CN114457079B

CN114457079B - Poplar flying-catkin development specific expression promoter ProMIXTA and application thereof

Info

Publication number: CN114457079B
Application number: CN202210169575.0A
Authority: CN
Inventors: 周芳伟; 李淑娴; 尹佟明
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2022-02-23
Filing date: 2022-02-23
Publication date: 2022-09-23
Anticipated expiration: 2042-02-23
Also published as: CN114457079A

Abstract

The invention discloses a poplar catkin development specific expression promoter ProMIXTA and application thereof, belonging to the technical field of biotechnology and plant genetic engineering. The nucleotide sequence is shown in SEQ ID NO. 1. The promoter of ProMIXTA is fused with a marker gene GUS, and the Arabidopsis thaliana is stably transformed through the mediation of agrobacterium GV3101 and has similar expression activity with a positive control. The combination of the expression mode of the PdeMIXTA gene among different tissues and the results of tissue in-situ hybridization experiments proves that the ProMIXTA promoter can start the specific expression of the corresponding target gene PdeMIXTA in poplar seed cells, and specifically knocks out or silences endogenous target genes in the poplar seed cells, thereby being beneficial to researching the regulation and control effect of the target genes on the differentiation and development of the poplar seed cells, further providing an effective gene tool for the creation of new varieties of populus without seed, and having important significance.

Description

Poplar flying-floc development specific expression promoter ProMIXTA and application thereof

Technical Field

The present invention belongs to the field of biotechnology and plant gene engineering technology. In particular to a promoter ProMIXTA capable of being specifically expressed in a flying tissue of a poplar and application of the promoter in genetic improvement of the poplar.

Background

Populus spp is a tree species which is distributed most widely and has the strongest adaptability in the world. The cultivation of poplar in China is mainly concentrated in the areas of North China, northeast China, northwest China and the like. The poplar is not only an important fast-growing high-yield timber forest tree species, but also an important tree species for environmental greening; it is also a suitable feedstock for second generation biofuel production due to its extremely fast growth rate and good cell wall chemistry; it has strong habitat adaptability, and can be widely used in ecological protection forest, three-north protection forest and agriculture and forestry protection forest. The economic value, the ecological value and the social value of the tree self reflect the superiority, and other tree species cannot possess and replace the superiority.

The problem of seasonal environmental pollution caused by poplar floating flocs becomes an important factor for restricting the development of the poplar industry at present. The poplar is a male and female heterostrain in reproductive characteristics, the female strain of the poplar has good quality and the growth performance of the female strain is generally superior to that of the male strain, so the excellent clone of the poplar popularized by breeding is mainly the female strain, however, after the capsule of the female strain of the poplar grows to be mature, the fruit cracks to generate a plurality of poplar catkins which fly around, and the duration of the catkins is long, and usually can reach half a month. The spread of poplar catkins can not only cause environmental pollution, but also cause unsmooth respiratory tract of people, and is easy to cause fire because of flammability. Therefore, how to exert the strong ecological benefit and the higher economic benefit of the poplar and control the seasonal floatage pollution of the poplar is a problem to be solved urgently in the current society.

A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription, thereby precisely and efficiently promoting expression of a gene. Commonly used promoters for plants are constitutive expression promoters, tissue-specific expression promoters and conditionally inducible expression promoters. The cotton fiber related promoter is mainly active in the initiation and elongation stages of the fiber and belongs to a tissue specific expression promoter. A tissue-specific promoter is one of the spatio-temporal specific promoters that only promotes expression in a specific cell, tissue or organ. The use of a tissue-specific promoter to control the expression of a target gene in genetic transformation of plants can more effectively avoid potential side effects associated with the use of constitutive promoters. Therefore, isolation and cloning of expression-specific promoters allows efficient regulation of gene expression at the transcriptional level, allowing expression of genes in different tissues, different organs, or under different environmental conditions.

The poplar catkin is flocculent fiber attached to the surface of the seeds, the length of the fiber is about 9.5-15.9 mm, the diameter of the fiber is about 8-11 mu m, and the poplar catkin is attached to the surface of the seeds and is mainly beneficial to the diffusion of the seeds after the seeds are mature. The current research proves that the plant MIXTA gene is a key gene for regulating the initial development of epidermal hair, and has the functions of regulating the initial development of cotton wool, the development of arabidopsis trichomes, the bulge differentiation of goldfish petal cells and the like. In poplar, the poplar seed is developed from placenta epidermal hair, similar to the development of cotton seed. At present, the MIXTA gene has been shown to play a key role in the initial development of cotton wool. Therefore, the research and analysis of the flying-catkin specific expression promoter of the poplar can start the specific expression of the target gene in the poplar catkin cell, thereby avoiding the influence of the target gene on the development of other tissues and organs of the poplar, realizing the specific high expression of the target gene in the poplar catkin cell through the poplar genetic transformation technology, specifically knocking out or silencing the target gene in the poplar catkin cell, being beneficial to researching the regulation and control function of the target gene on the differentiation and development of the poplar catkin cell, further providing an effective gene tool for the creation of a new species of the non-catkin poplar, and having important significance.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a poplar catkin specific expression promoter ProMIXTA. The invention also aims to solve another technical problem of providing the application of the poplar catkin specific promoter ProMIXTA in poplar transgenosis.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a promoter ProMIXTA for the specific expression of poplar catkin development has a nucleotide sequence shown in SEQ ID NO. 1.

The invention clones and verifies a poplar catkin development specific promoter ProMIXTA, the PdMEMIXTA gene started by the ProMIXTA is specifically expressed in the poplar catkin initial development period, and is not expressed in other tissues; in-situ hybridization further verifies that the PdEMIXTA gene is expressed in epidermal cells with raised placenta; the ProMIXTA promoter is fused with a GUS gene, arabidopsis thaliana is genetically transformed, a GUS signal is detected only in epidermal hairs, and the fact that the ProMIXTA promoter is a poplar catkin specific expression promoter is verified.

An expression cassette containing the poplar catkin development specific expression promoter ProMIXTA.

In the expression cassette, the downstream of the poplar catkin development specific expression promoter ProMIXTA is connected with a target gene for driving the specific expression of the target gene.

The recombinant expression vector containing the poplar catkin development specific expression promoter ProMIXTA.

The recombinant expression vector contains the expression cassette of the poplar catkin development specific expression promoter ProMIXTA.

The recombinant expression vector is a recombinant plant expression vector constructed by the expression cassette and plasmids, viruses or carrier expression vectors, and the plant expression vector used in the invention is pCAMBIA1301 vector plasmid.

The engineering bacteria containing the poplar catkin development specific expression promoter ProMIXTA.

The application of the poplar flying-floc development specific expression promoter ProMIXTA in obtaining transgenic poplar or transgenic arabidopsis thaliana.

The method specifically comprises the following steps: the poplar catkin development specific expression promoter ProMIXTA is operably connected with a gene sequence to be expressed in a vector, so as to construct a recombinant expression vector, and the recombinant expression vector is transformed into plant cells, tissues or organs for cultivation, wherein the plant is poplar or arabidopsis thaliana.

The application of the poplar flying-floc development specific expression promoter ProMIXTA in breeding new varieties of poplar with reduced flying-floc or no flying-floc.

The invention uses pCAMBIA1301 vector as backbone vector, uses the cloned promoter ProMIXTA to replace 35S promoter at GUS upstream in backbone vector by full length, uses promoter ProMIXTA to start GUS reporter gene, and uses agrobacterium GV3101 to mediate and stably transform arabidopsis thaliana, the result proves that promoter ProMIXTA starts GUS to express specifically in arabidopsis thaliana epidermal hair, and has similar expression activity with positive control (strong promoter CaMV 35S). By combining the expression mode of the PdEMIXTA gene among different tissues and the results of tissue in-situ hybridization experiments, the promIXTA promoter is proved to be capable of promoting the specific expression of the corresponding target gene in poplar catkin cells, so that the regulation and control effect of the target gene on the differentiation and development of the poplar catkin cells is verified, and the gene editing or overexpression transgenic technology is conveniently utilized to create a novel populus-free germplasm.

The method for obtaining the poplar catkin development specific expression promoter ProMIXTA comprises the following steps: taking genomic DNA of poplar catkin as a template, and carrying out PCR amplification on the genomic DNA by using primers consisting of SEQ ID NO.2 and SEQ ID NO.3 to obtain the promoter.

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

the invention discovers and positions a new poplar catkin specific expression promoter ProMIXTA for the first time. The promoter can realize the specific expression of the target gene PdeMIXTA in poplar seed cells through a poplar genetic transformation technology, and specifically knock out or silence endogenous target genes in the poplar seed cells, thereby being beneficial to researching the regulation and control effect of the target genes on the differentiation and development of the poplar seed cells, further providing an effective gene tool for the creation of new species of non-flocculent poplars and having important significance.

Drawings

FIG. 1 shows the expression of PdEMIXTA gene in different tissues of populus nigra, which is initiated by the specific promoter ProMIXTA for the growth of poplar catkin; selecting 7 different tissues of 3-day female inflorescences (-3DPA) before pollination, 0DPA on the day of pollination, 3-day female inflorescences (3DPA) after pollination, 5-day female inflorescences (5DPA) after pollination, leaves (Leaf), Leaf buds (Bud) and barks (Bark) for gene expression pattern analysis; histogram numbers represent the mean of three biological replicates, error bars are the standard error results for three biological replicates;

FIG. 2 is the tissue in situ hybridization analysis chart of PdEMIXTA gene initiated by the specific promoter ProMIXTA of poplar flying-floc development; in the figure, A is a diagram showing the results of in situ hybridization of the antisense probe; b is a graph of the result of in situ hybridization of the sense probe;

FIG. 3 is the electrophoresis gel diagram of the cloning of promoter ProMIXTA and the construction of expression vector; in the figure, A is an electrophoresis gel picture of promoter ProMIXTA full-length cloning by using DNA of a female inflorescence 1 day after pollination as a template; b is an electrophoresis gel picture of the constructed expression vector enzyme digestion result;

FIG. 4 is a schematic diagram of the construction of the ProMIXTA promoter genetic transformation vector; in the figure, LB and RB are left and right boundary sequences of T-DNA respectively, ProMIXTA is a specific expression promoter of the invention, and NOS is a terminator;

FIG. 5 is ProMIXTA: : GUS staining result chart of GUS transformed Arabidopsis thaliana; in the figure, A is a negative control; b is a positive control; c is the dominant expression of GUS in the epidermal hair of transgenic arabidopsis thaliana leaves.

Detailed Description

The invention is further described with reference to specific examples. The molecular biological experiments, which are not specifically described in the following examples, can be performed by methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or methods conventional in the art, or according to kits and product instructions.

Example 1

Analysis of PdeMIXTA gene expression pattern started by promixTA specific promoter for poplar catkin development

Collecting female inflorescences of different time points of the initial development of the poplar seed, wherein the female inflorescences comprise 4 time nodes of 3 days (-3DPA) before pollination, 3 days (3DPA) after pollination, and 5 days (5DPA) after pollination. In addition, 3 more vegetative organs were selected as controls, Leaf (Leaf), Leaf bud (Bud) and Bark (Bark). And extracting RNA of the collected sample to perform reverse transcription of cDNA. Designing a quantitative primer N00938F of the PdEMMIXTA gene: 5'-CGGCCACTGAATTTAATAGCGGCAAT-3', respectively; N00938R: 5'-TCCGAAAGTCCTTCAATGATCCCCATA-3' are provided. Taking PtUBQ gene as an internal reference gene, wherein the primer sequence of the internal reference gene is PtUBQF: 5'-GTTGATTTTTGCTGGGAAGC-3', respectively; PtUBQR: 5'-GATCTTGGCCTTCACGTTGT-3' are provided. The real-time quantitative PCR experiment procedure was as follows: prepare 20. mu.L reaction system including 100ng cDNA, 4pmol upstream and downstream primers, 10. mu.L AceQ qPCR SYBR Green Master Mix, sterile ultrapure water to make up to 20. mu.L. The reaction step, first 95 ℃ denaturation for 3 minutes, then 40 reaction cycles including 95 ℃ denaturation for 15 seconds, 60 ℃ annealing for 15 seconds, 72 ℃ extension for 30 seconds. Calculation of Gene expression 2 ^-ΔCT Method, i.e. Δ CT ═ CT _{Target gene} -CT _{Reference gene} . The relative expression pattern of PdMEMIXTA gene in different tissues of populus tremuloides is analyzed by using a T test method. The results show that PdeMIXTA is a specific expression gene within 5 days after pollination and is not expressed in other tissues (fig. 1). The specific expression time node of the gene is completely coincided with the time node of the poplar catkin initial development, and the ProMIXTA promoter is concluded to be the poplar catkin development specific promoter.

II, in-situ hybridization analysis of PdEMIXTA gene tissue initiated by specific promoter ProMIXTA in poplar flying-floc development

1) And (3) probe synthesis: carrying out homologous comparison on DNA sequences of PdeMIXTA of the poplar, finding out a specific sequence of the PdeMIXTA, and designing a sense RNA probe and an antisense RNA probe. PdeMIXTA: 5 '-DIG-CGAGUCGGUUUGGGGGAGGGUUAGGUAUAAUCAC-DIG-3', FF: 5 '-DIG-CUCUCUUUCCGUUUUCUGCUGUGGUCACUCUG-DIG-3', Probe Synthesis was performed according to the method of the DIG RNA Labeling Kit (Roche, USA) Kit.

2) Tissue fixation: cleaning female populus deltoides flower buds of pollinated 1DPA, and immediately placing into a fixing solution (prepared by DEPC water) for fixing for more than 12 h.

3) And (3) dehydrating: after the tissue is fixed, the tissue is dehydrated by gradient alcohol, and then is soaked in wax and embedded.

4) Slicing: slicing the paraffin by a slicer, taking out the slices by a spreading machine, and baking the slices for 2 hours by an oven at 62 ℃.

5) Paraffin section dewaxing to water: sequentially placing the slices in xylene I15 min-xylene II 15 min-absolute ethanol I5 min-absolute ethanol II 5min, air drying, and soaking in DEPC water.

6) The gene is circled, and is digested for 20min at 37 ℃ by adding protease K (20ug/m1) dropwise according to different index characteristics of different tissues. The membrane was washed with PBS 3 times with pure water and then 5 min.

7) Blocking endogenous peroxidase: 3% methanol-H is added dropwise ₂ O ₂ Incubate in the dark at room temperature for 15min, place the slide in PBS (pH7.4) and wash 3 times for 5min each time with shaking on a destaining shaker.

8) Pre-hybridization: add the prehybridization solution dropwise and incubate for 1h at 37 ℃.

9) And (3) hybridization: the prehybridization solution is poured out, and hybridization solution containing the probe FF/PdeMIXTA is dripped into the prehybridization solution at the concentration of 1uM, and the hybridization is carried out overnight in a thermostat at 42 ℃.

10) Washing after hybridization: the hybridization solution was washed off, 2 XSSC, 10min at 37 ℃, 2 XSSC, 2X 5min at 37 ℃ and 10min at 0.5 XSSC at room temperature. If there are more non-specific hybrids, formamide washing can be increased.

11) Dropwise adding confining liquid: blocking serum was added dropwise. Room temperature for 30 min.

12) Mouse anti-digoxin labeled peroxidase (anti-DIG-HRP) was added drop wise: the blocking solution was decanted and anti-DIG-HRP was added dropwise. Incubate at 37 ℃ for 50min, then wash 3 times in PBS × 5 min.

13) Adding CY3-TSA dropwise: adding CY3-TSA reagent dropwise, and reacting for 5min at room temperature in a dark place. Post TBST 3 times × 10 min. PBS wash 1 times × 5 min.

14) And (5) microscopic examination and photographing: the chromogenic solution (10. mu.L NBT/BCIP + 500. mu.L Buffer3) was added and incubated overnight at room temperature in the dark. The digoxin signal was then observed using an olympus microscope.

The result shows that PdMEMIXTA is mainly expressed in epidermal cells with raised placenta (figure 2), and the result is consistent with the result of qRT-PCR expression analysis, and further proves that the promoter of ProMIXTA is a specific promoter for poplar catkin development.

Third, cloning and expression vector construction of poplar catkin development specific promoter ProMIXTA

Primer F was designed based on the full-length sequence of ProMIXTA identified in the populus tremula genome: 5'-CTCTCTGACCTATCAAAATTAGGA-3', R: 5'-TTCTTCAATCCCACCTTATC-3', using DNA of 3 days inflorescence after pollination of NL895 poplar (Nanlin 895 poplar) as a template, homologous cloning ProMIXTA (A in figure 3), and connecting the target fragment to Blunt vector for sequencing (the total length is 2867bp, and the specific sequence is shown in SEQ ID NO. 1). The pCAMBIA1301 vector is used as a backbone vector, a 35S promoter at the upstream of GUS in the backbone vector is replaced by the promoter ProMIXTA obtained by cloning in a full length mode through a homologous recombination method, and the promoter ProMIXTA is used for starting a GUS reporter gene. Restriction enzyme validation using EcoRV yielded a clear restriction gel electrophoresis band (B in FIG. 3) confirming the completion of the vector construction, which was named ProMIXTA-GUS (FIG. 4).

Fourth, the promoter ProMIXTA transfers the Arabidopsis thaliana to carry on the functional verification

For plant transformationThe Columbia wild type Arabidopsis thaliana (col-0) that was transformed was obtained from laboratory storage. Arabidopsis seeds were surface-sterilized in 75% ethanol for 30 seconds, then washed in sterile water 3 times, then surface-sterilized in 10% NaClO (v/v) for 10 minutes, then washed again with sterile water 6 times, and then uniformly sown in 1/2MS medium containing 3% sucrose and 0.8% agar, pH 5.8. The seeds were first cultured in the dark at 4 ℃ for 3 days for vernalization, then transferred to a growth chamber (22-23 ℃, 16h light/8 h dark) for germination, and arabidopsis seedlings grown for about two weeks were transplanted into a nutrition pot with nutrient soil, black soil, perlite and vermiculite at 3: 1, grown under the same conditions (22-23 ℃, 16h light/8 h dark). The over-expression vector ProMIXTA-GUS which is constructed is transformed into Agrobacterium tumefaciens GV3101(pMP 90). Agrobacterium was grown up, and after collecting the cells, the cells were adjusted to an OD of 0.8 with a suspension (1/2MS + 0.5% sucrose) and used for transformation experiments with arabidopsis thaliana. The wild arabidopsis transformation adopts a floral organ dip-dyeing method. In the full-bloom stage (about 4 weeks of growth) of wild type arabidopsis, the inflorescence is soaked in the prepared agrobacterium suspension for 30s, dark culture is carried out in a growth chamber for 24h, and then normal growth conditions are restored until the seeds are harvested by division after maturation (T1). Screening T1 generation transgenic positive plants by using an MS culture medium containing 30mg/L hygromycin, transplanting the screened positive plants into soil, and placing the soil in a growth room (22-23 ℃, 16h light/8 h dark) for normal management. When the seedlings grow in the soil for 10 days, fresh Arabidopsis leaves are cut into small pieces and placed into a 1.5mL centrifuge tube, GUS staining solution (10mmol/LEDTA, 100mmol/L sodium phosphate buffer solution pH7.0, 0.1mol/L K) is added ₃ [Fe(CN) ₆ ]，0.1mol/L K ₄ [Fe(CN) ₆ ]0.1% (V/V) TritonX-100, 5mg/mL X-Glue). And putting the centrifuge tube containing the plant material and the GUS dye solution into a constant temperature incubator at 37.0 ℃ for dyeing for 3 h. After finally removing the staining solution and decolorizing with 70% ethanol, the staining result of the transformed material was observed under a microscope and photographed.

ProMIXTA conversion: : the GUS staining result in GUS Arabidopsis is shown in figure 5, and the result shows that GUS is preferentially expressed on the epidermal hair of transgenic Arabidopsis leaves and is not expressed in non-epidermal hair tissues, i.e., the promoter has the expression specificity of Arabidopsis epidermal hair. The above examples show that the cloned ProMIXTA promoter nucleotide length is 2867bp, and when the promoter is fused with a GUS reporter gene, the promoter can direct the expression of the GUS reporter gene in epidermal hair in arabidopsis thaliana.

In conclusion, a poplar catipital development specific promoter ProMIXTA is cloned through an expression mode of a poplar pdeMIXTA gene among different tissues and a tissue in-situ hybridization experiment. The transformation experiment of arabidopsis thaliana is carried out by fusing a ProMIXTA promoter and a marker gene GUS, and the function of the ProMIXTA promoter as a specific promoter for poplar catkin development is proved. The promoter can realize the specific expression of a target gene in poplar catkin cells through a poplar genetic transformation technology, specifically knock out or silence endogenous target genes in the poplar catkin cells, is beneficial to researching the regulation and control effect of the target genes on the differentiation and development of the poplar catkin cells, further provides an effective gene tool for the creation of new species of non-catkin poplar, and has important significance.

The above detailed description of the invention does not limit the invention, but the scope of the invention should be determined by the appended claims. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined in the appended claims.

Sequence listing

<110> Nanjing university of forestry

<120> poplar catkin development specific expression promoter ProMIXTA and application thereof

<130> 1

<160> 3

<170> SIPOSequenceListing 1.0

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<211> 2867

<212> DNA

<213> Poplar flying-floc development specific expression promoter ProMIXTA (artificial)

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tctaagaact ggtagtctcg ccagatgttg agaaatcatg tattaaacca gtatattcca 60

gatcacttgt aaaggaaagc cgcaacaaag ctagcaaatg gttgtatata cggaaagaca 120

gtacgatgga tagatatggc ctaagaagtt ctgtctagga ggttgggttt taagcgggac 180

ctgtgtgacc cctccaatct ttcctgggaa cttgcttagt ggaaggatta tccatacggt 240

actttttttg tatatatagt agtatgaaac agttgaaggc tagcaggatg acggcacaag 300

ggaacagttg ggttccaaag gttcaaggat ctgatggagt ggtgatttct tcaaagaagt 360

tagattcggt gactgtgatt tctcgagggg agaattgatg aagaccctga taatggaaga 420

ggaatacagc aagaggataa agattggcac cctattttga cttggacaag tgttatgaca 480

ggatgagctg ctgtcaaaca taagcaagga atagggagaa atctggagaa cagaaattgc 540

acgagggcac acggagattg tgcaggagta cccgtaggat ccaacgaggt actgtaatga 600

gacaacaggt gcaaggagaa gaagtgttcc cagatgaagc tcagagcaga gactgttaaa 660

gtttgtacaa caggaagtct cttatgctct tgatgaagac aacaggcgaa gacctttcca 720

atgcatgcaa ggatggaaag agagctgttg cagaggcacc taattgaggg ggtgcaaacg 780

cctgtgataa aaggcaaccg cctatgatga aaggcgaaga caccaaagag agttggtgta 840

ggtggagctg tcaagcagaa aggcacagaa gctccaaaca tagaaatcga ggtggaggat 900

tgcgaggagt ataccgcaac tttctctttc tatgtagtca gtggcagtaa tctctcccat 960

aagtgcacat ggtggtagag acttttggag ccactttgaa gcatctcagc tgaaggagga 1020

tgcgaccacc tatgaaaggt gaagacacct tagatggaag gtgtgtgagg gccatgatag 1080

gagcccggat cagaccgccc cataacaacg ggtgaagaca ccttagagaa gatgtgaggg 1140

ccgtgataga agccccagtt cagaccgccg tatggttacg agcggagaca cctaaggaga 1200

aggtgtgtgg gccacgatat gagcccagtt cagaacaacc ccagagccaa tgtaatggct 1260

gaatataagt ggacaggtgt atagccaatg aagtggctat gatgagtatg gagacagatg 1320

caggattgac tttcatggat attgccccca tgctaaagat caatgagcta gaagacattt 1380

tccttggaca ataagtgtgt gacttgtgga gcattaagac gcggctgcta tgaagaagca 1440

gaagggcatg cgcaggttcc gggaacgagt tgactcttgt caaggtggtg agctcggtaa 1500

tggcattgta atactcagag tatgaagaca gatatggatc aacctttatg ggctgccccc 1560

atgattaaag gtggagagct acctggactc ttacgactaa gagcgagaga ctcacggaga 1620

agtagggcgt ggttcctagg gtggaacaga gggcaggcgc aactcctgga tgagtagact 1680

cttggaagag tggtgagctc gtgattacat tgaaatactc aatgaccgtc gcacttggga 1740

atattcgttt gagggggtgt tgtaagcctt ggtgtaaggc ttgaaaaaac attccagacc 1800

gagcatgatt gatacactcg aaggggaatg ttgtgttgaa gacgctctca gaatggtaag 1860

cttggaagaa ctgcagaatg caaacttgtg ctgaagaagc aagaggacaa ttgcccacat 1920

aaatggcaaa gggggtgatt gttgggatat tgccatttag tggcaatacc ctaccactaa 1980

ttgtggctta gtggaagcat cactaatgcc acaattagtg gcataatttc aggaccattg 2040

tccccttcat gtgctggaga aaggtcacca tgtttgccta taaaagaggc aattatttga 2100

gagcaaaggt gagaagagtg agggtgtgag aacatgaaga gaagagaaaa ggagagaaag 2160

aggagctgct gccatgggca gcagccctgc tgccatatgc agcagagtgt gtgagctggg 2220

agttgagtga tcctcctcca tgtatttatt gtatccttcc tctatctcta aataatatgg 2280

actctctccc gtggatgtag gcggttttgc cgaaccacgt aaaatattgt gtcagtgtgc 2340

ttaagcctcc tatgagcaag tatcagtaca ccctcggtcc gcgcatgggg tgccggaatt 2400

ccccaacaat tatttcatct tataaaaata tatcattcat tgtttgataa ttaagaatag 2460

acttgggagt tttaatacta tcaactgccc aatgtattgc attagaaaat taatgacgaa 2520

ccgtaataga tcgagagtta ctgcaaacgg cttacatcac cggctgaaag agcaaggaaa 2580

taactgctgt ctgatgatca ttacacggaa cacgtctttc ctcccagcac atttcaagtt 2640

ttcagtacgt aaattgaact ttactacatt gctagtaagt tcaaggtgga tttgtactga 2700

tccatatcga tattaattta ttgcattgcc agtgcgccta tatattgtgc atgcgttttc 2760

tagttcactt gcccccccct cctcgcattt tccttacctt ccatattata aatcatcgat 2820

ctgacgtctt tctctctcaa ctgtcctaat tttgataggt cagagag 2867

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<212> DNA

<213> upstream primer of specific promoter ProMIXTA for poplar flying-floc development (Artificial)

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ctctctgacc tatcaaaatt agga 24

<210> 3

<211> 20

<212> DNA

<213> downstream primer of specific promoter ProMIXTA for poplar flying-floc development (Artificial)

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ttcttcaatc ccaccttatc 20

Claims

1. A promoter ProMIXTA specifically expressed in poplar catkin development is characterized in that the nucleotide sequence of the promoter ProMIXTA is shown as SEQ ID No. 1.

2. An expression cassette containing the poplar catkin development specific expression promoter ProMIXTA of claim 1.

3. A recombinant expression vector containing the poplar catkin development specific expression promoter ProMIXTA of claim 1.

4. The engineering bacteria containing the poplar flying-floc development specific expression promoter ProMIXTA of claim 1.

5. The application of the poplar flying-floc development specific expression promoter ProMIXTA of claim 1 in obtaining transgenic poplar or transgenic Arabidopsis.

6. Use according to claim 5, characterized in that it comprises the following steps: the poplar catkin development specific expression promoter ProMIXTA of claim 1 is operably linked to a gene sequence to be expressed in a vector to construct a recombinant expression vector, and the recombinant expression vector is transformed into a plant cell, tissue or organ for cultivation, wherein the plant is poplar or Arabidopsis thaliana.

7. The application of the poplar catkin development specific expression promoter ProMIXTA in the claim 1 in cultivating new varieties of poplar with reduced catkin or no catkin.