CN110734917A - Lycoris longituba LlDFRc genes, expressed protein and application thereof - Google Patents

Abstract

The invention discloses Lycoris longituba LlDFRc genes, expressed proteins and application thereof, and belongs to the field of plant molecular biology, wherein the nucleotide sequence of the LlDFRc 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 LlDFRc gene is an important regulation gene in a downstream path of anthocyanin synthesis, is expressed actively in the early bud period of Lycoris radiata, is released along with flowers, reduces the expression quantity, can deepen the flower color of genetically transformed tobacco, is changed from pink to light red, is DFR genes capable of changing the flower color, and can improve the flower color of plants so as to improve the ornamental value and has practical application value.

Description

Lycoris longituba LlDFRc genes, expressed protein and application thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to lycoris longituba LlDFRc genes, and expressed proteins and application thereof.

Background

The dihydroflavonol 4-reductase (DFR) is an important regulation gene in the downstream path of anthocyanin synthesis, catalyzes dihydroflavonol to generate respectively colorless pelargonium, colorless delphinidin and colorless cyanidin, is an important node for changing anthocyanidin from colorless to colored as a key enzyme in the flavonoid metabolic path, plays a crucial role for the final plant presentation, namely, the accumulation of the anthocyanidin with different colors mainly depends on the activity of a DFR gene, the activity of the modified DFR gene is lost, and the modified DFR gene generates a white chromogene mutant which is a useful regulation gene based on the expression mechanism of the DFR gene.

Disclosure of Invention

In view of the above problems of the prior art, the present invention has been made to solve the problems of Lycoris radiata-derived DFR genes LlDFRc, which are useful for improving flower color and improving ornamental value of plants, and is a technical problem of providing a method of using the LlDFRc gene, which is useful for improving flower color and improving ornamental value of plants.

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

Lycoris longituba LlDFRc genes, the nucleotide sequence of which is shown in SEQ ID NO. 1.

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

A vector comprising the Lycoris longituba LlDFRc gene according to claim 1.

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

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

A method for obtaining new plant varieties with changed flower color by using the Lycoris longituba LlDFRc gene, which comprises the following steps:

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

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

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

The invention has the advantages that compared with the prior art, the Lycoris longituba L1DFRc genes provided by the invention have the nucleotide sequence shown in SEQ ID NO.1 and the amino acid sequence of the expression protein shown in SEQ ID NO.2, the gene expression mode is determined to be active in the early bud period of Lycoris radiata, the expression quantity is reduced along with flower opening through cloning and fluorescence quantification, the gene function is identified through subcellular localization and transgenosis, the expression protein is a structural protein, the flower color of genetically transformed tobacco is deepened, and the flower color is changed from pink to red, so that the LDFRc is DFR genes capable of changing the flower color.

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 LlDFRc 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 LlDFRc in tobacco leaves; 35 s: : GFP: expression of epidermal cells under tobacco injection by agrobacterium GV3101 carrying empty vector pCAMBlAl 300; 35 s: : GFP-LlDFRc: expression profile of epidermal cells in tobacco injected with Agrobacterium GV3101 harboring plasmid pCAMBIA 1300-LlDFRc;

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 lines L11 and L35;

FIG. 6 is a graph showing the results of fluorescent quantitative analysis of the LlDFRc expression of the transgenic lines L11 and L35.

Detailed Description

The present invention is further described in with reference to the following examples the experimental methods of molecular biology not specifically described in the following examples can be performed according to the methods listed in molecular cloning guidelines (third edition) J. SammBruker or the 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 LlDFRc Gene

(1) Cloning of LlDFRc 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 Premier 5.0 software design 1 pair of specific primers (F: 5'-CAGTAATTCTACGTGCAGCTTC-3'; R: 5'-GTATTAAGATCTAAGTAGGATGGAGG-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: mu.L of 1st Strand cDNA, 2. mu.L of ORF Forward Primer (10mM), 2. mu.L of LORF Reverse Primer (10mM), 25. mu.L of 2 × Easypfu PCR Supermix, 20. mu.L of ddH₂O. reaction conditions comprise pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 20s, annealing at different temperatures for 20s, extension at 72 ℃ for 1min, 35 cycles, total extension at 72 ℃ for 10min and termination at 16 ℃, the obtained product is used for agarose electrophoresis detection, gel cutting and recovery of an electrophoresis band with an expected size of , pEASY-Blunt vector is connected and transformed into escherichia coli Trans1-T1 competent cells, 3 positive clones are selected and sent to Beijing Kingsry Biotechnology Limited company for sequencing, the nucleotide sequence of the LlDFRc gene sequence is determined to be shown as SEQ ID No.1 in a 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 is completed, 10 μ L of 5 × Loading Buffer is added, and all the 5 × Loading buffers are spotted into the glue 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 mu LWB (Wash buffer), centrifuging at 10000rpm for 1min, and discarding the effluent;

5) repeat the above steps;

6) centrifuging at 10000rpm for 2min, removing residual WB, placing the centrifugal column in new sterile 1.5 μ L centrifugal tubes, uncapping, and standing at room temperature for 3 min;

7) dripping 40 μ L ddH into the center of the centrifugal column₂Standing at room temperature for 2min, and centrifuging at 10000rpm for 1 min; 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 LlDFRc gene expression pattern

The non-conserved region was designed with fluorescent quantitative primers based on the full-length cDNA sequence of LlDFRc as follows:

F：5′-GAAGTGATAAAGCCAGCAATAGACG-3′；

R：5′-GATGATGTGAAAATAACTCGCTGG-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 toPremix Ex Taq^TMThe specification prepares different components according to the proportion of a reaction system. The total volume of the reaction system is about 10 mu L, and the forward and reverse primers are 0.4 mu L, SYBR5 mu L, cDNA1. mu.L, 0.2. mu.L of a calibration solution, and 3. mu.L of ultrapure water. 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 SPSS 20.0 software.

As shown in FIG. 2, the expression pattern of the LlDFRc gene is in a down-regulation trend in 3 different development periods of the lycoris radiata l petals, the LlDFRc gene is actively expressed in the early bud period and then is reduced along with the flower opening, and the expression quantity is presumed to be reduced, wherein the LlDFRc gene is involved in anthocyanin synthesis in the early bud period and is weakly expressed in the middle and later periods, and the trend is caused by rule that the flower color of the lycoris radiata is deeper in the early bud period and fades in the middle and later periods.

Example 2: LlDFRc vector construction and function verification

I. Subcellular localization observation of LlDFRc gene expression

(1) Cloning, recovering and sequencing target fragment

The complete ORF of the LlDFRc 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′-CAGTAATTCTACGTGCAGCTTC-3′；

R：5′-GTATTAAGATCTAAGTAGGATGGAGG-3′。

PCR amplification was performed using PrimeStarMax (TaKaRa) high fidelity enzyme using the sequenced pEASY-Blunt-LlDFRc 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 ddH₂O. reaction conditions comprise pre-denaturation at 98 ℃ for 1min, denaturation at 98 ℃ for 10s, annealing for 5s, extension at 72 ℃ for 15s, 35 cycles, total extension at 72 ℃ for 1min, termination of reaction at 4 ℃, and then performing second round of PCR enrichment on target fragments by taking products of the th round as templates according to the reaction system and the procedures, wherein the obtained products are used for agarose electrophoresis, and recovery, cloning and sequencing of the target fragments.

(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 anhydrous ethanol) into CP4, centrifuging at 12000rpm for 1min, discarding 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 new 1.5mL centrifuge tubes, 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 is subjected to plasmid duplication according to the following reaction systemEnzyme digestion (50 μ L): 10 μ L of LPLAStic DNA, 1 μ L of Kpn I, 1 μ L of Sma I, 5 μ L of 10 XQuickcut Buffer (Takara), 33 μ L of ddH₂O。

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

(4) Recombination reactions

The PCR product of the LlDFRc gene PCR fragment inserted into pEASY-Blunt vector and the pCAMBIA1300 vector after enzyme digestion are recombined to obtain pCAMBIA1300-LlDFRc, and the following recombination reaction system is configured in 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 Transl 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. LLB broth (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: 1 μ L of bacterial suspension, 10 μ L of 2 XTAQA Mix, 1 μ L of 1300Reverseprimer (10mM), 1 μ L of 1300Forward Primer (10mM), 7 μ 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, and extension at 72 ℃ for 2min, 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 amplification using the plasmid pEASY-Blunt-LlDFRc, which was sequenced correctly, as a template by the above-described specific method. Then the LlDFRc target fragment is inserted into a pCAMBIA1300 vector containing a reporter gene GFP by double digestion, and then is transferred into a Transl competent cell, and a large amount of recombinant plasmids are obtained through Transs 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 OD₆₀₀The bacterial liquid is heated at 4 deg.C and 5000 r.min to 0.6-1.0^-1The cells were collected by centrifugation for 5min, and the cells were washed with a buffer (containing 10 mM. 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 the 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 tobacco injected with plasmid pCAMBIA 1300-target fragment-agrobacterium GV3101 under a laser confocal microscope, respectively observing 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 lycoris longituba DFRc protein by taking the expression condition of the epidermal cells under the agrobacterium 3101 tobacco carrying plasmid no-load pCAMGVB 1Al300 as a control.

As shown in FIG. 3, the results of the experiments showed that the LlDFRc-GFP protein was a typical structural protein mainly localized in the nucleus, cell membrane and cytoplasm of the epidermal cell of Nicotiana benthamiana, as observed by confocal laser microscopy after injecting LlDFRc-GFP into the 5 th to 8 th tobacco leaves grown from Nicotiana benthamiana and culturing the leaf in an incubator for 2 days (FIG. 3).

II. Identification and phenotypic analysis of transgenic tobacco positive lines

Cloning the full-length coding region sequence of the LlDFRc 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-LlDFRc vector, transferring an expression vector containing a target gene into agrobacterium tumefaciens EHA105 by a freeze-thaw method, and transforming tobacco by a leaf disc method.

The extraction and preservation of anthocyanin are carried out by fully grinding tobacco corolla material preserved at minus 80 ℃ in liquid nitrogen, weighing dry powder 50mg, adding 1.5mL of 1% hydrochloric acid methanol extract, fully oscillating for 1min, extracting at 4 ℃ in dark place, oscillating for times every 8h, extracting for 24h, 4 ℃, 10000rpm, centrifuging for 10min, sucking supernatant, filtering with 0.22 μm filter membrane, storing in 1.5mL brown chromatographic bottle, and preserving at minus 20 ℃.

And (3) determining the content of anthocyanin:

weighing cyanidin-3-O-glucoside standard, 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 obtain 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, R²The concentration ug/mL is 0.9994, and the product is used for the quantitative analysis of anthocyanin.

In order to verify the function of the LlDFRc gene, the gene is transferred into tobacco through agrobacterium transformation, two transgenic strains L11 and L35 are selected from obtained positive plants, wild type tobacco is used as a control, phenotype analysis is carried out on the transgenic plants, the result is shown in figure 4, the color of the flower crown of the transgenic plants in the bud period is obviously reddened, the flower color is red from the bud period to the full bloom period, the flower crown of the wild type tobacco in the bud period is greenish, and the full bloom period is pink; the anthocyanin is also accumulated in the filaments of the transgenic plants, and the transgenic plants are obviously light red compared with wild plants. The result of anthocyanin content measurement also shows that the anthocyanin content in the corolla of the transgenic plant is obviously higher than that of the control (figure 5), and the average anthocyanin content in the corolla of 2 strains L11 and L35 is 6.9 times and 10.1 times of that of the control respectively.

The expression of LlDFRc in transgenic plants was confirmed by fluorescence quantification using wild-type tobacco plants as controls. LlDFRc expression analysis is carried out on transgenic strains, and the difference of expression levels also exists between L11 and L35 strains, the expression level of LlDFRc in the L35 strain is higher (figure 6), which is mutually verified with the result that anthocyanin has higher accumulation in the L35 strain, which shows that positive strains obtained by genetic transformation of tobacco by over-expressing LlDFRc gene also have weak gene expression, but anthocyanin is obviously accumulated compared with wild type. The above results indicate that LlDFRc significantly promotes the accumulation of anthocyanin in tobacco flowers.

Sequence listing

<110> Nanjing university of forestry

<120> lycoris longituba LlDFRc genes, expressed protein and application thereof

<130>100

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>1104

<212>DNA

<213>Lycoris longituba

<400>1

atgaaggggc ctgtggcagt gacgggagct ggtggttacg tcggttcatg gcttgtgatg 60

aagctcctcc aagacggtta caccgtcagg gccactctca gagacccatc aaacatgaag 120

aagagcaagc ccctgttaaa ccttccggga gcagaaaaga atctaaccct ctggaaagca 180

gatctcaatg aagaaggaag cttcgatgaa gccattaatg gatgcactgg ggttttccat 240

gttgcaactc caatggattt tgaatctaaa gatccagaga ttgaagtgat aaagccagca 300

atagacggag tgttaagcat tatgagatca tgtaagaaag caaggtcagt ccagcgagtt 360

attttcacat catctgcagg aactgtgaat gtggaggaac atcaaaagcc tgtatacgat 420

gaaaactcat ggagtgatgt cgagttctgc agacgcataa aaatgactgg atggatgtat 480

tttgtatcaa aatcgcttgc agagaaagct gcatgggctt ttgcaagaga aaatggcatt 540

gacctaatta ccatcatacc aacactggtg gtgggtccct tcatcacctc aaccatgcca 600

ccaagtatga tcactgcatt atcattgatc acaggaaatg aagctcacta ttcgataata 660

aagcaagctc agcttgttca cttagatgac ctgtgtgatg cccacattct actattgagc 720

catcctaaag cacaaggaag atacatatgt tcttctcatg atgctaccat ttatgattta 780

gcaaagatga tcacagaaaa gcatcctcag tattacattc ccaaaacatt tgaagggatc 840

gatgagaaaa ttcagcctgt gcgcttctct tcaaagaagc tcttggaact tggtttcagg 900

tacaagtaca gtatggctga aatgtttgat gatgccataa aatcatgcat tgagaagaag 960

ctcatacctc tccgaacagt ggaagaactt cctgaattga ttgaagaaca aactactgtg 1020

accaaagcta ttgttaatag gtcagaggag aaagtttcca ttgcaataca ctcggatttg 1080

tacacccaca atacaagctt atga 1104

<210>2

<211>367

<212>PRT

<213>Lycoris longituba

<400>2

Met Lys Gly Pro Val Ala Val Thr Gly Ala Gly Gly Tyr Val Gly Ser

1 5 10 15

Trp Leu Val Met Lys Leu Leu Gln Asp Gly Tyr Thr Val Arg Ala Thr

20 2530

Leu Arg Asp Pro Ser Asn Met Lys Lys Ser Lys Pro Leu Leu Asn Leu

35 40 45

Pro Gly Ala Glu Lys Asn Leu Thr Leu Trp Lys Ala Asp Leu Asn Glu

50 55 60

Glu Gly Ser Phe Asp Glu Ala Ile Asn Gly Cys Thr Gly Val Phe His

65 70 75 80

Val Ala Thr Pro Met Asp Phe Glu Ser Lys Asp Pro Glu Ile Glu Val

85 90 95

Ile Lys Pro Ala Ile Asp Gly Val Leu Ser Ile Met Arg Ser Cys Lys

100 105 110

Lys Ala Arg Ser Val Gln Arg Val Ile Phe Thr Ser Ser Ala Gly Thr

115 120 125

Val Asn Val Glu Glu His Gln Lys Pro Val Tyr Asp Glu Asn Ser Trp

130 135 140

Ser Asp Val Glu Phe Cys Arg Arg Ile Lys Met Thr Gly Trp Met Tyr

145 150 155 160

Phe Val Ser Lys Ser Leu Ala Glu Lys Ala Ala Trp Ala Phe Ala Arg

165 170 175

Glu Asn Gly Ile Asp Leu Ile Thr Ile Ile Pro Thr Leu Val Val Gly

180 185 190

Pro Phe Ile Thr Ser Thr Met Pro Pro Ser Met Ile Thr Ala Leu Ser

195 200 205

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

210 215 220

Leu Val His Leu Asp Asp Leu Cys Asp Ala His Ile Leu Leu Leu Ser

225 230 235 240

His Pro Lys Ala Gln Gly Arg Tyr Ile Cys Ser Ser His Asp Ala Thr

245 250 255

Ile Tyr Asp Leu Ala Lys Met Ile Thr Glu Lys His Pro Gln Tyr Tyr

260 265 270

Ile Pro Lys Thr Phe Glu Gly Ile Asp Glu Lys Ile Gln Pro Val Arg

275 280 285

Phe Ser Ser Lys Lys Leu Leu Glu Leu Gly Phe Arg Tyr Lys Tyr Ser

290 295 300

Met Ala Glu Met Phe Asp Asp Ala Ile Lys Ser Cys Ile Glu Lys Lys

305 310 315 320

Leu Ile Pro Leu Arg Thr Val Glu Glu Leu Pro Glu Leu Ile Glu Glu

325 330 335

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

340 345 350

Ser Ile Ala Ile His Ser Asp Leu Tyr Thr His Asn Thr Ser Leu

355 360 365

<210>3

<211>22

<212>DNA

<213> ORF Forward Primer sequence (Artificial)

<400>3

cagtaattct acgtgcagct tc 22

<210>4

<211>26

<212>DNA

<213> ORF Reverse Primer sequence (Artificial)

<400>4

gtattaagat ctaagtagga tggagg 26

<210>5

<211>25

<212>DNA

<213> QRT F primer sequence (Artificial)

<400>5

gaagtgataa agccagcaat agacg 25

<210>6

<211>24

<212>DNA

<213> QRT F primer sequence (Artificial)

<400>6

gatgatgtga aaataactcg ctgg 24

<210>7

<211>22

<212>DNA

<213> Recovery F primer sequence (artist)

<400>7

cagtaattct acgtgcagct tc 22

<210>8

<211>26

<212>DNA

<213> Recovery R primer sequence (artist)

<400>8

gtattaagat ctaagtagga tggagg 26

Claims (6)

1, kinds of lycoris longituba LlDFRc gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

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

3. A vector comprising the Lycoris longituba LlDFRc gene according to claim 1.

4. The vector for Lycoris longiradiata LlDFRc gene according to claim 3, wherein: the vector of the lycoris longituba LlDFRc gene is pEASY-Blunt-LlDFRc, pCAMBIA1300-LlDFRc or pCAMBIA 1304-LlDFRc.

5. Use of the lycoris longituba ldfrc gene of claim 1, the expression protein of the lycoris longituba ldfrc gene of claim 2, or the vector of the lycoris longituba ldfrc gene of any in claims 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 LlDFRc gene, which is characterized by comprising the following steps:

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

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

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

Images