CN109706156B - Application of an RNA interference vector FveDDM1-RNAi to promote early flowering of strawberries - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and specifically designs a gene, a vector and an application for advancing the flowering time of strawberry. The forest strawberry FveDDM1 gene was screened from the forest strawberry, and the plant suppression expression vector GN2300-FveDDM1 of the forest strawberry FveDDM1 gene was constructed, and then transfected into the transgenic engineering bacteria, and transferred into the diploid forest strawberry 'Ruegen' to obtain the RNAi of the FveDDM1 gene. Expression interference strains. Under the condition of in vitro leaf tissue culture, the flowering time of strawberry in the FveDDM1 gene-repressed expression line was significantly earlier than that of the wild type, indicating that down-regulation of FveDDM1 gene in forest strawberry could shorten the flowering time of strawberry plants. Genetically transformed forest strawberry transgenic lines with FveDDM1 interference vector The flowering time is significantly earlier. The present invention has great significance for advancing the flowering time of strawberries.

Description

Application of an RNA interference vector FveDDM1-RNAi for promoting early flowering of strawberries

technical field

The invention belongs to the technical field of genetic engineering, relates to a method for regulating the flowering period of strawberries, and in particular relates to the application of an RNA interference carrier FveDDM1-RNAi for promoting early flowering of strawberries.

Background technique

Strawberry is a perennial rose plant with high economic value in the world. Therefore, understanding the developmental regulation mechanism of strawberry can facilitate the development of strawberry breeding and production. Forest strawberry (Fragaria vesca L., 2n=2x=14), also known as wild strawberry, is a perennial herb of the Rosaceae family Strawberry. The diploid forest strawberry (Fragaria vesca) is the closest relative to the cultivated strawberry, and has the advantages of small genome (240Mbp), short regeneration period (about 4 months), and small plant size. It is an ideal model plant for genetic transformation.

Flowering is an important life transformation process of plants, which is a major transition from vegetative growth to reproductive growth. The transition from vegetative to reproductive growth in strawberries is a series of stages starting from flower induction. Flowering is controlled by environmental conditions and developmental regulation, and the complexity of this regulation results from a complex network of signaling pathways. Adjusting the flowering time of plants can regulate plant growth and development, which in turn affects yield. If the plants can be adjusted to the most suitable flowering period with the help of technology, the yield of the plants can be increased.

DNA methylation is one of the hotspots in epigenetic research. DNA methylation refers to the transfer of a methyl group on the donor S-adenosylmethionine to the 5th carbon atom of cytosine under the action of DNA methyltransferase using DNA as the acceptor. , thereby forming 5-methylcytosine (m5C). The level of DNA methylation is not constant, it is regulated by both DNA methylation and DNA demethylation. Cytosine methylation of DNA occurs in CG, CHG and CHH (H=A, C or T) sequences in plants and is a hallmark of epigenetic suppression of repetitive sequences. The chromatin remodeling factor DREREASE IN DNA METHYLATION1 (DDM1), a member of the SWI2/SNF2 protein family, is essential for DNA methylation, especially in CG and CHG sequences. DNA methylation is an important epigenetic mark. Too low or too high methylation levels can affect plant growth, development and differentiation, resulting in abnormal development and morphology.

At present, the regulation of strawberry flowering period mainly focuses on improving cultivation measures, hormone treatment, low temperature and short-day treatment, etc. There are few reports on the regulation of plant flowering by DDM1.

SUMMARY OF THE INVENTION

In the present invention, the RNAi transgenic material of FveDDM1 is obtained by constructing a gene silencing vector for DNA demethylation and using the Agrobacterium transformation method. It was proved that the flowering time was earlier than that of the wild type, and the FveDDM1 gene was verified to be related to strawberry flowering.

The technical scheme of the present invention is as follows:

The first object of the present invention is to provide the application of the forest strawberry FveDDM1 gene in advancing the flowering time of strawberry, and the nucleotide sequence of the forest strawberry FveDDM1 gene is shown in SEQ ID NO.2.

Further, inhibiting the expression of the forest strawberry FveDDM1 gene can advance the flowering time of the strawberry.

Further, the RNAi vector targeting the forest strawberry FveDDM1 gene is introduced into the forest strawberry, and the expression of the forest strawberry FveDDM1 gene is inhibited, which can advance the flowering time of the strawberry.

The second object of the present invention is to provide a kind of construction method of the RNAi carrier of forest strawberry FveDDM1 gene, and described method comprises the following steps:

S1: Design specific primers using the forest strawberry cDNA sequence as a template

FveDDM1-RNAi-F1:5'-ACTctgcaggTCGACTTGGCCCGAACTTCC-3' (SEQ ID NO. 4),

FveDDM1-RNAi-R1:5'-AATAATATGGTCGAcGAGAAGAAAGGGACG-3' (SEQ ID NO. 5), amplified to obtain the forward sequence of the forest strawberry FveDDM1 gene RNA interference fragment (SEQ ID NO. 8), the length is 392bp:

TTGGCCCGAACTTCCCAATAATTGTTACTTCATATGAAGTGGCGTTGGCTGATGCAAGAAGATGTTTAAGACACTACAACTGGAAATATCTCGTGGTTGATGAAGGACACAGATTGAAAAACTCCAAGTGCAAACTAGTGCAGCAGTTGAAGTACATACCTGTAGAGAATAAGATTCTGTTGACTGGAACACCTCTCCAGAATAATTTGGCTGAGCTTTGGTCGTTGTTGAACTTTATTTTGCCGGATATATTCTCATCCCATGAAGAATTTGAGTCGTGGTTTGACCTAGAAGGAAAGTGCCATAATGAAGCAATGAAGGAAGAATTAGAAGAGAAGAGAAGAGCTCAAGTGCTACCGAAACTCCATGCAATATTGCGTCCCTTTCTTCTC；

Design specific primers FveDDM1-RNAi-F2: 5'-TTGCAAAGCTctagaGAGAAGAAAGGGACG-3' (SEQ ID NO. 6), FveDDM1-RNAi-R2: 5'-TGAACGATCtctagATTGGCCCGAACTTCC-3' (SEQ ID NO. 7), amplify to obtain forest strawberry The reverse sequence of the RNA interference fragment of FveDDM1 gene (SEQ ID NO.9), the length is 392bp:

GAGAAGAAAGGGACGCAATATTGCATGGAGTTTCGGTAGCACTTGAGCTCTTCTCTTCTCTTCTAATTCTTCCTTCATTGCTTCATTATGGCACTTTCCTTCTAGGTCAAACCACGACTCAAATTCTTCATGGGATGAGAATATATCCGGCAAAATAAAGTTCAACAACGACCAAAGCTCAGCCAAATTATTCTGGAGAGGTGTTCCAGTCAACAGAATCTTATTCTCTACAGGTATGTACTTCAACTGCTGCACTAGTTTGCACTTGGAGTTTTTCAATCTGTGTCCTTCATCAACCACGAGATATTTCCAGTTGTAGTGTCTTAAACATCTTCTTGCATCAGCCAACGCCACTTCATATGAAGTAACAATTATTGGGAAGTTCGGGCCAA。

S2: The forward sequence of the forest strawberry FveDDM1 gene RNA interference fragment is connected to the GN2300 universal vector through the Sal I restriction site; the reverse sequence of the forest strawberry FveDDM1 gene RNA interference fragment is connected to the GN2300 universal vector through the XbaI restriction site;

S3: Transform the obtained recombinant vector into E. coli competent cell DH5α, extract the positive plasmid, detect by electrophoresis, and verify by sequencing, to obtain a forest containing the forward sequence of the forest strawberry FveDDM1 gene RNA interference fragment - the forest strawberry FveDDM1 gene RNA interference fragment reverse sequence Strawberry FveDDM1 gene RNAi vector GN2300-FveDDM1.

The third object of the present invention is to provide the RNAi vector of the forest strawberry FveDDM1 gene constructed by the aforementioned method.

The fourth object of the present invention is to provide the application of the aforementioned RNAi vector of the forest strawberry FveDDM1 gene in advancing the flowering time of strawberry.

The fifth object of the present invention is to provide a transgenic engineering bacterium transfected with the RNAi vector of the forest strawberry FveDDM1 gene.

The sixth object of the present invention is to provide the application of the aforementioned transgenic engineering bacteria transfected with the RNAi vector of the forest strawberry FveDDM1 gene in advancing the flowering time of strawberries.

The beneficial effect of the present invention is that

(1) The present invention can obtain strawberry plants with earlier flowering time by down-regulating the expression of the Fve DDM1 gene, which provides a new idea for regulating the flowering time of strawberries.

(2) The present invention advances the flowering time of strawberries by transgenic method, and overcomes the shortcoming of low efficiency in the currently commonly used method for regulating flowering time. By means of genetic transformation, a more efficient method for the regulation of strawberry flowering period is provided.

(3) The present invention transfers the Fve DDM1-RNAi vector into strawberry, successfully obtains plants with earlier flowering time, achieves the goal of obtaining early flowering by using RNA interference technology, and provides a reference for plant molecular breeding of RNA interference.

Description of drawings

Figure 1 is a comparison of the flowering (left) of the fifth Ruegen (right) of the 1-2/3 line after the GN2300-FveDDM1-RNAi vector of the forest strawberry FveDDM1 gene was transferred to it and the wild-type Ruegen;

Figure 2 is a comparison of the flowering (left) of the sixth Ruegen (right) of the 1-2/3 line after the GN2300-FveDDM1-RNAi vector of the forest strawberry FveDDM1 gene was transferred to the wild-type Ruegen;

Figure 3 is a comparison of the flowering (left) of the seventh Ruegen (right) of the 1-22/3 line after the GN2300-FveDDM1-RNAi vector of the forest strawberry FveDDM1 gene was transferred to the wild-type Ruegen;

Figure 4 shows the comparison of the flowering of the first Ruegen (right) and wild-type Ruegen (left) of the 1-②2/3 line after the GN2300-FveDDM1-RNAi vector of the forest strawberry FveDDM1 gene was transferred.

Detailed ways

Example 1 Cloning of FveDDM1 gene

According to the full-length CDS of Arabidopsis DDM1 gene published by Tair (https://www.arabidopsis.org/) (the sequence is shown in SEQ ID NO. edu/species/Fragaria_vesca/Fvesca-genome.v2.0.a2/genes/) to download the strawberry gene library, and use Bioedit software to locally compare the CDS of FveDDM1 corresponding to forest strawberry. Similarly, compare the protein sequence (CDS long 2178bp, the sequence is shown in SEQ ID NO.2, and the amino acid sequence encoded by the FveDDM1 gene is shown in SEQ ID NO.3)

The total RNA of wild-type forest strawberry was extracted, reverse transcribed into cDNA, and the CDS of the forest strawberry was obtained from the GDR website (download site is ftp://ftp.bioinfo.wsu.edu/species/Fragaria_vesca/Fvesca-genome.v2. 0.a2/genes/) Using the cDNA of wild-type forest strawberry as a template, PCR amplification of the FveDDM1 gene was performed, and a forward fragment was inserted. Snapgene software was used to design primers SEQ ID NO.4 and SEQ ID NO.5 to amplify the target fragment in the forward direction of the gene. The PCR reaction system is shown in Table 1.

Table 1:

The primer sequences are as follows:

FveDDM1-RNAi-F1:5'-ACTctgcaggTCGACTTGGCCCGAACTTCC-3' (SEQ ID NO. 4),

FveDDM1-RNAi-R1:5'-AATAATATGGTCGAcGAGAAGAAAGGGACG-3' (SEQ ID NO. 5)

The PCR program was set to: 94°C pre-denaturation for 2 min, 98°C denaturation for 10s, 55°C annealing for 30s, extension temperature 68°C, extension time calculated according to the length of the amplified fragment (1kb/min), extension time 25s, set 33 cycles with a final extension at 68°C for 10 min.

Amplify the forward sequence of the forest strawberry FveDDM1 gene RNA interference fragment as shown in SEQ ID NO.8, with a length of 392bp:

Using the cDNA of wild-type forest strawberry as a template, the FveDDM1 gene was amplified by PCR, and the reverse fragment was inserted. The PCR reaction system was the same as in Table 1. Primers SEQ ID NO.6 and SEQ ID NO.7 were designed with Snap gene software to amplify the reverse target fragment of the gene.

The primer sequences are as follows:

FveDDM1-RNAi-F2:5'-TTGCAAAGCTctagaGAGAAGAAAGGGACG-3' (SEQ ID NO. 6),

FveDDM1-RNAi-R2: 5'-TGAACGATCtctagATTGGCCCGAACTTCC-3' (SEQ ID NO. 7)

The PCR procedure is the same as when amplifying the forward target fragment of the gene.

Amplify the reverse sequence of the forest strawberry FveDDM1 gene RNA interference fragment as shown in SEQ ID NO.9, the length is 392bp:

Example 2 Construction of GN2300-FveDDM1-RNAi vector

1. Connection of forward fragments

Using the One Step Cloning Kit (Vazyme) recombinant cloning kit, Sal I was selected as the restriction site on the GN2300-35S-FAQ-NOS vector, and the cloned vector was linearized with restriction endonuclease, and placed at 37°C. In a water bath, the reaction was carried out for 4 hours. The reaction system is shown in Table 2:

Table 2:

After digestion, inactivate at 65°C for 20 minutes for the recombination reaction.

The reaction system was prepared in an ice-water bath, as shown in Table 3:

table 3:

After the above-mentioned mixed reaction solution was prepared, it was placed in a water bath at 37° C. for 30 min. The reaction solution was cooled on ice for 5 min;

2. Concatenation of Reverse Fragments

Select XbaI as the restriction site on the GN2300-35S-FAQ-NOS vector to which the forward fragment has been connected, and connect the reverse fragment. The ligation method is to replace the amplification product of the forward fragment in the reaction system with the amplification product of the reverse fragment. Others are the same as the forward fragment ligation reaction.

3. Conversion reaction

(1) Take out the E. coli competent cells DH5α from the -80°C refrigerator. When the E. coli competent cells are freeze-thawed, add 10 μL of the reaction solution in the second step obtained after forward and reverse ligation, and place on ice for 30 minutes. ;

(2) Put it into a 42°C constant temperature water bath for 90s;

(3) Place in ice water for 2min;

(4) Add 700 μL of LB liquid medium without any antibiotics to the ultra-clean workbench, and place Escherichia coli on a shaker at 37°C at a speed of 200 rpm for 1 h;

(5) 4,000 rpm, centrifuged for 1 min;

(6) Discard the supernatant in the ultra-clean workbench, leaving about 100 μl, resuspend the bacterial block, aspirate the bacterial liquid with a pipette tip, and put it on the LB solid medium containing 50 mg/L kanamycin, and use Spread the sterilized cooled applicator evenly, seal it with parafilm, and write your name. Invert it at 37°C overnight for 12-16 hours;

(7) After the colony is formed, pick a single colony, put it into the LB liquid medium containing Kana, and perform colony PCR verification. After electrophoresis detection, the cloned strains that may be positive were selected, and sent to the company for sequencing. After comparing their gene sequences to be completely correct, the clones containing the forward sequence of the forest strawberry FveDDM1 gene RNA interference fragment and the forest strawberry FveDDM1 gene RNA interference fragment reverse sequence were obtained. Forest strawberry FveDDM1 gene GN2300-FveDDM1-RNAi vector.

(8) Extract the recombinant plasmid of Escherichia coli, transform it into Agrobacterium strain GV3101, preserve the bacterial solution with 50% glycerol, and store it in a -80° refrigerator.

Embodiment 3 transforms strawberry to carry out functional verification

1. Preparation of Explants

The explants were selected from young leaves of the forest strawberry ecotype Ruegen.

Peel off the seeds of Ruegen's fruit with tweezers, put them on filter paper, and after air-drying, pick about 200 plump seeds and put them in a 5 ml centrifuge tube.

Perform the following operations on the ultra-clean workbench: add about 2 ml of 75% alcohol to the centrifuge tube, shake and invert for about 5 minutes and then suck out all the alcohol. Add sterile water to the centrifuge tube and wash 3-5 times to remove excess alcohol. Then add 2 ml of 10% sodium hypochlorite (sodium hypochlorite needs to be protected from light), and after shaking for 8 minutes, suck out the sodium hypochlorite. Add sterile water to the centrifuge tube and wash 5-7 times to remove sodium hypochlorite and avoid residue. After cleaning, pour the seeds onto sterile filter paper, use sterile cooled tweezers to place 4-5 seeds in each tissue culture bottle containing MS solid medium, mark the bottle cap, and place it in the bottle. Dark 28 ℃ constant temperature incubator, after germination, placed in the light to cultivate. The explants were selected from tender leaves with a leaf age of about 15-20 days, consistent growth, and fullness.

2. Agrobacterium infection and transformation

(1) Strain activation: Take out 100 μL of the GV3101 Agrobacterium tumefaciens strain containing the GN2300-FveDDM1-RNAi vector of the forest strawberry FveDDM1 gene in the -80°C refrigerator, streak on the LB solid medium plate, and incubate at 28°C Inverted culture for 1-2 days, after a single colony grows, the picked single colony is placed in LB liquid medium supplemented with 50 mg/L kanamycin and 100 mg/L rifampicin, and the temperature is kept at 28°C. Incubate at 200 rpm in a shaker for 1-2 days, until the color of the bacterial liquid is slightly shaken to light yellow. Aspirate 100-150 μL of the shaken bacterial liquid, add 50 mg/L kanamycin and 100 mg/L rifampicin in LB liquid medium, shake vigorously overnight at 200 rpm on a constant temperature shaker at 28°C, and 200 rpm. The shaken bacterial liquid was centrifuged at 4500rpm for 5min at room temperature, the filtrate was discarded, the bacterial mass was collected and resuspended with sterilized MS suspension (acetosyringone (AS) was added to the MS suspension to 100 μM), and the OD ₆₀₀ value was determined _. Adjusted to about 0.48, and then placed the bacterial liquid in a constant temperature shaker at 28°C, 100rpm, and suspended for 1h.

(2) Infected leaves: During the interstitial period of bacterial suspension, cut young leaves with a consistent leaf age of about 15-20 days from the tissue culture bottle, scratch the back of the strawberry leaves with a blade, and cut all the leaves. Afterwards, the leaves were put into the MS bacterial suspension, placed on a shaker at 28°C, and infected at 100 rpm for 20 minutes. After the infection was completed, the bacterial liquid was poured out, and the residual bacterial liquid on the leaves was blotted with sterile filter paper, and then transferred to the co-cultivation medium, and co-cultured for 3 days in the dark environment. After 15 days of sterilization in low light, the callus-grown explants were moved to the screening medium and cultured in the light at a temperature of 22°C, and the medium was replaced every 2 weeks.

(3) When adventitious buds are produced, the adventitious buds are cut into several small pieces and transferred to a budding medium. After the leaves grow out, transfer them to rooting medium, and when they grow into complete seedlings, slow down the seedlings and transfer them into flowerpots.

The medium formula used in the experiment is as follows (if there is no special note, the pH is adjusted to 5.8):

①1/2MS solid medium: sucrose 20g/L, 1/2MS medium powder 4.302g/L, agar 7g/L,

②LB solid medium: Tryptone 10g/L, Yeast Extract 5g/L, sodium chloride 10g/L, agar powder 10g/L, pH 7.0

③LB liquid medium: Tryptone 10g/L, Yeast Extract 5g/L, Sodium Chloride 10g/L, pH 7.0

④MS solid medium: MS medium powder (including vitamins) 4.406g/L, sucrose 30g/L, agar powder 7g/L

⑤MS-B5 solid medium: MS medium powder (without vitamins) 4.302g/L, sucrose 20g/L, B5 organic solution 5mL/L, agar powder 7g/L

⑥Strawberry co-culture medium: MS-B5 medium+2mg/L TDZ+0.2mg/L IBA+20mg/L AS

⑦Strawberry degerming medium: MS-B5 medium+2mg/LTDZ+0.2mg/L IBA+300mg/L Ti

⑧Strawberry screening medium: MS-B5 medium+3mg/L 6-BA+0.2mg/L IBA+300mg/L Ti+20mg/LKan

⑨Strawberry budding medium: MS-B5 medium+2mg/L 6-BA+0.2mg/L IBA+300mg/L Ti+20mg/LKan

⑩Strawberry rooting medium: MS-B5 medium+0.2mg/L IBA+300mg/L Ti

The hormone preparation method used in the experiment is as follows:

①TDZ (2mg/mL): Weigh 20mg TDZ powder, dissolve it in 200μL of 1M sodium hydroxide solution, add pure alcohol after complete dissolution, dilute to 10ml, and store at -20°C for later use.

②IBA (1 mg/mL): Weigh 10 mg of IBA powder, dilute to 10 mL with sterile distilled water, and store at -20°C for later use.

③AS (20 mg/mL): Weigh 20 mg of AS powder, dilute to 1 mL with DMSO, and store at -20°C for later use.

④Ti (300mg/mL): Weigh 3g Ti, dilute to 10mL with distilled water, and store at -20°C for later use.

⑤Kan (100mg/mL): Weigh 500mg of Kan, dilute to 5mL with distilled water, and store at -20°C for later use.

⑥MS-B5 organic solution: Weigh 0.2 g of niacin, 20 g of inositol, 2 g of vitamin B1, 0.2 g of vitamin B6, dissolve in distilled water and make up to 1 L, and store in a 4°C refrigerator for later use.

⑦ 1M sodium hydroxide solution: Weigh 6.0 g of sodium hydroxide flakes, dissolve in distilled water, dilute to 150 mL, and store at room temperature.

3. GUS staining verification of transgenic plants

GUS staining solution was prepared, the leaves of the transgenic strawberry plants were cut off, and the leaves were stained. If the staining is successful, it indicates that the GUS reporter gene has been successfully expressed in the plant and transferred into the strawberry genome.

4. Observation and Analysis of Transgenic Plants

Phenotype transplanting time number of flowers Statistics Time wild type strawberry September 12, 2018 0 November 22, 2018 Transgenic Line 1 September 12, 2018 4 November 22, 2018 Transgenic Line 2 September 12, 2018 2 November 22, 2018 Transgenic Line 3 September 12, 2018 1 November 22, 2018 Transgenic Line 4 September 12, 2018 1 November 22, 2018

It can be seen from the above results that, compared with wild-type strawberry plants, the flowering time of transgenic strawberry plants is earlier than that of wild-type plants. Under the same culture conditions, the wild-type strawberry plants have not bloomed for 71 days, while the transgenic strawberries transformed with the FveDDM1-RNAi vector have bloomed 71 days after transplantation. The above results show that the introduction of the RNAi vector targeting the FveDDM1 gene of the forest strawberry into the forest strawberry, thereby inhibiting the expression of the forest strawberry FveDDM1 gene, can shorten the flowering time of the strawberry. FveDDM1-RNAi vector can promote early flowering of strawberries. FveDDM1-RNAi vector is a functional vector, which can promote reproductive growth and promote early flowering of plants.

sequence listing

<110> Nanjing Agricultural University

<120> Application of an RNA interference vector FveDDM1-RNAi for promoting early flowering of strawberries

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2770

<212> DNA

<213> Arabidopsis thaliana

<400> 1

aaaaaataag aaaataggcg taaatatgag agtgtgtttt ttcaatatac cctcggtttt 60

gaatttgctc tcaaaagcga cggagacgac tgtttggctc ggtgatttct cccgccgttt 120

gggtttttct taccggaatt tccttctcct tcgatggtta gtctgcgctc cagaaaagtt 180

attccggctt cggaaatggt cagcgacggg aaaacggaga aagatgcgtc tggtgattca 240

cccacttctg ttctcaacga agaggaaaac tgtgaggaga aaagtgttac tgttgtagag 300

gaagagatac ttctagccaa aaatggagat tcttctctta tttctgaagc catggctcag 360

gaggaagagc agctgctcaa acttcgggaa gatgaagaga aagctaacaa tgctggatct 420

gctgttgctc ctaatctgaa tgaaactcag tttactaaac ttgatgagct cttgacgcaa 480

actcagctct actctgagtt tctccttgag aaaatggagg atatcacaat taatgggata 540

gaaagtgaga gccaaaaagc tgagcccgag aagactggtc gtggacgcaa aagaaaggct 600

gcttctcagt acaacaatac taaggctaag agagcggttg ctgctatgat ttcaagatct 660

aaagaagatg gtgagaccat caactcagat ctgacagagg aagaaacagt catcaaactg 720

cagaatgaac tttgtcctct tctcactggt ggacagttaa agtcttatca gcttaaaggt 780

gtcaaatggc taatatcatt gtggcagaat ggtttgaatg gaatattagc tgatcaaatg 840

ggacttggaa agacgattca aacgatcggt ttcttatcac atctgaaagg gaatgggttg 900

gatggtccat atctagtcat tgctccactg tctacacttt caaattggtt caatgagatt 960

gctaggttca cgccttccat caatgcaatc atctaccatg gggataaaaa tcaaagggat 1020

gagctcagga ggaagcacat gcctaaaact gttggtccca agttccctat agttattact 1080

tcttatgagg ttgccatgaa tgatgctaaa agaattctgc ggcactatcc atggaaatat 1140

gttgtgattg atgagggcca caggttgaaa aaccacaagt gtaaattgtt gagggaacta 1200

aaacacttga agatggataa caaacttctg ctgacaggaa cacctctgca aaataatctt 1260

tctgagcttt ggtctttgtt aaattttatt ctgcctgaca tctttacatc acatgatgaa 1320

tttgaatcat ggtttgattt ttctgaaaag aacaaaaacg aagcaaccaa ggaagaagaa 1380

gagaaaagaa gagctcaagt tgtttccaaa cttcatggta tactacgacc attcatcctt 1440

cgaagaatga aatgtgatgt tgagctctca cttccacgga aaaaggagat tataatgtat 1500

gctacaatga ctgatcatca gaaaaagttc caggaacatc tggtgaataa cacgttggaa 1560

gcacatcttg gagagaatgc catccgaggt caaggctgga agggaaagct taacaacctg 1620

gtcattcaac ttcgaaagaa ctgcaaccat cctgaccttc tccaggggca aatagatggt 1680

tcatatctct accctcctgt tgaagagatt gttggacagt gtggtaaatt ccgcttattg 1740

gagagattac ttgttcggtt atttgccaat aatcacaaag tccttatctt ctcccaatgg 1800

acgaaacttt tggacattat ggattactac ttcagtgaga aggggtttga ggtttgcaga 1860

atcgatggca gtgtgaagct ggatgaaagg agaagacaga ttaaagattt cagtgatgag 1920

aagagcagct gtagtatatt tctcctgagt accagagctg gaggactcgg aatcaatctt 1980

actgctgctg atacatgcat cctctatgac agcgactgga accctcaaat ggacttgcaa 2040

gccatggaca gatgccacag aatcgggcag acgaaacctg ttcatgttta taggctttcc 2100

acggctcagt cgatagagac ccgggttctg aaacgagcgt acagtaagct caagctggaa 2160

catgtggtta ttggccaagg gcagtttcat caagaacgtg ccaagtcttc aacaccttta 2220

gaggaagagg acatactggc gttgcttaag gaagatgaaa ctgctgaaga taagttgata 2280

caaaccgata taagcgatgc ggatcttgac aggttacttg accggagtga cctgacaatt 2340

actgcaccgg gagagacaca agctgctgaa gcttttccag tgaagggtcc aggttgggaa 2400

gtggtcctgc ctagttcggg aggaatgctg tcttccctga acagttagga cacattaata 2460

agccaggcct tgaaaccact tctgtgtttt tttttttttt ttccggaaca tgatcggtta 2520

cttttggctg ggaggattta attattagag ggctcggaag tttttgtaag ttaaagaact 2580

cacttaaaac cctgaaaaca tgacagttaa tggtgattag ctctcaatgt gatgaaaaca 2640

attggccctc tgattttgct gttgcggtaa tattatgact tgtgtacgtt tatagtcttt 2700

gtagtctgca attttggcat tgagctattt ctcacgaact tatgggatct tatgttttgg 2760

atttgggatt 2770

<210> 2

<211> 2178

<212> DNA

<213> Forest strawberry (Fragaria vesca)

<400> 2

atggcgacga agaccgagcc ggcggcggat tctcccactt cggttctcga ggaagaggat 60

ttgtgtgggg agattgatgt gaaattggtg aaggcggagg aggagctgct cgaggttcgg 120

gttaaggaag aggagacaga aagggagaag gagacacccg tcttgagtga gactcagttc 180

agcaagttgg atgagcttct cactaagact cagcttttta cagacttttt gctcgagaaa 240

atggatgaca tctcttttga tgttcccgag caactgaatg aacctgaacc tgtgcagaaa 300

aagagaggcc gcggtacgaa aagaaaggct cctacttaca ataatactaa ggccaagagg 360

gcagttgcgg ctatgcttac aagatctaaa gagggtgaga aaattgaaga tgtgaaccta 420

actgaggagg aaagacttga gaaacagcaa aaggaacttg tacctctact gactggtggc 480

aaattgaagt cttatcaact caaaggtgta aagtggttga tctctttatg gcaaaatggg 540

ctcaatggga tccttgcaga ccaaatggga cttggcaaga ctatccagac cataggtttt 600

ctttctcatc taaaatctat gggattggat gggccctact tggtgattgc tcctctttct 660

actctttcca actggattaa tgaaatctca aggtttacgc cttcaattaa ggctataatc 720

tatcatggta acaagaaaga aagggatgag ataataagga agcacatgcc caaatcagtt 780

ggcccgaact tcccaataat tgttacttca tatgaagtgg cgttggctga tgcaagaaga 840

tgtttaagac actacaactg gaaatatctc gtggttgatg aaggacacag attgaaaaac 900

tccaagtgca aactagtgca gcagttgaag tacatacctg tagagaataa gattctgttg 960

actggaacac ctctccagaa taatttggct gagctttggt cgttgttgaa ctttattttg 1020

ccggatatat tctcatccca tgaagaattt gagtcgtggt ttgacctaga aggaaagtgc 1080

cataatgaag caatgaagga agaattagaa gagaagagaa gagctcaagt gctaccgaaa 1140

ctccatgcaa tattgcgtcc ctttcttctc cgaagaatga agatagatgt tgagctgatg 1200

cttccaagaa agaaggaaat catactatat gcaaccatga cagagcatca aaagaagttt 1260

caggaacatc tgatcaataa gacactggag aaacatctaa tacttgaaaa gggaagccat 1320

gtaaatggcc tgaaagggaa gctgaacaat ttgatgatcc aacttcggaa gaactgcaat 1380

catcctgacc ttctagagtc ggcatttgat ggatcatatt tctacccgcc tgttgaccag 1440

atagttgagc aatgtgggaa atttagcttg cttgaaagac tgttgaagct gctgcttgcc 1500

ggcaaacata aggttctgat attctcgcag tggaccaaga ttttggatat aatggattac 1560

tattttagcg aaaaaggata tgaagtttgt agaattgatg gccatgtgaa actggatgat 1620

cggagaagac agattgcttc gttcaatgat ttagacagca cttgtagaat attcctactg 1680

agcacaagag ccggtggact aggtatcaac cttactgcag ctgatacctg tatactgtat 1740

gacagtgatt ggaaccctca aatggatttg caagccatgg atagatgtca caggattggg 1800

caaaccaagc ctgttcatgt ttaccggttg gcaacagcac aatctgtaga gggtcggatg 1860

ttaaaaagag cttttagcaa gttgaagctt gaacatgtag ttattggaaa agggaagttc 1920

catcaggaaa gagccaagcc tgaggcagat ttcttggagg aagaggatct catagcactt 1980

ctccgagatg aagaatctgc tgaagacaag atgatacaga cagatatcac tgatgaagag 2040

ctggagaaag tcttggatcg cagtgatctc attgggactc ctcctgatgc tgctgatgcg 2100

cttcccctga agggacctgg ctgggaagtg gtggttccta ccgctagtgg gggcatgctc 2160

tcctccctta atagttag 2178

<210> 3

<211> 725

<212> PRT

<213> Forest strawberry (Fragaria vesca)

<400> 3

Met Ala Thr Lys Thr Glu Pro Ala Ala Asp Ser Pro Thr Ser Val Leu

1 5 10 15

Glu Glu Glu Asp Leu Cys Gly Glu Ile Asp Val Lys Leu Val Lys Ala

20 25 30

Glu Glu Glu Leu Leu Glu Val Arg Val Lys Glu Glu Glu Thr Glu Arg

35 40 45

Glu Lys Glu Thr Pro Val Leu Ser Glu Thr Gln Phe Ser Lys Leu Asp

50 55 60

Glu Leu Leu Thr Lys Thr Gln Leu Phe Thr Asp Phe Leu Leu Glu Lys

65 70 75 80

Met Asp Asp Ile Ser Phe Asp Val Pro Glu Gln Leu Asn Glu Pro Glu

85 90 95

Pro Val Gln Lys Lys Arg Gly Arg Gly Thr Lys Arg Lys Ala Pro Thr

100 105 110

Tyr Asn Asn Thr Lys Ala Lys Arg Ala Val Ala Ala Met Leu Thr Arg

115 120 125

Ser Lys Glu Gly Glu Lys Ile Glu Asp Val Asn Leu Thr Glu Glu Glu

130 135 140

Arg Leu Glu Lys Gln Gln Lys Glu Leu Val Pro Leu Leu Thr Gly Gly

145 150 155 160

Lys Leu Lys Ser Tyr Gln Leu Lys Gly Val Lys Trp Leu Ile Ser Leu

165 170 175

Trp Gln Asn Gly Leu Asn Gly Ile Leu Ala Asp Gln Met Gly Leu Gly

180 185 190

Lys Thr Ile Gln Thr Ile Gly Phe Leu Ser His Leu Lys Ser Met Gly

195 200 205

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

210 215 220

Trp Ile Asn Glu Ile Ser Arg Phe Thr Pro Ser Ile Lys Ala Ile Ile

225 230 235 240

Tyr His Gly Asn Lys Lys Glu Arg Asp Glu Ile Ile Arg Lys His Met

245 250 255

Pro Lys Ser Val Gly Pro Asn Phe Pro Ile Ile Val Thr Ser Tyr Glu

260 265 270

Val Ala Leu Ala Asp Ala Arg Arg Cys Leu Arg His Tyr Asn Trp Lys

275 280 285

Tyr Leu Val Val Asp Glu Gly His Arg Leu Lys Asn Ser Lys Cys Lys

290 295 300

Leu Val Gln Gln Leu Lys Tyr Ile Pro Val Glu Asn Lys Ile Leu Leu

305 310 315 320

Thr Gly Thr Pro Leu Gln Asn Asn Leu Ala Glu Leu Trp Ser Leu Leu

325 330 335

Asn Phe Ile Leu Pro Asp Ile Phe Ser Ser His Glu Glu Phe Glu Ser

340 345 350

Trp Phe Asp Leu Glu Gly Lys Cys His Asn Glu Ala Met Lys Glu Glu

355 360 365

Leu Glu Glu Lys Arg Arg Ala Gln Val Leu Pro Lys Leu His Ala Ile

370 375 380

Leu Arg Pro Phe Leu Leu Arg Arg Met Lys Ile Asp Val Glu Leu Met

385 390 395 400

Leu Pro Arg Lys Lys Glu Ile Ile Leu Tyr Ala Thr Met Thr Glu His

405 410 415

Gln Lys Lys Phe Gln Glu His Leu Ile Asn Lys Thr Leu Glu Lys His

420 425 430

Leu Ile Leu Glu Lys Gly Ser His Val Asn Gly Leu Lys Gly Lys Leu

435 440 445

Asn Asn Leu Met Ile Gln Leu Arg Lys Asn Cys Asn His Pro Asp Leu

450 455 460

Leu Glu Ser Ala Phe Asp Gly Ser Tyr Phe Tyr Pro Pro Val Asp Gln

465 470 475 480

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

485 490 495

Leu Leu Leu Ala Gly Lys His Lys Val Leu Ile Phe Ser Gln Trp Thr

500 505 510

Lys Ile Leu Asp Ile Met Asp Tyr Tyr Phe Ser Glu Lys Gly Tyr Glu

515 520 525

Val Cys Arg Ile Asp Gly His Val Lys Leu Asp Asp Arg Arg Arg Gln

530 535 540

Ile Ala Ser Phe Asn Asp Leu Asp Ser Thr Cys Arg Ile Phe Leu Leu

545 550 555 560

Ser Thr Arg Ala Gly Gly Leu Gly Ile Asn Leu Thr Ala Ala Asp Thr

565 570 575

Cys Ile Leu Tyr Asp Ser Asp Trp Asn Pro Gln Met Asp Leu Gln Ala

580 585 590

Met Asp Arg Cys His Arg Ile Gly Gln Thr Lys Pro Val His Val Tyr

595 600 605

Arg Leu Ala Thr Ala Gln Ser Val Glu Gly Arg Met Leu Lys Arg Ala

610 615 620

Phe Ser Lys Leu Lys Leu Glu His Val Val Ile Gly Lys Gly Lys Phe

625 630 635 640

His Gln Glu Arg Ala Lys Pro Glu Ala Asp Phe Leu Glu Glu Glu Asp

645 650 655

Leu Ile Ala Leu Leu Arg Asp Glu Glu Ser Ala Glu Asp Lys Met Ile

660 665 670

Gln Thr Asp Ile Thr Asp Glu Glu Leu Glu Lys Val Leu Asp Arg Ser

675 680 685

Asp Leu Ile Gly Thr Pro Pro Asp Ala Ala Asp Ala Leu Pro Leu Lys

690 695 700

Gly Pro Gly Trp Glu Val Val Val Pro Thr Ala Ser Gly Gly Met Leu

705 710 715 720

Ser Ser Leu Asn Ser

725

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 4

actctgcagg tcgacttggc ccgaacttcc 30

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 5

aataatatgg tcgacgagaa gaaagggacg 30

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 6

ttgcaaagct ctagagagaa gaaagggacg 30

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 7

tgaacgatct ctagattggc ccgaacttcc 30

<210> 8

<211> 392

<212> DNA

<213> Artificial Sequence

<400> 8

ttggcccgaa cttcccaata attgttactt catatgaagt ggcgttggct gatgcaagaa 60

gatgtttaag acactacaac tggaaatatc tcgtggttga tgaaggacac agattgaaaa 120

actccaagtg caaactagtg cagcagttga agtacatacc tgtagagaat aagattctgt 180

tgactggaac acctctccag aataatttgg ctgagctttg gtcgttgttg aactttattt 240

tgccggatat attctcatcc catgaagaat ttgagtcgtg gtttgaccta gaaggaaagt 300

gccataatga agcaatgaag gaagaattag aagagaagag aagagctcaa gtgctaccga 360

aactccatgc aatattgcgt ccctttcttc tc 392

<210> 9

<211> 392

<212> DNA

<213> Artificial Sequence

<400> 9

gagaagaaag ggacgcaata ttgcatggag tttcggtagc acttgagctc ttctcttctc 60

ttctaattct tccttcattg cttcattatg gcactttcct tctaggtcaa accacgactc 120

aaattcttca tgggatgaga atatatccgg caaaataaag ttcaacaacg accaaagctc 180

agccaaatta ttctggagag gtgttccagt caacagaatc ttattctcta caggtatgta 240

cttcaactgc tgcactagtt tgcacttgga gtttttcaat ctgtgtcctt catcaaccac 300

gagatatttc cagttgtagt gtcttaaaca tcttcttgca tcagccaacg ccacttcata 360

tgaagtaaca attattggga agttcgggcc aa 392

Claims (2)

1. Application of an RNAi vector for inhibiting expression of FveDDM1 gene of forest strawberry in early flowering time of strawberry, the nucleotide sequence of said forest strawberry FveDDM1 gene is shown in SEQ ID NO.2. 2 . The application of the transgenic engineering bacteria transformed with the RNAi vector that inhibits the expression of the forest strawberry FveDDM1 gene in claim 1 in advancing the flowering time of strawberry. 3 .

Images