CN110372782B - Loquat flower organ development related transcription factor EjPI protein and coding gene and application thereof - Google Patents

Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to a loquat flower organ development related transcription factor gene EjPI and application thereof. The full length of the cDNA sequence of the EjPI gene is shown as SEQ ID NO.1, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 2. The EjPI gene of the present invention is expressed only in petals, filaments and anthers, but not in sepals and pistils. The EjPI gene overexpression vector is transferred into wild type Arabidopsis thaliana and pi-1 mutant by an agrobacterium-mediated inflorescence dip-dyeing method. The result shows that the EjPI gene is over-expressed in the wild type arabidopsis thaliana, so that sepals of the arabidopsis thaliana can be petalous and opened; the EjPI gene is overexpressed in Arabidopsis pi-1 mutants, and petals and stamens can be recovered. The transgenic plant material obtained by utilizing the EjPI gene overexpression vector can enable the wild type of angiosperm to generate sepal petaloid, and can be used for double petaloid breeding; the plant pi mutant recovers petals and stamens, and effectively completes the reproductive process. The gene can be used for plant petaloid flower modification to improve ornamental value and restore stamen organs for cross breeding, and has good application prospect.

Description

Loquat flower organ development related transcription factor EjPI protein and coding gene and application thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to loquat EjPI protein, and a coding gene and application thereof.

Background

Loquat (Eriobotrya japonica) is an important evergreen fruit tree in the subfamily Maloideae of Rosaceae and widely distributed. Flowering is one of the most important events in the life cycle of loquat. Unlike most fruit trees in the pattern of flower development, loquat flower organs begin to differentiate and develop in summer and flower in autumn and early winter. In the flower development process of the loquat, the apical meristem continuously grows into a conical inflorescence, so that flower organs are differentiated, and then the flower organ development process is regulated and controlled by a complex network of environment and endogenous genes. Therefore, the research on the loquat flower organ development is helpful for better understanding the rosaceous flower organ development process.

During the development of angiosperm floral organs, PISTILLATA(PI) homologous gene is the key B-type MADS-box transcription factor for regulating the development of petals and stamens. The PI homologous genes show different expression patterns in the development of floral organs of different angiosperm groups. In the model plants Arabidopsis thaliana and Goldwort, the PI homologous genes are expressed in both petals and stamens. In the rose plant taiwanensis, the TrPI gene is expressed in both the vegetative and reproductive organs, and the expression level is higher in petals and stamens. In corydalis impatiens and tobacco, which are core eudicots, PI homologous genes are expressed in petals and stamens. However, in the basal angiosperm magnolia denudata, MawuPI is mainly expressed in the perianth and stamen; in two basal plants of Ranunculaceae and Aristolochiaceae, PI homologous genes are respectively expressed in sepals, petals and stamens; in the basal angiosperm Eschschschschschschschschschschschschscholtzia californica, the ESCAGLO gene is expressed in petals and stamens. In the monocotyledon, musk lily, the PI homologous gene LMADS8 is only expressed in stamens at the early stage of flower bud differentiation and then is diffused to the periderm and the periderm to be expressed. At present, no report is available on the research of loquat PI homologous gene regulation of floral organ development.

Disclosure of Invention

The invention aims to provide loquat EjPI protein and a coding gene and application thereof.

First, the present invention provides loquat EjPI protein which is:

1) a protein consisting of the amino acids shown in SEQ ID No. 2; or

2) Protein which is derived from the protein 1) and has the same activity by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.2, wherein the protein derived from the protein 1) comprises 1 specific PI motif of a MADS domain, an I domain, a K domain, a C terminal domain and the C terminal domain.

The invention also provides a gene for coding the loquat EjPI protein.

The sequence of the gene is shown as SEQ ID No. 1.

The invention also provides an overexpression vector containing the gene, a host cell and an engineering bacterium.

The invention also provides the application of the gene in regulating and controlling the formation and development of angiosperm petals, filaments and anthers. .

In one embodiment of the invention, the EjPI gene is transferred into angiosperm plant genome and is overexpressed in the wild type of the transgenic plant, so that sepals can be petaloid and opened; alternatively, overexpression in transgenic plants with the pi-1 mutation restores petals and stamens and completes the reproductive process.

The invention also provides a construction method of the transgenic plant, which adopts an agrobacterium-mediated method to transfer the overexpression vector containing the gene into a plant genome and screen the overexpression vector to obtain the transgenic plant.

Wherein, compared with the wild type, the edge of the sepal of the transgenic plant is whitened and opened, and the sepal valvification occurs; compared with the transgenic mutant and the non-transgenic mutant, the method can restore petal and stamen organs.

The invention separates 1 loquat petal and the EjPI gene determined by stamen characteristics from loquat flower buds. The expression of EjPI gene in the petals, filaments and anthers of loquat was confirmed by semi-quantitative RT-PCR. The plant overexpression vector of EjPI gene is constructed by means of gene engineering, and is transferred into wild arabidopsis thaliana for overexpression, so that the edge of sepals can be whitened and opened, and the ornamental value is improved; when the mutant is transferred into an Arabidopsis thaliana pi-1 mutant, the EjPI transgenic Arabidopsis thaliana mutant recovers petal and stamen organs. The invention provides good application prospect for improving the floral organs of angiosperm.

Drawings

FIG. 1 is an electrophoretogram showing the 3'-RACE, 5' -RACE of the loquat EjPI gene and the sequence verification of the coding region of the gene in example 1. Wherein a is an electrophoresis picture of 3' -RACE, M is DL2000DNA marker, 1 is PCR product of step 1 of 3' RACE, and 2 is PCR product of step 2 of 3' RACE; b is an electrophoresis photograph of 5' RACE, M is DL2000DNA marker, 1 is PCR product of step 1 of 5' RACE, and 2 is PCR product of step 2 of 5' RACE; c is a PCR electrophoresis picture for verifying EjPI gene ORF, M is DL2000DNA marker, and 1 is a PCR product of EjPI gene ORF; the black arrows represent the location of the amplified gene of interest.

FIG. 2 is a cDNA nucleotide sequence diagram of loquat floral organ development related transcription factor gene EjPI.

FIG. 3 is an amino acid sequence and domain partition diagram of a protein encoded by EjPI gene.

FIG. 4 shows that the loquat EjPI gene of example 2 is expressed in loquat petals, filaments and anthers, but not in sepals and pistils.

FIG. 5 shows construction of EjPI gene overexpression vector of example 3. Wherein a is a pBI121 carrier enzyme cutting electrophoresis picture, M is DL15000DNA marker, and 1 is a pBI121 carrier after double enzyme cutting; b is double enzyme digestion verification electrophoretogram of an EjPI overexpression vector pBI121-EjPI, M is DL15000DNA marker, and 1 is a pBI121-EjPI vector after double enzyme digestion.

FIG. 6 is a photograph of floral organs of wild type Arabidopsis thaliana before and after the transgene of example 5. Wherein a is wild-type Arabidopsis thaliana; b and c are sepal valvularization of EjPI transgenic wild type Arabidopsis thaliana.

FIG. 7 is a photograph of floral organs of Arabidopsis thaliana pi-1 mutants before and after the transgene of example 5. Wherein a is an Arabidopsis thaliana pi-1 mutant; b and c are EjPI transgenic Arabidopsis thaliana pi-1 mutants.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory manual,2001), or the conditions suggested by the manufacturer's instructions.

EXAMPLE 1 cloning of cDNA sequence of EjPI Gene of Eriobotrya japonica

Extraction of loquat flower bud total RNA

Collecting fresh loquat flower buds with the length of about 1.0cm, quickly putting the loquat flower buds into a freezing tube, quickly freezing the loquat flower buds in liquid nitrogen for 3 hours, and putting the loquat flower buds into an ultra-low temperature refrigerator at the temperature of minus 80 ℃ for standby. Extracting total RNA of flower buds by adopting an RNA extraction kit: taking out flower bud material from-80 deg.C ultra-low temperature refrigerator, placing into a mortar which is pre-frozen and added with 2mL RLT lysate and 200 μ L PLAntaid, and grinding at room temperature; transferring the grinding fluid into a 2mL eppendorf centrifuge tube, centrifuging at 13000rpm for 15min, sucking 500 mu L of supernatant fluid, and transferring the supernatant fluid into a new 2mL centrifuge tube; adding 250 μ L of anhydrous alcohol into the supernatant, sucking, mixing, adding into adsorption column, and placing into collection tube; adding 600 μ L deproteinized solution into adsorption column. Adding 600 μ L of rinsing liquid, centrifuging at 13000rpm for 2min, pouring off waste liquid in the collecting pipe, adding rinsing liquid again and centrifuging; putting the adsorption column back into an empty collection tube, carrying out air centrifugation at 13000rpm for 4min, and removing residual rinsing liquid as much as possible; taking out the adsorption column, placing back into empty RNase-free eppendorf tube, adding 50 μ L RNase free water, standing at room temperature for 2min, and centrifuging at 13000rpm for 2 min; and adding the first eluent into the adsorption column again, and repeatedly centrifuging once again to improve the extraction concentration of RNA. mu.L of the diluted RNA sample was aspirated, and the RNA concentration was detected with a spectrophotometer.

3' RACE experiment of loquat flower bud total RNA

The extracted total RNA of the loquat flower buds is used as a 3' RACE experiment template, and 3' RACE adapter is used as a primer to carry out reverse transcription reaction to synthesize first chain cDNA of the 3' RACE experiment; taking 1 mu L of total RNA, 1 mu L of 3' RACE adapter and DEPC-ddH₂O4.5 μ L, mixing well, and denaturing at 70 deg.C for 10 min; ice-bath for 2 min; after the reaction is finished, sequentially adding 2 mu L of 5 XM-MLV buffer, 0.25 mu L of RNase inhibitor, 1 mu L of 10mM dNTP and 0.25 mu L of M-MLV, uniformly mixing, and reacting for 70min at 42 ℃; reacting for 10min at 70 ℃; ice-bath for 2min, and storing at-20 deg.C.

According to conserved regions of PI homologous gene sequences of loquat kindred species apples (AB081092) and sweet cherries (AB763910) published by NCBI website, upstream specific primers 3REjPIF1 and 3REjPIF2, 3REjPIF1 of 3' RACE experiment are directly designed: 5'-ATCACTGTTCTATGTGATGCTA-3' and 3REjPIF 2: 5'-GTGGATAGAGTCAAGAAAGACA-3' are provided. 3'RACE reverse transcription product was used as template, using high fidelity EX-taq enzyme, upstream Outer specific Primer 3REjPIF1 and 3' RACE Outer Primer: 5'-TACCGTCGTTCCACTAGTGATTT-3', performing first chain PCR reaction at 94 deg.C for 5 min; 30 cycles of 94 ℃ for 45s, 57 ℃ for 45s and 72 ℃ for 45 s; 10min at 72 ℃. Using the first strand PCR reaction product as template, using high fidelity EX-taq enzyme, upstream outer specific Primer 3REjPIF2 and 3' RACE Inner Primer: 5'-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3', performing second chain PCR reaction at 94 deg.C for 5 min; 30 cycles of 94 ℃ for 45s, 56 ℃ for 45s and 72 ℃ for 45 s; 10min at 72 ℃. After completion of the reaction, the fragment of interest was excised by 1% agarose gel electrophoresis (FIG. 1a), and the PCR product was recovered using an agarose gel DNA recovery kit according to the instructions. And connecting the recovered product to a pMD19-T vector, transferring the product into an escherichia coli competent cell, picking a monoclonal and sequencing the monoclonal.

5' RACE experiment of total RNA of loquat flower buds

Specific primers 5REjPIR1 and 5REjPIR2 for 5'RACE experiment were designed according to the sequence of 3' RACE experiment, wherein 5REPIR 1: 5'-AGATTTGGCTGAATAGGCTGCA-3', 5REjPIR 2: 5'-TATCTGCTGCTGGGTGGTGTTGTAG-3' are provided. According to the 5' RACE experimental procedure: first Strand Synthesis Buffer Mix was prepared by sequentially adding 2.0. mu.L of 5 XFirst-strand Buffer, 1.0. mu.L of dNTP Mix (10mM), and 1.0. mu.L of DTT (20mM), mixing well, and standing at room temperature.

To a 200. mu.L eppendorf tube, 1.75. mu.L of H was added₂O, 1.0. mu.L of 5 '-CDS primer A, 1.0. mu.L of total RNA, mixed well, subjected to instantaneous centrifugation, at 72 ℃ for 3min, cooled to 42 ℃ for 2min, cooled, and centrifuged at 14000g for 10s, 4.0. mu.L of Buffer Mix, 1. mu.L of SMARTER IIA oligo, 1.0. mu.L of SMARTscrube Reverse transcriptase (100U), 0.25. mu.L of RNase inhibitor (400U/. mu.L), and 10. mu.L in total volume, mixed well, centrifuged, reacted at 42 ℃ for 95min, and denatured at 70 ℃ for 10min to obtain control 5' -RACE-Ready cDNA.

5' RACE amplification System: 34.5. mu.L of PCR-Grade water, 1.0. mu.L of dNTP Mix, 5.0. mu.L of 10 × Advantage 2PCR Buffer, 1.0. mu.L of 50 × Advantage 2polymerase Mix, 2.5. mu.L of control 5' -RACE-Ready cDNA, 1.0. mu.L of 5REjPIR1 primer, 5.0. mu.L of UPM (10X). The procedure for touchdown PCR was: 30s at 94 ℃, 3min at 72 ℃ and 5 cycles; 30s at 94 ℃ and 30s at 70 ℃, 5 cycles; 3min at 72 ℃, 30s at 94 ℃ and 30s at 68 ℃ for 30 cycles; 5min at 72 ℃. After the reaction is finished, performing second-strand PCR reaction by using a PCR reaction product of first-strand 5' RACE as a template and high-fidelity EX-taq enzyme, an upstream outer side specific Primer 5REjPIR2 and a UPM Primer, wherein the reaction program is 94 ℃ for 5 min; 30 cycles of 94 ℃ for 45s, 56 ℃ for 45s and 72 ℃ for 45 s; 10min at 72 ℃. After the reaction, the result of the second strand PCR reaction was checked by electrophoresis on 1% agarose gel (FIG. 1 b). The target fragment was excised, and the PCR product was recovered using an agarose gel DNA recovery kit. After being connected to pMD19-T vector, the vector is transferred into Escherichia coli competent cells, and a single clone is picked up and sequenced.

The PCR sequencing results of 3'RACE and 5' RACE experiments are analyzed and spliced by using DNAMAN software to obtain the full-length cDNA sequence (SEQ ID No.1) of the loquat EjPI gene, and the sequence picture is shown in figure 2.

Designing primers FLEjPIF:5'-TAGAGAGAACAGGAATACTGAGAG-3' and FLEjPIR:5'-ATAATATAACTAGTCAGAAAGAGCA-3' in the full length of the loquat EjPI gene sequence, wherein the reaction condition is 94 ℃ for 4 min; 30 cycles of 94 ℃ for 45s, 56 ℃ for 45s and 72 ℃ for 45 s; 10min at 72 ℃. After completion of the reaction, the target fragment was excised after detection by 1% agarose gel electrophoresis (FIG. 1c), and the PCR product was recovered using an agarose gel DNA recovery kit. After being connected to a pMD19-T vector, the vector is transferred into an escherichia coli competent cell, a single clone is picked up and sequenced, and the CDS sequence of the EjPI gene functional region is verified.

Using primer 5 software, the full-length cDNA sequence of loquat EjPI gene was translated into protein sequence (SEQ ID No.2), and the loquat EjPI protein sequence was assigned 1 specific PI motif among MADS domain, I domain, K domain, C-terminal domain and C-terminal domain according to the characteristics of MADS-box protein (FIG. 3).

Example 2 analysis of semi-quantitative RT-PCR expression amount of EjPI Gene of Eriobotrya japonica

Respectively extracting total RNA of loquat sepals, petals, filaments, anthers and pistils, removing trace DNA in the total RNA, and performing reverse transcription to obtain cDNA. Semi-quantitative RT-PCR primers RTEjPIF:5'-TGCTAAGCATGAGAACCTCAGCAATGA-3' and RTEjPIR:5'-CAGCTGCCTCTGATGATACCCAT-3' were designed using oligo 6.0 software based on loquat cDNA as template. The specificity of the PCR is detected by using PCR, and a semi-quantitative RT-PCR experiment can be carried out on the premise of ensuring the specific amplification of the PCR. Taking loquat actin gene as reference gene, the primer is RTEjactinF: 5'-AATGGAACTGGAATGGTCAAGGC-3' and RTEjactinR: 5'-TGCCAGATCTTCTCCATGTCATCCCA-3', PCR amplifications were performed with 3 biological replicates per reaction. The PCR reaction program is pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 25 cycles. PCR products of EjPI gene and actin gene of sepals, petals, filaments, anthers and pistils, respectively, were detected by 1% agarose gel electrophoresis (FIG. 4). The results show that: in the loquat flower organ development process, the EjPI gene is expressed in petals, filaments and anthers, but not expressed in sepals and pistils, which shows that the EjPI gene is closely related to the formation and development of the petals, the filaments and the anthers.

Example 3 construction of plant transgenic vector pBI121-EjPI of loquat EjPI Gene

Introducing enzyme cutting sites at two ends of a CDS region of the loquat EjPI gene by adopting PCR amplification. Taking cDNA reverse transcribed by total RNA of loquat flower buds as a template, and taking TEjPI-F: 5' -TCTAGAATGGGGAGGGGTAAGATTGAGATCA-3' (introduction of Xba I cleavage site) and TEjPI-R: 5' -CCCGGGTTAGATTCTCTCCTGGAGATTTGG-3' (incorporating SmaI cleavage sites) as primers, was subjected to PCR amplification using EX-taq enzyme. PCR reaction procedure: 4min at 94 ℃; 30 cycles of 94 ℃ for 45s, 57 ℃ for 45s and 72 ℃ for 45 s; 10min at 72 ℃. After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit. And connecting the recovered PCR product with a pMD19-T vector, transferring into an escherichia coli competent cell, picking a monoclonal, and then, sending the sample for sequencing. According to the sequencing result, plasmids were extracted. The pMD19-EjPI recombinant plasmid and the pBI121 vector were double-digested with XbaI and SmaI restriction enzymes, respectively, detected by 1% agarose gel electrophoresis, and recovered using an agarose gel DNA recovery kit. Using T₄The EjPI gene after double enzyme digestion is connected with pBI121 by DNA ligase, and then transferred into escherichia coli competent cells to obtain a plant transgenic expression vector pBI 121-EjPI. pBI121 (control) and the pBI121-EjPI expression vector obtained were digested simultaneously with Xba I and Sma I, respectively, and only 1 band appeared in pBI121 after digestionpBI121-EjPI vector presents pBI121 vector and 2 EjPI gene bands (figure 5), respectively, and the successful connection of the EjPI gene and the pBI121 vector is proved.

Example 4 transformation of transgenic expression vector pBI121-EjPI into Arabidopsis thaliana pi-1 mutant

Taking 1 mu g of pBI121-EjPI plasmid, adding to 100 mu L of agrobacterium tumefaciens competent cells, sucking, beating and mixing uniformly; ice-cooling for 5min, transferring into liquid nitrogen, rapidly freezing for 1min, rapidly placing at 37 deg.C, and water-bathing for 5 min; adding 800 μ L LB liquid culture medium, oscillating at 28 deg.C and 250rpm for 5 h; the suspension was transferred to LB (50mL LB + 50. mu.g/mL Kan + 50. mu.g/mL Rif) solid selection medium, spread evenly and cultured in 28 ℃ for 2d in an inverted manner.

The Agrobacterium containing the pBI121-EjPI positive clone was streaked on 25mL solid plate medium (containing 25. mu.g/mL Kan + 25. mu.g/mL Rif), and cultured in an inverted state at 28 ℃ for 48 h; selecting a single clone, and inoculating the single clone into 10mL of liquid LB culture medium (containing 10 mu g/mL Kan +10 mu g/mL Rif); the cells were cultured overnight at 28 ℃ and 200rpm with shaking until OD was 0.7-0.8. Uniformly coating 1mL of culture broth on 25mL of solid plate culture medium (containing 25 mu g/mL Kan +25 mu g/mL Rif), and performing inverted culture at 28 ℃ for 48 h; agrobacterium was scraped off from the solid medium using a glass triangle rod, and the pellet was resuspended in 1/2MS liquid medium containing 5% sucrose and 3% Silwet L-77 to an OD of 0.2 for arabidopsis transgenesis.

Placing Arabidopsis seeds on wet filter paper, placing the filter paper at 4 ℃ for 48h, then sowing the seeds into nutrient soil (the nutrient soil is vermiculite: perlite: 5:4:1), and culturing the seeds under the conditions of temperature of 22 ℃, humidity of 75% and 14h darkness/10 h illumination; before transgenosis, plants of the identified arabidopsis pi-1 heterozygote (purchased from an arabidopsis mutant library) are watered thoroughly; cutting off existing siliques on an arabidopsis plant to be used during dip dyeing, and soaking flower buds into PBI121-EjPI agrobacterium tumefaciens dip dyeing solution for about 90 s; covering a black sealing film, maintaining a high-temperature and high-humidity environment in the film, and uncovering the film after dark culture for 2 d; the method is used for infecting 4 times with the interval time of 7 d.

Example 5 transgenic Arabidopsis thaliana screening and phenotypic characterization of the loquat EjPI Gene

And (4) collecting mature seeds of EjPI transgenic Arabidopsis, and cleaning the seeds. Performing vernalization treatment in a refrigerator at 4 deg.C for 2 weeks; putting the seeds into a collecting pipe, adding 900 μ L of absolute ethanol into the seeds, and shaking for 6 min; centrifuging at 5000rpm for 2 min; pouring off alcohol in the collecting pipe, adding 900 μ L70% ethanol into the collecting pipe, and shaking for 5 min; centrifuging at 5000rpm for 2 min; airing the seeds; the suspension was spread evenly on 1/2MS medium (pH 5.8, 50. mu.g/mL Kan, 3% sucrose and 0.8% agar) plates. Putting the inoculated flat plate into a refrigerator at 4 ℃ for vernalization for 2 d; and (4) placing the vernalized plate in an artificial climate box for normal culture. After 6 true leaves grow, the leaves are moved into nutrient soil, and after hardening and strengthening seedlings, the seedlings are managed according to conventional water and fertilizer until the flowers bloom.

Extracting EjPI transgenic Arabidopsis DNA, placing 1 piece of Arabidopsis leaf into a 1.5mL eppendorf tube, placing into liquid nitrogen for quick freezing, and grinding; adding 600 μ L of extraction buffer solution, vortex shaking, and placing on ice; after all samples are treated, placing the samples in a water bath at 65 ℃ for 25 min; taking out the sample from the water bath, placing the sample to room temperature, adding 340 mu L of potassium acetate solution after cooling to the room temperature, carrying out vortex oscillation and carrying out ice bath for 20 min; 12000rpm, high speed centrifugation for 10min, supernatant transferred to a new eppendorf tube; adding equal volume of isopropanol, centrifuging at 4 deg.C and 12000rpm for 20min, removing supernatant, and rinsing with ice anhydrous ethanol (the anhydrous ethanol is placed in a refrigerator at-20 deg.C 2h earlier); rinsing the precipitate with 70% and 100% ethanol in sequence; after the precipitate was blown dry, it was dissolved in 50. mu.L of sterile water.

And (3) carrying out PCR screening on the positive plant DNA of the transgenic arabidopsis thaliana by using the DNA of the non-transgenic wild arabidopsis thaliana and the pi-1 mutant as a control and using primers TEjPI-F and TEjPI-R constructed by a vector. PCR amplification procedure: 4min at 94 ℃; 30 cycles of 94 ℃ for 45s, 56 ℃ for 45s and 72 ℃ for 45 s; 10min at 72 ℃. 23 positive EjPI transgenic wild type Arabidopsis plants and 21 positive EjPI transgenic Arabidopsis pi-1 mutant plants are obtained together.

Floral organ phenotype identification was performed on the transgenic wild type Arabidopsis thaliana and the transgenic Arabidopsis thaliana pi-1 mutant, and the floral organ phenotype of Arabidopsis thaliana was photographed by observation under a Leica style microscope (FIGS. 6 and 7). The phenotypic results show that: overexpression of the EjPI gene in transgenic wild-type arabidopsis thaliana, compared to wild-type arabidopsis thaliana (fig. 6a), resulted in the sectioning and opening of the green sepal of arabidopsis thaliana (fig. 6b and c). Compared to the non-transgenic pi-1 mutants (without petals and stamens, FIG. 7a), there were 13 EjPI transgenic Arabidopsis thaliana pi-1 mutants that grew petals in the floral organ round 2 and stamens with shorter filaments in round 3 (FIG. 6 b); there were 8 EjPI transgenic Arabidopsis pi-1 mutants that grew petals in floral organ round 2 and normal stamens in round 3 (FIG. 7 c). The result shows that the EjPI gene has the function of replacing the PI gene, and the transgenic Arabidopsis material of the EjPI gene can be used for plant petaloid flower modification so as to improve ornamental value and restore stamen organs for cross breeding.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<210> 10

<211> 25

<212> DNA

<213> loquat (Eriobotrya japonica)

<400> 10

ataatataac tagtcagaaa gagca 25

<210> 11

<211> 27

<212> DNA

<213> loquat (Eriobotrya japonica)

<400> 11

tgctaagcat gagaacctca gcaatga 27

<210> 12

<211> 23

<212> DNA

<213> loquat (Eriobotrya japonica)

<400> 12

cagctgcctc tgatgatacc cat 23

<210> 13

<211> 23

<212> DNA

<213> loquat (Eriobotrya japonica)

<400> 13

aatggaactg gaatggtcaa ggc 23

<210> 14

<211> 26

<212> DNA

<213> loquat (Eriobotrya japonica)

<400> 14

tgccagatct tctccatgtc atccca 26

<210> 15

<211> 31

<212> DNA

<213> loquat (Eriobotrya japonica)

<400> 15

tctagaatgg ggaggggtaa gattgagatc a 31

<210> 16

<211> 30

<212> DNA

<213> loquat (Eriobotrya japonica)

<400> 16

cccgggttag attctctcct ggagatttgg 30

Claims (11)

1. Loquat EjPI protein, which is protein consisting of amino acids shown in SEQ ID number 2.

2. A gene encoding the loquat EjPI protein of claim 1.

3. The gene of claim 2, having the sequence shown in SEQ ID No. 1.

4. A vector containing the gene according to claim 2 or 3.

5. An engineered bacterium comprising the gene of claim 2 or 3.

6. Use of the gene of claim 2 or 3 for regulating the formation and development of petals, filaments and anthers of angiosperms.

7. Use according to claim 6, wherein the gene is transferred into the angiosperm genome, overexpressed in wild type plants, valvified and opened; alternatively, overexpression in Arabidopsis pi-1 mutants restores petals and stamens and completes the reproductive process.

8. A construction method of transgenic plant, adopting Agrobacterium mediated method, transferring the over-expression vector containing the gene of claim 2 or 3 into wild plant genome, and screening to obtain transgenic plant.

9. The method of claim 8, wherein the transgenic plant has a white sepal edge and is open compared to the wild type, and sepal valvularization occurs.

10. A construction method of transgenic plant, adopting Agrobacterium-mediated method, transferring the over-expression vector containing the gene of claim 2 or 3 into the arabidopsis thaliana pi-1 mutant genome, and screening to obtain transgenic mutant.

11. The method of claim 10, wherein said transgenic mutant restores petal and stamen organs compared to a non-transgenic mutant.

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