CN113999828B

CN113999828B - Application of strawberry methyltransferase MTA and MTB genes in control of strawberry maturation

Info

Publication number: CN113999828B
Application number: CN202110141699.3A
Authority: CN
Inventors: 秦国政; 王豫颖; 王威浩; 周磊磊; 汤仁坤
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2024-01-30
Anticipated expiration: 2041-02-02
Also published as: CN113999828A

Abstract

The invention provides application of strawberry methyltransferase genes in controlling strawberry maturation, wherein the methyltransferase genes code methyltransferase. The examples of the present invention demonstrate that methyltransferases MTA and MTB can alter fruit ripening processes by modulating fruit color and expression of softening key genes. Inhibiting the expression of the methyltransferase MTA gene or MTB gene in strawberry fruits can delay the progress of fruit ripening, and over-expression of the MTA gene or MTB gene can lead to premature fruit ripening. Therefore, the time to market of the strawberry fruits can be adjusted by reasonably utilizing the methyltransferase, and the strawberry fruits are served for strawberry production.

Description

Application of strawberry methyltransferase MTA and MTB genes in control of strawberry maturation

Technical Field

The invention relates to application of strawberry methyltransferase MTA and MTB genes in controlling strawberry maturation in the technical field of biology.

Background

The strawberry fruits are rich in nutrition and delicious in taste, and are popular with the public. However, the problems of concentrated supply season, difficult storage after picking, over single variety and the like exist in the production of the strawberries at present, so that the production cost of the strawberries is high. Therefore, searching the strawberry maturation control key genes, and further, applying genetic engineering to select and breed excellent strawberry varieties is an important way for solving the strawberry production problem in the future.

N ⁶ Methyl adenine (m) ⁶ A) Is the most common chemical modification of eukaryotic messenger RNA (mRNA), can enhance exon cleavage, promote small RNA (miRNA) processing, reduce mRNA stability and improve mRNA translation efficiency, and can regulate gene expression level at posttranscriptional level. m is m ⁶ The A modification is a reversible process that is coordinated by RNA methyltransferases and RNA demethylases. In mammals, m ⁶ Methyltransferases METTL3 and METTL14 in the a-modified complex as core components for mRNA binding and methyl catalysis, at m ⁶ Plays a key role in modification a. Heretofore, there has been no study of the gene function of strawberry methyltransferase or demethyltransferase.

Disclosure of Invention

The technical problem to be solved by the invention is how to control the maturity of strawberries.

In order to solve the technical problems, the invention provides application of a strawberry methyltransferase gene in controlling strawberry maturation, wherein the methyltransferase gene codes methyltransferase, and the methyltransferase is protein of A1 or A2 as follows:

a1, the amino acid sequence is protein of an amino acid sequence shown as a sequence 2 or a sequence 4 in a sequence table;

a2, N-terminal or/and C-terminal of A1) is linked to a protein tag.

In the application, the sequence 2 in the sequence table consists of 741 amino acid residues, and the sequence 4 in the sequence table consists of 1152 amino acid residues.

In the above application, the methyltransferase may be derived from strawberry.

In the above application, the methyltransferase gene may specifically be a gene as shown in the following D1 or D2:

the coding sequence of the coding chain is a DNA molecule of a sequence 1 or a sequence 3 in a sequence table;

d2 and nucleotide sequence are DNA molecules of sequence 1 or sequence 3 in the sequence table.

In the above application, the controlling strawberry maturation may delay strawberry maturation by inhibiting or reducing the expression of the methyltransferase gene. Specifically, the method can be realized by transferring methyltransferase genes to interfere with an expression vector. The methyltransferase gene interfering expression vector may be a recombinant expression vector obtained by ligating a methyltransferase gene into the interfering expression vector. The interfering expression vector may be pK7 gwwg 2D or pH7 gwwg 2D.

In the above application, the controlling strawberry maturation may promote strawberry maturation by promoting or enhancing expression of the methyltransferase gene. Specifically, the method can be realized by transferring a methyltransferase gene over-expression vector. The over-expression vector may be a recombinant expression vector obtained by ligating a methyltransferase gene into the over-expression vector. The over-expression vector can be any one of pCambia 2300, pCambia 1300 and pBI 121.

In order to solve the technical problems, the invention provides an agent for delaying strawberry maturation, wherein the active ingredient of the agent is a substance for inhibiting the expression of a gene encoding the methyltransferase, reducing the abundance of the methyltransferase and/or knocking out the gene encoding the methyltransferase.

The active ingredients of the agents may also contain other biological or/and non-biological ingredients, and the other active ingredients of the agents may be determined by one skilled in the art based on the effect of delaying strawberry maturation.

In order to solve the above technical problems, the present invention provides an agent for promoting strawberry maturation, the active ingredient of which is a substance that enhances the expression of a gene encoding the methyltransferase and/or increases the abundance of the methyltransferase.

The active ingredients of the above agents may also contain other biological or/and non-biological ingredients, and the other active ingredients of the above agents may be determined by one skilled in the art based on the effect of promoting strawberry maturation.

In order to solve the technical problems, the invention also provides a method for delaying strawberry maturation, which comprises the following steps: inhibiting expression of the methyltransferase in a recipient strawberry, reducing the abundance of the methyltransferase, and/or knocking out a gene encoding the methyltransferase to obtain a strawberry of interest having a maturity stage that is later than the recipient strawberry.

In order to solve the technical problems, the invention also provides a method for promoting strawberry maturation, which comprises the following steps: enhancing expression of the methyltransferase in a recipient strawberry and/or increasing the abundance of the methyltransferase, resulting in a strawberry of interest having a maturity earlier than the recipient strawberry.

The invention also provides a protein, which is methyltransferase, is a protein of A1 or A2 as follows:

a2, N-terminal or/and C-terminal of A1) is linked to a protein tag.

The invention also provides a nucleic acid molecule of the protein, which is specifically a nucleic acid molecule shown as the following D1 or D2:

In the invention, the control of the strawberry maturity stage can be to delay strawberry maturity or promote strawberry maturity.

The invention provides the use of a substance that modulates a strawberry methyltransferase gene encoding methyltransferase in controlling the maturation period of strawberry. The examples of the present invention demonstrate that methyltransferases MTA and MTB can alter fruit ripening processes by modulating fruit color and expression of softening key genes. Inhibiting the expression of the methyltransferase MTA gene or MTB gene in strawberry fruits can delay the progress of fruit ripening, and over-expression of the MTA gene or MTB gene can lead to premature fruit ripening. Therefore, the time to market of the strawberry fruits can be adjusted by reasonably utilizing the methyltransferase, and the strawberry fruits are served for strawberry production.

Drawings

FIG. 1 is an electropherogram of RT-PCR amplified products of the MTA and MTB genes of strawberry in example 1. Wherein 1-3 are amplification bands of strawberry MTA gene; 4-6 are amplification bands of strawberry MTB gene; 7-8 are controls; m is DNA molecular weight standard (Optimum, peking).

FIG. 2 is the expression of strawberry MTA and MTB genes in fruit ripening in example 2. Panel A of FIG. 2 shows the expression of MTA and MTB genes in diploid strawberry fruits at different maturity. In the figure, S6 is 15-20 days after flowers; RS1 is 30 days post-flowers; RS3 is 35 days post-flowers. Panel B of FIG. 2 shows the expression of the eight-fold strawberry fruits and MTA, MTB genes at different maturity stages. In the figure, SG is the small green fruit; wt is semen Ginkgo; IR is the color change fruit; FR is a fully ripe fruit.

FIG. 3 is a phenotypic chart of fruits transiently interfering with the expression of the octaploid strawberry MTA or MTB in example 3. After injection of the strawberry fruit with agrobacterium containing control vector, RNAi-MTA or RNAi-MTB vector, respectively (day 0 is the injection day), the RNAi fruit exhibits a delayed maturation phenotype at day 7.

FIG. 4 is a chart of fruit phenotypes of the transient overexpression of MTA or MTB in the octaloid strawberry in example 4. After injection of agrobacteria containing control vector, OE-MTA or OE-MTB vector, respectively, into strawberry fruit (day 0 is the day of injection), OE fruit exhibits accelerated maturation phenotype at day 5.

FIG. 5 shows the gene expression of MTA-RNAi fruit and MTB-RNAi fruit in example 5. Data are expressed as mean ± standard deviation, repetition number is 3, meaning that the significance analysis result is p.ltoreq.0.05, meaning that the significance analysis result is p.ltoreq.0.01.

FIG. 6 shows the gene expression of the OE-MTA fruit and OE-MTB fruit of example 5. Data are expressed as mean ± standard deviation, repetition number is 3, meaning that the significance analysis result is p.ltoreq.0.05, meaning that the significance analysis result is p.ltoreq.0.01.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples described below are all conventional biochemical reagents, unless otherwise specified, and are commercially available.

1 vector

The vector pMD in the following examples ^TM 19-T (product number: 6013) is a product of TaKaRa company.

In the following examples8/GW//>Vector cloning Vector (cat# k 250020) was a product of Invitrogen corporation.

The vector pK7 GWIGWG 2D in the examples described below is described in the literature "Cai J, qin G, chen T, et al, the mode of action of remorin1, in regulating fruit ripening at transcriptional and post-transcriptional levels [ J ]. NEW PHYTOLOGIST,2018,9", available to the public from the national academy of sciences of plant research.

The vector pCambia 2300 described in the examples below is described in the literature "Cai J, qin G, chen T, et al, the mode of action of remorin, 1 in regulating fruit ripening at transcriptional and post-transcriptional levels [ J ]. NEW PHYTOLOGIST,2018,9", available to the public from the national academy of sciences plant research.

2 plant lines

Strawberry variety 'Hawaii 4' in the examples below is described in non-patent documents "Luo H, dai C, li Y, et al reduced Anthocyanins in Petioles codes for a GST anthocyanin transporter that is essential forthe foliage and fruit coloration in strawberry" [ J ]. JOURNAL OF EXPERIMENTAL BOTANY,2018,3". The public is available from plant research institute of national academy of sciences to repeat the experiments of the present application, and is not available for other uses.

The strawberry variety `Hongyan` in the examples below is described in the non-patent literature `Riaz A, aadil RM, amousa AMO, et al application of chiposa-based apple peel polyphenols edible coating on the preservation ofstrawberry (Fragaria ananassa cv Hongyan) frui. JOURNAL OF FOOD PROCESSING AND PRESERVATION, 2021.DOI:10.1111/jfpp.15018`. The public is available from plant research institute of national academy of sciences to repeat the experiments of the present application, and is not available for other uses.

3 reagent

In the following examples, the preparation method of LB solid medium containing 100mg/L of ampicillin was as follows (200 mL for example): solid LB medium (200 mL), yeast extract 1g, pancreas protein 2g, sodium chloride 2g,3g agar powder, dissolved in 200mL deionized water, 120 ℃ high pressure sterilization 20min, in the medium temperature to 50 ℃, in the super clean bench added 200 u L ampicillin (100 mg/mL), mixing, pouring into the culture dish, solidification after 4 ℃ preservation.

In the following examples, LB liquid medium containing 50mg/L kanamycin, 50mg/L rifampicin and 50mg/L streptomycin was prepared by the following method (200 mL as an example): solid LB medium (200 mL) comprising yeast extract 1g, pancreatic protein 2g, sodium chloride 2g, dissolved in 200mL deionized water, autoclaved at 120℃for 20min, and when the medium temperature was reduced to 50℃100. Mu.L kanamycin (50 mg/mL), 100. Mu.L rifampicin (50 mg/mL), 100. Mu.L streptomycin (50 mg/mL) were added to the super clean bench, mixed well and stored at 4 ℃.

In the following examples, the preparation method of the conversion solution comprises the following steps: mgCl is added ₂ 2- (N-morpholinoethanesulfonic acid (MES), acetosyringone is dissolved in deionized water to obtain a solution with pH value of 5.6, mgCl is contained in the conversion solution ₂ Is 10mM, MES is 10mM, acetosyringone is 200. Mu.M.

In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

EXAMPLE 1 cloning and sequencing of the MTA and MTB Gene cDNA sequences of strawberry

Total RNA from fruits of wild strawberry (Fragaria vesca, 'Hawaii 4') was extracted using a plant total RNA isolation kit (Norviga biological Co., nanj), and after SimpliNano (thermo) quantification, 1. Mu.g of total RNA was taken and cDNA first strand was synthesized using a reverse transcription kit (PrimerScriptRT reagentKit, takara, dalian).

According to published genome sequence information of wild strawberries, primers specific to MTA genes are designed:

MTA-F:5′-ATGGAGGAGACCCAATCTGG-3′；

MTA-R:5′-TCCGGCCATCTCAACATCG-3′；

designing a primer specific to MTB genes:

MTB-F:5′-ATGGATTCGCCTGAACGTAG-3′；

MTB-R:5′-CAACAAATTCATGTGCCTGTGAG-3′。

primers were synthesized by Beijing engine biotechnology Co.

MTA and MTB genes were amplified separately using High fidelity enzymes (I-5M 2X High-FidelityMastermix, optimago).

The PCR reaction system is as follows: 100ng of cDNA template, 2. Mu.L of forward and reverse primers (10. Mu.M), 25. Mu.L of I-5M 2x High-FidelityMastermix, and water was added to 50. Mu.L.

The PCR reaction program was set as follows: 98 ℃ for 2min;98℃10s,56℃15s,72℃15s for a total of 35 cycles; finally, the extension is carried out for 5min at 72 ℃.

The amplified products were electrophoretically detected on a 1% agarose gel, and as shown in FIG. 1, the amplified products of MTA-F and MTA-R were found to have specific bands of interest at 2000bp, and the amplified products of MTB-F and MTB-R were found to have specific bands of interest at 3000 bp.

The amplified bands were recovered by using a DNA gel recovery kit (Norwegian biosystems, nanjing).

Purified MTA DNA fragment and vector pMD were subjected to DNA Ligation Kit (TaKaRa, daisy Co., ltd.) ^TM 19-T (TaKaRa, dalian) was ligated according to the TA cloning principle to construct pMD19T-MTA recombinant plasmid. The pMD19T-MTA plasmid was transformed into E.coli TOP10 strain (a strain of the family Optimaceae, beijing) Screening on LB solid medium containing 100mg/L ampicillin, picking positive single colony, and detecting pMD19T-MTA recombinant plasmid sequence by using M13-F universal sequencing primer.

M13-F：5′-ACTGGCCGTCGTTTTACAACG-3′。

Purified MTB DNA fragment and vector pMD were subjected to DNA Ligation Kit (TaKaRa, daisy Co., ltd.) ^TM 19-T (TaKaRa, dalian) was ligated according to the TA cloning principle to construct pMD19T-MTB recombinant plasmid. The pMD19T-MTA plasmid was transformed into E.coli TOP10 strain (a strain of the family Optimaceae, beijing) on LB solid medium containing 100mg/L of ampicillin, positive single colonies were picked and the pMD19T-MTB recombinant plasmid sequences were detected with M13-F universal sequencing primers.

The results showed that we obtained the cDNA sequences of the MTA and MTB genes of strawberry, since this gene was identical to the published gene sequence: the cDNA sequence of MTA is shown as sequence 1 in a sequence table, the locus number is gene01911 (ftp:// ftp. Bioinfo. Wsu. Edu/species/Fragaria_vesca/Fvesca-genome. V2.0.a 2), and the encoded protein contains 741 amino acid residues (shown as sequence 2 in the sequence table); the cDNA sequence of MTB is shown as sequence 3 in the sequence table, the locus number is gene31441 (ftp:// ftp. Bioinfo. Wsu. Edu/species/Fragaria_vesca/Fvesca-genome. V2.0.a 2), and the coded protein contains 1152 amino acid residues (shown as sequence 4 in the sequence table).

Example 2 expression characterization of methyltransferase MTA and MTB genes in strawberry fruit

1. Expression of methyltransferase MTA and MTB genes in diploid strawberries

Diploid wild strawberry (Fragaria vesca 'Hawaii 4') fruits were picked from the experimental greenhouse and divided into three maturity according to the number of days after flowering of the strawberry fruits (see panel A of FIG. 2): a green fruit period (S6), about 15-20 days after flowering; semen Ginkgo period (RS 1), 30 days after flower opening; stage of yellow fruit (RS 6), fruits 35 days after flowering. Immediately after picking, the fruits were snap frozen with liquid nitrogen and returned to the laboratory.

Three samples of strawberry of different maturity were RNA extracted and reverse transcribed to cDNA according to the method of example 1, the cDNA concentration was further diluted 20-fold for the template for the fluorescent quantitative PCR reaction. The change in expression of MTA and MTB genes was detected on a fluorescent quantitative PCR apparatus (STEPONE PLUS, ABI, USA) using a fluorescent quantitative reagent (SYBRPremix Ex TaqTM, taKaRa), respectively.

Primers used for detecting MTA gene are MTA-qRT-PCR-F and MTA-qRT-PCR-R:

MTA-qRT-PCR-F：5′-GGTCGATGGCGGAGGAGAA-3′；

MTA-qRT-PCR-R：5′-GTCTTGTTTGCTCTCCGGCAAC-3′。

primers used for detecting MTB gene are MTB-qRT-PCR-F and MTB-qRT-PCR-R:

MTB-qRT-PCR-F：5′-GGTTCAACCCAGAAGCCTGAAGAC-3′；

MTB-qRT-PCR-R：5′-GTTCCGGATGTAAGCCTCGGT-3′。

the reaction system is as follows: 2. Mu.L of template, 10. Mu.L of SYBR Mix, 0.8. Mu.L of forward and reverse primers each, 0.8. Mu.L of Rox, and 20. Mu.L of sterilized double distilled water were added.

The reaction procedure is: 98 ℃ for 10min; 15S at 95 ℃, 30S at 60 ℃ and 40 cycles; 72 ℃ for 5min; and (3) analyzing the melting curve at 95 ℃ for 1min to obtain a default setting of the system.

As shown in the A graph of FIG. 2, the expression of MTA gene gradually increases and then decreases with the increase of the maturity of the strawberry, the expression level is highest in the RS1 stage, and the MTB gene continuously increases with the increase of the maturity, which indicates that MTA and MTB possibly participate in the maturity regulation of the strawberry fruits.

2. Expression of methyltransferase MTA and MTB genes in octaloid strawberry

Strawberry (Fragaria ananassa cultivar 'Hongyan') fruits were picked from a strawberry base (commercial picking garden) greenhouse and fruit ripeness was divided according to the following characteristics (as in panel B of fig. 2): the small green fruits (SG) are characterized by dark green peel and green seeds; gingko (Wt) is characterized by green to white peel and the seeds begin to turn brown; the color-changing fruit (IR) is characterized in that one third of the peel surface is red, and the seeds are all brown; the fully ripe Fruit (FR) is characterized by the full red surface of the peel and the full dark brown color of the seed. Immediately after picking, the fruits were snap frozen with liquid nitrogen and returned to the laboratory.

The expression changes of MTA and MTB genes in the octaloid strawberry were detected according to the method of step 1 above, respectively.

As shown in the B graph of FIG. 2, the MTA and MTB genes are expressed in the highest amount in the IR period as the maturity of the strawberries is increased, and then the MTA and MTB genes are expressed in the gradually increased and then decreased, which indicates that the MTA and MTB can participate in the maturity regulation of the strawberries.

Example 3 construction of strawberry MTA and MTB interference expression vector and use thereof

1. Construction of MTA interference expression vector and MTB interference expression vector.

1.1 construction of MTA interference expression vector

According to the strawberry MTA gene sequence, a specific region of a conserved domain of a methyltransferase gene is selected and removed, and upstream and downstream primers MTA-RNAi-F and MTA-RNAi-R which can amplify the region are designed:

MTA-RNAi-F：5′-ATGGAGGAGACCCAATCTGGC-3′；

MTA-RNAi-R：5′-GTCCTCGTCCATTGAATCGGG-3′。

primers were synthesized by Beijing engine biotechnology Co.

The pMD19T-MTA plasmid was amplified using a High fidelity enzyme (I-5M 2X High-Fidelity MasterMix, a family of Optimus).

The PCR reaction system is as follows: pMD19T-MTA plasmid template 100ng, forward and reverse primers (10. Mu.M) 2. Mu.L, I-5M 2x High-Fidelity Master Mix. Mu.L, and water was made up to 50. Mu.L.

Recovery and purification of the target band, ligation of DNA fragments8/GW//>Vector cloning Vector (Invitrogen). The pK7 gwwg 2D vector was ligated by the method of GATEWAY cloning using LR recombinase (LR Clonase II Ezyme mix, invitrogen). MTA interference list was formed by E.coli transformation, antibiotic screening, double restriction identification and sequencingThe vector was designated RNAi-MTA.

1.2 construction of MTB interference expression vector

According to the MTB gene sequence of strawberry, selecting specific region for removing conserved domain of methyltransferase gene, designing upstream and downstream primers capable of amplifying said region

Primers used for the MTB gene were MTB-RNAi-F and MTB-RNAi-R:

MTB-RNAi-F：5′-ATGGATTCGCCTGAACGTAG-3′；

MTB-RNAi-R：5′-TCCCCATCCTGATACCAGT-3′。

primers were synthesized by Beijing engine biotechnology Co.

The pMD19T-MTB plasmid was amplified using a High fidelity enzyme (I-5M 2X High-Fidelity MasterMix, a family of Optimaceae).

The PCR reaction system is as follows: pMD19T-MTB plasmid template 100ng, forward and reverse primers (10. Mu.M) 2. Mu.L, I-5M 2x High-Fidelity Master Mix. Mu.L, and water was made up to 50. Mu.L.

Recovery and purification of the target band, ligation of DNA fragments8/GW//>Vector cloning Vector (Invitrogen). The pK7 gwwg 2D vector was ligated by the method of GATEWAY cloning using LR recombinase (LR Clonase II Ezyme mix, invitrogen). The MTB interference expression vector is formed through escherichia coli transformation, antibiotic screening, double enzyme digestion identification and sequencing, and is named RNAi-MTB.

2. RNAi-MTA and RNAi-MTB Agrobacterium preparation and transient transformation of strawberry fruits.

Transforming RNAi-MTA and RNAi-MTB recombinant expression vectors into agrobacterium GV3101 respectively by freeze thawing, picking positive clones, inoculating into 50mL LB liquid medium containing 50mg/L kanamycin, 50mg/L rifampicin and 50mg/L streptomycin,shaking at 200rpm (revolutions per minute) at 28℃to OD ₆₀₀ Bacterial liquid was collected by centrifugation at 5000g for 10min =0.6. Suspending the bacterial cells with 50mL of the transformation liquid, and centrifuging for 10min at 5000g under the room temperature condition to collect bacterial liquid; re-suspending the thallus with 50mL of conversion liquid, and standing at room temperature for more than 2h to obtain agrobacterium tumefaciens liquid for converting RNAi-MTA and agrobacterium tumefaciens liquid for converting RNAi-MTB respectively.

The agrobacterium liquid for transforming RNAi-MTA and the agrobacterium liquid for transforming RNAi-MTB are respectively infected with fruits of strawberry 'Hongyan' through an injection method. The specific method comprises the following steps: the fruit is injected at the central axis of the fruit in the green fruit period of the strawberry by using a 1ml sterile micro-injector until the fruit presents a water stain shape. Among them, the fruit of the agrobacterium liquid into which RNAi-MTA is injected is called MTA-RNAi fruit, and the fruit of the agrobacterium liquid into which RNAi-MTB is injected is called MTB-RNAi fruit. The fruit is placed at room temperature after injection, and the relative humidity is 70-90%, and observed every day.

The results are shown in FIG. 3, showing that the coloration of both strawberry fruits transformed with RNAi-MTA vector (MTA-RNAi fruits) and strawberry fruits transformed with RNAi-MTB vector (MTB-RNAi fruits) after injection is significantly retarded compared to control strawberry 'Hongyan' fruits transformed with empty vector pK7 GWIGWG 2D, indicating that transient MTA-RNAi or MTB-RNAi interference can delay the maturation of the octaloid strawberry fruits.

Example 4 construction of strawberry MTA and MTB overexpression vector and use thereof

1. Construction of MTA and MTB overexpression vectors.

1.1 construction of MTA overexpression vector

Specific primers MTA-2300-F and MTA-2300-R for amplifying the full-length gene of MTA (without termination codon sequence TAA) are designed according to the cDNA sequence of MTA (sequence 1 in the sequence table) and the sequence of the plant expression vector pCambia 2300:

MTA-2300-F:5′-GGTACCCGGGGATCCTCTAGAATGGAGGAGACCCAATCTGG-3′；

MTA-2300-R:5′-GCCCTTGCTCACCATTCTAGATCCGGCCATCTCAACATCG-3′。

the DNA fragment used to ligate the pCambia 2300 vector was amplified using the pMD19T-MTA recombinant plasmid of example 1 as a template.

The amplified fragment was purified and ligated to pCambia 2300 vector using homologous recombination cloning by homologous recombination (In-Fusion HD Cloning, taKaRa, dalian) to form OE-MTA vector.

1.2 construction of MTB overexpression vector

Specific primers MTB-2300-F and MTB-2300-R for amplifying the full-length MTB gene (without the stop codon sequence TAA) are designed according to the cDNA sequence (sequence 3) of MTB and the sequence of the plant expression vector pCambia 2300:

MTB-2300-F:5′-GGTACCCGGGGATCCTCTAGAATGGATTCGCCTGAACGTAG-3′；

MTB-2300-R:5′-GCCCTTGCTCACCATTCTAGACAACAAATTCATGTGCCTGTGAG-3′。

the DNA fragment used to ligate the pCambia 2300 vector was amplified using the pMD19T-MTB recombinant plasmid of example 1 as a template.

The amplified fragment was purified and ligated to pCambia 2300 vector using homologous recombination cloning by homologous recombination (In-Fusion HD Cloning, taKaRa, dalian) to form OE-MTB vector.

2. OE-MTA and OE-MTB Agrobacterium preparation and transient transformation of strawberry fruits.

An OE-MTA Agrobacterium solution and an OE-MTB Agrobacterium solution were prepared according to the Agrobacterium transformation method of example 3. The agrobacterial liquids were transformed into the fruits of strawberry 'Hongyan' according to the method of example 3 for transient transformation of strawberry fruits, respectively. Wherein, the fruit injected with the OE-MTA agrobacterium solution is called as OE-MTA fruit, and the fruit injected with the OE-MTB agrobacterium solution is called as OE-MTB fruit.

The results are shown in FIG. 4, showing that both the strawberry fruit transformed with OE-MTA (OE-MTA fruit) and the strawberry fruit transformed with OE-MTB vector (OE-MTB fruit) were significantly accelerated in coloration after injection, compared to the fruit of control strawberry 'Hongyan' transformed with empty vector (pCambia 2300), indicating that transient overexpression of MTA or MTB gene could accelerate the maturation of the octaloid strawberry fruit.

Example 5 molecular detection of MTA and MTB transgenic fruits

The sensory quality of strawberry fruits is determined by the color and texture, and the quality of strawberry with bright color and soft texture is generally better, so that the expression of color and soft key enzyme genes is an important molecular characteristic for measuring the quality change of strawberry. RNA was extracted from the MTA-RNAi fruit and MTB-RNAi fruit obtained in example 3, and the OE-MTA fruit and OE-MTB fruit obtained in example 4, and reverse transcribed into cDNA according to the method of example 2, and the expression of MTA, MTB, and anthocyanin synthesis key enzyme gene (CHS) and cell wall metabolism key enzyme gene (PG 1) in the MTA-RNAi fruit, MTB-RNAi fruit, OE-MTA fruit and OE-MTB fruit were detected by real-time fluorescent quantitative RT-PCR according to the method of example 2 and the primers.

The primers used for the CHS gene are CHS-qRT-PCR-F and CHS-qRT-PCR-R:

CHS-qRT-PCR-F：5′-CATACCCCGACTACTACTTTCGT-3′；

CHS-qRT-PCR-R：5′-CGCACATACTGGGATTCTCTT-3′。

the primers used for the PG1 gene are PG1-qRT-PCR-F and PG1-qRT-PCR-R:

PG1-qRT-PCR-F：5′-GCAAGTAGAGTCGCACAGTTTT-3′；

PG1-qRT-PCR-R：5′-TCAGTATTAGGCTTCCCACCA-3′。

as shown in fig. 5, compared with the control 'Hongyan' fruit transformed with RNAi empty vector pK7 gwiwawg 2D, MTA gene expression was significantly inhibited in MTA-RNAi fruit, expression of softening key enzyme gene PG1 and color key enzyme gene CHS was significantly reduced, MTB gene expression was significantly inhibited in MTB-RNAi fruit, and expression of PG1 and CHS was significantly reduced.

As shown in fig. 6, the expression of MTA gene in OE-MTA fruits was significantly increased, and the expression of softening key enzyme gene PG1 and color key enzyme gene CHS was significantly increased, compared to control 'Hongyan' fruits over-expressed empty vector pCambia 2300; in the OE-MTB fruits, the expression of MTB genes is obviously increased, and the expression of PG1 and CHS is obviously enhanced.

In conclusion, methyltransferases MTA and MTB can alter fruit ripening processes by regulating fruit color and softening key gene expression. Inhibiting the expression of MTA gene or MTB gene in strawberry fruit can delay the fruit ripening process, and over-expressing MTA gene or MTB gene can lead strawberry fruit to ripen in advance. Therefore, the time to market of strawberry fruits can be adjusted by reasonably utilizing the gene, and the strawberry fruits are served for strawberry production.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> institute of plant Material at national academy of sciences

<120> use of strawberry methyltransferase MTA and MTB genes in controlling strawberry maturation

<130> GNCSY210098

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2226

<212> DNA

<213> strawberry (Fragaria vesca)

<400> 1

atggaggaga cccaatctgg cggcaaagac gacaccgccg tgtccgccgt caaagcaatg 60

cgccaagacc tcgaaacccg aatcgaaacc caacgcgcca cccagctcga actcctctct 120

agtctccaat ccgaactcga ccccgccatc gtccccacca tcgatctctc cctcaaggtc 180

ctctcctcct tcaaccaccg ccccttcact cccacccctc ctctacccga cccgaaaccc 240

aacccgaccc gaaaccccgc cgcccgcctc ccctcccctc aaccacccct ccctcctcct 300

ccgctgcagc ccgaaagccc taatctttct ccccgacccg acccgacccg ggagtcctca 360

cccgattcaa tggacgagga cgcgaacccg ctctcggtga tccgggccat gattgcggtc 420

cggtttctgg agagggttcc gttcatgctg gtggagtcgt cggagctcct gaagcagctc 480

gagaccgacg cgaaggcctc gccggaggag aaggcggcgc tgcgggaggt ggggggagag 540

aacgggggaa ttctggcggt ggagatggcg ctgcggtcga tggcggagga gaacggcggc 600

gttatactgg aggagtacgc ggtcaacggg aagtctaggg ttacggtgag ggatattgac 660

cggactaggc tgatgaagga gttgccggag agcaaacaag actcgatttt ggatgggaat 720

gggagtttta atcagatggt gagtggtggc atggatggtg ggaacaatgt gggagggttt 780

atgggccgag gtgggccctg gggggttgcg ccggagcatt acatgggcgg gttgccgccg 840

ttgtttccgg ggataggtcc gagaggagga agagggatga tgggaatgcc gagagggatg 900

ttagggtctc caatgccgag gcctaatatg gggcagaatg gaggaatggt ggttagtccc 960

aatggaatgc cgaataagaa catgaagagc gaagaggatg agatgaagga tcttgaggca 1020

ttgctgaata agaagagttt taaggagttg cagaagtcga aaactgggga ggagcttttg 1080

gacctcatcc acaggccaac tgcaaaggag actagaacgg caaacaagtt caaaagcaaa 1140

ggtggttcac gagtgaagga atactgcagc tccctaacaa aagaggactg ccgacgtcaa 1200

tctaattcgt tgctcgcttg tgagaaggtt cattttaggc gcataattgc tctacatact 1260

gatgtcaatt taggagactg ttcttttcta gatacttgcc gtcacatgaa gacatgcaag 1320

tatgtacatt atgagctcga ttcaacacca gatgtgtcaa acatgatggc tccccctaga 1380

ccattaaagc agcgtgctga atattgttct gaggtggaac tcggtcaacc acaatggatt 1440

aactgtgata tccgcaattt tagaatggat attttgggac agtttggagt cataatggcc 1500

gatccaccgt gggacattca tatggagttg ccttatggga ctatggctga tgatgaaatg 1560

cgcactctta atgttcctgc attgcagact gatggtctga ttttcctttg ggttactggg 1620

cgtgcgatgg agcttggccg tgagtgttta gaactttggg gatacaagcg tatcgaggag 1680

atgatttggg tgaaaactaa tcagcttcaa cgaattatta gaactgggcg cactggccac 1740

tggctaaatc atagcaaaga gcattgccta gttggaataa aaggggatcc attagtgaat 1800

aggaatattg atactgatgt cattgttgcc gaggtcagag agacaagtcg taagccagat 1860

gagatgtacc ctttgctgga gaggattagt ccaaggacaa ggaagctgga gctgtttgct 1920

cgtatgcaca acactcatgc agggtggatg tcacttggta atcaactaag tggtgtacga 1980

ttggttgatg aaggcttgcg tgcacggttc aaggctgcgt acccagaggt ggaggtgcag 2040

ccagcatccc cacccagagc ttctgccatg gaagctgatt caaatgctac tcaaaccaga 2100

agtccctttt ccgaagcaaa acccacagag gctgctgctc cacaacaagc agagcctgca 2160

gggcctgatg cgcctgcttc tgaggtgaag ccaacgctta gcaccgatgt tgagatggcc 2220

ggatga 2226

<210> 2

<211> 741

<212> PRT

<213> strawberry (Fragaria vesca)

<400> 2

Met Glu Glu Thr Gln Ser Gly Gly Lys Asp Asp Thr Ala Val Ser Ala

1 5 10 15

Val Lys Ala Met Arg Gln Asp Leu Glu Thr Arg Ile Glu Thr Gln Arg

20 25 30

Ala Thr Gln Leu Glu Leu Leu Ser Ser Leu Gln Ser Glu Leu Asp Pro

35 40 45

Ala Ile Val Pro Thr Ile Asp Leu Ser Leu Lys Val Leu Ser Ser Phe

50 55 60

Asn His Arg Pro Phe Thr Pro Thr Pro Pro Leu Pro Asp Pro Lys Pro

65 70 75 80

Asn Pro Thr Arg Asn Pro Ala Ala Arg Leu Pro Ser Pro Gln Pro Pro

85 90 95

Leu Pro Pro Pro Pro Leu Gln Pro Glu Ser Pro Asn Leu Ser Pro Arg

100 105 110

Pro Asp Pro Thr Arg Glu Ser Ser Pro Asp Ser Met Asp Glu Asp Ala

115 120 125

Asn Pro Leu Ser Val Ile Arg Ala Met Ile Ala Val Arg Phe Leu Glu

130 135 140

Arg Val Pro Phe Met Leu Val Glu Ser Ser Glu Leu Leu Lys Gln Leu

145 150 155 160

Glu Thr Asp Ala Lys Ala Ser Pro Glu Glu Lys Ala Ala Leu Arg Glu

165 170 175

Val Gly Gly Glu Asn Gly Gly Ile Leu Ala Val Glu Met Ala Leu Arg

180 185 190

Ser Met Ala Glu Glu Asn Gly Gly Val Ile Leu Glu Glu Tyr Ala Val

195 200 205

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

210 215 220

Met Lys Glu Leu Pro Glu Ser Lys Gln Asp Ser Ile Leu Asp Gly Asn

225 230 235 240

Gly Ser Phe Asn Gln Met Val Ser Gly Gly Met Asp Gly Gly Asn Asn

245 250 255

Val Gly Gly Phe Met Gly Arg Gly Gly Pro Trp Gly Val Ala Pro Glu

260 265 270

His Tyr Met Gly Gly Leu Pro Pro Leu Phe Pro Gly Ile Gly Pro Arg

275 280 285

Gly Gly Arg Gly Met Met Gly Met Pro Arg Gly Met Leu Gly Ser Pro

290 295 300

Met Pro Arg Pro Asn Met Gly Gln Asn Gly Gly Met Val Val Ser Pro

305 310 315 320

Asn Gly Met Pro Asn Lys Asn Met Lys Ser Glu Glu Asp Glu Met Lys

325 330 335

Asp Leu Glu Ala Leu Leu Asn Lys Lys Ser Phe Lys Glu Leu Gln Lys

340 345 350

Ser Lys Thr Gly Glu Glu Leu Leu Asp Leu Ile His Arg Pro Thr Ala

355 360 365

Lys Glu Thr Arg Thr Ala Asn Lys Phe Lys Ser Lys Gly Gly Ser Arg

370 375 380

Val Lys Glu Tyr Cys Ser Ser Leu Thr Lys Glu Asp Cys Arg Arg Gln

385 390 395 400

Ser Asn Ser Leu Leu Ala Cys Glu Lys Val His Phe Arg Arg Ile Ile

405 410 415

Ala Leu His Thr Asp Val Asn Leu Gly Asp Cys Ser Phe Leu Asp Thr

420 425 430

Cys Arg His Met Lys Thr Cys Lys Tyr Val His Tyr Glu Leu Asp Ser

435 440 445

Thr Pro Asp Val Ser Asn Met Met Ala Pro Pro Arg Pro Leu Lys Gln

450 455 460

Arg Ala Glu Tyr Cys Ser Glu Val Glu Leu Gly Gln Pro Gln Trp Ile

465 470 475 480

Asn Cys Asp Ile Arg Asn Phe Arg Met Asp Ile Leu Gly Gln Phe Gly

485 490 495

Val Ile Met Ala Asp Pro Pro Trp Asp Ile His Met Glu Leu Pro Tyr

500 505 510

Gly Thr Met Ala Asp Asp Glu Met Arg Thr Leu Asn Val Pro Ala Leu

515 520 525

Gln Thr Asp Gly Leu Ile Phe Leu Trp Val Thr Gly Arg Ala Met Glu

530 535 540

Leu Gly Arg Glu Cys Leu Glu Leu Trp Gly Tyr Lys Arg Ile Glu Glu

545 550 555 560

Met Ile Trp Val Lys Thr Asn Gln Leu Gln Arg Ile Ile Arg Thr Gly

565 570 575

Arg Thr Gly His Trp Leu Asn His Ser Lys Glu His Cys Leu Val Gly

580 585 590

Ile Lys Gly Asp Pro Leu Val Asn Arg Asn Ile Asp Thr Asp Val Ile

595 600 605

Val Ala Glu Val Arg Glu Thr Ser Arg Lys Pro Asp Glu Met Tyr Pro

610 615 620

Leu Leu Glu Arg Ile Ser Pro Arg Thr Arg Lys Leu Glu Leu Phe Ala

625 630 635 640

Arg Met His Asn Thr His Ala Gly Trp Met Ser Leu Gly Asn Gln Leu

645 650 655

Ser Gly Val Arg Leu Val Asp Glu Gly Leu Arg Ala Arg Phe Lys Ala

660 665 670

Ala Tyr Pro Glu Val Glu Val Gln Pro Ala Ser Pro Pro Arg Ala Ser

675 680 685

Ala Met Glu Ala Asp Ser Asn Ala Thr Gln Thr Arg Ser Pro Phe Ser

690 695 700

Glu Ala Lys Pro Thr Glu Ala Ala Ala Pro Gln Gln Ala Glu Pro Ala

705 710 715 720

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

725 730 735

Val Glu Met Ala Gly

740

<210> 3

<211> 3459

<212> DNA

<213> strawberry (Fragaria vesca)

<400> 3

atgaagagtg atagggcggg ggacgacgag gagtgggagg ggagtgataa gaggaagcac 60

aggtcgagca ggtcaaggaa gtcggggaac ggagaggacg tggatggtgg tggaaggaga 120

agaagtcatg gggataggag tgagagccgt aaacggtcag gtggctctag caatgcggat 180

agcgaggagg aggactatga cttgagaaag gagtcgcggt ctaagatgat gaagaagaag 240

caagaggaga gtagtttgga gaagttgagc aactggtatc aggatgggga atttgataat 300

aggcaagatg gtggagataa gtcagggggt agagggctgg ttagagctga agaaaatgaa 360

agaaggaaac tggcttcaaa gctcgcacag cacgagattt cccagactaa aagtaagagc 420

aaagaagaaa agtctcatga tggagaacat gaaaagacac tggacagaga ttctaagtat 480

tcggacagga aagaaagcat tcgagagaag actcatgggt cttctgaaca ggtgaggact 540

tcaagaagaa aatgggatga atcagatggt ggcaagaaag cagaagaaat ttataatgaa 600

agatctgatt caagaagtag taagccttct gatcctaagt atgagccttc taaagagaaa 660

actgtgctag caaaaaatga accgagcgaa agtaaaatca ggggattaga ttcaagcatt 720

gaaaggggta ctaaatctaa taacaaggaa gagagaaaag ctgatgcgga aaagagtaag 780

agcaaaagca ggggagaaat acttgaagaa gataacaggg gcagtcctat cactcgtgaa 840

gacagatctg gcaaggagaa agctgaaaag catagacagc agagaactcc cactgctcgc 900

gatgctgctg agggcaggga gaggttgtct aatgcagatg atgatgcaag tgcagggatg 960

aatgataagg gtgctagaga atttggaaat accaccaggt caaggacacc tgagaggact 1020

gggaggcgat atcaggattc agaacacttt gagacggatt atgaccgaaa ttttaatctc 1080

aagcgaaaag aactggaaaa agatggctat agggacgacc gatctaaagg cagagatgat 1140

aactatagtg acaggagtag ggaccgggaa gtccccaaag aaaaaagacg gcaaccccca 1200

agtaatgaca aggattccaa aaatggggat atcagttatg atcatagcag ggaatggccg 1260

agatatggtc gtgagagggg tgacaacgag aggcctcatg gtcggtctgg taataggaaa 1320

gatggaaacc ggggtgaagc tgtgaaaact tcatcaaact ttggaatttc aaatgaaaac 1380

tatgatgtga ttgagatcca aactaagcca gattttgtaa gggcagagtt gggacccaac 1440

tttcctagga gaaacgaagt tggtcaacag tctgatggaa aatcagcacc aaatgatgaa 1500

gaatgtacaa ggaagagtga tatgtatggg tctggaccac ctagggaaga ttcaaaggaa 1560

agatatacag atgatactac ctcgcgagat cagagttcat ggaaggatga ctttgatgcc 1620

catggagtga agggaagggg acaaagaggt tctatgcctg gccgcagtgc tggtggccag 1680

agttctagtg gtggttcaca acctccatat ggaaatgcag agcagggacc cttcaacaga 1740

aatgcttctc aaggagtgaa aggaggtaga ggtgggagag gaggaagggg taggcctact 1800

ggaagagaca gccagcagat ggcaatccca attccgatga tgggatcacc atttggacct 1860

attggaatgc ctccgcctgg acccatgcag ccacttactc ctagcatgtc accagctcca 1920

ggtccgccaa tgtttccgtt ttctccacct gtgtggccgg gggctcgagg tgttgacatc 1980

agcatgttaa ccatcccacc tgttatgcct catggatcat ctggcccaag atttcctcct 2040

aatatggtga ctccaacaaa tccttccatg ttttgtggcc aatctggacc tggaagagga 2100

ggtcctccaa gcatatctag ccctggcttt aatccttctg ggccaatggg acgaggaact 2160

cctgctgata aaagtcaagg tggttgggtt cctcataaaa gtagtggacc tcctggtaaa 2220

gctccttcta gaggagagca gaatgattac tctcaaaatt ttgttgatac tggtatgcga 2280

ccccaaaatt tcatcaggga gctagagcta acaaatgttg tggaggatta ccccaagctt 2340

agggagctta tacagaaaaa agatgagatc gtggaaaaag ctgcttctaa tcccatgtat 2400

tataaatgca acctgaaaga gtttgagctg tctccagagt tctttggaac caagtttgat 2460

gtgattcttg ttgatccacc atgggaggaa tacgttcatc gtgctcctgg tgttgctgac 2520

catacagagt attggacatt tgaagaaata atgaatctta agatcgaggc aatagcagac 2580

acaccttcct ttatttttct ttgggtgggt gatggcatgg gtcttgagca gggccgtcaa 2640

tgtctgaaga agtggggatt tcgtagatgt gaagatatat gttgggtgaa gacaaacaaa 2700

actaatccga ctcctggttt gcggcatgat tctcatacat tgtttcagca ctcaaaggaa 2760

cattgcttga tgggaataaa gggtaccgtt cgtcgcagca ctgatggcca tataatccat 2820

gccaacattg atactgatgt aataatagct gaggaacccc catatggttc aacccagaag 2880

cctgaagaca tgtacaggat aattgagcat tttgccctcg gtcgcaggag gcttgagctc 2940

tttggtgaag accacaacat tcgggctggt tggctgactg ttggtaatgg attatcctca 3000

tcaaacttta ataccgaggc ttacatccgg aactttgctg acaaggatgg caaagtttgg 3060

caaggagggg gtggacgaaa tccacctccg gaagcacctc atctggttgt aactactcca 3120

gacatagaag cccttcgtcc caagtcacca atgaaaaacc agcagcagat gcagcagcag 3180

caatctgcat ctatttctct gacatcagta aattcctcca acagaaggcc tggaaactcc 3240

ccacaaaatc caaccggtct aagtatgaac caagaagctt caagttccaa tccatcaact 3300

ccagctcctt gggcggcttc accactggac ggctataaag ggagagaggg cagcattatg 3360

ccttcagatg ataagatttt tgatatgtat ggttacagtg gacagggaaa tggagactat 3420

atagattttg aggctcacag gcacatgaat ttgttgtaa 3459

<210> 4

<211> 1152

<212> PRT

<213> strawberry (Fragaria vesca)

<400> 4

Met Lys Ser Asp Arg Ala Gly Asp Asp Glu Glu Trp Glu Gly Ser Asp

1 5 10 15

Lys Arg Lys His Arg Ser Ser Arg Ser Arg Lys Ser Gly Asn Gly Glu

20 25 30

Asp Val Asp Gly Gly Gly Arg Arg Arg Ser His Gly Asp Arg Ser Glu

35 40 45

Ser Arg Lys Arg Ser Gly Gly Ser Ser Asn Ala Asp Ser Glu Glu Glu

50 55 60

Asp Tyr Asp Leu Arg Lys Glu Ser Arg Ser Lys Met Met Lys Lys Lys

65 70 75 80

Gln Glu Glu Ser Ser Leu Glu Lys Leu Ser Asn Trp Tyr Gln Asp Gly

85 90 95

Glu Phe Asp Asn Arg Gln Asp Gly Gly Asp Lys Ser Gly Gly Arg Gly

100 105 110

Leu Val Arg Ala Glu Glu Asn Glu Arg Arg Lys Leu Ala Ser Lys Leu

115 120 125

Ala Gln His Glu Ile Ser Gln Thr Lys Ser Lys Ser Lys Glu Glu Lys

130 135 140

Ser His Asp Gly Glu His Glu Lys Thr Leu Asp Arg Asp Ser Lys Tyr

145 150 155 160

Ser Asp Arg Lys Glu Ser Ile Arg Glu Lys Thr His Gly Ser Ser Glu

165 170 175

Gln Val Arg Thr Ser Arg Arg Lys Trp Asp Glu Ser Asp Gly Gly Lys

180 185 190

Lys Ala Glu Glu Ile Tyr Asn Glu Arg Ser Asp Ser Arg Ser Ser Lys

195 200 205

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

210 215 220

Lys Asn Glu Pro Ser Glu Ser Lys Ile Arg Gly Leu Asp Ser Ser Ile

225 230 235 240

Glu Arg Gly Thr Lys Ser Asn Asn Lys Glu Glu Arg Lys Ala Asp Ala

245 250 255

Glu Lys Ser Lys Ser Lys Ser Arg Gly Glu Ile Leu Glu Glu Asp Asn

260 265 270

Arg Gly Ser Pro Ile Thr Arg Glu Asp Arg Ser Gly Lys Glu Lys Ala

275 280 285

Glu Lys His Arg Gln Gln Arg Thr Pro Thr Ala Arg Asp Ala Ala Glu

290 295 300

Gly Arg Glu Arg Leu Ser Asn Ala Asp Asp Asp Ala Ser Ala Gly Met

305 310 315 320

Asn Asp Lys Gly Ala Arg Glu Phe Gly Asn Thr Thr Arg Ser Arg Thr

325 330 335

Pro Glu Arg Thr Gly Arg Arg Tyr Gln Asp Ser Glu His Phe Glu Thr

340 345 350

Asp Tyr Asp Arg Asn Phe Asn Leu Lys Arg Lys Glu Leu Glu Lys Asp

355 360 365

Gly Tyr Arg Asp Asp Arg Ser Lys Gly Arg Asp Asp Asn Tyr Ser Asp

370 375 380

Arg Ser Arg Asp Arg Glu Val Pro Lys Glu Lys Arg Arg Gln Pro Pro

385 390 395 400

Ser Asn Asp Lys Asp Ser Lys Asn Gly Asp Ile Ser Tyr Asp His Ser

405 410 415

Arg Glu Trp Pro Arg Tyr Gly Arg Glu Arg Gly Asp Asn Glu Arg Pro

420 425 430

His Gly Arg Ser Gly Asn Arg Lys Asp Gly Asn Arg Gly Glu Ala Val

435 440 445

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

450 455 460

Glu Ile Gln Thr Lys Pro Asp Phe Val Arg Ala Glu Leu Gly Pro Asn

465 470 475 480

Phe Pro Arg Arg Asn Glu Val Gly Gln Gln Ser Asp Gly Lys Ser Ala

485 490 495

Pro Asn Asp Glu Glu Cys Thr Arg Lys Ser Asp Met Tyr Gly Ser Gly

500 505 510

Pro Pro Arg Glu Asp Ser Lys Glu Arg Tyr Thr Asp Asp Thr Thr Ser

515 520 525

Arg Asp Gln Ser Ser Trp Lys Asp Asp Phe Asp Ala His Gly Val Lys

530 535 540

Gly Arg Gly Gln Arg Gly Ser Met Pro Gly Arg Ser Ala Gly Gly Gln

545 550 555 560

Ser Ser Ser Gly Gly Ser Gln Pro Pro Tyr Gly Asn Ala Glu Gln Gly

565 570 575

Pro Phe Asn Arg Asn Ala Ser Gln Gly Val Lys Gly Gly Arg Gly Gly

580 585 590

Arg Gly Gly Arg Gly Arg Pro Thr Gly Arg Asp Ser Gln Gln Met Ala

595 600 605

Ile Pro Ile Pro Met Met Gly Ser Pro Phe Gly Pro Ile Gly Met Pro

610 615 620

Pro Pro Gly Pro Met Gln Pro Leu Thr Pro Ser Met Ser Pro Ala Pro

625 630 635 640

Gly Pro Pro Met Phe Pro Phe Ser Pro Pro Val Trp Pro Gly Ala Arg

645 650 655

Gly Val Asp Ile Ser Met Leu Thr Ile Pro Pro Val Met Pro His Gly

660 665 670

Ser Ser Gly Pro Arg Phe Pro Pro Asn Met Val Thr Pro Thr Asn Pro

675 680 685

Ser Met Phe Cys Gly Gln Ser Gly Pro Gly Arg Gly Gly Pro Pro Ser

690 695 700

Ile Ser Ser Pro Gly Phe Asn Pro Ser Gly Pro Met Gly Arg Gly Thr

705 710 715 720

Pro Ala Asp Lys Ser Gln Gly Gly Trp Val Pro His Lys Ser Ser Gly

725 730 735

Pro Pro Gly Lys Ala Pro Ser Arg Gly Glu Gln Asn Asp Tyr Ser Gln

740 745 750

Asn Phe Val Asp Thr Gly Met Arg Pro Gln Asn Phe Ile Arg Glu Leu

755 760 765

Glu Leu Thr Asn Val Val Glu Asp Tyr Pro Lys Leu Arg Glu Leu Ile

770 775 780

Gln Lys Lys Asp Glu Ile Val Glu Lys Ala Ala Ser Asn Pro Met Tyr

785 790 795 800

Tyr Lys Cys Asn Leu Lys Glu Phe Glu Leu Ser Pro Glu Phe Phe Gly

805 810 815

Thr Lys Phe Asp Val Ile Leu Val Asp Pro Pro Trp Glu Glu Tyr Val

820 825 830

His Arg Ala Pro Gly Val Ala Asp His Thr Glu Tyr Trp Thr Phe Glu

835 840 845

Glu Ile Met Asn Leu Lys Ile Glu Ala Ile Ala Asp Thr Pro Ser Phe

850 855 860

Ile Phe Leu Trp Val Gly Asp Gly Met Gly Leu Glu Gln Gly Arg Gln

865 870 875 880

Cys Leu Lys Lys Trp Gly Phe Arg Arg Cys Glu Asp Ile Cys Trp Val

885 890 895

Lys Thr Asn Lys Thr Asn Pro Thr Pro Gly Leu Arg His Asp Ser His

900 905 910

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

915 920 925

Thr Val Arg Arg Ser Thr Asp Gly His Ile Ile His Ala Asn Ile Asp

930 935 940

Thr Asp Val Ile Ile Ala Glu Glu Pro Pro Tyr Gly Ser Thr Gln Lys

945 950 955 960

Pro Glu Asp Met Tyr Arg Ile Ile Glu His Phe Ala Leu Gly Arg Arg

965 970 975

Arg Leu Glu Leu Phe Gly Glu Asp His Asn Ile Arg Ala Gly Trp Leu

980 985 990

Thr Val Gly Asn Gly Leu Ser Ser Ser Asn Phe Asn Thr Glu Ala Tyr

995 1000 1005

Ile Arg Asn Phe Ala Asp Lys Asp Gly Lys Val Trp Gln Gly Gly Gly

1010 1015 1020

Gly Arg Asn Pro Pro Pro Glu Ala Pro His Leu Val Val Thr Thr Pro

1025 1030 1035 1040

Asp Ile Glu Ala Leu Arg Pro Lys Ser Pro Met Lys Asn Gln Gln Gln

1045 1050 1055

Met Gln Gln Gln Gln Ser Ala Ser Ile Ser Leu Thr Ser Val Asn Ser

1060 1065 1070

Ser Asn Arg Arg Pro Gly Asn Ser Pro Gln Asn Pro Thr Gly Leu Ser

1075 1080 1085

Met Asn Gln Glu Ala Ser Ser Ser Asn Pro Ser Thr Pro Ala Pro Trp

1090 1095 1100

Ala Ala Ser Pro Leu Asp Gly Tyr Lys Gly Arg Glu Gly Ser Ile Met

1105 1110 1115 1120

Pro Ser Asp Asp Lys Ile Phe Asp Met Tyr Gly Tyr Ser Gly Gln Gly

1125 1130 1135

Asn Gly Asp Tyr Ile Asp Phe Glu Ala His Arg His Met Asn Leu Leu

1140 1145 1150

Claims

1. Use of a strawberry methyltransferase gene for controlling strawberry ripening, wherein the controlling strawberry ripening is to promote strawberry ripening by promoting expression of the methyltransferase gene; the methyltransferase is a protein of A1 or A2 as follows:

a1, the amino acid sequence is protein of an amino acid sequence shown as a sequence 2 in a sequence table;

a2, a fusion protein obtained by linking protein tags at the N-terminal or/and the C-terminal of A1.

2. Use of a strawberry methyltransferase gene for controlling strawberry maturation, wherein said controlling strawberry maturation is delaying strawberry maturation by inhibiting expression of said methyltransferase gene; the methyltransferase is a protein with an amino acid sequence shown as a sequence 2 in a sequence table.

3. The use according to claim 1 or 2, wherein the methyltransferase gene is specifically a DNA molecule whose coding sequence of the coding strand is sequence 1 in the sequence listing.

4. A method of delaying strawberry ripening comprising the steps of: inhibiting the expression of the methyltransferase of claim 2 and/or reducing the abundance of the methyltransferase of claim 2 in a recipient strawberry, resulting in a strawberry of interest having a maturity period later than the recipient strawberry.

5. A method for promoting strawberry ripening comprising the steps of: enhancing the expression of the methyltransferase of claim 1 and/or increasing the abundance of the methyltransferase of claim 1 in a recipient strawberry to yield a strawberry of interest having a maturity earlier than the recipient strawberry.