CN113817766A - Gene expression cassette, recombinant expression vector, preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to a gene expression cassette, a recombinant expression vector and a preparation method and application thereof. The present invention provides a gene expression cassette comprising a promoter, an egfp reading frame, a coding sequence of a 2A peptide and a multi-site artificial linker sequence connected in sequence. Compared with the prior art, the vector in the present invention contains a GFP-2A expression cassette driven by a soybean endogenous promoter, and the screening of positive transgenic roots with GFP as a marker gene is a visual screening method, which avoids the need for chimeric plants. Defects sensitive to herbicides can be screened for transgenic roots and the positive rate of roots can be guaranteed.

Description

Gene expression cassette, recombinant expression vector, preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a gene expression cassette, a recombinant expression vector, and a preparation method and application thereof.

Background

Reverse genetics is one of the important methods for studying gene phenotypes, and can identify the phenotype of a gene by overexpressing a target gene. The common reverse genetics means is transgenosis, the time for obtaining T1 generation transgenic plants of soybean is 5 to 8 months, and the experimental period is long. In the research of root biology, researchers usually only pay attention to the phenotype of roots, but not to the whole plant. The method for quickly and specifically generating the transgenic soybean roots is explored, so that the experimental period can be shortened.

It has been shown that Agrobacterium rhizogenes K599 can induce soybean to produce hairy roots, and the subsequent studies have confirmed that introduction of plasmid K599 can induce regeneration of hairy roots of target gene at higher than normal expression level. However, because the hairy root and the normal adventitious root are highly similar in shape, how to distinguish the transgenic root from the adventitious root of the soybean becomes a new problem. In the experiment of transgenic plants, herbicide-resistant genes can be used for screening the transgenic plants, but because herbicides such as Basta and the like are conducted in plants through transpiration, the herbicides have lethality to overground part plants, and therefore, the herbicide-resistant genes are not suitable for screening hairy roots.

The use of Green Fluorescent Protein (GFP) is one approach to solve this problem. GFP is stable in structure, and green fluorescence can be observed in plants. Fluorescent proteins have found widespread use in research in cell biology, developmental biology and neuroscience.

Therefore, it is an urgent need to solve the problem of the art how to select hairy roots expressing a target gene by simultaneously using a fluorescent protein technology and a target gene screening technology and by observing fluorescence.

Disclosure of Invention

The invention aims to provide a recombinant expression vector and a preparation method and application thereof, wherein the recombinant expression vector is constructed by comprehensively using 2A peptide, a soybean endogenous promoter and a fluorescent protein label, and soybean hairy roots which simultaneously express two genes are obtained.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a gene expression cassette which comprises a promoter, an egfp reading frame, a 2A peptide coding sequence and a multi-site artificial joint sequence which are connected in sequence.

Preferably, the 2A peptide is one of F2A peptide, T2A peptide, P2A peptide and E2A peptide; the nucleotide sequence of the multi-site artificial linker sequence is shown as SEQ NO: 1 is shown.

Further preferred is F2A peptide, the nucleotide sequence of which is shown in SEQ NO: 2, respectively.

Preferably, the promoter is Gmubi or GmubiXL.

Further preferred is Gmubi, the nucleotide sequence of which is shown in SEQ NO: 3, respectively.

The invention also provides a recombinant expression vector, which comprises the gene expression cassette and an initial vector.

Preferably, the initial vector is pCAMBIA1303, pBI121 or pPZP 100.

Further preferably pCAMBIA 1303.

The invention also provides a preparation method of the recombinant expression vector, which comprises the following steps:

(1) when the initial Vector is pCAMBIA1303, a promoter is inserted between a CaMV poly (A) signal and an NOS terminator of the initial Vector to obtain Vector-P;

(2) inserting egfp reading frame between the promoter and the NOS terminator of the Vector-P obtained in the step (1) to obtain Vector-P-E;

(3) inserting a 2A peptide coding sequence between the egfp reading frame of the Vector-P-E obtained in the step (2) and the NOS terminator to obtain the Vector-P-E-2A;

(4) and (4) inserting a multi-site artificial linker sequence between the 2A peptide coding sequence of the Vector-P-E-2A obtained in the step (3) and the NOS terminator to obtain a recombinant expression Vector-P-E-2A-MCS.

The invention also provides a preparation method of the recombinant expression vector, which comprises the following steps:

(1) when the initial Vector is pBI121, inserting a promoter between the RB T-DNAREpeat and the NOS terminator of the initial Vector to obtain Vector-P;

(2) inserting egfp reading frame between the promoter and the NOS terminator of the Vector-P obtained in the step (1) to obtain Vector-P-E;

(3) inserting a 2A peptide coding sequence between the egfp reading frame of the Vector-P-E obtained in the step (2) and the NOS terminator to obtain the Vector-P-E-2A;

(4) and (4) inserting a multi-site artificial linker sequence between the 2A peptide coding sequence of the Vector-P-E-2A obtained in the step (3) and the NOS terminator to obtain a recombinant expression Vector-P-E-2A-MCS.

The invention also provides a preparation method of the recombinant expression vector, which comprises the following steps:

(1) when the initial Vector is pPZP100, inserting an NOS terminator into MCS of the initial Vector, and then inserting a promoter between the NOS terminator and LB T-DNAREAPEAT to obtain Vector-P;

(2) inserting egfp reading frame between the promoter and the NOS terminator of the Vector-P obtained in the step (1) to obtain Vector-P-E;

(3) inserting a 2A peptide coding sequence between the egfp reading frame of the Vector-P-E obtained in the step (2) and the NOS terminator to obtain the Vector-P-E-2A;

(4) and (4) inserting a multi-site artificial linker sequence between the 2A peptide coding sequence of the Vector-P-E-2A obtained in the step (3) and the NOS terminator to obtain a recombinant expression Vector-P-E-2A-MCS.

A host transformed or transfected with the recombinant expression vector of claim.

The invention also provides the gene expression cassette, the recombinant expression vector and the application of the host in preparing transgenic plants.

Preferably, the plant is soybean.

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

1. while traditional expression vectors typically use the CaMV promoter to drive downstream gene expression, studies have indicated that higher expression efficiencies can be achieved using endogenous promoters, which are about 40-fold higher in soybean than 35S. Meanwhile, 2A peptide, which is a short peptide sequence of 18 to 22 amino acids and can be used for dividing two recombinant proteins by self-shearing, is widely applied to gene editing. However, polycistronic expression using the self-splicing properties of the 2A peptide was not reported in the expression vector. The invention constructs a recombinant expression vector by comprehensively using the 2A peptide, the soybean endogenous promoter and the fluorescent protein label, and obtains the soybean hairy root for simultaneously expressing two genes.

2. Compared with the prior art, the vector contains a GFP-2A expression cassette driven by a soybean endogenous promoter, and the screening of the positive transgenic roots by taking GFP as a marker gene is a visual screening mode, so that the defect that chimeric plants are sensitive to herbicides is overcome, the transgenic roots can be screened, and the positive rate of screened roots can be ensured.

The recombinant expression vector can express target genes besides GFP, can express a plurality of proteins in one expression cassette, and can control the size of the vector and improve the transformation efficiency compared with the common double-expression cassette vector, so that positive transgenic hairy roots can be quickly screened by using the recombinant expression vector.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram of the structure of the recombinant expression vector obtained in example 1;

FIG. 2 is an electrophoretic image after PCR, wherein the Marker uses DL2,000, and the bands have molecular weights of 2000, 1000, 750, 500, 250, 100bp, four sets of repeats;

FIG. 3 is a hydroponic soybean expressing green fluorescence;

FIG. 4 is a hairy root expressing green fluorescence;

FIG. 5 shows the expression levels of the genes of interest in different experimental groups.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

A method for preparing a recombinant expression vector, comprising the steps of:

(1) inserting a promoter Gmpbi between a CaMVpoly (A) signal and an NOS terminator of an initial Vector pCAMBIA1303 to obtain Vector-P;

(2) inserting egfp reading frame between the promoter and the NOS terminator of the Vector-P obtained in the step (1) to obtain Vector-P-E;

(3) inserting a coding sequence of F2A peptide between the egfp reading frame of the Vector-P-E obtained in the step (2) and the NOS terminator to obtain Vector-P-E-2A;

(4) inserting a multi-site artificial linker sequence between the 2A peptide coding sequence of the Vector-P-E-2A obtained in the step (3) and the NOS terminator to obtain a recombinant expression Vector-P-E-2A-MCS, wherein the nucleotide sequence of the Vector is shown in SEQ NO: 4 is shown in the specification; the method for insertion is double digestion (T4 ligase is used for ligation).

Example 2

A method for preparing a recombinant expression vector, comprising the steps of:

(1) inserting a promoter GmubiXL between the RB T-DNAREpeat and the NOS terminator of the initial Vector pBI121 to obtain Vector-P;

(2) inserting egfp reading frame between the promoter and the NOS terminator of the Vector-P obtained in the step (1) to obtain Vector-P-E;

(3) inserting a coding sequence of T2A peptide between the egfp reading frame of the Vector-P-E obtained in the step (2) and the NOS terminator to obtain Vector-P-E-2A;

(4) inserting a multi-site artificial linker sequence between the 2A peptide coding sequence of the Vector-P-E-2A obtained in the step (3) and the NOS terminator to obtain a recombinant expression Vector-P-E-2A-MCS; the method for insertion is double digestion (T4 ligase is used for ligation).

Example 3

A method for preparing a recombinant expression vector, comprising the steps of:

(1) inserting an NOS terminator into MCS of an initial Vector pPZP100, and inserting a promoter Gmubi between the NOS terminator and LB T-DNAREpeat to obtain Vector-P;

(2) inserting egfp reading frame between the promoter and the NOS terminator of the Vector-P obtained in the step (1) to obtain Vector-P-E;

(3) inserting a coding sequence of P2A peptide between the egfp reading frame of the Vector-P-E obtained in the step (2) and the NOS terminator to obtain Vector-P-E-2A;

(4) inserting a multi-site artificial linker sequence between the 2A peptide coding sequence of the Vector-P-E-2A obtained in the step (3) and the NOS terminator to obtain a recombinant expression Vector-P-E-2A-MCS; the method for insertion is double digestion (T4 ligase is used for ligation).

Example 4 transformation of Agrobacterium rhizogenes k599 Using vectors

(1) The concentration of the recombinant expression vector obtained in example 1 was measured and 1mg was added to 100. mu.l of competent Agrobacterium rhizogenes k599 (purchased from Shanghai Weidi Biotech Ltd.) for transformation and activation; sucking 200 mu L of activated competent agrobacterium rhizogenes k599, coating the competent agrobacterium rhizogenes k599 on an LB culture medium containing kanamycin, and performing inverted culture at 28 ℃ for 36 hours;

(2) selecting the monoclonal (positive clone) obtained in the step (1) for liquid culture, detecting by PCR, and carrying out electrophoresis detection after PCR as shown in figure 2 (detecting the sequence of the vector expression frame in different clones);

verifying the primer sequence f: CCCTATAGGAACCCTAATTCCCTTATCTG (shown as SEQ NO: 5)

Verifying the primer sequence R: CATGCTTAACGTAATTCAACAGAAAT (shown in SEQ NO: 6).

EXAMPLE 5 transfer of vector into Agrobacterium rhizogenes and subsequent treatment of Soybean hypocotyls to obtain fluorescent hairy roots

(1) The positive clones obtained in example 1 were subjected to liquid scale-up culture.

(2) Selecting plump soybean seeds, sterilizing with chlorine, germinating in wet sterile vermiculite for 4-5 days, allowing seedling hypocotyl to extend to 3cm for use to obtain soybean hairy root, cleaning soybean hairy root with sterile water, cutting off at 45 ° slant at the white-green junction of hypocotyl, retaining green part, contacting cut surface with bacterial liquid, placing on 1/2MS slant plate, culturing in dark for 2 days, and maintaining at 23 deg.C.

(3) After the co-culture is finished, the seedlings are transferred to a tissue culture bottle filled with wet sterilized vermiculite, after the seedlings are cultured for two weeks under the condition of keeping humidity, and then the seedlings are irradiated by LUYOR-3415RG to observe GFP fluorescence, so that positive hairy roots expressing green fluorescence and non-transgenic adventitious roots produced by soybeans can be observed. (FIG. 3 is a photograph of hydroponics soybean, FIG. 4 is a photograph of newly grown fluorescent hairy roots)

Example 6 GUS Gene was inserted into vector, after induction, fluorescent expressed hairy roots were selected and expressed by qPCR detection

(1) The GUS gene was cloned from pCAMBIA1303 and used as a target gene to examine whether expression was normal.

Cloning primer sequence F: ATGTTACGTCCTGTAGAAACCCCAACC (shown as SEQ NO: 7)

Cloning primer sequence R: TTGTTTGCCTCCCTGCTGC (shown as SEQ NO: 8)

A linker sequence F: CCAGGACCCGAGCTCGTT (shown as SEQ NO: 9)

A linker sequence R: GTCACCAATTCATTAAGG (shown as SEQ NO: 10)

(2) The recombinant expression vector obtained in example 1 was subjected to double digestion with Hpa I and Stu I, followed by purification after PCR using a linker-containing cloning primer, followed by linearization, and the linearized recombinant expression vector was recovered.

(3) Connecting the PCR product with a linearized vector by using In-Fusion, screening positive clones, then transferring agrobacterium to infect soybean, screening positive hairy roots, extracting RNA of the screened hairy roots, then carrying out reverse transcription, and detecting the expression quantity of a target gene by using qPCR.

The results show that: compared with negative controls (Neg _ gfp and Neg _ gus), the expression level of the test sample (Q1 to Q4) in the gfp and gus indexes is improved by about 1000 times, and the difference is three orders of magnitude. The difference was 2 to 8-fold as compared with the positive control (Pos) (results are shown in FIG. 5) (it is known that the expression amount was doubled for each difference of 1 in Cq value, and the expression amount was higher for the smaller Cq value).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Shenyang agriculture university

<120> gene expression cassette, recombinant expression vector, preparation method and application thereof

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gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

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agtggaagcg gtgtgaagca aacactcaac tttgacctgc ttaaactcgc tggcgatgtt 780

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actctccgaa aatgcatcca ataccaaata ttacccgtgt cataggcacc aagtgacacc 180

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aaacgtaacc atagatcagg gctttttcat gaattacttc agatccgtta aacaacagcc 960

ttatttttta tacttctgtg gtttttcaag aaattgttca gatccgttga caaaaagcct 1020

tattcgttga ttctatatcg tttttcgaga gatattgctc agatctgtta gcaactgcct 1080

tgtttgttga ttctattgcc gtggattagg gttttttttc acgagattgc ttcagatccg 1140

tacttaagat tacgtaatgg attttgattc tgatttatct gtgattgttg actcgacagg 1200

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ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 1320

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gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 1680

gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 1740

cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 1800

ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 1860

ggtcctgctg gagttcgtga ccgccgccgg gatcactcac ggcatggacg agctgtacaa 1920

gactagtgga agcggtgtga agcaaacact caactttgac ctgcttaaac tcgctggcga 1980

tgttgaaagc aatccaggac ccgagctcgt taaccagctg aggccttaat gaattggtga 2040

ccagctcgaa tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc 2100

ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa 2160

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cgggatcaaa gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt 2520

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gccgccctgc ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat 2640

acttgcgact agaaccggag acattacgcc atgaacaaga gcgccgccgc tggcctgctg 2700

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gcggccggct gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg 2820

gagctggcca ggatgcttga ccacctacgc cctggcgacg ttgtgacagt gaccaggcta 2880

gaccgcctgg cccgcagcac ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc 2940

ggcgcgggcc tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc ggccggccgc 3000

atggtgttga ccgtgttcgc cggcattgcc gagttcgagc gttccctaat catcgaccgc 3060

acccggagcg ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc 3120

ctcaccccgg cacagatcgc gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg 3180

aaagaggcgg ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc 3240

agcgaggaag tgacgcccac cgaggccagg cggcgcggtg ccttccgtga ggacgcattg 3300

accgaggccg acgccctggc ggccgccgag aatgaacgcc aagaggaaca agcatgaaac 3360

cgcaccagga cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga 3420

tcgcggccgg gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa 3480

tcctggccgg tttgtctgat gccaagctgg cggcctggcc ggccagcttg gccgctgaag 3540

aaaccgagcg ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg 3600

cggtcgctgc gtatatgatg cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa 3660

ggttatcgct gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct 3720

agcccgcgcc ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg 3780

cagtgcccgc gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga 3840

ccgcccgacg attgaccgcg acgtgaaggc catcggccgg cgcgacttcg tagtgatcga 3900

cggagcgccc caggcggcgg acttggctgt gtccgcgatc aaggcagccg acttcgtgct 3960

gattccggtg cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt 4020

taagcagcgc attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc 4080

gatcaaaggc acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc 4140

cattcttgag tcccgtatca cgcagcgcgt gagctaccca ggcactgccg ccgccggcac 4200

aaccgttctt gaatcagaac ccgagggcga cgctgcccgc gaggtccagg cgctggccgc 4260

tgaaattaaa tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca 4320

aacacgctaa gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc 4380

ctggcagaca cgccagccat gaagcgggtc aactttcagt tgccggcgga ggatcacacc 4440

aagctgaaga tgtacgcggt acgccaaggc aagaccatta ccgagctgct atctgaatac 4500

atcgcgcagc taccagagta aatgagcaaa tgaataaatg agtagatgaa ttttagcggc 4560

taaaggaggc ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat 4620

gtgtggagga acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa 4680

tggcactgga acccccaagc ccgaggaatc ggcgtgagcg gtcgcaaacc atccggcccg 4740

gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc 4800

gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct 4860

gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag 4920

ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc 4980

cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga 5040

gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg 5100

gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc 5160

gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca 5220

cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac 5280

ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag 5340

gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag 5400

atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg 5460

taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt 5520

ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag 5580

gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc 5640

aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat 5700

ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc 5760

gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta 5820

gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca 5880

aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac 5940

cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac 6000

tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag 6060

cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc 6120

gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca 6180

ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat 6240

caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 6300

cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 6360

cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 6420

cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 6480

atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc 6540

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 6600

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 6660

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 6720

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 6780

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 6840

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 6900

gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 6960

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 7020

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 7080

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 7140

tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 7200

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 7260

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 7320

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 7380

cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt ctcccaatca 7440

ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc gatatcctcc 7500

ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc gcttctccca 7560

agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc tcccaggtcg 7620

ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc atacagctcg 7680

cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc ggccagatcg 7740

ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt atagggacaa 7800

tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc gataatcttt 7860

tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc ctcactcatg 7920

agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac aggcagcttt 7980

ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt ccctttatac 8040

cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt atatacctta 8100

gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag ttttttcaat 8160

tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta cagtatttaa 8220

agatacccca agaagctaat tataacaaga cgaactccaa ttcactgttc cttgcattct 8280

aaaaccttaa ataccagaaa acagcttttt caaagttgtt ttcaaagttg gcgtataaca 8340

tagtatcgac ggagccgatt ttgaaaccgc ggtgatcaca ggcagcaacg ctctgtcatc 8400

gttacaatca acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt 8460

tgccgttctt ccgaatagca tcggtaacat gagcaaagtc tgccgcctta caacggctct 8520

cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag 8580

ctgccggtcg gggagctgtt ggctggctgg 8610

<210> 5

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccctatagga accctaattc ccttatctg 29

<210> 6

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

catgcttaac gtaattcaac agaaat 26

<210> 7

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgttacgtc ctgtagaaac cccaacc 27

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttgtttgcct ccctgctgc 19

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccaggacccg agctcgtt 18

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gtcaccaatt cattaagg 18

Claims (10)

1. A gene expression cassette is characterized by comprising a promoter, an egfp reading frame, a 2A peptide coding sequence and a multi-site artificial joint sequence which are connected in sequence.

2. The gene expression cassette of claim 1, wherein the 2A peptide is one of F2A peptide, T2A peptide, P2A peptide, and E2A peptide; the nucleotide sequence of the multi-site artificial linker sequence is shown as SEQ NO: 1 is shown.

3. The gene expression cassette of claim 1 or 2, wherein the promoter is Gmubi or GmubiXL.

4. A recombinant expression vector comprising the gene expression cassette of any one of claims 1 to 3 and an initial vector.

5. The recombinant expression vector according to claim 4, wherein the initial vector is pCAMBIA1303, pBI121 or pPZP 100.

6. The method for producing the recombinant expression vector according to claim 5, comprising the steps of:

(1) when the initial Vector is pCAMBIA1303, a promoter is inserted between a CaMV poly (A) signal and an NOS terminator of the initial Vector to obtain Vector-P;

(2) inserting egfp reading frame between the promoter and the NOS terminator of the Vector-P obtained in the step (1) to obtain Vector-P-E;

(3) inserting a 2A peptide coding sequence between the egfp reading frame of the Vector-P-E obtained in the step (2) and the NOS terminator to obtain the Vector-P-E-2A;

(4) and (4) inserting a multi-site artificial linker sequence between the 2A peptide coding sequence of the Vector-P-E-2A obtained in the step (3) and the NOS terminator to obtain a recombinant expression Vector-P-E-2A-MCS.

7. The method for producing the recombinant expression vector according to claim 5, comprising the steps of:

(1) when the initial Vector is pBI121, inserting a promoter between the RB T-DNA repeat and the NOS terminator of the initial Vector to obtain Vector-P;

(2) inserting egfp reading frame between the promoter and the NOS terminator of the Vector-P obtained in the step (1) to obtain Vector-P-E;

(3) inserting a 2A peptide coding sequence between the egfp reading frame of the Vector-P-E obtained in the step (2) and the NOS terminator to obtain the Vector-P-E-2A;

(4) and (4) inserting a multi-site artificial linker sequence between the 2A peptide coding sequence of the Vector-P-E-2A obtained in the step (3) and the NOS terminator to obtain a recombinant expression Vector-P-E-2A-MCS.

8. The method for producing the recombinant expression vector according to claim 5, comprising the steps of:

(1) when the initial Vector is pPZP100, inserting an NOS terminator into MCS of the initial Vector, and then inserting a promoter between the NOS terminator and LB T-DNA repeat to obtain Vector-P;

(2) inserting egfp reading frame between the promoter and the NOS terminator of the Vector-P obtained in the step (1) to obtain Vector-P-E;

(3) inserting a 2A peptide coding sequence between the egfp reading frame of the Vector-P-E obtained in the step (2) and the NOS terminator to obtain the Vector-P-E-2A;

(4) and (4) inserting a multi-site artificial linker sequence between the 2A peptide coding sequence of the Vector-P-E-2A obtained in the step (3) and the NOS terminator to obtain a recombinant expression Vector-P-E-2A-MCS.

9. A host transformed or transfected with the recombinant expression vector of claim 4 or 5.

10. Use of the gene expression cassette of any one of claims 1 to 3, the recombinant expression vector of claim 4 or 5, or the host of claim 9 for the production of transgenic plants.

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