CN109706156B - Application of RNA interference vector FveDDM1-RNAi for promoting early flowering of strawberry - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly designs a gene and a vector for advancing flowering time of strawberries and application thereof. Screening forest strawberry FveDDM1 genes from forest strawberries, constructing a plant suppression expression vector GN2300-FveDDM1 of the forest strawberry FveDDM1 genes, further transfecting transgenic engineering bacteria, and transferring the transgenic engineering bacteria into diploid forest strawberries 'Ruegen' to obtain RNAi expression interference strains of the FveDDM1 genes. Under the condition of tissue culture of the in vitro leaves, the flowering time of the strawberry of the FveDDM1 gene suppression expression system is obviously earlier than that of the wild type, which indicates that the flowering time of strawberry plants can be advanced by reducing the FveDDM1 gene of the forest strawberries, and the flowering time of a forest strawberry transgenic line of a genetic transformation FveDDM1 interference vector is obviously earlier. The method has important significance for advancing the flowering time of the strawberries.

Description

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

Technical Field

The invention belongs to the technical field of genetic engineering, relates to a regulation and control method of strawberry flowering phase, and particularly relates to application of an RNA interference vector FveDDM1-RNAi for promoting early flowering of strawberries.

Background

Strawberry is a perennial rose plant and has a high economic value in the world. Therefore, understanding the developmental regulation mechanism of strawberry can promote the development of strawberry breeding and production. Forest strawberries (Fragaria viscosa l.,2n =2x = 14), also called wild strawberries, are perennial herbaceous plants of the rosaceous genus, rosaceous, belonging to diploid, short and small plants, growing in half-horizontal clusters. The diploid forest strawberry (Fragaria viscosa) has the closest relationship with the cultivated strawberry, has the advantages of small genome (240 Mbp), short regeneration period (about 4 months), small plant and the like, and is an ideal model plant suitable for genetic transformation.

Flowering is an important life transformation process for plants, a major transformation from vegetative to reproductive growth. The transition from vegetative to reproductive growth of strawberries is a series of stages beginning with flower induction. Flowering is controlled by environmental conditions and developmental regulation, the complexity of which is caused by a complex network of signalling pathways. The growth and development of the plants can be regulated and controlled by regulating the flowering time of the plants, so that the yield is influenced. The yield of plants can be increased if the plants can be adjusted to the most suitable flowering stage by means of techniques.

DNA methylation (DNA methylation) is one of the hot spots in epigenetic studies at present. DNA methylation refers to the process of transferring 1 methyl group on a donor S-adenosylmethionine to the 5 th carbon atom of cytosine by the action of DNA methyltransferase using DNA as an acceptor, thereby forming 5-methylcytosine (m 5C). The level of DNA methylation is not constant and 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 (DDM 1) is a member of the SWI2/SNF2 protein family and is critical for DNA METHYLATION, particularly IN the CG and CHG sequences. DNA methylation is an important epigenetic marker, and the low or high methylation level can affect the growth, development and differentiation of plants, and cause abnormal development and morphology.

At present, the regulation and control of the flowering period of the strawberries mainly focus on the aspects of improvement of cultivation measures, hormone treatment, low-temperature and short-day treatment and the like, and reports about the regulation and control of plant flowering by DDM1 are newly found.

Disclosure of Invention

The invention obtains the RNAi transgenic material of the FveDDM1 by constructing a gene silencing vector of DNA demethylation and using an agrobacterium transformation method. The flowering time is proved to be earlier than that of the wild type, and the correlation between the FveDDM1 gene and strawberry flowering is verified.

The technical scheme of the invention is as follows:

the first purpose of the invention is to provide application of a forest strawberry FVEDDM1 gene in advancing flowering time of strawberries, wherein the nucleotide sequence of the forest strawberry FVEDDM1 gene is shown as SEQ ID NO. 2.

Furthermore, the expression of the forest strawberry FVEDDM1 gene is inhibited, so that the flowering time of the strawberries can be advanced.

Furthermore, an RNAi vector aiming at the forest strawberry FveDDM1 gene is introduced into the forest strawberry, so that the expression of the forest strawberry FveDDM1 gene is inhibited, and the flowering time of the strawberry can be advanced.

The second purpose of the invention is to provide a method for constructing an RNAi vector of a forest strawberry FVEDDM1 gene, which comprises the following steps:

s1: designing specific primer by taking forest strawberry cDNA sequence as template

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

FveDDM1-RNAi-R1:5 'AATAATATATGGTCGAcGAcGAAGAAAAGGGACG-3' (SEQ ID NO. 5), and the forward sequence (SEQ ID NO. 8) of the RNA interference fragment of the forest strawberry FveDDM1 gene is obtained through amplification, and the length is 392bp:

TTGGCCCGAACTTCCCAATAATTGTTACTTCATATGAAGTGGCGTTGGCTGATGCAAGAAGATGTTTAAGACACTACAACTGGAAATATCTCGTGGTTGATGAAGGACACAGATTGAAAAACTCCAAGTGCAAACTAGTGCAGCAGTTGAAGTACATACCTGTAGAGAATAAGATTCTGTTGACTGGAACACCTCTCCAGAATAATTTGGCTGAGCTTTGGTCGTTGTTGAACTTTATTTTGCCGGATATATTCTCATCCCATGAAGAATTTGAGTCGTGGTTTGACCTAGAAGGAAAGTGCCATAATGAAGCAATGAAGGAAGAATTAGAAGAGAAGAGAAGAGCTCAAGTGCTACCGAAACTCCATGCAATATTGCGTCCCTTTCTTCTC；

designing a specific primer of FveDDM1-RNAi-F2: 5-:

GAGAAGAAAGGGACGCAATATTGCATGGAGTTTCGGTAGCACTTGAGCTCTTCTCTTCTCTTCTAATTCTTCCTTCATTGCTTCATTATGGCACTTTCCTTCTAGGTCAAACCACGACTCAAATTCTTCATGGGATGAGAATATATCCGGCAAAATAAAGTTCAACAACGACCAAAGCTCAGCCAAATTATTCTGGAGAGGTGTTCCAGTCAACAGAATCTTATTCTCTACAGGTATGTACTTCAACTGCTGCACTAGTTTGCACTTGGAGTTTTTCAATCTGTGTCCTTCATCAACCACGAGATATTTCCAGTTGTAGTGTCTTAAACATCTTCTTGCATCAGCCAACGCCACTTCATATGAAGTAACAATTATTGGGAAGTTCGGGCCAA。

s2: connecting the forward sequence of RNA interference fragment of forest strawberry FveDDM1 gene to GN2300 universal vector through Sal I enzyme cutting site; the reverse sequence of the forest strawberry FveDDM1 gene RNA interference fragment is connected to a GN2300 universal vector through an XbaI enzyme cutting site;

s3: and transforming the obtained recombinant vector into an escherichia coli competent cell DH5 alpha, extracting positive plasmids, carrying out electrophoresis detection and sequencing verification to obtain an RNAi vector GN2300-FVEDDM1 of the forest strawberry FVEDDM1 gene containing a forest strawberry FVEDDM1 gene RNA interference fragment forward sequence-forest strawberry FVEDDM1 gene RNA interference fragment reverse sequence.

The third purpose of the invention is to provide an RNAi vector of the forest strawberry FveDDM1 gene constructed by the method.

The fourth purpose of the invention is to provide the application of the RNAi vector of the forest strawberry FVEDDM1 gene in advancing the flowering time of the strawberries.

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

The sixth purpose of the invention is to provide the application of the transgenic engineering bacteria of the RNAi vector transfected with the forest strawberry FVEDDM1 gene in advancing the flowering time of the strawberries.

The invention has the beneficial effects that

(1) According to the invention, the strawberry plant with early flowering time can be obtained by down-regulating the expression of the FVe DDM1 gene, so that a new idea is provided for regulating and controlling the flowering time of the strawberry.

(2) The invention advances the flowering time of the strawberries by a transgenic mode and overcomes the defect of low efficiency in the commonly used method for regulating and controlling the flowering time at present. Through a genetic transformation mode, a more efficient method is provided for regulating and controlling the flowering phase of the strawberries.

(3) The invention transfers the FVe DDM1-RNAi vector into the strawberry, successfully obtains the plant with earlier flowering time, realizes the aim of obtaining early flowering by using an RNA interference technology, and provides reference for RNA interference plant molecular breeding.

Drawings

FIG. 1 is a comparison of flowering (left) of the fifth Ruegen (right) of the 2/3 line of 1- (2) after the GN2300-FveDDM1-RNAi vector transferred into the forest strawberry FveDDM1 gene with the wild type Ruegen;

FIG. 2 shows the comparison of the flowering (left) of the sixth Ruegen (right) of line 1- (2) and 2/3 after the GN2300-FveDDM1-RNAi vector transferred into the forest strawberry FveDDM1 gene with the wild type Ruegen;

FIG. 3 is a comparison of flowering (left) of the seventh Ruegen (right) of 2/3 line 1- (2) after the GN2300-FveDDM1-RNAi vector transferred into the forest strawberry FveDDM1 gene with the wild type Ruegen;

FIG. 4 shows comparison of flowering (left) of the first Ruegen of the 2/3 line 1- (2) after the GN2300-FveDDM1-RNAi vector transferred into the forest strawberry FveDDM1 gene with that of the wild type Ruegen.

Detailed Description

EXAMPLE 1 cloning of the FveDDM1 Gene

According to the full-length CDS (shown in SEQ ID NO. 1) of Arabidopsis thaliana DDM1 gene published by Tair (https:// www.arabidopsis.org /), a strawberry gene library is downloaded on a GDR webpage (ftp:// ftp. Bioinfo.wsu.edu/species/Fragaria _ visco/Fvesca-gene.v 2.0.a2/genes /), the CDS of the FVEDDM1 corresponding to forest strawberry is locally aligned by using Bioedit software, and similarly, a protein sequence is aligned (the CDS is 2178bp in length, and is shown in SEQ ID NO.2, and the amino acid sequence encoded by the FVEDDM1 gene is shown in SEQ ID NO. 3)

Extracting total RNA of wild forest strawberry, reversely transcribing the total RNA into cDNA, acquiring the CDS of the forest strawberry from a GDR website (the download website address is ftp:// ftp. Bioinfo. Wsu. Edu/species/Fragaria _ visco/Fvesca-gene. V2.0.a2/genes /) and carrying out PCR amplification on the FveDDM1 gene by taking the cDNA of the wild forest strawberry as a template, and inserting a forward fragment. Primers SEQ ID NO.4 and SEQ ID NO.5 are designed by Snapgene software to amplify a gene forward target fragment, and a 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: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension temperature for 68 ℃, extension time calculated according to the length of the amplified fragment (1 kb/min), extension time for 25s, set for 33 cycles, and finally extension at 68 ℃ for 10min.

Amplifying to obtain a forward sequence of the RNA interference fragment of the forest strawberry FveDDM1 gene shown as SEQ ID NO.8, wherein the length of the forward sequence is 392bp:

and (3) carrying out PCR amplification on the FveDDM1 gene by taking cDNA of wild forest strawberries as a template, and inserting a reverse fragment. The PCR reaction system was the same as in Table 1. Snap gene software is used to design primers SEQ ID NO.6 and SEQ ID NO.7 for amplification of the gene reverse target fragment.

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 was the same as when the gene was amplified forward to the desired fragment.

Amplifying to obtain a reverse sequence of the forest strawberry FveDDM1 gene RNA interference fragment shown as SEQ ID NO.9, wherein the length is 392bp:

example 2 construction of GN2300-FveDDM1-RNAi vector

1. Ligation of Forward fragments

Using One Step Cloning Kit (Vazyme) recombinant Cloning Kit, sal I was selected as an enzyme cleavage site on GN2300-35S-FAQ-NOS vector, the Cloning vector was linearized with restriction enzymes, placed in a 37 ℃ water bath, and reacted for 4 hours. The reaction system is shown in table 2:

table 2:

after enzyme digestion, the mixture is inactivated at 65 ℃ for 20 minutes and used for recombination reaction.

The reaction system was formulated in an ice-water bath as shown in table 3:

table 3:

the mixed reaction solution is prepared and then placed in a water bath kettle at 37 ℃ for water bath for 30min. Cooling the reaction solution on ice for 5min;

2. ligation of reverse fragments

XbaI is selected as an enzyme cutting site on a GN2300-35S-FAQ-NOS vector connected with a forward segment, and a reverse segment is connected.

3. Conversion reaction

(1) Taking out Escherichia coli competent cell DH5 alpha from a refrigerator at-80 ℃, adding 10 mu L of reaction liquid obtained in the step 2 after forward and reverse connection when the Escherichia coli competence is frozen and thawed, and placing on ice for 30min;

(2) Putting into a constant temperature water bath kettle at 42 ℃ for 90s;

(3) Standing in ice water for 2min;

(4) Adding 700 μ L LB liquid medium without any antibiotic on a clean bench, and shaking the Escherichia coli on a shaker at 37 deg.C at 200rpm for 1h;

(5) 4,000 revolutions per minute, and centrifuging for 1min;

(6) The supernatant was discarded in an ultraclean bench, leaving about 100. Mu.l, the pellet was resuspended, aspirated with a pipette tip, blotted onto LB solid medium containing 50mg/L kanamycin, spread evenly with a cooled sterilized spreading stick, sealed with a sealing film, and the name written. Culturing the culture medium in an inverted state at 37 deg.C overnight for 12-16 hr;

(7) After the colony is formed, picking a single colony, putting the single colony into an LB liquid culture medium containing Kana, and carrying out colony PCR verification. And (3) selecting clone strains which are possibly positive after electrophoresis detection, sending the clone strains to a company for sequencing, and comparing the gene sequences of the clone strains to be completely correct to obtain the GN2300-FVEDDM1-RNAi vector of the forest strawberry FVEDDM1 gene, which contains the forward sequence of the forest strawberry FVEDDM1 gene RNA interference fragment-the forest strawberry FVEDDM1 gene RNA interference fragment reverse sequence.

(8) Extracting recombinant plasmid of Escherichia coli, transforming into GV3101 Agrobacterium strain, preserving the strain with 50% glycerol, and preserving in-80 deg.C refrigerator.

Example 3 transformation of strawberries for functional validation

1. Preparation of explants

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

Seeds of Ruegen fruits were peeled off with tweezers, placed on filter paper, and after air-drying, approximately 200 filled seeds were picked and placed in a 5ml centrifuge tube.

The following operations are performed on an ultra-clean bench: add about 2 ml of 75% alcohol to the centrifuge tube, shake and invert for about 5 minutes and aspirate all alcohol. Sterile water was added to the centrifuge tube and washed 3-5 times to remove excess alcohol. Then 2 ml of 10% sodium hypochlorite (sodium hypochlorite needs to be kept away from light) is added, and the sodium hypochlorite is sucked out after shaking for 8 minutes. Sterile water is added into the centrifuge tube, and the centrifuge tube is washed for 5-7 times to remove sodium hypochlorite and avoid residues. After the washing, the seeds are poured on sterile filter paper, 4-5 seeds are placed in each tissue culture bottle filled with MS solid culture medium by using sterile cooled tweezers, the bottle cover is marked, the tissue culture bottles are placed in a dark constant-temperature incubator at 28 ℃, and after germination, the tissue culture bottles are placed in the light for culture. The explant selects tender leaves with the leaf age of about 15-20 days, consistent growth vigor and plump.

2. Transformation by infection with Agrobacterium

(1) Activating strains: 100 mu L of GV3101 Agrobacterium tumefaciens strain containing GN2300-FveDDM1-RNAi vector of forest strawberry FveDDM1 gene is taken out from a refrigerator at minus 80 ℃, streaked on an LB solid culture medium plate, inversely cultured in a thermostat at 28 ℃ for 1-2 days, after the single bacterium grows out, the picked single bacterium colony is placed in an LB liquid culture medium added with 50mg/L kanamycin and 100mg/L rifampicin, cultured in a constant temperature shaker at 28 ℃ for 1-2 days at 200rpm, and the color of the bacterium liquid is slightly shaken to light yellow. 100-150. Mu.L of the shake-down bacteria solution was aspirated, and 50mg/L kanamycin and 100mg/L rifampicin in LB liquid medium were added thereto, and shaken overnight at 200rpm on a constant temperature shaker at 28 ℃ and 200rpm. Centrifuging the shaken bacterial solution at 4500rpm at room temperature for 5min, discarding the filtrate, collecting the bacterial mass, resuspending with sterilized MS suspension (prepared by adding Acetosyringone (AS) to 100 μ M in MS suspension), and determining OD ₆₀₀ Value, OD ₆₀₀ After adjusting to about 0.48, the bacterial solution was suspended for 1h on a constant temperature shaker at 28 ℃ and 100 rpm.

(2) Infecting the leaves: and (3) cutting off tender leaves with consistent leaf age of about 15-20 days from the tissue culture bottle in the gap time of bacterial liquid suspension, scratching the back of strawberry leaves by using a blade, putting the leaves into MS bacterial liquid suspension after all the leaves are cut, putting the MS bacterial liquid suspension into a shaking table at 28 ℃, and infecting for 20 minutes at 100 rpm. After infection, the bacterial liquid is poured out, the residual bacterial liquid on the leaves is absorbed by sterile filter paper, transferred to a co-culture medium and co-cultured for 3 days in a dark environment. After the explants are degermed for 15 days under the weak light, the explants growing the calluses are moved to a screening culture medium and are moved to the light for culture, the temperature is set to be 22 ℃, and the culture medium is replaced every 2 weeks.

(3) When the adventitious bud is produced, the adventitious bud is cut into several small segments and transferred to a germination medium. Transferring the leaves into a rooting culture medium after the leaves grow out, and transferring the seedlings into a flowerpot after the seedlings are recovered when the leaves grow into complete seedlings.

The following media were used in the experiments (pH 5.8 unless otherwise specified):

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

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

(3) LB liquid medium: tryptone (Tryptone) 10g/L, yeast Extract (Yeast Extract) 5g/L, sodium chloride 10g/L, pH 7.0

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

(5) MS-B5 solid medium: 4.302g/L MS culture medium powder (containing no vitamin), 20g/L sucrose, 5mL/L B5 organic solution and 7g/L agar powder

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

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

(8) Strawberry screening culture medium: MS-B5 culture medium +3 mg/L6-BA +0.2mg/L IBA +300mg/L Ti +20mg/L Kan

(9) Strawberry germination medium: MS-B5 culture medium +2 mg/L6-BA +0.2mg/L IBA +300mg/L Ti +20mg/L Kan

Red strawberry rooting medium: MS-B5 medium +0.2mg/L IBA +300mg/L Ti

The formulation of the hormones used in the experiments was as follows:

(1) TDZ (2 mg/mL): weighing 20mg of TDZ powder, dissolving with 200 μ L of 1M sodium hydroxide solution, adding pure alcohol after complete dissolution, metering to 10ml, and storing at-20 deg.C for use.

(2) IBA (1 mg/mL): weighing 10mg IBA powder, diluting to 10mL with sterile distilled water, and storing at-20 deg.C for use.

(3) AS (20 mg/mL): weighing 20mg AS powder, adding DMSO to a constant volume of 1mL, and storing at-20 deg.C for use.

(4) Ti (300 mg/mL): weighing 3g of Ti, using distilled water to fix the volume to 10mL, and storing at-20 ℃ for later use.

(5) Kan (100 mg/mL): weighing Kan 500mg, adding distilled water to a constant volume of 5mL, and storing at-20 ℃ for later use.

(6) MS-B5 organic solution: weighing nicotinic acid 0.2g, inositol 20g, vitamin B1 g, vitamin B6.2 g, dissolving with distilled water to constant volume of 1L, and storing in refrigerator at 4 deg.C for use.

(7) Weighing 6.0g of sodium hydroxide sheet-shaped object in 1M sodium hydroxide solution, dissolving with distilled water, fixing the volume to 150mL, and storing at room temperature.

3. GUS staining verification of transgenic plants

Preparing GUS dye solution, cutting off leaves of transgenic strawberry plants, and dyeing the leaves. If the staining is successful, the GUS reporter gene is successfully expressed in the plant and is transferred into the strawberry genome.

4. Observing and analyzing transgenic plants

Phenotype	Time of transplantation	Number of flowers	Counting time
				Wild type strawberry	9/12/2018	0	11 and 22 months in 2018
Transgenic line 1	9/12/2018	4	11 months in 201822 days
				Transgenic line 2	9/12/2018	2	11/2018 and 22/month
Transgenic line 3	9/12/2018	1	11/2018 and 22/month
				Transgenic line 4	9/12/2018	1	11/2018 and 22/month

From the above results, it can be seen that the transgenic strawberry plants exhibited earlier flowering time than the wild type strawberry plants. Under the same culture condition, the wild strawberry plant does not flower after 71 days, and the transgenic strawberry transferred into the FveDDM1-RNAi vector flowers after 71 days of transplantation. According to the results, the RNAi vector aiming at the forest strawberry FveDDM1 gene is introduced into the forest strawberry, so that the expression of the forest strawberry FveDDM1 gene is inhibited, and the flowering time of the strawberry can be advanced. The FveDDM1-RNAi vector can promote early flowering of strawberries, and the FveDDM1-RNAi vector is a functional vector and can promote reproductive growth and early flowering of plants.

Sequence listing

<110> Nanjing university of agriculture

<120> application of RNA interference vector FveDDM1-RNAi for promoting early flowering of strawberry

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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 visco)

<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 (Artificial Sequence)

<400> 4

actctgcagg tcgacttggc ccgaacttcc 30

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aataatatgg tcgacgagaa gaaagggacg 30

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttgcaaagct ctagagagaa gaaagggacg 30

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tgaacgatct ctagattggc ccgaacttcc 30

<210> 8

<211> 392

<212> DNA

<213> Artificial Sequence (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 (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. Forest-inhibiting strawberryFveDDM1Application of RNAi vector for gene expression in advancing flowering time of strawberries, and forest strawberriesFveDDM1The nucleotide sequence of the gene is shown in SEQ ID NO. 2.

2. Transformed forest-inhibiting strawberries as claimed in claim 1FveDDM1The transgenic engineering bacteria of the RNAi vector of the gene expression is applied to the early flowering time of the strawberry.

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