CN113999828A - Application of MTA and MTB genes of strawberry methyltransferase in controlling strawberry maturity - Google Patents

Abstract

The present invention provides the use of a strawberry methyltransferase gene encoding a methyltransferase for controlling strawberry maturation. The examples of the present invention demonstrate that methyltransferases MTA and MTB can alter the ripening process of fruits by regulating the color of fruits and the expression of key softening genes. The inhibition of the expression of the methyltransferase MTA gene or MTB gene in strawberry fruits can delay the fruit ripening process, while overexpression of the MTA gene or MTB gene can lead to premature fruit ripening. Therefore, the marketing time of strawberry fruits can be adjusted by reasonably utilizing the methyltransferase, and the strawberry production is served.

Description

Application of MTA and MTB genes of strawberry methyltransferase in controlling strawberry maturity

Technical Field

The invention relates to application of strawberry methyltransferase MTA and MTB genes in the field of biotechnology in controlling strawberry maturity.

Background

The strawberry fruit is rich in nutrition, delicious in taste and popular with the public. However, the production cost of strawberries is high due to the problems of concentrated supply seasons, difficult storage after collection, single variety and the like in the production of the strawberries at present. Therefore, finding key genes for strawberry maturation regulation and further applying genetic engineering to breed excellent strawberry varieties is an important way for solving the production problem of strawberries in the future.

N⁶-methyladenine (m)⁶A) Is the most common chemical modification of eukaryotic messenger RNA (mRNA), can enhance exon shearing, promote small RNA (miRNA) processing, reduce mRNA stability and improve translation efficiency of mRNA, and can regulate the expression level of genes at the post-transcriptional level. m is⁶A modification is a reversible process coordinated by RNA methyltransferase and RNA demethyltransferase. In mammals, m⁶Methyltransferases METTL3 and METTL14 in the a-modified complex as core components for mRNA binding and methyl catalysis at m⁶Plays a key role in the A modification. To date, no studies have been made on the functions of strawberry methyltransferase or demethylase genes.

Disclosure of Invention

The technical problem to be solved by the invention is how to control the mature period of the strawberry.

In order to solve the above technical problems, the present invention provides use of a strawberry methyltransferase gene encoding a methyltransferase which is a protein of a1 or a2 as follows for controlling strawberry maturation:

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

a2, protein tag linked to the N-terminus or/and C-terminus of A1).

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

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

In the above application, the methyltransferase gene may be specifically a gene represented by D1 or D2:

d1, the coding sequence of the coding chain is a DNA molecule of sequence 1 or sequence 3 in the sequence table;

d2, DNA molecule with the nucleotide sequence of sequence 1 or sequence 3 in the sequence table.

In the application, the strawberry maturation is controlled, and the strawberry maturation can be delayed by inhibiting or reducing the expression of the methyltransferase gene. Specifically, the method can be realized by transferring a methyltransferase gene to interfere an expression vector. The methyltransferase gene interference expression vector can be a recombinant expression vector obtained by connecting a methyltransferase gene to an interference expression vector. The interfering expression vector may be pK7GWIWG2D or pH7GWIWG 2D.

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

In order to solve the above technical problems, the present invention provides an agent for delaying the maturation of strawberry, the agent having as an active ingredient a substance that inhibits the expression of a gene encoding the methyltransferase, reduces the abundance of the methyltransferase, and/or knocks out the gene encoding the methyltransferase.

The active ingredient of the agent may also contain other biological or/and non-biological components, and the other active ingredients of the agent can be determined by the skilled person in the art according to the effect of delaying the ripening of the strawberry.

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

The active ingredient of the agent may also contain other biological or/and non-biological components, and the other active ingredients of the agent can be determined by those skilled in the art according to the effect of promoting the ripening of strawberry.

In order to solve the above technical problem, the present invention further provides a method for delaying strawberry ripening, comprising the following steps: inhibiting the expression of the methyltransferase in a recipient strawberry, reducing the abundance of the methyltransferase, and/or knocking out a gene encoding the methyltransferase, resulting in a strawberry of interest having a later maturity stage than the recipient strawberry.

In order to solve the above technical problem, the present invention further provides a method for promoting strawberry ripening, comprising the following steps: enhancing the expression of the methyltransferase and/or increasing the abundance of the methyltransferase in a recipient strawberry to obtain a strawberry of interest with a maturation stage earlier than the recipient strawberry.

The invention also provides a protein, being a methyltransferase, which is a protein of a1 or a2 as follows:

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

a2, protein tag linked to the N-terminus or/and C-terminus of A1).

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

d1, the coding sequence of the coding chain is a DNA molecule of sequence 1 or sequence 3 in the sequence table;

d2, DNA molecule with the nucleotide sequence of sequence 1 or sequence 3 in the sequence table.

In the invention, the controlling of the strawberry maturation period can be delaying of strawberry maturation or promoting of strawberry maturation.

The present invention provides the use of a substance that regulates the strawberry methyltransferase gene, which encodes a methyltransferase, in controlling the maturation phase of strawberries. The examples of the present invention demonstrate that methyltransferases MTA and MTB can alter the ripening process of fruits by regulating the color of fruits and the expression of key softening genes. The inhibition of the expression of the methyltransferase MTA gene or MTB gene in strawberry fruits can delay the fruit ripening process, while overexpression of the MTA gene or MTB gene can lead to premature fruit ripening. Therefore, the marketing time of strawberry fruits can be adjusted by reasonably utilizing the methyltransferase, and the strawberry production is served.

Drawings

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

FIG. 2 shows the expression of the strawberry MTA and MTB genes in fruit ripening in example 2. FIG. 2A is a graph showing the expression of MTA and MTB genes in diploid strawberry fruits at different maturity stages. In the figure, S6 represents fruits 15-20 days after the flower; RS1 is fruit 30 days after blossom; RS3 is 35 days after blossom. FIG. 2B is a graph showing the expression of MTA and MTB genes in the octaploid strawberry fruits at different maturity stages. In the figure, SG is small green fruit; wt is ginkgo seed; IR is color-changing fruit; FR is fully ripe fruit.

Fig. 3 is a graph of fruit phenotype transiently interfering with MTA or MTB expression in the octaploid strawberries of example 3. After injecting Agrobacterium containing the control vector, RNAi-MTA or RNAi-MTB vector, respectively, into strawberry fruits (day 0 is the day of injection), the RNAi fruits show a delayed maturation phenotype at day 7.

FIG. 4 is a fruit phenotype plot of transient overexpression of MTA or MTB in the octaploid strawberry of example 4. After injection of the Agrobacterium containing the control vector, OE-MTA or OE-MTB vector, respectively, into the strawberry fruit (day 0 is the day of injection), the OE fruit displays an accelerated ripening phenotype on day 5.

FIG. 5 shows the gene expression profiles of MTA-RNAi fruit and MTB-RNAi fruit in example 5. Data are presented as mean ± sd, with 3 repeats, indicating a significance analysis result of P ≦ 0.05, and P ≦ 0.01.

FIG. 6 shows the gene expression profiles of OE-MTA fruit and OE-MTB fruit in example 5. Data are presented as mean ± sd, with 3 repeats, indicating a significance analysis result of P ≦ 0.05, and P ≦ 0.01.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.

1 vector

Vector pMD in the following examples^TM19-T (cat # 6013) is a product of TaKaRa Co.

The following examples

8/GW/

Vector cloning Vector (cat # k250020) was manufactured by Invitrogen corporation.

The vector pK7GWIWG2D in the examples described below is described in the documents "Cai J, Qin G, Chen T, et al, the mode of action of remorin1 in regulating free dependent at a transcriptional and post-translational levels [ J ]. NEW PHYTOLOGIST,2018, 9", publicly available from the plant research institute of Chinese academy of sciences.

The vector pCambia 2300 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 the lateral dependence at a translational and post-translational levels [ J ]. NEW PHYTOLOGIST,2018, 9", publicly available from the plant research institute of Chinese academy of sciences.

2 plant lines

The strawberry variety ` Hawaii 4 ` in the following examples is described in non-patent documents "Luo H, Dai C, Li Y, et al reduced Anthocyanocyanines in peptides codes for a GST anticancer in transporter at a site for the food and front color in strawberry. [ J ]. JOURNAL OF EXPERIMENTAL BOTANY,2018, 3". The public is available from the plant institute of the Chinese academy of sciences to repeat the experiments of the present application and not for other uses.

The strawberry variety ` Hongyan ` in the following examples is described in non-patent document "Riaz A, Aardil RM, Amoussa AMO, et al. application OF the chip-based apple polyphenol coating on the PRESERVATION OF the strawberry (Fragaria ananassa cv Hongyan) from JOURNAL OF FOOD PROCESSING AND 2021.DOI:10.1111/jfpp.15018. The public is available from the plant institute of the Chinese academy of sciences to repeat the experiments of the present application and not for other uses.

3 reagent

In the following examples, LB solid medium containing 100mg/L ampicillin was prepared (in 200mL): solid LB culture medium (200mL), yeast extract 1g, trypsin 2g, sodium chloride 2g, agar powder 3g, dissolved in 200mL deionized water, 120 ℃ high pressure sterilization for 20min, when the culture medium temperature is reduced to 50 ℃, adding 200. mu.L of ampicillin (100mg/mL) into a clean bench, mixing, pouring into a culture dish, solidifying and storing at 4 ℃.

In the following examples, LB liquid medium containing 50mg/L kanamycin, 50mg/L rifampicin, and 50mg/L streptomycin was prepared as follows (in 200mL): solid LB medium (200mL), yeast extract 1g, trypsin 2g, sodium chloride 2g, dissolved in 200mL deionized water, 120 ℃ autoclaving for 20min, when the medium temperature is reduced to 50 ℃, 100. mu.L kanamycin (50mg/mL), 100. mu.L rifampicin (50mg/mL), 100. mu.L streptomycin (50mg/mL) are added to the clean bench, mixed, and stored at 4 ℃.

In the following examples, the transformation liquid was prepared by the following method: mixing MgCl₂2- (N-morpholine) ethanesulfonic acid (MES) and acetosyringone in deionized water to obtain a solution with pH of 5.6, MgCl in the conversion solution₂Has a concentration of 10mM, MES concentration of 10mM and acetosyringone concentration of 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 cDNA sequences of strawberry MTA and MTB genes

Total RNA of wild strawberry (Fragaria vesca, 'Hawaii 4') fruits was extracted using a plant Total RNA isolation kit (Novozan Biotech., Nanjing), and after quantification by SimpliNano (thermo), 1. mu.g of each total RNA was taken, and a first strand cDNA was synthesized using a reverse transcription kit (PrimeScriptRT reagentKit, Takara, Dalian).

Designing specific primers of MTA gene according to published wild strawberry genome sequence information:

MTA-F:5′-ATGGAGGAGACCCAATCTGG-3′；

MTA-R:5′-TCCGGCCATCTCAACATCG-3′；

design of primers specific for the MTB gene:

MTB-F:5′-ATGGATTCGCCTGAACGTAG-3′；

MTB-R:5′-CAACAAATTCATGTGCCTGTGAG-3′。

the primers were synthesized by Biotech, Inc. of Ongbenaceae, Beijing.

The MTA and MTB genes were amplified separately using High fidelity enzyme (I-5TM2x High-Fidelity MasterMix, Scophtalae).

The PCR reaction system is as follows: cDNA template 100ng, forward and reverse primers (10. mu.M) 2. mu.L, I-5TM2x High-Fidelity MasterMix 25. mu.L, water to 50. mu.L.

The PCR reaction program was set up as follows: 2min at 98 ℃; 35 cycles of 98 ℃ for 10s, 56 ℃ for 15s and 72 ℃ for 15 s; final extension at 72 ℃ for 5 min.

The amplification products were electrophoresed on 1% agarose gel, and as a result, as shown in FIG. 1, it was found that the amplification products of MTA-F and MTA-R had a specific band of interest at 2000bp, and the amplification products of MTB-F and MTB-R had a specific band of interest at 3000 bp.

The amplified bands were collected separately using a DNA gel collection kit (Novovodka Biotech., Nanjing).

The purified MTA DNA piece was purified using DNA Ligation Kit (TaKaRa, Dalian)Fragment and vector pMD^TM19-T (TaKaRa, Dalian) was ligated according to the principle of TA cloning to construct pMD19T-MTA recombinant plasmid. The Escherichia coli TOP10 strain (organisms of the family Populaceae, Beijing) transformed by the pMD19T-MTA plasmid is screened on LB solid medium containing 100mg/L ampicillin, a positive single colony is selected, and the pMD19T-MTA recombinant plasmid sequence is detected by using M13-F universal sequencing primer.

M13-F：5′-ACTGGCCGTCGTTTTACAACG-3′。

The purified MTB DNA fragment and the vector pMD were ligated using the DNA Ligation Kit (TaKaRa, Large Scale)^TM19-T (TaKaRa, Dalian) was ligated according to the principle of TA cloning to construct a pMD19T-MTB recombinant plasmid. The Escherichia coli TOP10 strain (organisms of the family Populaceae, Beijing) transformed with pMD19T-MTA plasmid was selected on LB solid medium containing 100mg/L ampicillin, a positive single colony was picked, and the pMD19T-MTB recombinant plasmid sequence was detected with M13-F universal sequencing primer.

The results show that we obtained cDNA sequences of the MTA and MTB genes of strawberry, since the genes are identical to the published gene sequences: the cDNA sequence of MTA is shown as sequence 1 in the sequence table, the locus number is gene01911(ftp:// ftp. biolin. fo. wsu. edu/species/Fragaria _ vesca/Fvesca-genome. v2.0.a2), 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. biolin. fo. wsu. edu/species/Fragaria _ vesca/Fvesca-genome. v2.0.a2), and the encoded protein contains 1152 amino acid residues (shown as sequence 4 in the sequence table).

Example 2 characterization of the expression of the 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 ripeness degrees according to the distance of the strawberry fruit from the days after flowering (see panel a of fig. 2): green fruit stage (S6), approximately 15-20 days after flowering; ginkgo period (RS1), fruits 30 days after flowering; yellow fruit stage (RS6), fruit 35 days after flowering. After the fruits were picked, they were immediately frozen with liquid nitrogen and transported back to the laboratory.

According to the method of example 1, three strawberry samples of different maturity were RNA extracted and reverse transcribed into cDNA, and the cDNA concentration was further diluted 20-fold for use as a template for fluorescent quantitative PCR reactions. Changes in expression of MTA and MTB genes were detected on a fluorescent quantitative PCR instrument (STEPONE PLUS, ABI, USA) using a fluorescent quantitation reagent (SYBRPremix Ex TaqTM, TaKaRa), respectively.

The primers used for detecting the 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: mu.L of template, 10. mu.L of SYBR Mix, 0.8. mu.L of forward and reverse primers, 0.8. mu.L of Rox, and sterile double distilled water to make up to 20. mu.L.

The reaction procedure is as follows: 10min at 98 ℃; 15S at 95 ℃, 30S at 60 ℃ and 40 cycles; 5min at 72 ℃; and (4) at 95 ℃ for 1min, and analyzing a melting curve as a default setting of the system.

As shown in the A diagram of FIG. 2, the expression of MTA gene is gradually increased and then decreased along with the increase of the strawberry maturity, the expression level is highest in the RS1 stage, and the MTB gene is continuously increased along with the increase of the maturity, which indicates that MTA and MTB are possibly involved in the regulation of strawberry fruit maturity.

2. Expression of methyltransferase MTA and MTB genes in octaploid strawberries

Fruits of strawberries (Fragaria ananassa cubvar 'Hongyan') were picked from a strawberry base (commercial picking garden) greenhouse and divided for fruit maturity according to the following characteristics (as in panel B of fig. 2): small green fruit (SG) is characterized by dark green peel and green seeds; gingko (Wt) is characterized in that the peel turns white and the seeds turn brown; color-changing fruits (IR) are characterized in that one third of the surface of the peel is red, and the seeds are all brown; the fully ripe Fruit (FR) is characterized by a red overall peel surface and a dark brown overall seed. After the fruits were picked, they were immediately frozen with liquid nitrogen and transported back to the laboratory.

Changes in expression of the MTA and MTB genes in the octaploid strawberries were detected separately according to the method of step 1 above.

As shown in the B diagram of FIG. 2, the expression of MTA and MTB genes gradually increases and then decreases with the increase of the strawberry maturity, and the expression levels are highest in the IR stage, which indicates that MTA and MTB are possibly involved in the strawberry fruit maturation control.

Example 3 construction of strawberry MTA and MTB interference expression vectors 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 sequence of the strawberry MTA gene, selecting a specific region with a conserved structural domain of the methyltransferase gene removed, and designing upstream and downstream primers MTA-RNAi-F and MTA-RNAi-R which can amplify the region:

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

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

the primers were synthesized by Biotech, Inc. of Ongbenaceae, Beijing.

The pMD19T-MTA plasmid was amplified using High Fidelity enzyme (I-5TM2x High-Fidelity MasterMix, Scophyton).

The PCR reaction system is as follows: pMD19T-MTA plasmid template 100ng, forward and reverse primers (10. mu.M) 2. mu.L, I-5TM2x High-Fidelity Master Mix 25. mu.L, water to 50. mu.L.

The PCR reaction program was set up as follows: 2min at 98 ℃; 35 cycles of 98 ℃ for 10s, 56 ℃ for 15s and 72 ℃ for 15 s; final extension at 72 ℃ for 5 min.

Recovering and purifying the desired band, ligating the DNA fragment

8/GW/

Vector cloning Vector (Invitrogen). Using LR Recombinase (LR)Clonase II enzyme mix, Invitrogen) was ligated into pK7GWIWG2D vector by the method of GATEWAY cloning. An MTA interference expression vector is formed by escherichia coli transformation, antibiotic screening, double enzyme digestion identification and sequence determination, and is named as RNAi-MTA.

1.2 construction of MTB interference expression vectors

According to the strawberry MTB gene sequence, selecting a specific region for removing the conserved domain of the methyltransferase gene, and designing an upstream primer and a downstream primer capable of amplifying the 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′。

the primers were synthesized by Biotech, Inc. of Ongbenaceae, Beijing.

The pMD19T-MTB plasmid was amplified using High Fidelity enzymes (I-5TM2x High-Fidelity MasterMix, Scophyton).

The PCR reaction system is as follows: pMD19T-MTB plasmid template 100ng, forward and reverse primers (10. mu.M) 2. mu.L, I-5TM2x High-Fidelity Master Mix 25. mu.L, water to 50. mu.L.

The PCR reaction program was set up as follows: 2min at 98 ℃; 35 cycles of 98 ℃ for 10s, 56 ℃ for 15s and 72 ℃ for 15 s; final extension at 72 ℃ for 5 min.

Recovering and purifying the desired band, ligating the DNA fragment

8/GW/

Vector cloning Vector (Invitrogen). The pK7GWIWG2D vector was ligated by the method of GATEWAY cloning using LR recombinase (LR clone II Ezyme mix, Invitrogen). And forming an MTB interference expression vector named RNAi-MTB through escherichia coli transformation, antibiotic screening, double enzyme digestion identification and sequence determination.

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

(ii) isolation of RN by Freeze-thawingThe Ai-MTA and RNAi-MTB recombinant expression vectors are respectively transformed into Agrobacterium GV3101, positive clones are picked and inoculated into 50mL LB liquid medium containing 50mg/L kanamycin, 50mg/L rifampicin and 50mg/L streptomycin, and shaken at 200rpm (rpm) at 28 ℃ to OD₆₀₀The cells were centrifuged at 5000g for 10min at 0.6 g to collect the cells. Suspending thalli by using 50mL of transformation liquid, and centrifuging for 10min at 5000g under the condition of room temperature to collect bacterial liquid; then 50mL of transformation liquid is used for suspending the thalli again, and the thalli is kept stand for more than 2h at room temperature, so that the agrobacterium liquid for transforming RNAi-MTA and the agrobacterium liquid for transforming RNAi-MTB are obtained respectively.

And respectively infecting the fruits of the strawberry 'Hongyan' with the agrobacterium liquid for transforming RNAi-MTA and the agrobacterium liquid for transforming RNAi-MTB by an injection method. The specific method comprises the following steps: injecting the strawberry fruit in the green fruit period at the central axis of the fruit by using a 1ml sterile micro-injector until the fruit is in a water stain shape. Among them, the fruit injected with the Agrobacterium solution transformed with RNAi-MTA is called MTA-RNAi fruit, and the fruit injected with the Agrobacterium solution transformed with RNAi-MTB is called MTB-RNAi fruit. The injection results were kept at room temperature with a relative humidity of 70-90% and observed daily.

The results are shown in fig. 3, and compared to the control strawberry 'Hongyan' fruit transformed with the empty vector pK7GWIWG2D, the coloration of both the strawberry fruit transformed with the RNAi-MTA vector (MTA-RNAi fruit) and the strawberry fruit transformed with the RNAi-MTB vector (MTB-RNAi fruit) after injection is significantly slowed down, suggesting that transient MTA-RNAi or MTB-RNAi interference can delay the ripening of the octaploid strawberry fruit.

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

1. Construction of MTA and MTB overexpression vectors.

1.1 construction of MTA overexpression vector

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

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

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

a DNA fragment for ligation with pCambia 2300 vector was amplified using pMD19T-MTA recombinant plasmid of example 1 as a template.

The PCR reaction system is as follows: pMD19T-MTA plasmid template 100ng, forward and reverse primers (10. mu.M) 2. mu.L, I-5TM2x High-Fidelity Master Mix 25. mu.L, water to 50. mu.L.

The PCR reaction program was set up as follows: 2min at 98 ℃; 35 cycles of 98 ℃ for 10s, 56 ℃ for 15s and 72 ℃ for 15 s; final extension at 72 ℃ for 5 min.

After the amplified fragment was purified, it was ligated to pCambia 2300 vector using homologous recombinase (In-Fusion HD Cloning, TaKaRa, Dalian) according to the homologous recombination Cloning principle to form OE-MTA vector.

1.2 construction of MTB overexpression vector

Based on the cDNA sequence of MTB (sequence No. 3 in the sequence table) and the sequence of a plant expression vector pCambia 2300, specific primers MTB-2300-F and MTB-2300-R for amplifying the MTB full-length gene (without a termination codon sequence TAA) were designed:

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

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

a DNA fragment for ligation with pCambia 2300 vector was amplified using pMD19T-MTB recombinant plasmid of example 1 as a template.

The PCR reaction system is as follows: pMD19T-MTB plasmid template 100ng, forward and reverse primers (10. mu.M) 2. mu.L, I-5TM2x High-Fidelity Master Mix 25. mu.L, water to 50. mu.L.

The PCR reaction program was set up as follows: 2min at 98 ℃; 35 cycles of 98 ℃ for 10s, 56 ℃ for 15s and 72 ℃ for 15 s; final extension at 72 ℃ for 5 min.

After purification, the amplified fragment was ligated to pCambia 2300 vector using homologous recombinase (In-Fusion HD Cloning, TaKaRa, Mass.) according to the homologous recombination Cloning principle to form OE-MTB vector.

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

OE-MTA Agrobacterium solution and OE-MTB Agrobacterium solution were prepared according to the Agrobacterium transformation method of example 3. The fruit of strawberry 'Hongyan' was transformed with the above-mentioned Agrobacterium solution according to the transient transformation method for strawberry fruit of example 3. Wherein, the fruit injected with OE-MTA agrobacterium liquid is called OE-MTA fruit, and the fruit injected with OE-MTB agrobacterium liquid is called OE-MTB fruit.

As shown in FIG. 4, compared to the fruits of control strawberry 'Hongyan' transferred with empty vector (pCambia 2300), both the strawberry fruit transferred with OE-MTA (OE-MTA fruit) and the strawberry fruit transferred with OE-MTB vector (OE-MTB fruit) were significantly accelerated in coloring after injection, suggesting that the maturation of the octaploid strawberry fruit can be accelerated by the transient overexpression of MTA or MTB gene.

Example 5 molecular testing of MTA and MTB transgenic fruits

The sensory quality of strawberry fruits is determined by color and texture, and usually, the strawberry with bright color and soft texture has better quality, so that the expression of color and softening key enzyme genes is an important molecular characteristic for measuring the quality change of the strawberries. 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 were subjected to RNA extraction and reverse transcription into cDNA according to the method of example 1, and the expression of MTA, MTB, anthocyanin synthesis key enzyme gene (CHS) and cell wall metabolism key enzyme gene (PG1) in the MTA-RNAi fruit, MTB-RNAi fruit, OE-MTA fruit and OE-MTB fruit was examined by real-time fluorescent quantitative RT-PCR according to the method and primers of example 2.

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 PG 1-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 of the RNAi-transferred empty vector pK7GWIWG2D, the MTA-RNAi fruit inhibiting MTA significantly inhibited the expression of MTA gene, softening key enzyme gene PG1 and color key enzyme gene CHS significantly reduced the expression of MTB gene, and the MTB-RNAi fruit inhibiting the expression of MTB gene significantly inhibited the expression of MTB gene, and significantly reduced the expression of PG1 and CHS.

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

In conclusion, the methyltransferases MTA and MTB can change the fruit ripening process by regulating the fruit color and softening the expression of key genes. The fruit ripening process can be delayed by inhibiting the expression of the MTA gene or MTB gene in strawberry fruits, and the strawberry fruits are ripened in advance by over-expressing the MTA gene or MTB gene. Therefore, the gene can be reasonably utilized to adjust the marketing time of strawberry fruits, thereby serving for strawberry production.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of plant of Chinese academy of sciences

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

<130> GNCSY210098

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2226

<212> DNA

<213> strawberry (Fragaria visco)

<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 visco)

<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 visco)

<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 visco)

<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 (10)

1. Use of a strawberry methyltransferase gene for controlling strawberry maturation, wherein the methyltransferase is a protein of a1 or a2 as follows:

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

a2, and a fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of A1.

2. Use according to claim 1, wherein the methyltransferase gene is in particular the gene D1 or D2:

d1, the coding sequence of the coding chain is a DNA molecule of sequence 1 or sequence 3 in the sequence table;

d2, DNA molecule with the nucleotide sequence of sequence 1 or sequence 3 in the sequence table.

3. The use according to claim 1 or 2, wherein the controlling of strawberry maturation is delaying of strawberry maturation by inhibiting or reducing the expression of the methyltransferase gene of claim 1.

4. The use according to claim 1 or 2, wherein the controlling of strawberry maturation is promoting of strawberry maturation by promoting or enhancing the expression of the methyltransferase gene of claim 1.

5. An agent for delaying the maturation of strawberry, wherein the active ingredient of the agent is a substance that inhibits the expression of a gene encoding the methyltransferase of claim 1, decreases the abundance of the methyltransferase of claim 1, and/or knocks out a gene encoding the methyltransferase of claim 1.

6. An agent for promoting strawberry maturation, wherein the active ingredient of the agent is a substance that enhances the expression of a gene encoding the methyltransferase of claim 1 and/or increases the abundance of the methyltransferase of claim 1.

7. A method for delaying the ripening of strawberries, which is characterized by comprising the following steps: inhibiting the expression of the methyltransferase of claim 1 in a recipient strawberry, reducing the abundance of the methyltransferase of claim 1, and/or knocking out a gene encoding the methyltransferase of claim 1 to yield a strawberry of interest with a later maturity than the recipient strawberry.

8. A method for promoting strawberry ripening is characterized by comprising the following steps: enhancing the expression of the methyltransferase of claim 1 in a recipient strawberry and/or increasing the abundance of the methyltransferase of claim 1 to obtain a strawberry of interest with a maturation stage earlier than that of the recipient strawberry.

9. Protein, characterized in that it is a protein of a1 or a2 as follows:

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

a2, and a fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of A1.

10. Nucleic acid molecule encoding a protein according to claim 9, in particular a nucleic acid molecule as depicted in D1 or D2 as follows:

d1, the coding sequence of the coding chain is a DNA molecule of sequence 1 or sequence 3 in the sequence table;

d2, DNA molecule with the nucleotide sequence of sequence 1 or sequence 3 in the sequence table.

Images