CN110747179A - Lycoris longituba LlDFRb gene and protein expressed by same and application of gene - Google Patents

Abstract

The invention discloses a lycoris longituba LlDFRb gene, an expressed protein and application thereof, belonging to the field of plant molecular biology, wherein the nucleotide sequence of the LlDFRb gene is shown as SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2. The LlDFRb gene is an important regulation gene in a downstream path for synthesizing anthocyanin, is expressed actively in the early bud stage of lycoris radiata, and then is released along with flowers, the expression quantity is reduced, so that the genetically transformed tobacco flower color is deepened and is changed from pink to light red, and the LlDFRb gene is a DFR gene capable of changing the flower color. The LlDFRb gene is applied to transgenic plants, can improve the flower color of the plants so as to improve the ornamental value of the plants, and has practical application value.

Description

Lycoris longituba LlDFRb gene and protein expressed by same and application of gene

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to a lycoris longituba LlDFRb gene, and expressed protein and application thereof.

Background

Lycoris longituba (Lycoris longitauba) belongs to Amaryllidaceae (Lycoris), is a unique species in China, is mainly produced in Jiangsu Anhui, has extremely rich intraspecies flower color variation, such as pink, yellow, orange, purple red, reddish blue, rare emerald green and the like, has very large flower shape variation, can emit faint aroma, and has very large research value and application potential. The flavanonol 4-reductase (DFR) is an important regulation gene in a downstream pathway for synthesizing anthocyanin, catalyzes flavanonol to generate colorless pelargonidin, colorless delphinidin and colorless cyanidin respectively, is an important node for leading anthocyanidin to be colored from colorless, and plays an important role in the final flower color of plants as a key enzyme in the downstream of a flavonoid metabolic pathway, namely in the process of forming the flower color, the accumulation of the anthocyanidin with different colors mainly depends on the activity of the DFR gene, and mutants losing the activity of the DFR generate ivory color or white color. Based on the DFR functional identification, the transgenic technology is utilized to achieve remarkable results in the research of a plurality of plant flower color modifications. The function and expression regulation mode of the DFR gene in plants are variable, but the expression regulation mechanism of the gene is less analyzed in monocotyledons at present, the function of the DFR gene of the lycoris longituba in flower color regulation is researched, and a useful molecular tool can be provided for improving the ornamental characters of the plants by utilizing genetic engineering.

Disclosure of Invention

In view of the above problems in the prior art, the present invention is directed to provide a DFR gene LlDFRb derived from lycoris radiate, which can be used for improving flower color and plant ornamental traits. Another object of the present invention is to provide a method for applying the ldfrb gene, which can improve the flower color of plants and thus improve their ornamental value.

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

a lycoris longituba LlDFRb gene has a nucleotide sequence shown in SEQ ID No. 1.

The amino acid sequence of the expression protein of the lycoris longituba LlDFRb gene is shown in SEQ ID NO. 2.

A vector comprising the Lycoris longituba LlDFRb gene of claim 1.

Preferably, the vector of the lycoris longituba LlDFRb gene is pEASY-Blunt-LlDFRb, pCAMBIA1300-LlDFRb or pCAMBIA 1304-LlDFRb.

The lycoris longituba LlDFRb gene, or the expression protein of the lycoris longituba LlDFRb gene, or the application of the lycoris longituba LlDFRb gene vector in improving plant flower color.

A method for obtaining a new plant variety with changed flower color by using lycoris longituba LlDFRb gene comprises the following steps:

1) constructing a vector of the lycoris longituba LlDFRb gene of claim 3 or 4;

2) transforming the constructed lycoris longituba LlDFRb gene vector into plants or plant cells;

3) breeding and screening to obtain new plant variety with changed flower color.

Has the advantages that: compared with the prior art, the invention has the advantages that: the nucleotide sequence of the lycoris longituba LlDFRb gene provided by the invention is shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO. 2. The gene expression mode is determined by cloning and fluorescence quantification to be that the lycoris radiata is actively expressed in the early bud stage and then the expression quantity is reduced along with the flower opening; the gene function is identified through subcellular localization and transgenosis, and the expressed protein is found to be structural protein, so that the flower color of the genetically transformed tobacco is deepened and turns from pink to light red, which indicates that the LlDFRb is a DFR gene capable of changing the flower color. The LlDFRb gene serving as an important regulatory gene DFR family member in the anthocyanin synthesis process can be used for improving plant flower color in genetic engineering so as to improve the ornamental character of plants and has practical application value.

Drawings

FIG. 1 is a diagram of petals of pink long tube lycoris at different development stages; s1: in the bud period; bud period in S2; s3: full bloom period;

FIG. 2 is a graph showing the results of LlDFRb expression analysis of pink Bulbus Lycoridis Radiatae in different developmental stages; s1: in the bud period; bud period in S2; s3: full bloom period;

FIG. 3 is a graph of the subcellular localization of LlDFRb in tobacco leaves; 35 s: : GFP: expression of epidermal cells under tobacco injection by agrobacterium GV3101 carrying empty vector pCAMBlAl 300; 35 s: : GFP-LlDFRb: expression of epidermal cells in tobacco after Agrobacterium GV3101 carrying plasmid pCAMBIA1300-LlDFRb is injected;

FIG. 4 is a graph comparing the phenotype of transgenic lines with wild-type tobacco; a: and (3) wild type B: transgenic plants;

FIG. 5 is a graph of anthocyanin content in transgenic line L3.

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.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The material used in the experiment is collected from an lycoris germplasm resource library of garden plant discipline of Nanjing forestry university, 3 lycoris longituba pink color lines (bud period in S1, bud period in S2, full-bloom period in S3) (figure 1) petals of flowers in the development period are selected in 2016 year and 8 month, the petals are put into a sterilized centrifuge tube, and the centrifuge tube is immediately put into liquid nitrogen for quick freezing, and then the centrifuge tube is placed in a refrigerator at minus 80 ℃ for storage.

Example 1: cloning and expression analysis of LlDFRb Gene

(1) Cloning of LlDFRb Gene

This example uses the TIANGEN plant RNA extraction kit (DP432) to extract total plant RNA. Using TaKaRaPrimeScript^TMThe RT Master Mix (Perfect Real Time) reverse transcription kit reversely transcribes the extracted RNA into cDNA, and the finally obtained cDNA is diluted by 10 times with water and stored in a refrigerator at-20 ℃.

1 Unigene segment with high DFR protein homology is obtained by screening according to the Lycoris longituba petal transcriptome database obtained by the previous subject group, and the full-length integrity of the screened genes is preliminarily determined to be good by comparing the Unigene segments with the high DFR protein homology through an NCBI database. And the coding region sequence was cloned using Primer Premier5.0 software design 1 pair of specific primers (F: 5'-GAGAGAGAGAGATGAAGGGGC-3'; R: 5'-GTCATAAGCTTGTATTGTGGGTG-3').

Using 1st Strand cDNA as a template, a PCR amplification reaction was performed using Easypfu Mix high fidelity enzyme from Seikagaku corporation, in a reaction system of 50. mu.L: 1 μ L of 1st Strand cDNA, 2 μ L of ORF Forward Primer (10mM), 2 μ L of ORFREverser Primer (10mM), 25 μ L of 2xEasypfu PCR Supermix, 20 μ L of ddH₂And O. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 20s, annealing at different temperatures for 20s, extension at 72 ℃ for 1min, and 35 cycles; the total extension was carried out at 72 ℃ for 10min and the reaction was stopped at 16 ℃. The obtained product is used for agarose electrophoresis detection, gel cutting is carried out, an electrophoresis band with the same size as the expected size is recovered, a pEASY-Blunt vector is connected and transformed into an escherichia coli Trans1-T1 competent cell, and 3 positive clones are picked and sent to Beijing Kingsry biotechnology limited for sequencing. The nucleotide sequence of the LlDFRb gene sequence is shown as SEQ ID No.1 in the sequence table, and the amino acid sequence of the expressed protein is shown as SEQ ID No.2 in the sequence table.

And (3) recovering and purifying a target fragment: after PCR amplification was complete, 10. mu.L of 5 × Loading Buffer was added and the whole was spotted into wells; taking out the gel after electrophoresis for 35min by adopting 1.5 percent agarose gel at 160V and 200 mA; taking a picture by using a gel imager, taking out the gel, and cutting down the target fragment under an ultraviolet lamp; the glue recovery is carried out, and the specific steps are as follows:

1) the band of interest was carefully cut into a sterile centrifuge tube (1.5. mu.L) and weighed (the centrifuge tube was removed from its weight prior to weighing).

2) Adding 3 times volume of GSB (gel solution buffer) (converting the weight of the gel block into volume, roughly calculating 100mg and 100 mu L), melting the gel block completely in a water bath at 55 ℃ for 10min, and shaking up by reversing every 2-3 min;

3) after the gel block is completely melted, adding isopropanol with the volume of 1 time into the solution, uniformly mixing, adding all the isopropanol into a centrifugal column, standing for 1min, centrifuging at 10000rpm for 1min, and discarding an effluent;

4) adding 650 μ L WB (Wash Buffer), centrifuging at 10000rpm for 1min, and discarding the effluent;

5) repeating the previous step;

6) centrifuging at 10000rpm for 2min, removing residual WB, placing the column in a new sterile 1.5 μ L centrifuge tube, opening the cover, and standing at room temperature for 3 min;

7) dripping 40 μ L ddH into the center of the centrifugal column₂O, standing at room temperature for 2min, centrifuging at 10000rpm

lmin; eluted ddH₂Suspending O again and dripping into the centrifugal column, standing at room temperature for 2min, and centrifuging at 10000rpm for 1 min; the spin column was discarded and the resulting DNA solution was stored in a freezer at-20 ℃ for further use.

(2) Fluorescent real-time quantitative PCR analysis of LlDFRb gene expression pattern

Fluorescent quantitative primers were designed in the non-conserved region based on the full-length cDNA sequence of LlDFRb as follows:

F：5′-AAGAACAAACTACTGTGACCAAAGC-3′；

R：5′-CCATAAGCTTGTATTGTGGGTGT-3′。

taking cDNA of 3 development stages of pink long-tube lycoris petals as a template, and selecting the long-tube lycoris eIF gene as an internal reference gene. According to

Premix Ex Taq^TMThe specification prepares different components according to the proportion of a reaction system. The reaction system had a total volume of about 10. mu.L, forward and reverse primers of 0.4. mu. L, SYBR 5. mu. L, cDNA 1. mu.L, calibrator solution of 0.2. mu.L, and ultrapure water of 3. mu.L. The amplification procedure was pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 45s, for 40 cycles.Each sample was assigned 3 biological replicates and three technical replicates. Use after ensuring that experimental data are reliable 2^-ΔΔCTThe expression difference of the target gene is calculated, and the obtained data are subjected to significance analysis by using SPSS20.0 software.

The result is shown in figure 2, the expression pattern of the LlDFRb gene is in a down-regulation trend in 3 different development periods of the lycoris radiata petal, the LlDFRb gene is expressed actively in the early bud period and then is reduced along with the flower opening, and the expression quantity is reduced.

Example 2: vector construction and function verification of LlDFRb

I. Subcellular localization observation of LlDFRb gene expression

(1) Cloning, recovering and sequencing target fragment

The complete ORF of the LlDFRb gene was subjected to cleavage site analysis in BioXM software, and the restriction enzymes Kpn I and Sma I that could be used were finally selected. Designing a primer containing the enzyme cutting site as follows:

F：5′-GAGAGAGAGAGATGAAGGGGC-3′；

R：5′-GTCATAAGCTTGTATTGTGGGTG-3′。

PCR amplification was performed using PrimeStarMax (TaKaRa) high fidelity enzyme using the sequenced pEASY-Blunt-LlDFRb plasmid as template in 50. mu.L: mu.L of 1st Strand cDNA, 2. mu.L of ORF Forward Primer (10mM), 2. mu.L of ORF Reverse Primer (10mM), 25. mu.L of 2 XPrimerStar Max (Takara), 20. mu.L of ddH₂And O. Reaction conditions are as follows: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10s, annealing at 5s, extension at 72 ℃ for 15s, 35 cycles; and (3) total extension is carried out at 72 ℃ for 1min, reaction is terminated at 4 ℃, then the first round of products are used as templates, second round of PCR enrichment of target fragments is carried out according to the reaction system and the procedures, and the obtained products are used for agarose electrophoresis, recovery of the target fragments, cloning and sequencing.

(2) Extraction of pCAMBIA1300 plasmid

Extraction of plasmids was accomplished using a small-to-medium-amount Tiangen plasmid kit:

1) melting the subject group on pCAMBIA1300 plasmid bacterial liquid stored in a refrigerator at-80 ℃ on ice, sucking 100 mu L of the melted liquid, adding the melted liquid into 20ml of fresh LB culture medium (containing kan), shaking the liquid at 37 ℃ and 200rpm for 12-14 h;

2) adding 5-10 microliters of the shaken bacterial liquid into a 2mL centrifuge tube in several times, centrifuging at 12000rpm for 1min, and discarding the supernatant;

3) adding 500 μ L of equilibrium liquid BL into adsorption column CP4, centrifuging at 12000rpm for 1min, and pouring off waste liquid;

4) adding 500 mu L of solution P1 (added with RNaseA) into a centrifuge tube, and shaking by vortex shaking until bacterial plaque at the bottom is completely dissolved to completely suspend the bacteria;

5) adding 500 μ L of solution P2, and turning gently up and down for 6-10 times to make the thallus fully split (the process doesn't shake violently to avoid genome DNA pollution);

6) adding 700 μ L of solution P2, turning gently for 6-8 times, mixing well, observing whether white flocculent precipitate appears, centrifuging at 12000rpm for 10min, and forming precipitate at the bottom of the centrifuge tube;

7) adding the supernatant collected in the previous step into a filtering column CS in batches, centrifuging at 12000rpm for 2min, adding the solution obtained in the collecting tube into CP4 in batches, centrifuging at 12000rpm for 1min by an adsorption column, and discarding the waste liquid;

8) adding 500 μ L deproteinized solution PD into CP4, centrifuging at 12000rpm for 1min, and discarding the waste liquid;

9) adding 600 μ L of rinsing solution PW (added with absolute ethanol) into CP4, centrifuging lmin at 12000rpm, discarding the eluate, and repeating the step for 1 time;

10) the CP4 is put back into the collection tube and centrifuged at 12000rpm for 2 min;

11) placing CP4 in a new 1.5mL centrifuge tube, suspending and dripping 40-60 μ LddH to the middle part of the adsorption membrane₂O (preheating effect is best at 65-70 ℃), standing for 2min at room temperature, centrifuging for 1min at 12000rpm, and then determining the plasmid concentration.

(3) pCAMBIA1300 plasmid double digestion

The extracted pCAMBIA1300 plasmid was subjected to double digestion (50. mu.L) according to the following reaction system: 10 μ L of LPLAStic DNA, 1 μ L of KpnI, 1 μ L of Sma I，5μL 10×QuickCut Buffer(Takara)，33μL ddH₂O。

Reaction conditions are as follows: 1-2h at 37 ℃.

(4) Recombination reactions

Carrying out recombination reaction on a PCR product of the LlDFRb gene PCR fragment inserted into the pEASY-Blunt vector and the digested pCAMBIA1300 vector to obtain pCAMBIA1300-LlDFRb, and configuring the following recombination reaction system in an ice water bath: 2 μ L

II, 40ng of the amplified product of the insert, 60ng of the vector plasmid, 4. mu.L of 5 XCE IIBuffer, ddH₂O was made up to 20. mu.L.

Before preparing the system, the concentration and purity of the PCR product of the insert and the vector after enzyme digestion are respectively determined, and appropriate amount is taken according to the optimal mole ratio of the cloning vector to the insert of 1: 2.

After the system is prepared, the mixture is lightly blown and beaten by a pipettor to be uniformly mixed, so that bubbles are prevented from being generated and violent oscillation is avoided. Reacting at 37 deg.C for 30min, cooling in ice water bath for 5min, and storing in refrigerator at-20 deg.C.

(5) Ligation transformation

Add more than 5. mu.L of the cooled reaction solution to 50. mu.L of Trans1 competent cells, mix them by gentle pipetting, and ice-wash for 30 min. Heat shock at 42 deg.C for 90 s, ice bath for 2 min. Add 300. mu.L LB liquid medium (without antibiotics) and shake the bacteria for 1h at 37 ℃. Centrifuging at 3000rpm for 1min, removing 150 μ L of supernatant, blowing off the remaining liquid, spreading on a plate containing Kna, and culturing overnight at 37 deg.C by inversion.

(6) Identification and sequencing of Positive clones

12 white monoclonals are picked from each plate by a sterile toothpick, are backed up on a new plate, and are dipped into 7 mu L of sterile water to prepare a 20 mu L PCR reaction system: mu.L of bacterial suspension, 10. mu.L of 2 XTAQA Mix, L. mu.L of 1300 revertprimer (10mM), 1. mu.L of 1300Forward Primer (10mM), 7. mu.L of ddH₂And O. PCR detection reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; extension at 72 ℃ for 10 min.

PCR products were detected with 1% agarose gel, three positive clones were selected from the backup, dipped into 800. mu.L of liquid LB screening medium with sterile toothpicks, incubated at 37 ℃ 200rpm for 5-6h, and then sequenced. And (5) after sequencing identification is correct, preserving bacteria and backing up.

The PCR product was recovered by amplifying the plasmid pEASY-Blunt-LlDFRb, which was sequenced correctly, as a template by the above-described specific method. Then the LlDFRb target fragment is inserted into a pCAMBIA1300 vector containing reporter gene GFP by double digestion, and then is transferred into a Trans1 competent cell, and a large amount of recombinant plasmids are obtained through Trans 1. After enzyme digestion and identification, the obtained recombinant plasmid is introduced into agrobacterium tumefaciens GV3101 by a freeze-thaw method. Then, the vector-containing Agrobacterium and the auxiliary expression vector P19 were cultured in a liquid medium containing LB (100 mg. L.)^-1Kanamycin) was cultured with shaking (28 ℃, 200 r.min)^-1) To the bacterial liquid OD₆₀₀Centrifuging the bacterial solution at 4 deg.C under 5000r min-1 for 5min at 0.6-1.0, collecting thallus, and adding buffer solution (containing 10mM L)^-1MgCl₂，10mM·L^-1Biological buffer MES, 150. mu.M.L^-1Acetosyringone) to re-suspend the thallus, and finally, the re-suspended bacteria liquid is used according to the proportion (OD)₆₀₀The ratio is 7: 5), standing for 2-3h at room temperature. Injecting mixed bacteria-containing liquid into the back of a Benedict tobacco leaf by using a 1mL medical injector, putting the tobacco leaf into an incubator to culture for 2-3d, observing the expression condition of epidermal cells under the condition of injecting tobacco carrying plasmid pCAMBIA 1300-LlDFRb-Agrobacterium GV3101 under a laser confocal microscope, respectively observing the expression condition of epidermal cells under a GFP green fluorescence Field (GFP), a Chloroplast pink fluorescence Field (Chloroplast), a white light Field (Bright Field) and a mixed Field of the former three (Merged), and simultaneously determining the subcellular localization condition of the LlDFRb protein of the lycoris longituba by taking the expression condition of epidermal cells under the tobacco carrying plasmid-unloaded pCAMBlAl300 Agrobacterium 3101 as a control.

As shown in FIG. 3, the results of the experiments showed that LlDFRb-GFP was injected into the 5 th to 8 th tobacco leaves grown from Nicotiana benthamiana, cultured in an incubator for 2 days, and then observed by confocal laser microscopy to show that GFP signals can be detected in the nucleus, cell membrane and cytoplasm of the epidermal cells of Nicotiana benthamiana, which primarily demonstrated that LlDFRb protein is localized in the nucleus, cell membrane and cytoplasm and is a typical structural protein.

(2) Identification and phenotypic analysis of transgenic tobacco positive lines

Cloning the full-length coding region sequence of the LlDFRb gene to a pCAMBIA1304 vector containing a CaMV 35S promoter through XbaI and KpnI enzyme cutting sites, connecting the transformation step with a specific method used in the construction of the pCAMBIA1300-LlDFRb vector, transferring an expression vector containing a target gene into agrobacterium tumefaciens EHA105 by adopting a freeze-thaw method, and transforming tobacco by a leaf disc method. The expression of LlDFRb in the transgenic plants is verified by fluorescence quantification by taking wild tobacco plants as a control.

The extraction and preservation method of anthocyanin comprises the following steps: sufficiently grinding tobacco corolla material preserved at-80 ℃ in liquid nitrogen, weighing 50mg of dry powder, adding 1.5mL of 1% hydrochloric acid methanol extract, sufficiently oscillating for 1min, extracting at 4 ℃ in a dark place, oscillating once every 8h, extracting for 24h, centrifuging at 4 ℃, 10000rpm for 10min, sucking supernatant, filtering with 0.22 mu m filter membrane, storing in a 1.5mL brown chromatographic bottle, and preserving at-20 ℃.

The anthocyanin content determination method comprises the following steps: weighing cyanidin-3-O-glucoside standard substance, dissolving with 1% hydrochloric acid methanol solution by volume fraction, and respectively preparing into 0.01, 0.025, 0.05, 0.075 and 0.1mg/mL to make standard curve. The chromatographic column used in the liquid chromatogram is C18, the diode array detector, the mobile phase A liquid is acetonitrile, the B liquid is phosphoric acid solution with volume fraction of 0.4%, the liquid A, B volume ratio is 20: 80, the flow rate is 1.0mL/min, the column temperature is 30 ℃, the sample injection volume is 10 mu L, and the detection wavelength is 526 nm. Drawing a standard curve by taking the standard mass concentration (X) as a horizontal coordinate and taking the peak area (Y) as a vertical coordinate to obtain a linear regression equation and a correlation coefficient: 0.0225x-2.5427, wherein R2 is 0.9994, and the concentration is ug/mL, and the reagent is used for the quantitative analysis of anthocyanin.

In order to verify the function of the LlDFRb gene, the LlDFRb gene is transferred into tobacco through agrobacterium transformation, a strain L3 with an obvious phenotype is selected from obtained positive plants, wild tobacco is used as a control, and phenotype analysis is carried out on the transgenic plants to find out that the transgenic plants have slightly red corolla in the bud period compared with the control color, and the flower color is obviously red in the full-bloom period compared with the control color and is light red as shown in figure 4; while the flower crown of the wild tobacco in the bud period is greenish, and the full bloom period is pink; the transgenic plants did not produce an alteration in anther compared to the wild type, whereas the tip of the filament was slightly reddish compared to the wild type. The result of anthocyanin content measurement also shows that the anthocyanin content in the corolla of the transgenic plant is higher than that of the control (figure 5) and is 2.1 times of that of the control. The above results indicate that the LlDFRb significantly promotes the accumulation of anthocyanin in tobacco flowers.

Sequence listing

<110> Nanjing university of forestry

<120> Lycoris longituba LlDFRb gene, protein expressed by same and application thereof

<130>100

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>801

<212>DNA

<213>Lycoris longituba

<400>1

atgagatcat gtaagaaagc aaggtcagtc cagcgagtta ttttcacatc atctgcagga 60

actgtgaatg tggaggaaca tcaaaagcct gtatacgatg aaaactcatg gagtgatgtc 120

gagttctgca gacgcataaa aatgactgga tggatgtatt ttgtatcaaa atcgcttgca 180

gagaaagctg catgggcttt tgcaagagaa aatggcattg acctaattac catcatacca 240

acactggtgg tgggtccctt catcacctca accatgccac caagtatgat cactgcatta 300

tcattgatca caggaaatga agctcactat tcgataataa agcaagctca gcttgttcac 360

ttagatgacc tgtgtgatgc ccacattcta ctattgagcc atcctaaagc acaaggaaga 420

tacatatgtt cttctcatga tgctaccatt tatgatttag caaagatgat cacagaaaag 480

catcctcagt attacattcc caaaacattt gaagggatcg atgagaaaat tcagcctgtg 540

cgcttctctt caaagaagct cttggaactt ggtttcaggt acaagtacag tatggctgaa 600

atgtttgatg atgccataaa atcatgcatt gagaagaagc tcatacctct ccgaacagtg 660

gaagaacttc ctgaattgat tgaagaacaa actactgtga ccaaagctat tgttaatagg 720

tcagaggaga aagtttccat tgcaatacac tcggatttgt acacccacaa tacaagctta 780

tgacaagaga tgagtttcta g 801

<210>2

<211>265

<212>PRT

<213>Lycoris longituba

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Met Arg Ser Cys Lys Lys Ala Arg Ser Val Gln Arg Val Ile Phe Thr

1 5 10 15

Ser Ser Ala Gly Thr Val Asn Val Glu Glu His Gln Lys Pro Val Tyr

20 25 30

Asp Glu Asn Ser Trp Ser Asp Val Glu Phe Cys Arg Arg Ile Lys Met

35 40 45

Thr Gly Trp Met Tyr Phe Val Ser Lys Ser Leu Ala Glu Lys Ala Ala

50 55 60

Trp Ala Phe Ala Arg Glu Asn Gly Ile Asp Leu Ile Thr Ile Ile Pro

65 70 75 80

Thr Leu Val Val Gly Pro Phe Ile Thr Ser Thr Met Pro Pro Ser Met

85 90 95

Ile Thr Ala Leu Ser Leu Ile Thr Gly Asn Glu Ala His Tyr Ser Ile

100 105 110

Ile Lys Gln Ala Gln Leu Val His Leu Asp Asp Leu Cys Asp Ala His

115120 125

Ile Leu Leu Leu Ser His Pro Lys Ala Gln Gly Arg Tyr Ile Cys Ser

130 135 140

Ser His Asp Ala Thr Ile Tyr Asp Leu Ala Lys Met Ile Thr Glu Lys

145 150 155 160

His Pro Gln Tyr Tyr Ile Pro Lys Thr Phe Glu Gly Ile Asp Glu Lys

165 170 175

Ile Gln Pro Val Arg Phe Ser Ser Lys Lys Leu Leu Glu Leu Gly Phe

180 185 190

Arg Tyr Lys Tyr Ser Met Ala Glu Met Phe Asp Asp Ala Ile Lys Ser

195 200 205

Cys Ile Glu Lys Lys Leu Ile Pro Leu Arg Thr Val Glu Glu Leu Pro

210 215 220

Glu Leu Ile Glu Glu Gln Thr Thr Val Thr Lys Ala Ile Val Asn Arg

225 230 235 240

Ser Glu Glu Lys Val Ser Ile Ala Ile His Ser Asp Leu Tyr Thr His

245 250 255

Asn Thr Ser Leu Gln Glu Met Ser Phe

260 265

<210>3

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<213> ORF Forward Primer sequence (Artificial)

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gagagagaga gatgaagggg c 21

<210>4

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<212>DNA

<213> ORF Reverse Primer sequence (Artificial)

<400>4

gtcataagct tgtattgtgg gtg 23

<210>5

<211>25

<212>DNA

<213> QRT F primer sequence (Artificial)

<400>5

aagaacaaac tactgtgacc aaagc 25

<210>6

<211>23

<212>DNA

<213> QRT primer sequence (Artificial)

<400>6

ccataagctt gtattgtggg tgt 23

<210>7

<211>21

<212>DNA

<213> Recovery F primer sequence (artist)

<400>7

gagagagaga gatgaagggg c 21

<210>8

<211>23

<212>DNA

<213> Recovery R primer sequence (artist)

<400>8

gtcataagct tgtattgtgg gtg 23

Claims (6)

1. A lycoris longituba LlDFRb gene has a nucleotide sequence shown in SEQ ID No. 1.

2. The expression protein of the lycoris longituba LlDFRb gene of claim 1, wherein the amino acid sequence of the expression protein is shown in SEQ ID No. 2.

3. A vector comprising the Lycoris longituba LlDFRb gene of claim 1.

4. The vector of lycoris longituba LlDFRb gene of claim 3, wherein: the vector of the lycoris longituba LlDFRb gene is pEASY-Blunt-LlDFRb, pCAMBIA1300-LlDFRb or pCAMBIA 1304-LlDFRb.

5. Use of the lycoris longituba LlDFRb gene of claim 1, or the expression protein of the lycoris longituba LlDFRb gene of claim 2, or the vector of the lycoris longituba LlDFRb gene of claim 3 or 4 for improving plant flower color.

6. A method for obtaining a new plant variety with changed flower color by using lycoris longituba LlDFRb gene is characterized by comprising the following steps:

1) constructing a vector of the lycoris longituba LlDFRb gene of claim 3 or 4;

2) transforming the constructed lycoris longituba LlDFRb gene vector into plants or plant cells;

3) breeding and screening to obtain new plant variety with changed flower color.

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