CN111826394A - Construction and application of plant transient expression vector - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to the construction and application of a plant transient expression vector. The present invention uses artificially synthesized sequences to increase the transcription amount of the vector, and at the same time selectively adds amino acid sequences to improve the translation level and stability of the target protein, and finally achieves the effect of increasing the accumulation of the target protein in plant leaf cells as a whole.

Description

Construction and application of plant transient expression vector

technical field

The invention relates to the field of biotechnology, in particular to the construction and application of a plant transient expression vector.

Background technique

Like mammalian cell expression system and microbial fermentation system, plant expression system is a biological protein expression reactor with broad application prospects. Mammalian cell expression system has the advantages of high yield, mature system, and similar to human cells, and is widely promoted, but the operating price is relatively expensive; the technology of microbial fermentation system is also quite complete, at the same time, the yield is high and the price is low, but due to the difference of cell types , microbial fermentation systems cannot express target proteins that require specific protein modifications. Plant expression systems have inherent advantages, such as less biomass restriction, fast growth, low cost and mammalian expression systems, and plant cells can perform various post-translational modifications on proteins, which can make up for the limitations of microbial fermentation systems and insect expression systems. Insufficient, has good production prospects, commercial application is a relatively popular research direction.

For target protein expression in plants, an efficient plant-exogenous protein expression vector is crucial. At the current stage, domestic plant exogenous protein expression vectors are mainly used in the field of basic scientific research, and there are few explorations of application vectors that can be efficiently commercialized. International plant recombinant protein expression companies Medicago and IconGenetic have developed unique expression vectors based on basic research and their own research progress. Therefore, it is of great practical significance to transform and improve the expression efficiency of plant exogenous expression vectors by means of genetic engineering, so that they can meet the needs of commercial production of target proteins.

SUMMARY OF THE INVENTION

In view of this, the present invention provides the construction and application of plant transient expression vector. The present invention uses artificially synthesized sequences to increase the transcription amount of the vector, and at the same time selectively adds amino acid sequences to improve the translation level and stability of the target protein, and finally achieves the effect of increasing the accumulation of the target protein in plant leaf cells as a whole.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a plant transient expression vector, including the 5'UTR region, the MCS region and the SEKDEL sequence.

In some specific embodiments of the present invention, the sequences of the 5'UTR region, the MCS region and the SEKDEL sequence are shown in SEQ ID No.1.

In some specific embodiments of the present invention, the plant transient expression vector takes the plant expression vector pEAQ as the basic backbone.

In some specific embodiments of the present invention, the plant transient expression vector further includes a target gene, the N-terminal of the target gene is added with a PacI restriction site, and the C-terminal is added with an XhoI restriction site.

In some specific embodiments of the present invention, in the construction method of the plant transient expression vector, the upstream primer shown in SEQ ID No. 2 and the downstream primer shown in SEQ ID No. 3 are used to amplify the Sequence shown in ID No.1.

In some specific embodiments of the present invention, the amplification system for the amplification comprises 22 μl ddH ₂ O, 25 μl KOD OneTM PCRMasterMix, 1 μl 10 μM upstream primer shown in SEQ ID No.2, 1 μl 10 μM shown in SEQ ID No.3 The downstream primer shown, 1 μl of 100 ng/μl plasmid template containing the synthetic sequence of SEQ ID No. 1.

In some specific embodiments of the present invention, the amplification procedure is: 98°C for 30 seconds; 28 cycles: 98°C for 10 seconds; 55°C for 30 seconds; 68°C for 10 seconds; 68°C for 5 minutes; 4°C for 10 seconds minute.

The present invention also provides the application of the plant transient expression vector in expressing foreign protein.

In some specific embodiments of the invention, the exogenous protein comprises GFP.

On the basis of the above research, the present invention also provides a method for expressing exogenous protein from a plant transient expression vector, comprising the following steps:

Step 1: construct the plant transient expression vector;

Step 2: insert the gene of the exogenous protein into the plant transient expression vector prepared in step 1; obtain a recombinant plasmid;

Step 3: transform the recombinant plasmid obtained in step 2 into a competent host, culture, collect bacterial liquid, infiltrate plant leaves, and extract protein.

At present, there are two main ways to improve the expression of plant exogenous protein expression vectors. One is to increase the transcription of the target gene expressed by the vector in plant cells, and the other is to increase the translation and accumulation of proteins through some methods. The present invention uses artificially synthesized sequences to increase the transcription amount of the vector, and at the same time selectively adds amino acid sequences to improve the translation level and stability of the target protein, and finally achieves the effect of increasing the accumulation of the target protein in plant leaf cells as a whole.

The transformed pEAQ vector provided by the present invention is named pEAQE, and the ability to express foreign proteins is significantly improved compared with the original pEAQ vector. This type of improvement is mainly achieved by enhancing the transcription level of the target gene in the cell by the vector, and at the same time increasing the accumulation of the protein in the cell. Especially for some exogenous proteins whose expression in plant cells may be less stable, the selective fusion expression of SEKDEL can help the target protein to be more stably expressed and accumulated in plant cells. It can provide a high-quality expression vector platform basis for the development of commercial protein expression in the later stage. At the same time, the ingenious restriction site design of the pEAQE plant expression vector provides a very simple cloning method for protein operations that need to add fusion expression SEKDEL peptides. It saves the labor time and reagent cost of laboratory operations on molecular cloning.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

Fig. 1 shows the plasmid map of inserted GFP;

Figure 2 shows the photos of UV fluorescence comparing the GFP fluorescence difference of different vectors;

Figure 3 shows the difference in the transcription level of GFP mRNA in different vectors compared by Q-PCR;

Figure 4 shows the difference of GFP protein expression data in different vectors compared by WB.

Detailed ways

The invention discloses the construction and application of a plant transient expression vector, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The raw materials and reagents used in the construction and application of the plant transient expression vector provided by the present invention can be purchased from the market.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1 Construction of pEAQE vector

(1), take the plant expression vector pEAQ as the basic skeleton, carry out 2 hours of digestion with two restriction sites of PacI and XhoI, and then reclaim the 9200bp fragment by DNA gel electrophoresis;

(2), synthesis contains artificial sequence as shown in SEQ ID No.1, including 5'UTR region, MCS region, SEKDEL sequence;

(3), design amplification primers for amplifying artificial sequence gene fragments, the upstream primers are shown in SEQ ID No.2, and the downstream primers are shown in SEQ ID No.3;

(4), the KOD One ^TM PCRMasterMix of TOYOBO carries out the amplification reaction, and the amplification system is: 22ul ddH ₂ O, 25ul KOD One ^TM PCRMasterMix, 1ul 10uM upstream primer, 1ul 10uM downstream primer, 1ul 100ng/ul containing synthetic sequence SEQ Plasmid template of ID No. 1. PCR amplification program: 98 degrees for 30 seconds; 28 cycles: 98 degrees for 10 seconds; 55 degrees for 30 seconds; 68 degrees for 10 seconds; final 68 degrees for 5 minutes; 4 degrees for 10 minutes;

(5) A PacI restriction site was added to the N-terminal of the final amplified gene fragment, and an XhoI restriction site was added to the C-terminal. After gel electrophoresis, the fragments were recovered by tapping the gel; the single-fragment homologous recombination was performed with Novozan's Infusion enzyme to link the vector backbone and the amplified fragments;

(6) After heat-shock transformed DH5α competent, coated with Kana resistance medium, colony PCR verification was performed on the next day, positive clones were selected and the corresponding plasmids were extracted and sequenced. The plasmid with completely correct sequencing results is stored, and the sequence is shown in SEQ ID No. 6.

SEQ ID No. 1: Synthetic artificial sequence

CAGTGAATTGTTAATTAAGAATTCGAGCTCCACCGCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGAGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGTTTaAAGAGtCGCAcCCAaAACGCTCaAACcCAATtatTCaACATcTATTtAAgGTCcCTAAAAATGGCCACCCCCGGGAGTGAAAAAGACGAATTATAGCTCGAGGCCTTTAACTCTGGT

SEQ ID No. 2: Amplification of artificial sequence upstream primers

CAGTGAATTGTTAATTAAGGAAAACCTC

SEQ ID No. 3: Amplification of artificial sequence downstream primers

ACCAGAGTTAAAGGCCTCGAGCTAGTG

Example 2 Insertion of GFP gene into pEAQE vector

(1), take the plant expression vector pEAQE as the basic skeleton, carry out the digestion with SmaI restriction site for 2 hours, and then recover the 10469bp fragment by DNA gel electrophoresis;

(2), design amplification primers for amplifying artificial sequence gene fragments, upstream primer SEQ ID No.4, downstream primer SEQ ID No.5;

(3) The KOD One ^TM PCRMasterMix of TOYOBO carries out the amplification reaction, and the amplification system is: 22ul ddH ₂ O, 25ul KOD One ^TM PCRMasterMix, 1ul 10uM upstream primer, 1ul 10uM downstream primer, 1ul 100ng/ul plasmid template containing GFP . PCR amplification program: 98 degrees for 30 seconds; 28 cycles: 98 degrees for 10 seconds; 55 degrees for 30 seconds; 68 degrees for 10 seconds; final 68 degrees for 5 minutes; 4 degrees for 10 minutes;

(4), in the final amplified gene fragment, carry out gel electrophoresis to recover the fragment; use Novozan's Infusion enzyme to carry out single-segment homologous recombination to link the vector backbone and the amplified fragment;

(5) After heat-shock transformed DH5α competent, coated with kana-resistant medium, carried out colony PCR verification on the next day, selected positive clones, and extracted the corresponding plasmids for sequencing. The plasmid with completely correct sequencing results was stored, the sequence is shown in SEQ ID No. 6, and the plasmid map is shown in Figure 1 .

SEQ ID No.4: Amplify GFP upstream primer

AAAATGGCCACCCCCATGGTGAGCAAGGGGCGAGGAG

SEQ ID No. 5: Amplify GFP downstream primer

CTTTTTCACTCCCTTACTTGTACAGCTCGTCCATGCCG

SEQ ID No.6: pEAQE-GFP plasmid sequence

AGCGGTGAGTTCGCGGGGCGCACGCATGACGGTGCGGCTTGCGATGGTTTCGGCATCCTCGGCGGAAAACCCCGCGTCGATCAGTTCTTGCCTGTATGCCTTCCGGTCAAACGTCCGATTCATTCACCCTCCTTGCGGGATTGCCCCGACTCACGCCGGGGCAATGTGCCCTTATTCCTGATTTGACCCGCCTGGTGCCTTGGTGTCCAGATAATCCACCTTATCGGCAATGAAGTCGGTCCCGTAGACCGTCTGGCCGTCCTTCTCGTACTTGGTATTCCGAATCTTGCCCTGCACGAATACCAGCGACCCCTTGCCCAAATACTTGCCGTGGGCCTCGGCCTGAGAGCCAAAACACTTGATGCGGAAGAAGTCGGTGCGCTCCTGCTTGTCGCCGGCATCGTTGCGCCACATCTAGGTACTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAAC AGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATTTATTATTTCCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATAACAAGACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTATAACATAGTATCGACGGAGCCGATTTTGAAACCACAATTATGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTATCTCTGCTCTCACTGCCGTAAAACATGGCAACTGCAGTTCACTTACACCGCTTCTCAACCCGGTACGCACCAGAAAATCATTGATATGGCCATGAATGGCGTTGGATGCCGGGCAACAGCCCGCATTATGGGCGTTGGCCTCAACACGATTTTACGTCACTTAAAAAACTCAGGCCGCAGTCGGTAACTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGA CGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGGTAACCTCGCGCATACAGCCGGGCAGTGACGTCATCGTCTGCGCGGAAATGGACGGGCCCCCGGCGCCAGATCTGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCACGCCCTTTTAAATATCCGATTATTCTAATAAACGCTCTTTTCTCTTAGGTTTACCCGCCAATATATCCTGTCAAACACTGATAGTTTGTGAACCATCACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTTAATTAAGAATTCGAGCTCCACCGCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGAGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGC CTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGTTTaAAGAGtCGCAcCCAaAACGCTCaAACcCAATtatTCaACATcTATTtAAgGTCcCTAAAAATGGCCACCCCCatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaGGGAGTGAAAA AGACGAATTATAGCTCGAGGCCTTTAACTCTGGTTTCATTAAATTTTCTTTAGTTTGAATTTACTGTTATTCGGTGTGCATTTCTATGTTTGGTGAGCGGTTTTCTGTGCTCAGAGTGTGTTTATTTTATGTAATTTAATTTCTTTGTGAGCTCCTGTTTAGCAGGTCGTCCCTTCAGCAAGGACACAAAAAGATTTTAATTTTATTAAAAAAAAAAAAAAAAAAAAGACCGGGAATTCGATATCAAGCTTATCGACCTGCAGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCTCTAGAGTCTCAAGCTTGGCGCGCCAGCTTGGCGTAATCATGGTCATAGCTGTTGCGATTAAGAATTCGAGCTCGGTACCCCCCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACAGCCCAAGC TTCGACTCTAGAGGATCCCCTTAAATCGATATGGAACGAGCTATACAAGGAAACGACGCTAGGGAACAAGCTAACAGTGAACGTTGGGATGGAGGATCAGGAGGTACCACTTCTCCCTTCAAACTTCCTGACGAAAGTCCGAGTTGGACTGAGTGGCGGCTACATAACGATGAGACGAATTCGAATCAAGATAATCCCCTTGGTTTCAAGGAAAGCTGGGGTTTCGGGAAAGTTGTATTTAAGAGATATCTCAGATACGACAGGACGGAAGCTTCACTGCACAGAGTCCTTGGATCTTGGACGGGAGATTCGGTTAACTATGCAGCATCTCGATTTTTCGGTTTCGACCAGATCGGATGTACCTATAGTATTCGGTTTCGAGGAGTTAGTATCACCGTTTCTGGAGGGTCTCGAACTCTTCAGCATCTCTGTGAGATGGCAATTCGGTCTAAGCAAGAACTGCTACAGCTTGCCCCAATCGAAGTGGAAAGTAATGTATCAAGAGGATGCCCTGAAGGTACTCAAACCTTCGAAAAAGAAAGCGAGTAAGTCGAGGGCGAGCTCGAATTCGGTACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCCATAAATAATGTGTGAGTAGTTTCCCGATAAGGGAAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCTCCTAAAGTCCCTATAGATCTTTGTCGTGAATATAAACCAGACACGAGACGACTAAACCTGGAGCCCAGACGCCGTTCGAAGCTAGAAGTACCGCTTAGGCAGGAGGCCGTTAGGGAAAAGATGCTAAGGCAGGGTTGGTTACGTTGACTCCCCCGTAGGTTTGGTTTAAATATGATGAAGTGGACGGAAGGAAGGAGGAAGACA AGGAAGGATAAGGTTGCAGGCCCTGTGCAAGGTAAGAAGATGGAAATTTGATAGAGGTACGCTACTATACTTATACTATACGCTAAGGGAATGCTTGTATTTATACCCTATACCCCCTAATAACCCCTTATCAATTTAAGAAATAATCCGCATAAGCCCCCGCTTAAAAATTGGTATCAGAGCCATGAATAGGTCTATGACCAAAACTCAAGAGGATAAAACCTCACCAAAATACGAAAGAGTTCTTAACTCTAAAGATAAAAGATGGCGCGTGGCCGGCCATGAGCGGAGAATTAAGGGAGTCACGTTATGACCCCCGCCGATGACGCGGGACAAGCCGTTTTACGTTTGGAACTGACAGAACCGCAACGTTGAAGGAGCCACTCAGCCGCGGGTTTCTGGAGTTTAATGAGCTAAGCACATACGTCAGAAACCATTATTGCGCGTTCAAAAGTCGCCTAAGGTCACTATCAGCTAGCAAATATTTCTTGTCAAAAATGCTCCACTGACGTTCCATAAATTCCCCTCGGTATCCAATTAGAGTCTCATATTCACTCTCAATCCAAATAATCTGCACCGGATCTGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCA CCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGATGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACGGGATCTCTGCGGAACAGGCGGTCGAAGGTGCCGATATCATTACGACAGCAACGGCCGACAAGCACAACGCCACGATCCTGAGCGACAATATGATCGCGGCGTCCACATCAACGGCGTCGGCGGCGACTGCCCAGGCAAGACCGAGATGCACCGCGATATCTTGCTGCGTTCGGATATTTTCGTGGAGTTCCCGCCACAGACCCGGATGATCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATC GCGCGCGGTGTCATCTATGTTACTAGATCGGGGGCCGGCCCTCACTGGTGAAAAGAAAAACCACCCCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACACCACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCTGTGATCAAATATCATCTCCCTCGCAGAGATCCGAATTATCAGCCTTCTTATTCATTTCTCGCTTAACCGTGACAGAGTAGACAGGCTGTCTCGCGGCCGAGGGGCGCAGCCCCTGGGGGGATGGGAGGCCCGCGTTAGCGGGCCGGGAGGGTTCGAGAAGGGGGGGCACCCCCCTTCGGCGTGCGCGGTCACGCGCACAGGGCGCAGCCCTGGTTAAAAACAAGGTTTATAAATATTGGTTTAAAAGCAGGTTAAAAGACAGGTTAGCGGTGGCCGAAAAACGGGCGGAAACCCTTGCAAATGCTGGATTTTCTGCCTGTGGACAGCCCCTCAAATGTCAATAGGTGCGCCCCTCATCTGTCAGCACTCTGCCCCTCAAGTGTCAAGGATCGCGCCCCTCATCTGTCAGTAGTCGCGCCCCTCAAGTGTCAATACCGCAGGGCACTTATCCCCAGGCTTGTCCACATCATCTGTGGGAAACTCGCGTAAAATCAGGCGTTTTCGCCGATTTGCGAGGCTGGCCAGCTCCACGTCGCCGGCCGAAATCGAGCCTGCCCCTCATCTGTCAACGCCGCGCCGGGTGAGTCGGCCCCTCAAGTGTCAACGTCCGCCCCTCATCTGTCAG TGAGGGCCAAGTTTTCCGCGAGGTATCCACAACGCCGGCGGCCGCGGTGTCTCGCACACGGCTTCGACGGCGTTTCTGGCGCGTTTGCAGGGCCATAGACGGCCGCCAGCCCAGCGGCGAGGGCAACCAGCCCGGTGAGCGTCGGAAAGGCGCTCGGTCTTGCCTTGCTCGTCGGTGATGTACACTAGTCGCTGGCTGCTGAACCCCCAGCCGGAACTGACCCCACAAGGCCCTAGCGTTTGCAATGCACCAGGTCATCATTGACCCAGGCGTGTTCCACCAGGCCGCTGCCTCGCAACTCTTCGCAGGCTTCGCCGACCTGCTCGCGCCACTTCTTCACGCGGGTGGAATCCGATCCGCACATGAGGCGGAAGGTTTCCAGCTTGAGCGGGTACGGCTCCCGGTGCGAGCTGAAATAGTCGAACATCCGTCGGGCCGTCGGCGACAGCTTGCGGTACTTCTCCCATATGAATTTCGTGTAGTGGTCGCCAGCAAACAGCACGACGATTTCCTCGTCGATCAGGACCTGGCAACGGGACGTTTTCTTGCCACGGTCCAGGACGCGGAAGCGGTGCAGCAGCGACACCGATTCCAGGTGCCCAACGCGGTCGGACGTGAAGCCCATCGCCGTCGCCTGTAGGCGCGACAGGCATTCCTCGGCCTTCGTGTAATACCGGCCATTGATCGACCAGCCCAGGTCCTGGCAAAGCTCGTAGAACGTGAAGGTGATCGGCTCGCCGATAGGGGTGCGCTTCGCGTACTCCAACACCTGCTGCCACACCAGTTCGTCATCGTCGGCCCGCAGCTCGACGCCGGTGTAGGTGATCTTCACGTCCTTGTTGACGTGGAAAATGACCTTGTTTTGCAGCGCCTCGCGCGGGATTTTCTTGTTGCGCGTGGTGAACAGGGCAGAGCGGGCCGTGTCGTTTGGCATCGCTCGCATCGTGTCCGGCCACGGCGCAATATCGAACAAGGAAAGCTGCATTTCCTTGATCTGCTG CTTCGTGTGTTTCAGCAACGCGGCCTGCTTGGCCTCGCTGACCTGTTTTGCCAGGTCCTCGCCGGCGGTTTTTCGCTTCTTGGTCGTCATAGTTCCTCGCGTGTCGATGGTCATCGACTTCGCCAAACCTGCCGCCTCCTGTTCGAGACGACGCGAACGCTCCACGGCGGCCGATGGCGCGGGCAGGGCAGGGGGAAGCCAGTTGCACGCTGTCGCGCTCGATCTTGGCCGTAGCTTGCTGGACCATCGAGCCGACGGACTGGAAGGTTTCGCGGGGCGCACGCATGACGGTGCGGCTTGCGATGGTTTCGGCATCCTCGGCGGAAAACCCCGCGTCGATCAGTTCTTGCCTGTATGCCTTCCGGTCAAACGTCCGATTCATTCACCCTCCTTGCGGGATTGCCCCGACTCACGCCGGGGCAATGGGCCCTTATTCCTGATTTGACCCGCCTGGGGGCTTGGGTGTCCAGAA

Example 3 Infiltration

(1) The pEAQE-GFP plasmid was introduced into Agrobacterium competent cells by electric shock, and smeared on the double antibody medium of kana and rifampicin.

(2), pick a single clone and use the medium to shake the bacteria, when OD ₆₀₀ =0.8, centrifuge at 8000rpm for 2 minutes, and use the infiltration buffer (10mM MES, 10mM Mgcl ₂ , 1uM acetosyringone) to precipitate the bacteria solution. Resuspend and adjust to _OD600 = 0.6.

(3), infiltrate the bacterial liquid into the tobacco leaves with a negative pressure vacuum pump, and observe the fluorescence after 2 days.

Example 4 Fluorescence Photography

The leaf infiltration area was irradiated with a portable UV fluorescent lamp and photographed under a yellow filter (as shown in Figure 2). In the area with pEAQE-GFP infiltration and expression, bright green leaves were observed, while in the area without pEAQE-GFP infiltration and expression, the leaves appeared red.

From the data in the figure, we can see that the three groups of repeated experiments we conducted maintained good stability and consistency. Through the data Students' T-test test, it was found that the fluorescence of pEAQE was stronger than that of pEAQ, and the difference was significant.

Example 5Q-PCR

(1), extract RNA. Take 0.1 g of the infiltrated part of the leaf, grind it with liquid nitrogen, add 1 ml of TRIZOL and 300 ul of chloroform, mix well, and let it stand at room temperature for 10 minutes. Centrifuge at 15,000 rpm for 5 minutes, take the supernatant and transfer it to a new centrifuge tube, add an equal volume of isopropanol, stand at room temperature for 10 minutes, and then centrifuge at 15,000 rpm for 5 minutes. After washing twice with 70% ethanol, the pellet was air-dried and dissolved in 100 ul of DEPC water.

(2) cDNA was obtained by reverse transcription using TOYOBO's RT-PCR QuickMasterMix kit.

(3) The Q-PCR reaction system was 10ul of SYBRMIX, 0.8ul of Forwardprimer, 0.8ul of Reverse primer, 2ul of DNA template, and 6.4ul of ddH ₂ O. Reaction conditions: PCR cycle parameters: pre-denaturation at 95°C for 3 min; followed by 30 cycles of denaturation at 95°C for 30s, low-temperature annealing at 55°C for 45s, and extension at 72°C for 30s; melting curve analysis selected according to the operating instructions of the instrument: 95°C for 15s, 60°C for 15s , 95℃ for 15s.

The results are shown in Figure 3. GFP expressed from pEAQ and pEAQE vectors was analyzed at the transcriptional level. RT-qPCR method was used to compare the difference in the accumulation of GFP mRNA expressed by the two vectors. Through data analysis, it can be found that the mRNA content of GFP expressed by pEAQE is 120% of that of pEAQ, and the Students' T-test test found that the mRNA content of pEAQE GFP is significantly higher than that of pEAQ.

Example 6 SDS-PAGE gel and WB operation

(1) Take 0.1 g of leaves in the infiltrated area, grind with liquid nitrogen, add 200 ul of protein extraction buffer, centrifuge at 15,000 rpm for 10 minutes, take 200 ul of supernatant and add 50 ul of protein for loading. Boil at 100 degrees for 10 minutes and cool on ice.

(2), take 10ul of the sample and load the sample, electrophoresis at 80V for half an hour, and electrophoresis at 100V for 1 hour.

(3), remove the gel stacking gel and put it in the transfer buffer. Cut the nitrocellulose (NC) membrane and clean filter paper of the same size as the gel and wet them in transfer buffer for 5-10 min. Open the electric transfer clip, the black side of the cathode is facing down, wet the sponge pad, filter paper, gel, NC membrane, filter paper, and sponge pad in the order, and then lay it flat, remove the air bubbles between the middle layers and clamp the electric transfer clip. 100V transfer film for 60min.

(4) After the NC membrane was rinsed with PBST buffer, it was immersed in blocking solution and incubated at room temperature for 1 hour; the membrane was immersed in the dilution of GFP primary antibody, and incubated at room temperature for 1-2 hours by shaking slowly on a horizontal shaker; after washing the membrane 3 times with PBST, Transfer the membrane to the dilution of the desired secondary antibody, and slowly shake the reaction at room temperature for 1-2 hours; wash the membrane 4 times with PBST, 5 min each time, and briefly wash with PBS to remove the Tween-20 on the membrane surface;

(5) Alkaline phosphatase (AP) color development: add 66 μL NBT and 33 μL BCIP to 10 mL color development buffer, respectively, immerse the membrane in the color developing solution and react in the dark until the specific bands are clear, non-specific bands and background. So far, it generally takes 3 to 30 minutes. The fully developed membrane was placed in deionized water to stop the reaction.

(6) Coomassie brilliant blue staining: stain the SDS-PAGE gel with Coomassie brilliant blue staining solution (40% methanol; 10% acetic acid; 0.25% Coomassie brilliant blue G250) on a shaking table for 30 minutes; ; 10% acetic acid) until the bands are clearly visible.

As shown in Figure 4. The accumulation of GFP protein expressed by pEAQ-GFP and pEAQE-GFP was compared by SDS-PAGE gel staining and WB. It can be known that the expression level of pEAQE-GFP is higher than that of pEAQ-GFP. Students' T-test test shows that P<0.001, indicating that the difference in the increase of this expression level is very significant.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Zhejiang Huadi Pharmaceutical Group Pharmaceutical Development Co., Ltd.

<120> Construction and Application of Plant Transient Expression Vector

<130> MP2012471

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 472

<212> DNA

<213> Artificial Sequence

<400> 1

cagtgaattg ttaattaaga attcgagctc caccgcggaa acctcctcgg attccattgc 60

ccagctatct gtcactttat tgagaagata gtggaaaagg aaggtggctc ctacaaatgc 120

catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa 180

gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca 240

aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat 300

ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag gtttaaagag 360

tcgcacccaa aacgctcaaa cccaattatt caacatctat ttaaggtccc taaaaatggc 420

cacccccggg agtgaaaaag acgaattata gctcgaggcc tttaactctg gt 472

<210> 2

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 2

cagtgaattg ttaattaagg aaacctc 27

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 3

accagagtta aaggcctcga gctagtg 27

<210> 4

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 4

aaaatggcca cccccatggt gagcaagggc gaggag 36

<210> 5

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 5

ctttttcact cccttacttg tacagctcgt ccatgccg 38

<210> 6

<211> 10472

<212> DNA

<213> Artificial Sequence

<400> 6

agcggtgagt tcgcggggcg cacgcatgac ggtgcggctt gcgatggttt cggcatcctc 60

ggcggaaaac cccgcgtcga tcagttcttg cctgtatgcc ttccggtcaa acgtccgatt 120

cattcaccct ccttgcggga ttgccccgac tcacgccggg gcaatgtgcc cttattcctg 180

atttgacccg cctggtgcct tggtgtccag ataatccacc ttatcggcaa tgaagtcggt 240

cccgtagacc gtctggccgt ccttctcgta cttggtattc cgaatcttgc cctgcacgaa 300

taccagcgac cccttgccca aatacttgcc gtgggcctcg gcctgagagc caaaacactt 360

gatgcggaag aagtcggtgc gctcctgctt gtcgccggca tcgttgcgcc acatctaggt 420

actaaaacaa ttcatccagt aaaatataat attttatttt ctcccaatca ggcttgatcc 480

ccagtaagtc aaaaaatagc tcgacatact gttcttcccc gatatcctcc ctgatcgacc 540

ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc gcttctccca agatcaataa 600

agccacttac tttgccatct ttcacaaaga tgttgctgtc tcccaggtcg ccgtgggaaa 660

agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc atacagctcg cgcggatctt 720

taaatggagt gtcttcttcc cagttttcgc aatccacatc ggccagatcg ttattcagta 780

agtaatccaa ttcggctaag cggctgtcta agctattcgt atagggacaa tccgatatgt 840

cgatggagtg aaagagcctg atgcactccg catacagctc gataatcttt tcagggcttt 900

gttcatcttc atactcttcc gagcaaagga cgccatcggc ctcactcatg agcagattgc 960

tccagccatc atgccgttca aagtgcagga cctttggaac aggcagcttt ccttccagcc 1020

atagcatcat gtccttttcc cgttccacat cataggtggt ccctttatac cggctgtccg 1080

tcatttttaa atataggttt tcattttctc ccaccagctt atatacctta gcaggagaca 1140

ttccttccgt atcttttacg cagcggtatt tttcgatcag ttttttcaat tccggtgata 1200

ttctcatttt agccatttat tatttccttc ctcttttcta cagtatttaa agatacccca 1260

agaagctaat tataacaaga cgaactccaa ttcactgttc cttgcattct aaaaccttaa 1320

ataccagaaa acagcttttt caaagttgtt ttcaaagttg gcgtataaca tagtatcgac 1380

ggagccgatt ttgaaaccac aattatgggt gatgctgcca acttactgat ttagtgtatg 1440

atggtgtttt tgaggtgctc cagtggcttc tgtttctatc agctgtccct cctgttcagc 1500

tactgacggg gtggtgcgta acggcaaaag caccgccgga catcagcgct atctctgctc 1560

tcactgccgt aaaacatggc aactgcagtt cacttacacc gcttctcaac ccggtacgca 1620

ccagaaaatc attgatatgg ccatgaatgg cgttggatgc cgggcaacag cccgcattat 1680

gggcgttggc ctcaacacga ttttacgtca cttaaaaaac tcaggccgca gtcggtaact 1740

atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc 1800

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 1860

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 1920

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1980

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 2040

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 2100

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 2160

gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 2220

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 2280

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagg taacctcgcg 2340

catacagccg ggcagtgacg tcatcgtctg cgcggaaatg gacgggcccc cggcgccaga 2400

tctggggaac cctgtggttg gcatgcacat acaaatggac gaacggataa accttttcac 2460

gcccttttaa atatccgatt attctaataa acgctctttt ctcttaggtt tacccgccaa 2520

tatatcctgt caaacactga tagtttgtga accatcaccc aaatcaagtt ttttggggtc 2580

gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 2640

gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgccat 2700

tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc 2760

tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt 2820

cacgacgttg taaaacgacg gccagtgaat tgttaattaa gaattcgagc tccaccgcgg 2880

aaacctcctc ggattccatt gcccagctat ctgtcacttt attgagaaga tagtggaaaa 2940

ggaaggtggc tcctacaaat gccatcattg cgataaagga aaggccatcg ttgaagatgc 3000

ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg tggaaaaaga 3060

agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca ctgacgtaag 3120

ggatgacgca caatcccact atccttcgca agacccttcc tctatataag gaagttcatt 3180

tcatttggag aggtttaaag agtcgcaccc aaaacgctca aacccaatta ttcaacatct 3240

atttaaggtc cctaaaaatg gccaccccca tggtgagcaa gggcgaggag ctgttcaccg 3300

gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt 3360

ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca 3420

ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt 3480

gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg 3540

aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg 3600

ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact 3660

tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg 3720

tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca 3780

acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg 3840

acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag 3900

accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca 3960

ctctcggcat ggacgagctg tacaagtaag ggagtgaaaa agacgaatta tagctcgagg 4020

cctttaactc tggtttcatt aaattttctt tagtttgaat ttactgttat tcggtgtgca 4080

tttctatgtt tggtgagcgg ttttctgtgc tcagagtgtg ttattttat gtaatttaat 4140

ttctttgtga gctcctgttt agcaggtcgt cccttcagca aggacacaaa aagattttaa 4200

ttttattaaa aaaaaaaaaa aaaaaaagac cgggaattcg atatcaagct tatcgacctg 4260

cagatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 4320

cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 4380

gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 4440

acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 4500

ctatgttact agatctctag agtctcaagc ttggcgcgcc agcttggcgt aatcatggtc 4560

atagctgttg cgattaagaa ttcgagctcg gtacccccct actccaaaaa tgtcaaagat 4620

acagtctcag aagaccaaag ggctattgag acttttcaac aaagggtaat ttcgggaaac 4680

ctcctcggat tccattgccc agctatctgt cacttcatcg aaaggacagt agaaaaggaa 4740

ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ctatcattca agatgcctct 4800

gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac 4860

gttccaacca cgtcttcaaa gcaagtggat tgatgtgaca tctccactga cgtaagggat 4920

gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag ttcatttcat 4980

ttggagagga cagcccaagc ttcgactcta gaggatcccc ttaaatcgat atggaacgag 5040

ctatacaagg aaacgacgct agggaacaag ctaacagtga acgttgggat ggaggatcag 5100

gaggtaccac ttctcccttc aaacttcctg acgaaagtcc gagttggact gagtggcggc 5160

tacataacga tgagacgaat tcgaatcaag ataatcccct tggtttcaag gaaagctggg 5220

gtttcgggaa agttgtattt aagagatatc tcagatacga caggacggaa gcttcactgc 5280

acagagtcct tggatcttgg acgggagatt cggttaacta tgcagcatct cgatttttcg 5340

gtttcgacca gatcggatgt acctatagta ttcggtttcg aggagttagt atcaccgttt 5400

ctggagggtc tcgaactctt cagcatctct gtgagatggc aattcggtct aagcaagaac 5460

tgctacagct tgccccaatc gaagtggaaa gtaatgtatc aagaggatgc cctgaaggta 5520

ctcaaacctt cgaaaaagaa agcgagtaag tcgagggcga gctcgaattc ggtacgctga 5580

aatcaccagt ctctctctac aaatctatct ctctctattt tctccataaa taatgtgtga 5640

gtagtttccc gataagggaa attagggttc ttatagggtt tcgctcatgt gttgagcata 5700

taagaaaccc ttagtatgta tttgtatttg taaaatactt ctatcaataa aatttctaat 5760

tcctaaaacc aaaatccagt actaaaatcc agatctccta aagtccctat agatctttgt 5820

cgtgaatata aaccagacac gagacgacta aacctggagc ccagacgccg ttcgaagcta 5880

gaagtaccgc ttaggcagga ggccgttagg gaaaagatgc taaggcaggg ttggttacgt 5940

tgactccccc gtaggtttgg tttaaatatg atgaagtgga cggaaggaag gaggaagaca 6000

aggaaggata aggttgcagg ccctgtgcaa ggtaagaaga tggaaatttg atagaggtac 6060

gctactatac ttatactata cgctaaggga atgcttgtat ttatacccta taccccctaa 6120

taacccctta tcaatttaag aaataatccg cataagcccc cgcttaaaaa ttggtatcag 6180

agccatgaat aggtctatga ccaaaactca agaggataaa acctcaccaa aatacgaaag 6240

agttcttaac tctaaagata aaagatggcg cgtggccggc catgagcgga gaattaaggg 6300

agtcacgtta tgacccccgc cgatgacgcg ggacaagccg ttttacgttt ggaactgaca 6360

gaaccgcaac gttgaaggag ccactcagcc gcgggtttct ggagtttaat gagctaagca 6420

catacgtcag aaaccattat tgcgcgttca aaagtcgcct aaggtcacta tcagctagca 6480

aatatttctt gtcaaaaatg ctccactgac gttccataaa ttcccctcgg tatccaatta 6540

gagtctcata ttcactctca atccaaataa tctgcaccgg atctggatcg tttcgcatga 6600

ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 6660

atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 6720

aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 6780

acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 6840

acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 6900

tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 6960

ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 7020

agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 7080

atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 7140

atgatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 7200

gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 7260

cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 7320

tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 7380

agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 7440

atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 7500

ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 7560

cgggatctct gcggaacagg cggtcgaagg tgccgatatc attacgacag caacggccga 7620

caagcacaac gccacgatcc tgagcgacaa tatgatcgcg gcgtccacat caacggcgtc 7680

ggcggcgact gcccaggcaa gaccgagatg caccgcgata tcttgctgcg ttcggatatt 7740

ttcgtggagt tcccgccaca gacccggatg atccccgatc gttcaaacat ttggcaataa 7800

agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 7860

aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 7920

tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 7980

gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg ggggccggcc 8040

ctcactggtg aaaagaaaaa ccaccccagt acattaaaaa cgtccgcaat gtgttattaa 8100

gttgtctaag cgtcaatttg tttacaccac aatatatcct gccaccagcc agccaacagc 8160

tccccgaccg gcagctcggc acaaaatcac cactcgatac aggcagccca tcagtccggg 8220

acggcgtcag cgggagagcc gttgtaaggc ggcagacttt gctcatgtta ccgatgctat 8280

tcggaagaac ggcaactaag ctgccgggtt tgaaacacgg atgatctcgc ggagggtagc 8340

atgttgattg taacgatgac agagcgttgc tgcctgtgat caaatatcat ctccctcgca 8400

gagatccgaa ttatcagcct tcttattcat ttctcgctta accgtgacag agtagacagg 8460

ctgtctcgcg gccgaggggc gcagcccctg gggggatggg aggcccgcgt tagcgggccg 8520

ggagggttcg agaagggggg gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc 8580

agccctggtt aaaaacaagg tttataaata ttggtttaaa agcaggttaa aagacaggtt 8640

agcggtggcc gaaaaacggg cggaaaccct tgcaaatgct ggattttctg cctgtggaca 8700

gcccctcaaa tgtcaatagg tgcgcccctc atctgtcagc actctgcccc tcaagtgtca 8760

aggatcgcgc ccctcatctg tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac 8820

ttatccccag gcttgtccac atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc 8880

cgatttgcga ggctggccag ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg 8940

tcaacgccgc gccgggtgag tcggcccctc aagtgtcaac gtccgcccct catctgtcag 9000

tgagggccaa gttttccgcg aggtatccac aacgccggcg gccgcggtgt ctcgcacacg 9060

gcttcgacgg cgtttctggc gcgtttgcag ggccatagac ggccgccagc ccagcggcga 9120

gggcaaccag cccggtgagc gtcggaaagg cgctcggtct tgccttgctc gtcggtgatg 9180

tacactagtc gctggctgct gaacccccag ccggaactga ccccacaagg ccctagcgtt 9240

tgcaatgcac caggtcatca ttgacccagg cgtgttccac caggccgctg cctcgcaact 9300

cttcgcaggc ttcgccgacc tgctcgcgcc acttcttcac gcgggtggaa tccgatccgc 9360

acatgaggcg gaaggtttcc agcttgagcg ggtacggctc ccggtgcgag ctgaaatagt 9420

cgaacatccg tcgggccgtc ggcgacagct tgcggtactt ctcccatatg aatttcgtgt 9480

agtggtcgcc agcaaacagc acgacgattt cctcgtcgat caggacctgg caacgggacg 9540

ttttcttgcc acggtccagg acgcggaagc ggtgcagcag cgacaccgat tccaggtgcc 9600

caacgcggtc ggacgtgaag cccatcgccg tcgcctgtag gcgcgacagg cattcctcgg 9660

ccttcgtgta ataccggcca ttgatcgacc agcccaggtc ctggcaaagc tcgtagaacg 9720

tgaaggtgat cggctcgccg ataggggtgc gcttcgcgta ctccaacacc tgctgccaca 9780

ccagttcgtc atcgtcggcc cgcagctcga cgccggtgta ggtgatcttc acgtccttgt 9840

tgacgtggaa aatgaccttg ttttgcagcg cctcgcgcgg gattttcttg ttgcgcgtgg 9900

tgaacagggc agagcgggcc gtgtcgtttg gcatcgctcg catcgtgtcc ggccacggcg 9960

caatatcgaa caaggaaagc tgcatttcct tgatctgctg cttcgtgtgt ttcagcaacg 10020

cggcctgctt ggcctcgctg acctgttttg ccaggtcctc gccggcggtt tttcgcttct 10080

tggtcgtcat agttcctcgc gtgtcgatgg tcatcgactt cgccaaacct gccgcctcct 10140

gttcgagacg acgcgaacgc tccacggcgg ccgatggcgc gggcagggca gggggaagcc 10200

agttgcacgc tgtcgcgctc gatcttggcc gtagcttgct ggaccatcga gccgacggac 10260

tggaaggttt cgcggggcgc acgcatgacg gtgcggcttg cgatggtttc ggcatcctcg 10320

gcggaaaacc ccgcgtcgat cagttcttgc ctgtatgcct tccggtcaaa cgtccgattc 10380

attcaccctc cttgcgggat tgccccgact cacgccgggg caatgggccc ttattcctga 10440

tttgacccgc ctgggggctt gggtgtccag aa 10472

Claims (10)

1. Plant transient expression vector, is characterized in that, comprises 5' UTR region, MCS region and SEKDEL sequence. 2. The plant transient expression vector of claim 1, wherein the sequences of the 5'UTR region, the MCS region and the SEKDEL sequence are shown in SEQ ID No.1. 3. The plant transient expression vector according to claim 1 or 2, wherein the plant expression vector pEAQ is used as the basic backbone. 4. plant transient expression vector as described in any one of claim 1 to 3, is characterized in that, also comprises target gene, the N end of described target gene increases PacI restriction enzyme site, and C end increases XhoI restriction enzyme restriction site . 5. the plant transient expression vector as described in any one of claim 2 to 4, is characterized in that, in its construction method, adopts upstream primer as shown in SEQ ID No.2 and as shown in SEQ ID No.3 Downstream primers amplify the sequence shown in SEQ ID No. 1. 6. The plant transient expression vector of claim 5, wherein the amplified amplification system comprises 22 μl ddH ₂ O, 25 μl KOD OneTM PCR MasterMix, 1 μl 10 μM upstream primers as shown in SEQ ID No.2 , 1 μl 10 μM downstream primer shown in SEQ ID No.3, 1 μl 100 ng/μl plasmid template containing artificially synthesized sequence SEQ ID No.1. 7. The plant transient expression vector according to claim 5 or 6, wherein the amplification procedure is: 98°C for 30 seconds; 28 cycles: 98°C for 10 seconds; 55°C for 30 seconds; 68°C for 10 seconds seconds; 5 minutes at 68°C; 10 minutes at 4°C. 8. The application of the plant transient expression vector according to any one of claims 1 to 7 in expressing foreign proteins. 9. The use of claim 8, wherein the exogenous protein comprises GFP. 10. The method for expressing exogenous protein of plant transient expression vector, is characterized in that, comprises the steps: Step 1: construct the plant transient expression vector as claimed in claim 1; Step 2: insert the gene of the exogenous protein into the plant transient expression vector prepared in step 1; obtain a recombinant plasmid; Step 3: transform the recombinant plasmid obtained in step 2 into a competent host, culture, collect bacterial liquid, infiltrate plant leaves, and extract protein.

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