CN111732663B - Fusion protein for marking microtubule framework, encoding gene and construction method of transgenic leguminous plant - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to a fusion protein for marking a microtubule framework, an encoding gene and a construction method of a transgenic leguminous plant. The amino acid sequence of the fusion protein is shown as SEQ ID No.2, and meanwhile, the invention also relates to a nucleotide sequence for coding the fusion protein, a plant expression vector containing the sequence, recombinant agrobacterium and a construction method of a transgenic leguminous plant.

Description

Fusion protein for marking microtubule framework, encoding gene and construction method of transgenic leguminous plant

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a fusion protein for marking a microtubule framework, an encoding gene and a construction method of a transgenic leguminous plant.

Background

The plant microtubule skeleton is formed by combining alpha tubulin and beta tubulin, has high dynamic characteristics, and can quickly recombine microtubule arrays after cells are stimulated by external environmental signals, thereby playing a vital role in the aspects of plant cell growth, cell division, vesicle transport, organelle transport, cell wall synthesis, biotic and abiotic stress reaction and the like. Plant-specific microtubule arrays are formed in plant cells, such as periplasmic Microtubules (CMTs), prophase bands (PPB) and membrane formers (Phragmoplast). The PPB, centrosome, spindle microtubules and membrane-forming bodies are involved in mitosis of cells, and the periplasmic microtubules are closely related to the cell morphology.

The sequences of the tubulin of different plants have certain differences, the microtubule skeleton in Arabidopsis has the main function of participating in the elongation growth of various tissues and organs and the like, the microtubule skeleton in leguminous plants has the main function in the symbiotic nitrogen fixation process, and researches show that the mutation of the protein MtDREPP which regulates both microfilaments and microtubules does not influence the elongation growth of alfalfa root hairs but influences the infection efficiency of rhizobia so as to influence the nodulation number, and the fact that the microtubule skeleton in alfalfa is possibly marked by the gene in Arabidopsis is presumed not completely representative. In addition, the 35S strong promoter used for marking the microtubule skeleton in most of the plants at present can cause the situation of chimeric expression after being transferred into the plants, and can not mark the microtubule skeleton in root and root hair cells. Although transient expression can label microtubules in alfalfa roots and root hair cells, microtubule labeling elsewhere is not achieved with transient expression techniques.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a fusion protein, a coding gene and a construction method of a transgenic leguminous plant.

In order to achieve the purpose, the invention adopts the following technical scheme:

one of the purposes of the invention is to provide a fusion protein for marking a microtubule framework, wherein the fusion protein is GFP-MtTUA6-GFP, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 2; constructing an expression vector for coding the fusion protein gene, and transforming leguminous plants to obtain transgenic leguminous plants for marking microtubule skeletons in leguminous plants.

Another object of the present invention is to provide a nucleotide sequence encoding the fusion protein, wherein the nucleotide sequence is shown as 2520-5303 of SEQ ID NO. 1.

It is a further object of the present invention to provide a plant expression vector comprising said nucleotide sequence.

The fourth purpose of the invention is to provide a recombinant agrobacterium which comprises the plant expression vector.

Further, the construction method of the recombinant agrobacterium is as follows:

mixing the agrobacterium tumefaciens EHA105 competent cells with the plant expression vector, placing on ice for incubation for 30min, quickly freezing for 2min by liquid nitrogen, recovering for 3-5min at 37 ℃, adding 1mL of liquid YEB culture medium, performing shake culture at 28 ℃ and 180rpm for 4h, collecting thalli, and performing culture for 36h at 28 ℃ on YEB solid culture medium containing 50mg/L spectinomycin and 100mg/L rifampicin. Selecting monoclonal shake bacteria, and identifying positive clone by PCR and enzyme digestion detection to obtain recombinant agrobacterium.

The fifth purpose of the invention is to provide a construction method of a transgenic leguminous plant, which comprises the following steps:

s1, transforming leguminous plants by using the recombinant agrobacterium, recording as T0 generations, and recording seeds as T1 generations;

resistant seedlings are obtained by screening seeds of S2 and T1 generations through kanamycin and are recorded as T2 generations;

and (3) screening seeds of S3 and T2 generations by kanamycin, and obtaining a survival plant which is a homozygous T3 generation transgenic plant, namely a transgenic leguminous plant, when a full survival proportion appears.

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

1. the invention uses the tubulin MtTUA6 gene self promoter of the leguminous plant medicago truncatula to start the expression of tubulin, constructs the stable expression plant material which can mark the microtubule skeleton in the leguminous plant cell, and the leguminous plant endogenous gene marks the plant microtubule, and has strong specificity.

2. The invention overcomes the problem of expression mosaic caused by the start of 35S promoter in model plant Arabidopsis thaliana, uses the gene promoter to start gene expression, has no mosaic expression, more comprehensively marks the tissue dynamic change of microtubule skeleton in leguminous plants, and can observe the state of microtubule skeleton in each tissue organ of leguminous plants.

3. The two green fluorescent protein labels are connected, so that green fluorescent signals are stronger, and microtubule skeleton marks are clearer.

4. The transgenic leguminous plant constructed by the invention can more accurately mark a microtubule skeleton in the leguminous plant so as to conveniently research the mechanisms in the aspects of symbiotic nitrogen fixation, biomass change, root system development mode and the like.

Drawings

FIG. 1 is a schematic diagram of vector map of PZP211-TUA6Pro: GFP-MtTUA 6-GFP.

FIG. 2 is a graph showing the results of expression of GFP-MtTUA6-GFP in different cells of transgenic alfalfa.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.

The Medicago truncatula R108 in the examples described below is publicly available from the institute of bioscience and technology, Shenyang university of agriculture, and the biomaterial is used only for repeating the experiments related to the present invention, and is not used for other purposes.

Agrobacterium tumefaciens EHA105(CraneC, Wright E, DixonRA, & WangZY. (2006). Trans genic media genetic and transformed plant gene and transformed DNA bacteria genes. plant, 223(6), 1344. 1354.)

GFP-PZP211 and GFP-PUC vector vectors were obtained as a gift from the subject group of the Proc of King Tree, Shiyang university, publicly available from the institute of bioscience and technology, Shenyang university, and the biomaterials were used only for repeating the experiments related to the present invention, and were not used for other purposes.

Example 1

Plant expression vector

The construction method comprises the following steps:

a DNA molecule shown in SEQ ID NO.1 and named TUA6Pro GFP-MtTUA6-GFP-PZP211 is artificially synthesized. In SEQ ID NO.1, positions 1-2519 are the promoter of the TUA6 gene; the 2520-5303 position of SE Q ID NO.1 is a GFP-MtTUA6-GFP fusion gene, the coding sequence of which encodes the fusion protein GFP-MtTUA6-GFP shown in SEQ ID NO. 2; the 2520-3236 th position of SEQ ID NO.1 is a gene encoding an N-terminal GFP encoding the GFP shown in the 1 st-239 th positions of SEQ ID NO. 2; the 3257-4586 locus of SEQ ID NO.1 is the coding gene for the alfalfa tubulin MtTUA6, which encodes the alfalfa TUA6 protein shown at positions 240-689 of SEQ ID NO. 2; the 4587-5303-position of SE Q ID NO.1 is the coding gene for C-terminal GFP, which codes for the GFP shown in the 690-928-position of SEQ ID NO. 2.

The method comprises the following specific steps:

(1) construction of the intermediate vector MtTUA6-T

The MtTUA6 gene sequence was cloned using alfalfa R108 wild-type cDNA as a template, and after agarose gel electrophoresis, the gel block with the MtTUA6 fragment was cut back. The purified MtTUA 6-purpose fragment was recovered using the SanPrep column DNA gel recovery kit. The intermediate vector MtTUA6-T was obtained by ligating the vector Mt TUA6 with the cloning vector T using the PGM-T kit (Tiangen).

Taking out DH5 alpha colibacillus competent cells frozen at-80 ℃, and slowly thawing the cells on ice (about 10 min); adding intermediate carrier MtTUA6-T, and standing on ice for 30 min; rapidly cooling on ice for 2min at 42 deg.C for 90 s; adding LB culture medium, culturing at 37 deg.C and 200rpm for 1 h; the cells were collected and cultured overnight at 37 ℃ on LB solid medium with a final concentration of 50mg/L carboxybenzyl. Single colonies were picked in 3mL LB medium and cultured overnight at 200 rpm. And (3) detecting positive clones by using PCR (polymerase chain reaction) by taking the bacterial liquid as a template. The positive strain is selected, the plasmid MtTUA6-T is extracted by using a SanPrep column type plasmid DNA small-scale extraction kit (Shanghai worker), the plasmid MtTUA6-T is sent to a company for sequencing (Shanghai worker), and the sequencing result shows that the MtTUA6-T contains the DNA molecule of MtTUA 6.

(2) The plasmid containing MtTUA6 and GFP-PUC vector were recovered by double-digesting MtTUA6-T and GFP-PUC with restriction enzymes Nde I and Sac I, and T was used as a restriction enzyme₄-DNA Ligase (TAKA RA) fragments of MtTUA6 were ligated to the GFP-PUC vector.

Taking out DH5 alpha colibacillus competent cells frozen at-80 ℃, and slowly thawing the cells on ice (about 10 min); adding the vector GFP-MtTUA6-PUC, and placing on ice for 30 min; heating at 42 deg.C for 90s, rapidly cooling on ice for 2 min; adding LB culture medium, culturing at 37 deg.C and 200rpm for 1 h; the cells were collected and cultured on LB solid medium with a final concentration of 50mg/L carboxybenzyl at 37 ℃ for 12 hours. Colonies were picked in 3mL LB medium and cultured overnight at 200 rpm. And (3) detecting positive clones by using PCR (polymerase chain reaction) by taking the bacterial liquid as a template. Selecting positive strains, extracting plasmids GFP-MtTUA6-PUC by using a plasmid extraction kit (SanPrep), digesting GFP-MtTUA6-PUC plasmids by using restriction enzymes Nde I and Sac I, and observing the size of an MtT UA6 band and the size of a GFP-PUC band after agarose gel electrophoresis. GFP-MtTUA6-PUC was sent to the company for sequencing (Shanghai, Japan), and the sequencing result indicated that the GFP-PUC vector contained the DNA molecule of MtTUA 6.

(3) The 35S promoter in the GFP-MtTUA6-PUC vector is replaced by the promoter of Medicago truncatula TUA6Pro to construct the TUA6Pro vector, wherein the GFP-MtTUA6-PUC vector comprises the following specific steps:

extracting the genome DNA of the medicago truncatula R108 plant, cloning a promoter of the MtTUA6 gene, namely 2519bp at the upstream of ATG of the MtTUA6 gene, carrying out agarose gel electrophoresis on the cloned sequence, cutting gel to recover TUA6Pro fragments, and recovering and purifying TUA6Pro target fragments by using a SanPrep column type DNA gel recovery kit (Shanghai's worker). The GFP-MtTUA6-PUC vector is subjected to double digestion by PstI and NcoI, agarose gel electrophoresis is carried out, the GFP-MtTUA6-PUC vector is recovered by cutting gel, and the recovered and purified TUA6Pro fragment and the GFP-MtTUA6-PUC vector utilize T₄DNA Ligase ligation, transformation of the ligation product into E.coli DH5 alpha competent cells, selection of single clones, identification of positive clones by PCR and enzyme digestion detection, obtaining TUA6Pro: GFP-MtTUA6-PUC vector.

(4) The TUA6Pro vector including GFP-MtTUA6-PUC and GFP-PZP211 vector is subjected to double digestion with restriction enzymes PstI and SacI, and the digestion reaction is carried out at 37 ℃ for 4h, thus recovering the TUA6Pro vector including GFP-MtTUA6 fragment and GFP-PZP211 vector, and utilizing T₄DNA ligase (Takara) ligation of TUA6Pro: GFP-MtTUA6 with GFP-PZP 211. Taking out DH5 frozen at-80 deg.CCompetent cells of alpha E.coli were thawed slowly on ice (about 10 min); adding plasmid PZP211-TUA6Pro GFP-MtTUA6-GFP, and standing on ice for 30 min; heating at 42 deg.C for 90s, rapidly cooling on ice for 2 min; LB medium was added thereto, cultured at 37 ℃ and 200rpm for 1 hour, and the cells were collected and cultured on LB solid medium with a final concentration of 50mg/L spectinomycin at 37 ℃ for 12 hours. Colonies were picked in 3mL LB medium and cultured overnight at 200 rpm. The bacterial liquid is used as a template, PCR and double enzyme digestion are utilized to detect positive clones, and a PZP211-TUA6Pro vector, GFP-MtTU A6-GFP vector is obtained, and the map is schematically shown in figure 1.

Example 2

Recombinant agrobacterium tumefaciens

The construction method comprises the following steps:

mixing Agrobacterium tumefaciens EHA105 competent cells with PZP211-TUA6Pro, GFP-MtTUA6-GF P plasmid, placing on ice for incubation for 30min, liquid nitrogen quick freezing for 2min, recovering for 3-5min at 37 ℃, adding 1mL liquid YEB culture medium, shaking and culturing at 28 ℃ and 180rpm for 4h, collecting the thallus, and culturing at 28 ℃ for 36h on YEB solid culture medium containing spectinomycin with the final concentration of 50mg/L and rifampicin with the final concentration of 100 mg/L. Selecting monoclonal shake bacteria, identifying positive clone by PCR and enzyme digestion detection, obtaining recombinant agrobacterium containing PZP211-TUA6Pro, GFP-MtTUA6-GFP expression vector, named PZP211-TUA6Pro, GFP-Mt TUA6-GFP/EHA105, and obtaining the infecting bacteria for genetic transformation of alfalfa.

Example 3

Transgenic leguminous plant-transgenic alfalfa

The construction method comprises the following steps:

the genetic transformation of alfalfa adopts a leaf disc transformation method. Activated PZP211-TUA6Pro GFP-MtTU A6-GFP/EHA105 was shake-cultured overnight at 30 ℃ (200rpm) in liquid medium containing spectinomycin at a final concentration of 50mg/L and rifampicin 2mLYE B at a final concentration of 100 mg/L; transferring 1-2mL of overnight bacterial liquid to 200mL conical flask containing 30mLYEB liquid culture medium for amplification culture, and performing OD₆₀₀nm should be up to 0.6. The cells were collected by centrifugation at 3000g for 2min and resuspended in 50mL sterile SH3a medium. Leaves of plants grown ex vivo or in a greenhouse at 4 to 6 weeks of age were selected for transformation. The greenhouse grown leaves were placed in 50mL centrifuge tubes (20-30 leaves one)Tube) leaves were washed in a first pass with water 2-3 times with an ionic detergent (Teepol) added. The tube was inverted several times to ensure that the leaves were fully submerged and flushed with tap water. Replace water with sodium hypochlorite solution, mix gently, and cover the mouth of pipe on the test-tube rack and place 7min, put the pipe back to the workstation and wait 7min again. Under sterile conditions, the leaves were rinsed three times with sterile water in the same 50mL centrifuge tube. The leaves were placed in a petri dish containing 30ml of sterile water, the edges of the leaves were removed with a sterile knife and cut into squares. The cut leaves are put into the agrobacterium tumefaciens bacterial solution, the operation is carried out in a sterile space (20-30 leaves are put into 50mL of agrobacterium tumefaciens bacterial solution), and the leaves are uniformly scattered in the bacterial solution by shaking the tube. Vacuum was applied at 650psi for 20 minutes using leaf explants of Agrobacterium in S H3a solution. After releasing the vacuum, the vacuum flask was placed on a shaking (50-60rpm) table at room temperature for 1-2 hours to allow the tissue to recover from the infusion process. In the clean bench, the explants were transferred to empty 9cm petri dishes and the inoculum was maximally aspirated with a pipette gun. Leaf explants were transferred to solid SH3a medium without antibiotics. The underside of the leaf explants (AB axis) should be in contact with the culture medium. The plates were then sealed with medical tape and incubated in a plant growth chamber (24 ℃) for 2 days under dark conditions. Leaf explants were removed from the medium and gently wiped on fresh solid (SH3a or SH9) medium to remove excess bacteria growing on the explants. The leaf explants were transferred to fresh SH3a medium with 800mg/L Ampere sterilization (to eradicate Agrobacterium). Sealing with a plastic plate, placing in the dark (24 ℃), keeping for 5-6 weeks, and checking whether the bacteria are infected or not at regular intervals. Callus material appeared after 2 weeks of infection. These calli were transferred to new SH3a medium every 2-3 weeks. The calli were then transferred every 3 weeks to new SH9 medium until pre-embryos appeared (between 3-6 weeks in this medium). From the callus, pre-embryos will develop into true embryos on SH9 medium within 20-30 days. Plants develop from the embryo about 2-3 weeks after embryo formation. It was transferred to 1/SH9 medium to induce rooting. At the beginning of rooting, the plants were transferred to 1/2SH9 petri dishes. It is vertically placed (or inclined at 45 deg.) in the growth chamber, and the amount of agar is raised to 9g/L, or moreThe medium was well solidified. Rooting takes 2-6 weeks on this medium. Plant material that is transferred from in vitro conditions to greenhouses is very sensitive to changes in humidity conditions. Under water saturation conditions, plants were kept growing and then gradually acclimated to normal greenhouse conditions. Plantlets of several leaves and roots developed on 1/2S H9 medium were transplanted into sterilized (or cleaned) trays containing a transparent cover. During the first two weeks, the plants were rinsed with tap water, followed by watering of the plants with the medium liquid. A tray is placed under the basin to keep it in the water. The soil remains moist. The lid was kept closed for the first 5 days of growth in the pot to keep the plants in an environment filled with humid gas. The lid was then gradually opened to slowly reduce the humidity. At the end of the second week, the lid can be completely removed. Then alternately watering with culture solution and water. When these plants grow new leaves under greenhouse conditions, they can be moved into pots where soil and sand are mixed. We refer to these plants as T0 generation plants. If they develop normally, they should be sown after 2-3 months. The temperature of the culture room is 24 ℃, the illumination is 16h, and the darkness is 8 h. The seeds of T0 generation transgenic plants are T1 generation seeds, T1 generation seeds are planted on a culture medium with kanamycin and hygromycin resistance to obtain T2 generation plants with a separation ratio of 3:1, the surviving plants are moved to soil for normal condition culture, seeds are collected from individual plants, and the collected seeds are T2 generation seeds. When each T2 generation seed is planted on a resistant culture medium, and the proportion of the whole survival rate appears, the obtained survival plant is the homozygous T3 generation transgenic plant.

Example 4

Phenotype study of transgenic leguminous plants

The growth state of the transgenic alfalfa plant is not obviously different from that of a wild-type medicago truncatula R108 plant, and the result shows that the growth of the plant is not influenced by the GFP-MtTUA6-GFP expression vector for transforming PZP211-TUA6 Pro.

The 8 obtained PZP211-TUA6Pro strains GFP-MtTUA6-GFP alfalfa were subjected to the following observation by selecting 3 strains each

Leaf epidermal cells, hypocotyl cells, root tip elongation cells and root hair cells of the plants were observed under a laser confocal microscope (Leica A1, Leica Co.) (488 nm excitation light). As shown in FIG. 2, it was found that 8T 3 homozygous PZP211-TUA6Pro transformants were expressed with GFP-MtTUA6-GFP fusion protein in all leaf epidermal cells, hypocotyl cells, elongation cells at root tips and root hair cells of all plants of the GFP-MtTUA6-GFP alfalfa R108 strain, and that the fluorescence signal was strong.

It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any numerical value between the two ends can be selected, and the preferred embodiments of the present invention are described for the purpose of avoiding redundancy.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

<110> Shenyang agriculture university

Fusion protein for marking microtubule skeleton, encoding gene and construction method of transgenic leguminous plant

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5303

<212> DNA

<213> Artificial sequence

<400> 1

atgcaaatcg catagcaaat gaaacaacat ttattaatcg catttgaatt tgagatgatt 60

gttatgatgt gtgcaatcca acattcagat aagatcaaag gttgatttga tagtgatgct 120

caaggataga gatccaacgg tcaacattga ttggagccaa aaagattagg attagagttg 180

acacaatttc ttaacattgt tattagtatt agaattaagt aaaataagat ggttattaga 240

cacttaattt gacatcaaca tcatttttaa tataatttac cctctcacta tgtccaaaga 300

caacatctat ttaatgagta tcttatatgt ctaatacaat gtgttcctgt atagacaagg 360

tttacctatt gaatatatgg tgcctcctgt ctccagtgat tataatcacc gtgactgatc 420

ttatgctccc tctctaaacg ggagggtgtg tgcatgcaaa agaggcacta acgcccaatt 480

gttgtcattt aacagagtat gtgagtgggg tataaatgag tcagatgatt atatctagag 540

gcgagacccc tatttacaat gacgcctcta gtgtgatatt ccttgactgt tgctatcatt 600

cctcttgcga tttgttacat tatgttacga ggtactctag ttattttgga gtaattacat 660

ctaaaacagg tggaaacact tatttgattt aagtgaaccc ccgtttaagt atgagtgact 720

cagtgcatat cacatggttt gtttaatgta agtaggaacg acctgatagc tactatccat 780

aagttggttt tgctcaggat agtggagcac aacaatgccc tgataggaat atggatgaca 840

aaataggttg gacccgtcgg gtcaacccgt ttaacccctt tttaagtggg gtgtatatgt 900

attttcaatc caacactaaa agggtgtccg ccctgcttaa atcgttaaaa aacaaggttg 960

tgatgaggcg ccatgggttg aaattaaaaa aaacttcttt tcacttcttt tatttatagg 1020

tcacaatcaa tttttttcct ttagttttct cctccatata gtatatataa gaaagtcatt 1080

taatttaatc acttttgttt tatgatttta gcctcatgtt tttaatagtt agtcaattgg 1140

tgaactcatg aaatttttat tttggatatc ttgttttttt ttttttttta atgtcaaatg 1200

ttcgtagtta gctactagat taattttttt tttcctcctt catacgatta tgaacccatg 1260

gtctccaact ccttgtcatt tgacttaaac tagttgagcc gcccaatccc tccattttgg 1320

ttgtgggtta tgtgatacac aatatgattg tttttcacaa agaacgaaac aatgcatcta 1380

aaatgaaatt ccaacactca agtcaataag aactcgagga gccttgtggt cttctcataa 1440

tgagagtgca tacatttttt ttttactatg aatgaattct taaatatggg ttgaggttgc 1500

caaatagata tgtagtggtt aggcgagtcc ttttttaccg acgcattgtt agggtcatag 1560

gtgggcaaaa aaaccaaaaa actgaattga actgctgaac taaactgaac aaaataaaac 1620

tgaacattac taacaattct gaaactgaac tgaaacagtt cagtacaaaa taatttagtt 1680

cgattcgaaa caattcggaa cagtccagtt cagttcggta actaaaattc aatttcgttt 1740

gttttttttc ttcaacgata ccaatattaa cagttcagaa cagttcaatt tggtttgata 1800

gcagtccggt tcaattcgaa atgcaaacag ttcggttcat tttaattatt ttaaatttag 1860

caaacaattc agtttgattc gacctgaata gctatcttat cttatagctc agttggtagg 1920

gatattgcat attatatgca ggggctgggg ttcgaacctc ggatacccca cttcttcaca 1980

attaaattgt gtgagctcta accactaggc tacttgataa aaaaaaaact atcttatctt 2040

gtttaccaac attgttttgt aaatttttac caacacaact ctctttgtag ttgtattcga 2100

gacaaaaaat gtcaaaacaa atcaaataat ttcattaatc aaatttcaaa aaataagact 2160

ttactaataa aatgatattc ttttaattca ttaatccatt taaaaatcga aaaaaactgt 2220

gtaaaatatc aatctatata aaaacaagat ttcttttctt ctacttttta agttctctta 2280

ataaataacg tatctaattt atattaaaaa ctagatattg aaagaattta atataaataa 2340

tttagagaat gaattcaact ttcttttatc atgtagaatt aattgagtat gaaacacatt 2400

tgaaattgaa aatagagagt aaagcaacgg gcctgaagcg gcaaaaaata aataaaaaat 2460

aaaaatgaaa aaggacgtta accatggttt ctaagtttag ttactttgta cagatgcaaa 2520

tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 2580

gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 2640

gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 2700

tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 2760

agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 2820

tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 2880

tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 2940

agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 3000

gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 3060

accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 3120

acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 3180

tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagatga 3240

gagagtgcat ttcagttcac attggtcaag ccggtattca ggttggaaat gcttgctggg 3300

agctttactg tctcgaacac ggcatcggcc ctgatggcca aatgccgagt gacaagacta 3360

ttggaggcgg tgatgacgct ttcaacacct tcttcagtga gaccggtgct ggaaagcatg 3420

tcccccgtgc tgtttttgta gatcttgagc ccactgttat cgatgaggtg aggactggaa 3480

cctatcgcca gctcttccat cccgagcagc tcatcagtgg caaagaagat gctgccaaca 3540

acttcgcccg tggtcattat accattggga aagagatcgt ggatctgtgt ttggaccgca 3600

tcagaaagct tgctgataac tgcactggtc tccaagggtt tttggttttt aatgctgttg 3660

gaggaggaac tggttctggt cttggttctc tgctccttga gcgtctgtct gttgattatg 3720

gcaagaaatc caagcttggg ttcactgtct atccctcccc tcaggtttcc acctctgttg 3780

ttgagccata caacagtgtc ctctccaccc actctctctt ggagcacact gatgtagctg 3840

ttcttctgga caatgaagct atctatgaca tctgtaggcg ctccctcgac attgagcgtc 3900

ccacttacac caaccttaac cgtcttgttt ctcaggtgat ttcatccttg actgcttcct 3960

tgaggtttga tggtgctctc aatgttgatg tgactgaatt ccagaccaac ttggtcccat 4020

atcccagaat ccatttcatg ctttcttcat atgctccagt tatctctgct gagaaggctt 4080

atcatgagca gctttcagtt gctgaaatta ccaacagtgc ttttgagcca tcatctatga 4140

tggctaagtg tgaccctcgc catggaaagt acatggcttg ctgtttgatg taccgtggtg 4200

atgttgttcc caaggatgtg aatgctgctg tcgctacaat caaaaccaag aggaccattc 4260

agtttgtgga ttggtgccct actggtttca agtgtggtat taactaccag cctcctactg 4320

ttgttcctgg tggtgacctt gctaaggtac agagggcagt ttgcatgatt tcaaactcca 4380

caagtgtagc tgaggtgttt ggtcgcattg atcacaagtt tgatctgatg tatgccaagc 4440

gtgcttttgt tcactggtat gtgggtgagg gtatggaaga aggagaattt tctgaagccc 4500

gtgaggatct tgctgctttg gaaaaggatt atgaagaagt tggtgccgag tctggtgagg 4560

gcggtgatga agatatggat gactacatgg tgagcaaggg cgaggagctg ttcaccgggg 4620

tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg 4680

gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg 4740

gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct 4800

tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag 4860

gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg 4920

aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca 4980

aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct 5040

atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca 5100

tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg 5160

gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc 5220

ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc 5280

tcggcatgga cgagctgtac aag 5303

<210> 2

<211> 928

<212> PRT

<213> Artificial sequence

<400> 2

MET Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

1 5 10 15

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly

20 25 30 35

Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu

40 45 50

Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe

55 60 65 70

Ser Arg Tyr Pro Asp His MET Lys Gln His Asp Phe Phe Lys Ser Ala MET Pro

75 80 85 90

Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys

95 100 105

Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu

110 115 120 125

Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile MET Ala Asp Lys Gln Lys Asn Gly Ile

145 150 155 160

Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala

165 170 175 180

Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp

185 190 195

Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg

200 205 210 215

Asp His MET Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly MET

220 225 230

Asp Glu Leu Tyr Lys MET Arg Glu Cys Ile Ser Val His Ile Gly Gln Ala Gly

235 240 245 250

Ile Gln Val Gly Asn Ala Cys Trp Glu Leu Tyr Cys Leu Glu His Gly Ile Gly

255 260 265 270

Pro Asp Gly Gln MET Pro Ser Asp Lys Thr Ile Gly Gly Gly Asp Asp Ala Phe

275 280 285

Asn Thr Phe Phe Ser Glu Thr Gly Ala Gly Lys His Val Pro Arg Ala Val Phe

290 295 300 305

Val Asp Leu Glu Pro Thr Val Ile Asp Glu Val Arg Thr Gly Thr Tyr Arg Gln

310 315 320

Leu Phe His Pro Glu Gln Leu Ile Ser Gly Lys Glu Asp Ala Ala Asn Asn Phe

325 330 335 340

Ala Arg Gly His Tyr Thr Ile Gly Lys Glu Ile Val Asp Leu Cys Leu Asp Arg

345 350 355 360

Ile Arg Lys Leu Ala Asp Asn Cys Thr Gly Leu Gln Gly Phe Leu Val Phe Asn

365 370 375

Ala Val Gly Gly Gly Thr Gly Ser Gly Leu Gly Ser Leu Leu Leu Glu Arg Leu

380 385 390 395

Ser Val Asp Tyr Gly Lys Lys Ser Lys Leu Gly Phe Thr Val Tyr Pro Ser Pro

400 405 410

Gln Val Ser Thr Ser Val Val Glu Pro Tyr Asn Ser Val Leu Ser Thr His Ser

415 420 425 430

Leu Leu Glu His Thr Asp Val Ala Val Leu Leu Asp Asn Glu Ala Ile Tyr Asp

435 440 445 450

Ile Cys Arg Arg Ser Leu Asp Ile Glu Arg Pro Thr Tyr Thr Asn Leu Asn Arg

455 460 465

Leu Val Ser Gln Val Ile Ser Ser Leu Thr Ala Ser Leu Arg Phe Asp Gly Ala

470 475 480 485

Leu Asn Val Asp Val Thr Glu Phe Gln Thr Asn Leu Val Pro Tyr Pro Arg Ile

490 495 500

His Phe MET Leu Ser Ser Tyr Ala Pro Val Ile Ser Ala Glu Lys Ala Tyr His

505 510 515 520

Glu Gln Leu Ser Val Ala Glu Ile Thr Asn Ser Ala Phe Glu Pro Ser Ser MET

525 530 535 540

MET Ala Lys Cys Asp Pro Arg His Gly Lys Tyr MET Ala Cys Cys Leu MET Tyr

545 550 555

Arg Gly Asp Val Val Pro Lys Asp Val Asn Ala Ala Val Ala Thr Ile Lys Thr

560 565 570 575

Lys Arg Thr Ile Gln Phe Val Asp Trp Cys Pro Thr Gly Phe Lys Cys Gly Ile

580 585 590

Asn Tyr Gln Pro Pro Thr Val Val Pro Gly Gly Asp Leu Ala Lys Val Gln Arg

595 600 605 610

Ala Val Cys MET Ile Ser Asn Ser Thr Ser Val Ala Glu Val Phe Gly Arg Ile

615 620 625 630

Asp His Lys Phe Asp Leu MET Tyr Ala Lys Arg Ala Phe Val His Trp Tyr Val

635 640 645

Gly Glu Gly MET Glu Glu Gly Glu Phe Ser Glu Ala Arg Glu Asp Leu Ala Ala

650 655 660 665

Leu Glu Lys Asp Tyr Glu Glu Val Gly Ala Glu Ser Gly Glu Gly Gly Asp Glu

670 675 680

Asp MET Asp Asp Tyr MET Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val

685 690 695 700

Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser

705 710 715 720

Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

725 730 735

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr

740 745 750 755

Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His MET Lys Gln His Asp Phe Phe

760 765 770

Lys Ser Ala MET Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp

775 780 785 790

Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val

795 800 805 810

Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly

815 820 825

His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile MET Ala Asp Lys

830 835 840 845

Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly

850 855 860

Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

865 870 875 880

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp

885 890 895 900

Pro Asn Glu Lys Arg Asp His MET Val Leu Leu Glu Phe Val Thr Ala Ala Gly

905 910 915

Ile Thr Leu Gly MET Asp Glu Leu Tyr Lys

920 925

Claims (5)

1. The application of the fusion gene in constructing transgenic alfalfa plants is characterized in that the fusion gene has a sequence shown as SEQ ID NO.1, and in the SEQ ID NO.1, the 1 st to 2519 th sites areTUA6The promoter of the gene, wherein the 2520-3236 position is the coding gene of the N-terminal GFP, the 3237-4586 position is the coding gene of the alfalfa tubulin MtTUA6, and the 4587-5303 position is the coding gene of the C-terminal GFP;

all leaf epidermal cells, hypocotyl cells, root tip elongation region cells and root hair cells of the transgenic alfalfa plant express GFP-MtTUA6-GFP fusion protein shown in SEQ ID No.2 and emit fluorescent signals.

2. A plant expression vector comprising the sequence of SEQ ID No.1 of claim 1.

3. A recombinant agrobacterium comprising the plant expression vector of claim 2.

4. The method for constructing recombinant Agrobacterium according to claim 3, which comprises:

mixing Agrobacterium tumefaciens EHA105 competent cells with the plant expression vector of claim 2, incubating on ice for 30min, liquid nitrogen quick freezing for 2min, recovering at 37 ℃ for 3-5min, adding 1mL liquid YEB medium, shake culturing at 28 ℃ and 180rpm for 4h, collecting the thallus, culturing at 28 ℃ for 36h on YEB solid medium containing 50mg/L spectinomycin and 100mg/L rifampicin; selecting monoclonal shake bacteria, and identifying positive clone by PCR and enzyme digestion detection to obtain recombinant agrobacterium.

5. A construction method of a transgenic alfalfa plant is characterized by comprising the following steps:

s1, using the recombinant Agrobacterium constructed in claim 4 to transform alfalfa plants, recording as T₀Generation, its seed is marked as T₁Generation;

S2、T₁the generation seeds are screened by kanamycin to obtain resistant seedlings which are recorded as T₂Generation;

S3、T₂the generation seeds are screened by kanamycin, and when the full survival proportion appears, the obtained survival plants are homozygous T₃And generating transgenic plants, namely transgenic alfalfa plants.

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