 3. Mu.L of each of the upstream and downstream primers, 0.5. Mu.L of KOD Plus Neo, 1. Mu.L of the template and the balance of ddH ₂ O. The procedure for amplification is preferably as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 58℃for 30s, elongation at 68℃for 1min,34 cycles; extending at 72℃for 5min. The double cleavage reaction system is preferably 10. Mu.L, 10 XBuffer 2. Mu.L, bamHI 1. Mu.L, sacI 1. Mu.L and the balance ddH of the PCR recovered product ₂ O. The reaction conditions for the double cleavage are preferably 37℃overnight. The ligation is preferably performed with a T4 ligase. The conditions for the ligation are preferably 16℃for 4 hours. After the ligation, verification of the recombinant vector is preferably performed. The method of the present invention is not particularly limited, and the method of the present invention may be performed by any method known in the art, such as culturing in transformed E.coli, extracting plasmids, and performing colony PCR amplification or sequencing. The source of the commercial vector pBI121 is not particularly limited in the present invention, and the commercial vector pBI121 known in the art may be used. The commercial vector pBI121 was purchased from Clontech in the examples of the present invention.

In the invention, the specific primers with KpnI and HindIII cleavage sites at the two ends are shown as SEQ ID NO. 17 and SEQ ID NO. 18. When the fragment of the insect-resistant gene expression cassette obtained by amplification is connected with the p3301-NF carrier after the cleavage of KpnI and HindIII, the connection condition is preferably that the connection is carried out at 16 ℃ for 4 hours. After the ligation, verification of the recombinant vector is preferably performed. The method of the present invention is not particularly limited, and the method of the present invention may be performed by any method known in the art, such as culturing in transformed E.coli, extracting plasmids, and performing colony PCR amplification or sequencing.

In the invention, the g2m-epsps fragment containing the XhoI enzyme cutting site synthesized by the sequence and the p3301-NF-cry1C vector are connected after being cut by the XhoI enzyme, so as to form the plant recombinant expression vector pGR containing the herbicide-resistant gene expression frame. The present invention is not particularly limited to sequence synthesis of g2m-epsps fragments containing XhoI cleavage sites, and may employ sequence synthesis techniques well known in the art. In the examples of the present invention, the g2m-epsps fragment containing the XhoI cleavage site was synthesized by Songhong Biotech Co., ltd.

In the present invention, the transgenic plant is preferably a transgenic plant with herbicide-resistant, insect-resistant and stress-tolerant complex traits.

In the present invention, the transgenic plants preferably include screening. The screening includes PCR detection of the target gene. The primer for PCR detection of the target gene comprises g2m-F/g2m-R, cry1C-F/cry1C-R, NFYB1-F/NFYB1-R. The PCR reaction conditions: denaturation at 94℃for 3min, denaturation at 94℃for 30s, annealing at 61-63℃for 30s, elongation at 72℃for 30s,35 cycles, and elongation at 72℃for 8min after the cycle.

In the invention, the transgenic plants obtained by screening are subjected to character screening, and herbicide resistance identification results show that the transgenic soybeans are not inhibited in growth, the leaves are not fading, shrink and have no glyphosate phytotoxicity reactions such as new leaf yellowing, and the transgenic soybeans show high resistance to glyphosate; the insect resistance identification result shows that the damage of the receptor contrast leaf is serious and is expressed as insect sensing; the transgenic soybean leaves remain intact and show high resistance to prodenia litura; the stress tolerance identification result shows that the drought resistance of the transgenic soybean in the seedling stage is obviously higher than that of a receptor.

The following describes a plant recombinant expression vector, its construction method and application in detail with reference to examples, but they should not be construed as limiting the scope of the invention.

Example 1

Construction method of plant expression vector pGR20

(1) The GmNFYB1 (Glyma.10G192000.1) gene was amplified by PCR from the cDNA of the soybean variety Williams 82 using gene specific primers with BglII and BstEII cleavage sites. The amplified GmNFYB1 gene fragment and a commercial vector pCAMBIA3301 are respectively connected after BglII and BstEII double enzyme digestion, a p3301-NF vector is constructed, a stress-tolerant gene expression frame containing CaMV35S-GmNFYB1-NOS elements is formed, and the nucleotide sequence is shown in SEQ ID NO:3.

(2) The insect-resistant gene cry1C and a commercial vector pBI121 are respectively digested with BamHI and SacI and then connected to construct a pBI121-crylC vector, so as to form an insect-resistant gene expression frame containing CaMV 35S-cry1C-NOS components, and the nucleotide sequence is shown in SEQ ID NO:2.

(3) From the constructed pBI121-crylC vector, a fragment of an insect-resistant gene expression frame with the size of 3.5kb and containing CaMV 35S-cry1C-NOS components is amplified by utilizing a specific primer with KpnI and HindIII cleavage sites at two ends, and is connected with a p3301-NF vector which is cleaved by KpnI and HindIII, so as to construct a p3301-NF-cry1C plant expression vector.

(4) The g2m-epsps fragment containing XhoI cleavage site synthesized by the sequence and the p3301-NF-cry1C vector are connected after being digested by XhoI to form a herbicide-resistant gene expression frame containing CaMV 35S-cry1C-PolyA element, and the nucleotide sequence is shown in SEQ ID NO:1. finally obtaining pGR plant expression vector, wherein the schematic diagram of the vector is shown in figure 1, the nucleotide sequence is shown in SEQ ID NO:4.

example 2

Application of plant expression vector in cultivation of transgenic plants

1. pGR20 vector transformed soybean

Adopts an agrobacterium-mediated soybean cotyledonary node transformation method, and the agrobacterium strain is Ag10. The soybean cotyledonary node is sterilized, then the callus is induced, and is co-cultured with agrobacterium to infect the callus, and the transformed callus is selected, and then plant regeneration is performed on selective culture.

Obtaining of sterile explants: explants are prepared by selecting normal sprouting pollution-free seedlings. Cutting soybean along hypocotyl with a surgical knife in an ultra clean workbench, reserving 3-4mm hypocotyl, placing in a sterile culture dish, adding appropriate amount of co-culture solution to facilitate peeling seed coat, cutting the hypocotyl vertically along cotyledon hypocotyl, removing clean true leaf tissue, making 5-7 cuts axially at the joint of cotyledon and cotyledon hypocotyl, and making cuts about 3-4mm long. Each explant consists of a cotyledon connected with a hypocotyl, and two explants can be formed by one seed.

Preparation of agrobacterium: the agrobacterium strain preserved at low temperature is taken out from the ultra-low temperature refrigerator and frozen and thawed on ice, a small amount of strain is dipped by an inoculating loop or a sterile gun head to be inoculated on an LB plate, and the LB solid culture medium contains 50mg/L of corresponding antibiotics. The cells were cultured upside down at 28℃for 1-2d to obtain a monoclonal antibody. After 2d, the monoclonal was picked up and inoculated in 5ml of YEP medium (containing the corresponding antibiotic) and grown at 220rpm at 28℃overnight (about 12 h). When the bacterial liquid is activated to a saturated state for the first time, 1ml of bacterial liquid is extracted from the bacterial liquid, inoculated into a triangular shaking flask containing 100ml of YEP (containing corresponding antibiotics) and activated for the second time under the conditions of 28 ℃ and 220 rpm. After the agrobacterium is fully activated to OD ₆₀₀ When about 1.0, the bacterial solution was centrifuged at 4000rpm at 4℃for 10min, the supernatant was discarded to collect the pellet, and the pellet was suspended in an equal volume of co-culture solution at the bottom of the tube, at which time the OD ₆₀₀ About 0.5 to about 0.8 for standby.

Co-culturing the explant and agrobacterium: when the explant is prepared, every 40-60 pieces of the explant are placed in a 100ml triangular flask, about 50ml of re-suspended agrobacterium liquid is added into each flask, and the bacterial liquid needs to be over the explant. Co-infects under dark or dim light for 30-35min, shake the flask once every 5min to allow sufficient contact of Agrobacterium with the explant. After infection is completed, the excess agrobacterium liquid is carefully poured out, a layer of sterile filter paper is paved on a co-culture medium, the impregnated explant is paved on the filter paper downwards towards one side of the axis, and co-culture is carried out for 3d under the condition of darkness or low light at 24 ℃.

Resistance screening and regeneration: after co-culturing for 3d, the explant is subjected to the stages of resistance cluster bud induction, elongation bud induction, rooting and the like to obtain a regenerated plant; and respectively adding a proper resistance screening agent in the clustered bud induction and elongation stage, and adding a proper concentration of agrobacterium bacteriostat and IBA (IBA) to induce rooting according to the practical requirement in an experiment in the rooting stage, wherein the clustered bud induction and elongation bud induction stage is carried out once every two weeks for the explant, and a new incision is prepared on the back of the explant during the subculture so that the explant can absorb nutrients better. The elongated buds are elongated to 4-6cm and can induce rooting, and after rooting, the sealing film opening on the culture dish is uncovered for hardening seedlings for 1-3 days; transplanting the plants to a pot plant or a field for growth to obtain transgenic plants.

2. PCR detection of transgenic soybean

Extraction of genomic DNA: taking 0.1g of homogenized sample into a 1.5ml centrifuge tube, adding 500 μl of CTAB extract, and incubating at 65deg.C for 30min; adding equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, V/V), uniformly mixing, centrifuging for 10min at 13000r/min, taking supernatant, and repeating the above steps for 1-2 times; adding twice volume of CTAB precipitation solution into the supernatant, and incubating for 30min at room temperature; after centrifugation, the precipitate was dissolved with 350. Mu.l NaCl (1.2 mol/L), chloroform was added, and mixed well; transferring the supernatant to a new centrifuge tube after centrifugation; adding 0.6 times volume of isopropanol and 1/10 times volume of 3mol/L NH ₄ Ac (pH 6.8), fully and uniformly mixing, and standing at low temperature for 15min; washing the precipitate with 70% ethanol after centrifugation, and drying at room temperature; dissolving DNA in 50. Mu.l TE buffer, adding 1. Mu.l RNaseA (10 mg/ml), incubating at 37℃for 15min, and preserving at-20 ℃; the DNA concentration was measured by Spectrophotometer ND-1000 and the amount to be applied was 1.5. Mu.l each time. Agarose electrophoresis to detect DNA concentration: preparing 0.8% agarose electrophoresis liquid, and adding proper EB. Electrophoresis conditions u=120v, i=140 mA.

Transgenic strain PCR detection: the target genes of the transgenic soybean lines were detected using specific primers (table 1). PCR reaction conditions: denaturation at 94℃for 3min, denaturation at 94℃for 30s, annealing at 61-63℃for 30s, elongation at 72℃for 30s,35 cycles, and elongation at 72℃for 8min after the cycle. The PCR amplification product was taken in 10. Mu.l, and subjected to electrophoresis detection (containing ethidium bromide) by 1.5% agarose gel, electrophoresis at 5V/cm for 40min, and observation under an ultraviolet lamp was performed.

TABLE 1 list of transgenic detection primers

And extracting genome DNA of the transgenic soybean plant as a template, and carrying out PCR detection on g2m-epsps, cry1C and GmNFYB1 target genes. The detection results are shown in FIG. 2. The results indicate that the presence of the gene of interest can be detected in the transgenic material, indicating that the exogenous gene has been stably integrated into the soybean genome.

Example 3

Transgenic plant herbicide resistance, insect resistance and stress tolerance function detection

To identify the resistance of transgenic plants, herbicide, pest and drought resistance assays were performed on transgenic plants that were positive for PCR detection.

1. Herbicide resistance identification

The transgenic soybeans and the receptor seeds thereof with positive PCR detection are sown in a pot, and after the seedlings grow to 2 leaves, the glyphosate herbicide is sprayed in the period of complete unfolding, the application amount of the glyphosate herbicide is 2 times of the dosage in pesticide registration (1800 g/hectare of active ingredient), and the seedlings are investigated after 2 weeks of pesticide application. The results show that the receptor varieties are not resistant under the spraying dosage, and the plants die after 2 weeks; the transgenic soybeans are not inhibited in growth, leaves are not faded, shrink and have no glyphosate phytotoxicity reaction such as new leaf yellowing, and the transgenic soybeans show that the transgenic soybeans show high resistance to glyphosate (figure 3).

2. Insect resistance identification

The method comprises the steps of collecting the unfolding leaves with consistent health and fresh tenderness on transgenic soybean and receptor plants, punching the leaves into 2.5cm wafers by a puncher, selecting 6 leaves, placing the wafers into a culture dish paved with sponge, moisturizing the wafers by 13mL of sterile water, inoculating 2-year-old prodenia litura larvae according to the proportion of 2 heads/dish, and carrying out result investigation analysis after 7 days. The result shows that the damage of the receptor control leaves is serious and is expressed as pest sensing; while the transgenic soybean leaves remained intact and exhibited high resistance to prodenia litura (figure 4).

3. Stress tolerance identification

The transgenic soybeans and the recipients are sown in a greenhouse in a potting mode, so that the soil content in each pot is consistent, and 10 seeds of each material are obtained. Thinning after emergence of seedlings, and ensuring that the number of seedlings in each pot is the same. Stopping watering when the plants grow to two three-leaf compound period, and starting natural drought treatment. The results indicate that the drought resistance of transgenic soybean in seedling stage is significantly higher than that of the receptor (figure 5).

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

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cgctgctctc gctaagggca cttcaaggct gtccggggct ctcaagtctg acgatacccg 1500

gcacatgtca gtcgccctgc gccagatggg cgttaccatc gacgagcccg acgataccac 1560

gttcgtggtc acaagccagg gctcgctgca gctcccagct cagcctctgt tcctcggcaa 1620

cgctgggact gcgatgaggt tcctgaccgc tgctgtcgct actgttcagg gcaccgttgt 1680

gctcgacggg gatgagtaca tgcagaagag gcccatcggc ccactgctcg ctacgctcgg 1740

ccagaatggg attcaggtcg actccccaac gggctgccca ccagttacag tgcacggcat 1800

ggggaaggtg caggcgaagc gcttcgagat cgacggcggg ctgtccagcc agtacgtcag 1860

cgctctgctc atgctcgctg cttgcggcga ggctcctatc gaggtggctc tgacagggaa 1920

ggacattggc gctaggggct acgtcgacct gaccctcgat tgcatgaggg ctttcggcgc 1980

tcaggtggat gctgtcgacg atacaacttg gagggtggct ccaactggct acaccgctca 2040

tgactacctc atcgagcctg atgcttccgc tgctacttac ctgtgggctg ctgaggttct 2100

caccggcggg aggatcgaca ttggcgtggc tgctcaggac ttcacccagc cagatgctaa 2160

ggcccaggcg gtcatcgcgc agttccccaa catgcaggct actgtcgttg gctcccagat 2220

gcaggacgct attccaacgc tggccgtgct cgccgcgttc aacaatacgc ctgtccgctt 2280

cacagagctg gcgaacctcc gcgttaagga gtgcgacagg gtgcaggccc tgcatgatgg 2340

cctcaatgag atcaggccag gcctggctac gattgagggg gacgatctgc tcgtggcttc 2400

ggacccagct ctggctggca cggcgtgcac agctctcatc gacacccacg cggatcatcg 2460

gattgctatg tgcttcgcgc tggctggcct caaagtcagc gggatccgca ttcaggaccc 2520

ggattgcgtt gccaagactt accctgatta ctggaaggct ctggcgtccc tgggcgttca 2580

cctcaatgat tagcgatcga caagctcgag 2610

<210> 2

<211> 3075

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

gagctcggta ccagattagc cttttcaatt tcagaaagaa tgctaaccca cagatggtta 60

gagaggctta cgcagcaggt ctcatcaaga cgatctaccc gagcaataat ctccaggaaa 120

tcaaatacct tcccaagaag gttaaagatg cagtcaaaag attcaggact aactgcatca 180

agaacacaga gaaagatata tttctcaaga tcagaagtac tattccagta tggacgattc 240

aaggcttgct tcacaaacca aggcaagtaa tagagattgg agtctctaaa aaggtagttc 300

ccactgaatc aaaggccatg gagtcaaaga ttcaaataga ggacctaaca gaactcgccg 360

taaagactgg cgaacagttc atacagagtc tcttacgact caatgacaag aagaaaatct 420

tcgtcaacat ggtggagcac gacacacttg tctactccaa aaatatcaaa gatacagtct 480

cagaagacca aagggcaatt gagacttttc aacaaagggt aatatccgga aacctcctcg 540

gattccattg cccagctatc tgtcacttta ttgtgaagat agtggaaaag gaaggtggct 600

cctacaaatg ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca 660

gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa 720

ccacgtcttc aaagcaagtg gattgatgtg atatctccac tgacgtaagg gatgacgcac 780

aatcccacta tccttcgcaa gacccttcct ctatataagg aagttcattt catttggaga 840

gaacacgggg gactctagag gatccaccat ggaggagaac aatcagaacc agtgtatccc 900

ttacaattgt ctttctaatc ctgaagaagt tcttttggat ggagaaagga tctcaactgg 960

taactcatca attgacatct ctctctcact tgttcagttc ttggtttcta actttgtgcc 1020

aggaggagga ttccttgttg gacttatcga cttcgtttgg ggaatcgttg gaccttctca 1080

atgggatgca tttctcgttc agatcgaaca gctcatcaac gaaagaatcg ctgagttcgc 1140

taggaatgct gctattgcta accttgaagg acttggaaac aacttcaaca tctacgtgga 1200

ggcattcaag gaatgggaag aagatcctaa caacccagca accaggacca gagtgatcga 1260

taggttccgt atccttgatg gacttcttga aagggacatt cctagcttta ggatctctgg 1320

atttgaagtt ccacttctct ctgtttacgc tcaagctgct aatctccatc ttgctatcct 1380

tagagattct gtgatcttcg gagaaagatg gggattgaca accatcaacg tgaacgagaa 1440

ctacaacaga ctcatcaggc acatcgatga gtacgctgat cactgtgcta acacttacaa 1500

ccgtggactc aacaaccttc ctaagtctac ctatcaagat tggatcacat acaaccgact 1560

taggagagac cttacattga ctgttcttga tatcgctgct ttctttccaa actatgacaa 1620

taggagatat ccaattcagc cagttggtca acttacaagg gaagtttaca ctgacccact 1680

catcaacttc aacccacagc ttcagtctgt tgctcagctt cctaccttca acgttatgga 1740

gagcagcgca atcagaaatc ctcacctctt cgacatcttg aacaacctta caatctttac 1800

cgattggttt agtgttggac gtaacttcta ctggggagga catcgagtga tctctagcct 1860

catcggaggt ggtaacatca catctcctat ctacggaaga gaggctaacc aggagcctcc 1920

aagatcattc actttcaacg gacctgtgtt caggactctt tcaaatccta ctcttcgact 1980

tcttcagcaa ccttggccag ctccaccatt caaccttcgt ggtgttgaag gagttgagtt 2040

ctctacacct acaaacagct tcacctatcg tggaagaggt actgttgatt ctcttactga 2100

acttccacct gaggaaacag tgtgccacct cgtgaaggat acagtcatcg tctttgtcat 2160

gcaaccttcg ttcaaagatc tggaacacct ttccttacaa ctggtgttgt gttctcttgg 2220

actcatcgta gtgcaactct taccaacaca attgatccag agaggatcaa ccagatccct 2280

cttgtgaaag gattcagagt ttggggagga acctctgtga ttacaggacc aggattcaca 2340

ggaggtgata tccttcgaag aaacaccttt ggtgacttcg tttctcttca agtgaacatc 2400

aactcaccaa tcacccaaag ataccgtctt agatttcgtt acgcttctag tagggatgca 2460

cgagttatcg ttcttacagg agctgcatct acaggagtgg gaggtcaagt tagtgtgaac 2520

atgcctcttc agaaaactat ggagatcgga gagaacctca catctagaac attcagatac 2580

accgacttca gtaatccttt ctcattcaga gctaatccag acatcatcgg tatcagtgaa 2640

caacctctct tcggtgcagg ttctatcagt agcggtgaac tttacatcga caagatcgag 2700

atcatccttg cagatgcaac atttgaagca gaatctgacc ttgaaagagc acaaaagtag 2760

tgaccaacgt atttatatca gaaaatagat gagtcgaaat taaaagctta tacccgttaa 2820

tgagatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt 2880

gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa 2940

tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa 3000

tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca 3060

tctatgttac tagat 3075

<210> 3

<211> 1319

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ccctaggcat ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg 60

gcgaacagtt catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca 120

tggtggagca cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc 180

aaagggcaat tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt 240

gcccagctat ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat 300

gccatcattg cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca 360

aagatggacc cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt 420

caaagcaagt ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact 480

atccttcgca agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg 540

ggactatgtc ggatgcaccg gcgagtccga gtcacgagag tggtggcgag cagagccctc 600

gcggctcgtt gtccggcgcg gctagagagc aggaccggta ccttcccatt gccaacatca 660

gccgcatcat gaagaaggct ctgcctccca atggcaagat tgcgaaggat gcaaaagaca 720

caatgcaaga atgcgtttct gaattcatca gcttcattac cagcgaggcg agtgagaaat 780

gccagaagga gaagagaaag acaatcaatg gagacgattt actatgggcc atggcaactt 840

tagggtttga agactacatt gagccgctta aggtgtacct ggctaggtac agagaggcgg 900

agggtgacac taaaggatct gctagaagtg gtgatggatc tgctagacca gatcaagttg 960

gccttgcagg tcaaaatgct cagcttgttc atcagggttc gctgaactat attggtttgc 1020

aggtgcaacc acaacatctg gttatgcctt caatgcaagg ccatgaatag cgttcaaaca 1080

tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat 1140

aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg acgttattta 1200

tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg atagaaaaca 1260

aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg ttactagat 1319

<210> 4

<211> 13578

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60

atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120

agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180

gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240

agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300

ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360

ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420

acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480

ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540

acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600

agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660

tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720

tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780

ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840

gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900

gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960

cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020

ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080

gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140

tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200

aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260

aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320

ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380

ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440

cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500

atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560

accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620

gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680

gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740

ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800

cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860

aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920

gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980

agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2040

ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100

atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160

accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220

tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280

cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340

gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400

tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460

cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520

gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 2580

tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640

cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 2700

gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760

gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820

tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880

tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940

agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000

gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060

gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120

ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180

cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240

aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3300

catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360

gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420

tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480

cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540

aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600

ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660

gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720

aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780

actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840

aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900

ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960

tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020

gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080

ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140

gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200

ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260

ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320

acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380

tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440

ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500

ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620

actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680

gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740

tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800

caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860

atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920

acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980

atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040

ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100

gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160

gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220

ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280

gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340

taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400

cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460

gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520

gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580

atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640

tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700

tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760

tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820

aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880

ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940

caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000

gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060

tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120

cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180

tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240

taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300

gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360

ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420

ggaactactc acacattatt atggagaaac tcgagcttgt cgatcgctaa tcattgaggt 6480

gaacgcccag ggacgccaga gccttccagt aatcagggta agtcttggca acgcaatccg 6540

ggtcctgaat gcggatcccg ctgactttga ggccagccag cgcgaagcac atagcaatcc 6600

gatgatccgc gtgggtgtcg atgagagctg tgcacgccgt gccagccaga gctgggtccg 6660

aagccacgag cagatcgtcc ccctcaatcg tagccaggcc tggcctgatc tcattgaggc 6720

catcatgcag ggcctgcacc ctgtcgcact ccttaacgcg gaggttcgcc agctctgtga 6780

agcggacagg cgtattgttg aacgcggcga gcacggccag cgttggaata gcgtcctgca 6840

tctgggagcc aacgacagta gcctgcatgt tggggaactg cgcgatgacc gcctgggcct 6900

tagcatctgg ctgggtgaag tcctgagcag ccacgccaat gtcgatcctc ccgccggtga 6960

gaacctcagc agcccacagg taagtagcag cggaagcatc aggctcgatg aggtagtcat 7020

gagcggtgta gccagttgga gccaccctcc aagttgtatc gtcgacagca tccacctgag 7080

cgccgaaagc cctcatgcaa tcgagggtca ggtcgacgta gcccctagcg ccaatgtcct 7140

tccctgtcag agccacctcg ataggagcct cgccgcaagc agcgagcatg agcagagcgc 7200

tgacgtactg gctggacagc ccgccgtcga tctcgaagcg cttcgcctgc accttcccca 7260

tgccgtgcac tgtaactggt gggcagcccg ttggggagtc gacctgaatc ccattctggc 7320

cgagcgtagc gagcagtggg ccgatgggcc tcttctgcat gtactcatcc ccgtcgagca 7380

caacggtgcc ctgaacagta gcgacagcag cggtcaggaa cctcatcgca gtcccagcgt 7440

tgccgaggaa cagaggctga gctgggagct gcagcgagcc ctggcttgtg accacgaacg 7500

tggtatcgtc gggctcgtcg atggtaacgc ccatctggcg cagggcgact gacatgtgcc 7560

gggtatcgtc agacttgaga gccccggaca gccttgaagt gcccttagcg agagcagcga 7620

gcaggagagc cctatttgtg atagacttcg acccaggtgg agccaccttg cccgtcagtg 7680

ggaagttggg cggtgtgacc gtcacggtct tctggctgct gagagtgcaa ggctcctggt 7740

cgaggcgagg cggcgtgcta tgcccaacgt gaggccccga atcatctggg aggcaagcca 7800

tgcatgccgt ggaaacagaa gacatgacct taagaggacg aagctcagag ccaattaacg 7860

tcatcccact cttcttcaat ccccacgacg acgaaatcgg ataagctcgt ggatgctgct 7920

gcgtcttcag agaaaccgat aagggagatt tgcgttgact ggatttcgag agattggaga 7980

taagagatgg gttctgcaca ccattgcaga ttctgctaac ttgcgccata gagatagatt 8040

tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac ttccttatat 8100

agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc agtggagata 8160

tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc 8220

ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg aacgatagcc 8280

tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg 8340

atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt 8400

tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag 8460

acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga agacgtggtt 8520

ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg 8580

tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg 8640

ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg 8700

aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag 8760

actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat gttggcaagc 8820

tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 8880

ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 8940

agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 9000

gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattcg 9060

agctcggtac cagattagcc ttttcaattt cagaaagaat gctaacccac agatggttag 9120

agaggcttac gcagcaggtc tcatcaagac gatctacccg agcaataatc tccaggaaat 9180

caaatacctt cccaagaagg ttaaagatgc agtcaaaaga ttcaggacta actgcatcaa 9240

gaacacagag aaagatatat ttctcaagat cagaagtact attccagtat ggacgattca 9300

aggcttgctt cacaaaccaa ggcaagtaat agagattgga gtctctaaaa aggtagttcc 9360

cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt 9420

aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt 9480

cgtcaacatg gtggagcacg acacacttgt ctactccaaa aatatcaaag atacagtctc 9540

agaagaccaa agggcaattg agacttttca acaaagggta atatccggaa acctcctcgg 9600

attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc 9660

ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag 9720

tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 9780

cacgtcttca aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca 9840

atcccactat ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag 9900

aacacggggg actctagagg atccaccatg gaggagaaca atcagaacca gtgtatccct 9960

tacaattgtc tttctaatcc tgaagaagtt cttttggatg gagaaaggat ctcaactggt 10020

aactcatcaa ttgacatctc tctctcactt gttcagttct tggtttctaa ctttgtgcca 10080

ggaggaggat tccttgttgg acttatcgac ttcgtttggg gaatcgttgg accttctcaa 10140

tgggatgcat ttctcgttca gatcgaacag ctcatcaacg aaagaatcgc tgagttcgct 10200

aggaatgctg ctattgctaa ccttgaagga cttggaaaca acttcaacat ctacgtggag 10260

gcattcaagg aatgggaaga agatcctaac aacccagcaa ccaggaccag agtgatcgat 10320

aggttccgta tccttgatgg acttcttgaa agggacattc ctagctttag gatctctgga 10380

tttgaagttc cacttctctc tgtttacgct caagctgcta atctccatct tgctatcctt 10440

agagattctg tgatcttcgg agaaagatgg ggattgacaa ccatcaacgt gaacgagaac 10500

tacaacagac tcatcaggca catcgatgag tacgctgatc actgtgctaa cacttacaac 10560

cgtggactca acaaccttcc taagtctacc tatcaagatt ggatcacata caaccgactt 10620

aggagagacc ttacattgac tgttcttgat atcgctgctt tctttccaaa ctatgacaat 10680

aggagatatc caattcagcc agttggtcaa cttacaaggg aagtttacac tgacccactc 10740

atcaacttca acccacagct tcagtctgtt gctcagcttc ctaccttcaa cgttatggag 10800

agcagcgcaa tcagaaatcc tcacctcttc gacatcttga acaaccttac aatctttacc 10860

gattggttta gtgttggacg taacttctac tggggaggac atcgagtgat ctctagcctc 10920

atcggaggtg gtaacatcac atctcctatc tacggaagag aggctaacca ggagcctcca 10980

agatcattca ctttcaacgg acctgtgttc aggactcttt caaatcctac tcttcgactt 11040

cttcagcaac cttggccagc tccaccattc aaccttcgtg gtgttgaagg agttgagttc 11100

tctacaccta caaacagctt cacctatcgt ggaagaggta ctgttgattc tcttactgaa 11160

cttccacctg aggaaacagt gtgccacctc gtgaaggata cagtcatcgt ctttgtcatg 11220

caaccttcgt tcaaagatct ggaacacctt tccttacaac tggtgttgtg ttctcttgga 11280

ctcatcgtag tgcaactctt accaacacaa ttgatccaga gaggatcaac cagatccctc 11340

ttgtgaaagg attcagagtt tggggaggaa cctctgtgat tacaggacca ggattcacag 11400

gaggtgatat ccttcgaaga aacacctttg gtgacttcgt ttctcttcaa gtgaacatca 11460

actcaccaat cacccaaaga taccgtctta gatttcgtta cgcttctagt agggatgcac 11520

gagttatcgt tcttacagga gctgcatcta caggagtggg aggtcaagtt agtgtgaaca 11580

tgcctcttca gaaaactatg gagatcggag agaacctcac atctagaaca ttcagataca 11640

ccgacttcag taatcctttc tcattcagag ctaatccaga catcatcggt atcagtgaac 11700

aacctctctt cggtgcaggt tctatcagta gcggtgaact ttacatcgac aagatcgaga 11760

tcatccttgc agatgcaaca tttgaagcag aatctgacct tgaaagagca caaaagtagt 11820

gaccaacgta tttatatcag aaaatagatg agtcgaaatt aaaagcttat acccgttaat 11880

gagatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 11940

cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 12000

gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 12060

acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 12120

ctatgttact agatccctag gcatggagtc aaagattcaa atagaggacc taacagaact 12180

cgccgtaaag actggcgaac agttcataca gagtctctta cgactcaatg acaagaagaa 12240

aatcttcgtc aacatggtgg agcacgacac acttgtctac tccaaaaata tcaaagatac 12300

agtctcagaa gaccaaaggg caattgagac ttttcaacaa agggtaatat ccggaaacct 12360

cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg 12420

tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc 12480

cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 12540

tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg taagggatga 12600

cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt 12660

ggagagaaca cgggggacta tgtcggatgc accggcgagt ccgagtcacg agagtggtgg 12720

cgagcagagc cctcgcggct cgttgtccgg cgcggctaga gagcaggacc ggtaccttcc 12780

cattgccaac atcagccgca tcatgaagaa ggctctgcct cccaatggca agattgcgaa 12840

ggatgcaaaa gacacaatgc aagaatgcgt ttctgaattc atcagcttca ttaccagcga 12900

ggcgagtgag aaatgccaga aggagaagag aaagacaatc aatggagacg atttactatg 12960

ggccatggca actttagggt ttgaagacta cattgagccg cttaaggtgt acctggctag 13020

gtacagagag gcggagggtg acactaaagg atctgctaga agtggtgatg gatctgctag 13080

accagatcaa gttggccttg caggtcaaaa tgctcagctt gttcatcagg gttcgctgaa 13140

ctatattggt ttgcaggtgc aaccacaaca tctggttatg ccttcaatgc aaggccatga 13200

atagcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 13260

gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 13320

catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 13380

cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 13440

tatgttacta gatcgtttaa actatcagtg tttgacagga tatattggcg ggtaaaccta 13500

agagaaaaga gcgtttatta gaataacgga tatttaaaag ggcgtgaaaa ggtttatccg 13560

ttcgtccatt tgtatgtg 13578

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

cgtaagggat gacgcacaat 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cgaggaacag aggctgagct 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ttctactggg gaggacatcg 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

cggtatcttt gggtgattgg 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cgtaagggat gacgcacaat 20

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

caatatagtt cagcgaaccc tga 23

<210> 11

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gaagatctat gtcggatgca ccggcga 27

<210> 12

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gggttaccct attcatggcc ttgcattga 29

<210> 13

<211> 541

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

gaagatctat gtcggatgca ccggcgagtc cgagtcacga gagtggtggc gagcagagcc 60

ctcgcggctc gttgtccggc gcggctagag agcaggaccg gtaccttccc attgccaaca 120

tcagccgcat catgaagaag gctctgcctc ccaatggcaa gattgcgaag gatgcaaaag 180

acacaatgca agaatgcgtt tctgaattca tcagcttcat taccagcgag gcgagtgaga 240

aatgccagaa ggagaagaga aagacaatca atggagacga tttactatgg gccatggcaa 300

ctttagggtt tgaagactac attgagccgc ttaaggtgta cctggctagg tacagagagg 360

cggagggtga cactaaagga tctgctagaa gtggtgatgg atctgctaga ccagatcaag 420

ttggccttgc aggtcaaaat gctcagcttg ttcatcaggg ttcgctgaac tatattggtt 480

tgcaggtgca accacaacat ctggttatgc cttcaatgca aggccatgaa tagggtaacc 540

c 541

<210> 14

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

cgggatccat ggaggagaac aatcaga 27

<210> 15

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

cgagctccta cttttgtgct ctttcaa 27

<210> 16

<211> 1907

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cgggatccat ggaggagaac aatcagaacc agtgtatccc ttacaattgt ctttctaatc 60

ctgaagaagt tcttttggat ggagaaagga tctcaactgg taactcatca attgacatct 120

ctctctcact tgttcagttc ttggtttcta actttgtgcc aggaggagga ttccttgttg 180

gacttatcga cttcgtttgg ggaatcgttg gaccttctca atgggatgca tttctcgttc 240

agatcgaaca gctcatcaac gaaagaatcg ctgagttcgc taggaatgct gctattgcta 300

accttgaagg acttggaaac aacttcaaca tctacgtgga ggcattcaag gaatgggaag 360

aagatcctaa caacccagca accaggacca gagtgatcga taggttccgt atccttgatg 420

gacttcttga aagggacatt cctagcttta ggatctctgg atttgaagtt ccacttctct 480

ctgtttacgc tcaagctgct aatctccatc ttgctatcct tagagattct gtgatcttcg 540

gagaaagatg gggattgaca accatcaacg tgaacgagaa ctacaacaga ctcatcaggc 600

acatcgatga gtacgctgat cactgtgcta acacttacaa ccgtggactc aacaaccttc 660

ctaagtctac ctatcaagat tggatcacat acaaccgact taggagagac cttacattga 720

ctgttcttga tatcgctgct ttctttccaa actatgacaa taggagatat ccaattcagc 780

cagttggtca acttacaagg gaagtttaca ctgacccact catcaacttc aacccacagc 840

ttcagtctgt tgctcagctt cctaccttca acgttatgga gagcagcgca atcagaaatc 900

ctcacctctt cgacatcttg aacaacctta caatctttac cgattggttt agtgttggac 960

gtaacttcta ctggggagga catcgagtga tctctagcct catcggaggt ggtaacatca 1020

catctcctat ctacggaaga gaggctaacc aggagcctcc aagatcattc actttcaacg 1080

gacctgtgtt caggactctt tcaaatccta ctcttcgact tcttcagcaa ccttggccag 1140

ctccaccatt caaccttcgt ggtgttgaag gagttgagtt ctctacacct acaaacagct 1200

tcacctatcg tggaagaggt actgttgatt ctcttactga acttccacct gaggaaacag 1260

tgtgccacct cgtgaaggat acagtcatcg tctttgtcat gcaaccttcg ttcaaagatc 1320

tggaacacct ttccttacaa ctggtgttgt gttctcttgg actcatcgta gtgcaactct 1380

taccaacaca attgatccag agaggatcaa ccagatccct cttgtgaaag gattcagagt 1440

ttggggagga acctctgtga ttacaggacc aggattcaca ggaggtgata tccttcgaag 1500

aaacaccttt ggtgacttcg tttctcttca agtgaacatc aactcaccaa tcacccaaag 1560

ataccgtctt agatttcgtt acgcttctag tagggatgca cgagttatcg ttcttacagg 1620

agctgcatct acaggagtgg gaggtcaagt tagtgtgaac atgcctcttc agaaaactat 1680

ggagatcgga gagaacctca catctagaac attcagatac accgacttca gtaatccttt 1740

ctcattcaga gctaatccag acatcatcgg tatcagtgaa caacctctct tcggtgcagg 1800

ttctatcagt agcggtgaac tttacatcga caagatcgag atcatccttg cagatgcaac 1860

atttgaagca gaatctgacc ttgaaagagc acaaaagtag gagctcg 1907

<210> 17

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ctcggtacca gattagcctt ttcaatttca 30

<210> 18

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

gtataagctt ttaatttcga ctcatcta 28

Claims

1. A plant recombinant expression vector pGR, which is characterized by comprising a herbicide-resistant gene expression frame, an insect-resistant gene expression frame and a stress-resistant gene expression frame;

the herbicide-resistant gene expression frame sequentially comprises a CaMV35S promoter, a glyphosate-resistant gene with an optimized codon and a CaMV PolyA terminator;

the glyphosate-tolerant gene comprisesg2m-epsps；

The nucleotide sequence of the herbicide-resistant gene expression frame is a DNA fragment shown in SEQ ID NO. 1;

the insect-resistant gene expression frame sequentially comprises a CaMV35S promoter, an insect-resistant gene with optimized codons and an NOS terminator; the insect-resistant gene comprisescry1C；

The nucleotide sequence of the insect-resistant gene expression frame is a DNA fragment shown in SEQ ID NO. 2;

the stress-tolerant gene expression frame sequentially comprises a CaMV35S promoter, a soybean stress-tolerant gene and an NOS terminator; the stress-tolerant gene comprisesGmNFYB1；

The nucleotide sequence of the stress-resistant gene expression frame is a DNA fragment shown in SEQ ID NO. 3.

2. The plant recombinant expression vector pGR according to claim 1, wherein the multiple cloning site of the herbicide-resistant gene expression cassette isXhoI；

The multiple cloning site of the insect-resistant gene expression frame isKpnI/HindIII；

The multiple cloning site of the stress-resistant gene expression frame isBglII/BstEII；

3. The plant recombinant expression vector pGR according to claim 2, wherein the nucleotide sequence of pGR20 is set forth in SEQ ID No. 4.

4. A method for constructing the plant recombinant expression vector pGR20 according to any one of claims 1 to 3, comprising the steps of:

containingBglII and IIBstEII cleavage siteGmNFYB1The gene fragment and the commercial vector pCAMBIA3301 were used separatelyBglII and IIBstThe EII double enzyme is connected after cutting, and a p3301-NF carrier is constructed;

insect-resistant genecry1CAnd commercial vector pBI121BamHI andSaci, connecting after double enzyme digestion, and constructing a pBI121-crylC vector;

the pBI121-crylC vector is used as a template, and two ends are used for carryingKpnI andHindIII, specific primer of enzyme cutting site, amplifying to obtain fragment of insect-resistant gene expression frame, and utilizingKpnI andHindIII, connecting the digested p3301-NF vectors to construct a p3301-NF-cry1C plant expression vector;

containing by sequence synthesisXhoI cleavage siteg2m-epspsFragments and the p3301-NF-cry1C vectorXhoAnd I, connecting after enzyme digestion to form a plant recombinant expression vector pGR containing the herbicide-resistant gene expression frame.

5. Use of the plant recombinant expression vector pGR according to any one of claims 1 to 3 or the plant recombinant expression vector pGR according to the construction method of claim 4 for cultivating transgenic plants, wherein the transgenic plants are soybean.

6. The use according to claim 5, wherein the transgenic plant is a herbicide-resistant, pest-resistant and stress-tolerant complex trait-tolerant transgenic plant.

7. A method of breeding herbicide, pest and stress tolerant transgenic plants comprising the steps of: a transgenic plant obtained by introducing the plant recombinant expression vector pGR20 according to any one of claims 1 to 3 into a recipient plant.