Strawberry PAL2 mutant gene and application thereof

Abstract

The invention discloses a strawberry PAL2 mutant gene and application thereof. Compared with the nucleotide sequence of a wild strawberry PAL2 gene, the nucleotide sequence of the PAL2 mutant gene comprises one or two of the following mutations: 1) C1054T and 2) G371A. The PAL2 mutant gene is introduced into strawberries, so that the strawberries with pink fruits and reduced anthocyanin content can be obtained, the PAL2 mutant gene can be used for selective breeding of the pink strawberries with reduced anthocyanin content and identification of the pink strawberries, the genetic improvement process of pink strawberry varieties can be accelerated, and the yield of the strawberries is increased. The method has important theoretical and practical guiding significance for improving the breeding selection efficiency.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, in particular to a strawberry PAL2 mutant gene and application thereof. Background Art

[0002] Strawberry fruits are loved by consumers because they are rich in nutrients and delicious. The color of strawberry fruits is an important factor affecting consumers' market choices. At present, the main strawberry varieties grown in China, such as Hongyan, Xiangye, and Zhangji, are all from Japan, and the color of these fruits is red. With the diversification of consumer demand, special strawberry varieties with pink or white fruit colors have been deeply loved by consumers in recent years.

[0003] As an important component of the appearance quality of strawberry fruit, color determines its commercial value to a certain extent. In view of this value pursuit, breeders have also bred a number of pink or white varieties. For example, the "Pink Jade" bred by the Hangzhou Academy of Agricultural Sciences is a pink variety. Due to its unique color and taste, this variety is the preferred specialty variety by strawberry growers. In addition, breeders have also bred strawberry varieties with darker colors, which are usually called black strawberries. For example, "Black Pearl" and "True Red Meiling" belong to this type of strawberry variety.

[0004] With the introduction of new varieties and the progress of breeding, consumers' demand for the color diversity of fresh strawberry fruits continues to increase. However, there are few genes that have been reported to regulate the color of strawberries. For example, after the FvMYB10 gene in diploid forest strawberries mutates, its fruit appears white; studies in cultivated strawberries have also found that FaMYB10 is the main QTL locus that controls the color of strawberry fruits; for example, CN115058399 discloses that a single point mutation of F3H can make strawberry fruits change from red to pink; and CN110819603 discloses that knocking out the RAP gene in octoploid strawberries can make the fruit appear white. These studies provide good candidate genes for creating strawberry varieties with different fruit colors through biotechnology.

[0005] The different phenotypes of strawberry fruit color are mainly determined by the different levels and components of anthocyanidins in strawberry fruits of different genotypes. Anthocyanidins or anthocyanins in plants are synthesized from phenylalanine as a direct precursor through a series of enzyme-catalyzed reactions (see Fig.11). In this pathway, phenylalanine is converted into 4-coumaryl CoA under the action of phenylalanine ammonia lyase (PAL), cinnamyl-4-hydroxylase (C4H) and 4-coumarate: CoA ligase (4CL), and then chalcone is formed with malonyl CoA under the catalysis of chalcone synthase (CHS), thus forming the basic skeleton of anthocyanidins. Next, naringenin is generated under the action of chalcone isomerase, and dihydroflavonol is generated under the catalysis of flavanone-3-hydroxylase (F3H). Subsequently, a portion of the dihydroflavonols is converted into dihydrocodole under the action of flavonoid 3'-hydroxylase (F3'H) and enters the cyanidin synthesis branch; another portion of the dihydroflavonols is converted into dihydromyricetin under the action of flavonoid 3',5'-hydroxylase (F3'5'H) and enters the delphinidin synthesis branch. Furthermore, dihydroflavonols can also enter the pelargonidin synthesis branch under the catalysis of dihydroflavonol 4-reductase (DFR). Each branch then synthesizes three anthocyanidins, namely pelargonidin, delphinidin and cyanidin, under the catalysis of anthocyanidin synthase (ANS) or leucoanthocyanidin dioxygenase (LDOX). Subsequently, glycosylation modification is completed by UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT), methylation modification and acetyl modification are completed by oxygen-methyltransferase (OMTs) and acetyltransferase (Acethy transferase, ACTs), respectively, ultimately generating different types of stable anthocyanins, which make the plants present different colors.

[0006] Although studies have shown that interfering with the gene expression level of UFGT or CHS in strawberries can affect the accumulation of anthocyanins in fruits. However, these existing findings have focused on changing the expression of related enzyme genes to affect anthocyanin synthesis, and have not studied the key sites and functions of related genes. As for strawberries, although there are many strawberry varieties such as red, pink, white, and black and red, there are few studies on the key genes that control related color phenotypes. Therefore, exploring genes that control color can lay a theoretical foundation and clear target genes for creating new strawberry varieties of different colors through molecular breeding. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a strawberry PAL2 mutant gene and use thereof.

[0008] To achieve the above objectives and other related objectives, the present invention is achieved through the following technical solutions.

[0009] The first aspect of the present invention protects a strawberry PAL2 mutant gene, wherein the nucleotide sequence of the PAL2 mutant gene comprises one or two of the following mutations compared with the nucleotide sequence of the wild-type strawberry PAL2 gene: 1) C1054T, 2) G371A.

[0010] In certain embodiments, the nucleotide sequence of the wild-type strawberry PAL2 gene comprises SEQ ID NO.1.

[0011] In certain embodiments, the nucleotide sequence of the PAL2 mutant gene comprises the sequence shown in SEQ ID NO.3 or SEQ ID NO.5.

[0012] The second aspect of the present invention protects biological materials related to the PAL2 mutant gene as described above, and the biological materials are any of the following: a) a recombinant expression vector containing the nucleotide sequence as described above; b) a bioengineering bacterium containing the nucleotide sequence as described above, or a bioengineering bacterium containing the recombinant expression vector as described in a); c) a protein encoded by the nucleotide sequence as described above.

[0013] In certain embodiments, the amino acid sequence of the protein comprises the sequence shown in SEQ ID NO.4 or SEQ ID NO.6.

[0014] The third aspect of the present invention protects the use of the PAL2 mutant gene as described above or the biological material as described above in any of the following:

[0015] A1) Regulates the synthesis of strawberry anthocyanins;

[0016] A2) Regulate the color of strawberry organs;

[0017] A3) Develop a reagent for identifying the color of strawberry organs;

[0018] A4) Strawberry breeding or germplasm improvement;

[0019] A5) preparing a strawberry assisted breeding kit;

[0020] A6) Preparation of transgenic strawberries.

[0021] The fourth aspect of the present invention protects the amplification primers for the PAL2 mutant gene as described above, comprising primers with sequences shown in SEQ ID NO.7 and SEQ ID NO.8.

[0022] The fifth aspect of the present invention protects a method for identifying pink strawberries, comprising the following steps:

[0023] 1) Identifying whether the strawberry seeds or strawberry seedlings to be tested contain the PAL2 mutant gene as described above; or,

[0024] 2) Identify whether the strawberry seeds or strawberry seedlings to be tested express the protein described above.

[0025] The sixth aspect of the present invention protects a method for inhibiting anthocyanin synthesis in strawberries, comprising introducing the PAL2 mutant gene as described above into strawberries to obtain strawberries with inhibited anthocyanin synthesis.

[0026] A seventh aspect of the present invention protects a method for cultivating strawberries with pink fruits, which comprises cultivating strawberries containing the PAL2 mutant gene as described above to obtain strawberries with pink fruits.

[0027] In certain embodiments, the method further comprises breeding, and the breeding comprises the following steps: hybridizing the strawberries with inhibited anthocyanin synthesis and / or strawberries with pink fruits or strawberries with green petioles as parents.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The invention discloses a gene related to the synthesis of strawberry anthocyanins, phenylalanine ammonia lyase (PAL2), and its mutants. Six non-synonymous mutations affecting the amino acid coding sequence were identified in the mutant rg120 obtained by EMS mutagenesis by Mutmap analysis method. Among them, the PAL2 gene on the Fvb6 chromosome is a key gene related to the synthesis of anthocyanins in strawberry. When the 1054th nucleotide of the PAL2 coding region mutates from C to T, a point mutation is caused, resulting in the premature termination of amino acid translation. At the same time, in the rg40 mutant, it is found that when the 371st nucleotide of the PAL2 coding region mutates from G to A, a point mutation is caused, resulting in the mutation of glycine at the 124th position to aspartic acid. These mutation forms will block the synthesis of anthocyanins, making the strawberry fruit pink and the petiole yellow-green. Molecular markers are developed based on the key mutation sites of candidate genes, which can realize the rapid, efficient and accurate identification of red strawberry / pink strawberry materials, and have a promoting effect on the selection and breeding of new strawberry varieties and molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shown is the phenotypic analysis of the wild-type RG, mutant rg40, mutant rg120, and hybrid F1 generation (rg40♂×rg120♀) in Example 1 of the present application. Among them, A is the phenotype of wild-type RG strawberry plants; B is the phenotype of mutant rg40 strawberry plants; C is the phenotype of mutant rg120 strawberry plants; D is the phenotype of the hybrid F1 generation plants of mutant rg40 and mutant rg120; E is the petiole phenotype of wild-type RG, mutant rg40, mutant rg120 and F1 generation plants; F is the fruit phenotype of wild-type RG, mutant rg40, mutant rg120 and hybrid F1 generation; G is the result of the determination of anthocyanin content in petioles of wild-type RG, mutant rg40, mutant rg120 and hybrid F1 generation; H is the result of the determination of anthocyanin content in fruits of wild-type RG, mutant rg40, mutant rg120 and hybrid F1 generation. The scale bars in E and F are 1 cm.

[0031] Figure 2 Shown is the distribution diagram of the whole genome SNP-Index in the mutant pool analyzed by BSA-seq in Example 2 of the present application.

[0032] Figure 3 The figure shows the identification of the mutant gene of the mutant rg120 in Example 2 of the present application. A is the distribution of the SNP-Index value in the mutant pool of the mutant rg120 in chromosome VI; B is a schematic diagram of the structure of PAL2 in the wild-type RG and the mutant rg120. SNP2 is located in the second exon of the PAL2 gene; the mutation from base C to base T results in the conversion of glutamine to a stop codon.

[0033] Figure 4 Shown are the results of agarose gel electrophoresis detection of the PAL2 (CDS region) gene of the mutant rg40 in Example 2 of the present application.

[0034] Figure 5 Shown is an alignment diagram of the PAL2 protein sequences of the wild-type RG, mutant rg40 and mutant rg120 in Example 2 of the present application.

[0035] Figure 6 It is shown that the expression of PAL2 in Example 3 of the present application can complement the fruit color phenotype of the mutant rg120. Among them, A is a real shot of the phenotype of the mutant rg120 fruit after injection of 35S::PAL2-YFP (upper) and 35S::YFP (lower); B is a result diagram of the relative content of anthocyanins after the mutant rg120 fruit was injected with 35S::PAL2-YFP and 35S::YFP; C is a result diagram of the PAL2 gene expression level after the mutant rg120 fruit was injected with 35S::PAL2-YFP and 35S::YFP. The scale bar in A is 1 cm. DAI: Days after injection.

[0036] Figure 7 The wild-type PAL1 protein (PAL1 RG -His), RG wild-type PAL2 protein (PAL2 RG -His), rg40 mutant PAL2 protein (PAL2 rg40 -His), rg120 mutant PAL2 protein (PAL2 rg120 -His) Coomassie blue staining.

[0037] Figure 8 The HPLC results of the reaction of RG wild-type PAL1 protein, RG wild-type PAL2 protein, rg40 mutant PAL2 protein, and rg120 mutant PAL2 protein with the substrate L-phenylalanine in Example 4 of the present application are shown. RG represents wild-type PAL1 protein; PAL2 RG Represents wild-type PAL2 protein; mPAL2 rg40 PAL2 protein representing mutant rg40; mPAL2 rg120 Represents the PAL2 protein of mutant rg120; L-phenylalanine represents L-phenylalanine; trans-cinnamic acid represents trans-cinnamic acid; blue arrows indicate the peaks corresponding to phenylalanine and trans-cinnamic acid.

[0038] Fig. 9The results of the protease activity assays of RG wild-type PAL1 protein, RG wild-type PAL2 protein, rg40 mutant PAL2 protein, and rg120 mutant PAL2 in Example 4 of the present application are shown. RG represents wild-type PAL1 protein; PAL2 RG Represents wild-type PAL2 protein; mPAL2 rg40 PAL2 protein representing mutant rg40; mPAL2 rg120 Represents the PAL2 protein of mutant rg120.

[0039] Fig.10 The diagram shows the comparison analysis of the amino acid sequences of PAL between different species in Example 4 of the present application, wherein the arrows indicate the glycine at position 124 and the glutamine at position 352.

[0040] Fig.11 Shown is a schematic diagram of the anthocyanin synthesis pathway. DETAILED DESCRIPTION

[0041] The inventor accidentally discovered that after the strawberry phenylalanine ammonia lyase (PAL2) mutated, the color of the organs of the obtained strawberry mutant became lighter, the fruit color changed from red to light red (pink), the anthocyanin content decreased, and the color of the petiole changed from purple red to yellow green. Then, by complementing the recombinant expression vector expressing PAL2 in the strawberry mutant, it was found that the strawberry mutant restored the properties of the wild-type strawberry, the light red strawberry turned red, the anthocyanin content in the fruit increased, and the expression level of the PAL2 gene increased. Further, the wild-type PAL2 protein and the mutated protein were subjected to enzyme activity experiments in vitro, and it was found that the wild-type PAL2 protein had a higher enzyme activity, while the enzyme activity of the mutated PAL2 protein was significantly reduced. In addition, the present invention also compared and analyzed the PAL2 proteins of various plants, and found that the 124th glycine (G) and the 352nd glutamine (Q) of the PAL2 protein were both conserved sites. Therefore, the research on the PAL2 gene in the present invention provides a new gene resource for the subsequent regulation of strawberry organ color or regulation of strawberry anthocyanin synthesis, and can cultivate strawberry varieties with low anthocyanin content or lighter organ color through targeted changes to the PAL2 gene, providing gene site information for molecular breeding of strawberries and providing new options for strawberry variety selection, which is conducive to accelerating the promotion of strawberries with reduced anthocyanin content or lighter organ color, and has important application value in accelerating the strawberry breeding process.

[0042] The first aspect of the present invention protects a strawberry PAL2 mutant gene, the nucleotide sequence of which contains one or two of the following mutations compared to the nucleotide sequence of the wild-type strawberry PAL2 gene: 1) C1054T, 2) G371A. Compared with the wild-type strawberry PAL2, the protein encoded by the strawberry PAL2 mutant gene provided by the present invention cannot convert the substrate L-phenylalanine into cinnamic acid, resulting in the inhibition of anthocyanin synthesis and the production of strawberries with pink fruits, yellow-green petioles and reduced anthocyanin content. Therefore, by introducing the mutant strawberry PAL2 gene into strawberries, strawberries with pink fruits and reduced anthocyanin content can be obtained, meeting the market's demand for diversified strawberry varieties.

[0043] In certain embodiments, the nucleotide sequence of the wild-type strawberry PAL2 gene comprises SEQ ID NO.1.

[0044] The nucleotide sequence of PAL2 comprises the sequence shown in SEQ ID NO.1, and the encoded amino acid sequence comprises the sequence shown in SEQ ID NO.2.

[0045] PAL2 nucleotide sequence, SEQ ID NO.1.

[0046]

[0047] PAL2 amino acid sequence, SEQ ID NO.2.

[0048] METVTQNGYHQNGIQNGSLDILCVKTESLKAVSDPLNWGAAAESMTGSLDEVKRMVAEYRKPVVKLGGKTLTIAQVAAIANHDSGVKVELDESARAAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGAALQKELIRFLNAGVFGKGTESHTLPHPTTRAAMLVRINTLLQGYSG IRFEILEAITKFLNHNITPCLPLRGTISSSGDLVPLSYIAGLLTGRPNSKSVGPKGETLNAAQAFEQVGISSGFFELQPKEGLALVNGTAVGSGLASTVLFETNILTLLSEILSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEYILDGSDYVKAAKKLHEQDPLQKPKQDRYALR TSPQWLGPLIEVIRFSTKSIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNTRLAIASIGKLMFAQFSELVNDYYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSSLGLISSRKTAEAVDILKLMSSTFLVALCQAIDLRHLEENLKST VKNTVSQLAKRLLTTGVNGELHPSRFCEKDLLMVVEREYLFAYIDDPCSATYPLMQRLRQVLVEHALTNGENEKNANTSIFQKISAFEEELKTILPKEVESARAACESGNAAIPNRIIECRSYPLYKFVREELGGEFLTGEKVRSPGEECDKVFTAMCQGKIIDPILDCLSGWNGEPLPIC

[0049] Furthermore, compared with the nucleotide sequence of the wild-type strawberry PAL2 gene, the mutant gene has two genotypes, one of which is that the nucleotide at position 1054 mutates from C to T, causing a point mutation, which terminates protein translation prematurely, that is, the glutamine at position 352 of the PAL2 protein mutates to a terminator, and its nucleotide sequence contains SEQ ID NO.3, and the amino acid sequence contains SEQ ID NO.4.

[0050] One of the nucleotide sequences of the PAL2 mutant gene, SEQ ID NO. 3, the mutated bases are in bold and framed.

[0051]

[0052]

[0053] One of the amino acid sequences of the PAL2 mutant gene, SEQ ID NO.4.

[0054] METVTQNGYHQNGIQNGSLDILCVKTESLKAVSDPLNWGAAAESMTGSLDEVKRMVAEYRKPVVKLGGKTLTIAQVAAIANHDSGVKVELDESARAAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGAALQKELIRFLNAGVFGKGTESHTLPHPTTRAAMLVRINTL LQGYSGIRFEILEAITKFLNHNITPCLPLRGTISSSGDLVPLSYIAGLLTGRPNSKSVGPKGETLNAAQAFEQVGISSGFFELQPKEGLALVNGTAVGSGLASTVLFETNILTLLSEILSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEYILDGSDYVKAAKKLHEQDPL

[0055] Another genotype of the PAL2 mutant gene is that the nucleotide at position 371 mutates from G to A, causing the amino acid at position 124 encoded by it to mutate from glycine (G) to aspartic acid (D), and its nucleotide sequence comprises the sequence shown in SEQ ID NO.5, and the amino acid sequence comprises the sequence shown in SEQ ID NO.6.

[0056] The second nucleotide of the PAL2 mutant gene, SEQ ID NO.5, the mutated bases are in bold and framed.

[0057]

[0058]

[0059]

[0060] The second nucleotide amino acid of the PAL2 mutant gene, SEQ ID NO.6.

[0061] METVTQNGYHQNGIQNGSLDILCVKTESLKAVSDPLNWGAAAESMTGSLDEVKRMVAEYRKPVVKLGGKTLTIAQVAAIANHDSGVKVELDESARAAGVKASSDWVMDSMNKGTDSYGVTTGFDATSHRRTKQGAALQKELIRFLNAGVFGKGTESHTLPHPTTRAAMLVRINTLLQGYSG IRFEILEAITKFLNHNITPCLPLRGTISSSGDLVPLSYIAGLLTGRPNSKSVGPKGETLNAAQAFEQVGISSGFFELQPKEGLALVNGTAVGSGLASTVLFETNILTLLSEILSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEYILDGSDYVKAAKKLHEQDPLQKPKQDRYALR TSPQWLGPLIEVIRFSTKSIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNTRLAIASIGKLMFAQFSELVNDYYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSSLGLISSRKTAEAVDILKLMSSTFLVALCQAIDLRHLEENLKST VKNTVSQLAKRLLTTGVNGELHPSRFCEKDLLMVVEREYLFAYIDDPCSATYPLMQRLRQVLVEHALTNGENEKNANTSIFQKISAFEEELKTILPKEVESARAACESGNAAIPNRIIECRSYPLYKFVREELGGEFLTGEKVRSPGEECDKVFTAMCQGKIIDPILDCLSGWNGEPLPIC

[0062] Another aspect of the present invention protects biological materials related to the PAL2 mutant gene as described above, and the biological materials include any of the following: a) a recombinant expression vector containing the nucleotides as described above; b) a bioengineering bacterium containing the nucleotides as described above, or a bioengineering bacterium containing the recombinant expression vector as described in a); c) a protein encoded by the nucleotides as described above.

[0063] In certain embodiments, the amino acid sequence of the protein comprises the sequence shown in SEQ ID NO.4 or SEQ ID NO.6.

[0064] In certain embodiments, the recombinant expression vector is selected from plasmid, cosmid, phage or viral vector. The recombinant expression vector refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors well known in the art.

[0065] In some embodiments, the bioengineering bacteria can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples include: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, etc. The bioengineering bacteria contains the recombinant expression vector as described above or has the nucleotides as described above integrated into the genome. The bioengineering bacteria are cultured so that they can express proteins.

[0066] Another aspect of the present invention protects the use of the PAL2 mutant gene as described above or the biological material as described above in any of the following:

[0067] A1) Regulates the synthesis of strawberry anthocyanins;

[0068] A2) Regulate the color of strawberry organs;

[0069] A3) Develop a reagent for identifying the color of strawberry organs;

[0070] A4) Strawberry breeding or germplasm improvement;

[0071] A5) preparing a strawberry assisted breeding kit;

[0072] A6) Preparation of transgenic strawberries.

[0073] In certain embodiments, when the PAL2 gene in strawberry is mutated into a PAL2 mutant gene using genetic engineering methods such as homologous recombination or CRISPR, the synthesis of anthocyanins in strawberry can be inhibited, making the fruit pink and the petioles yellow-green; overexpressing the PAL2 gene in strawberry can promote anthocyanin synthesis, making the fruit redder.

[0074] In certain embodiments, the development of a strawberry organ color identification reagent refers to designing a reagent based on the PAL2 mutant gene, wherein the reagent comprises primers, and amplification is performed using the primers. The color of the strawberry organ is judged based on the amplification product, that is, the color of the fruit is red or pink, and the color of the petiole is purple-red or yellow-green.

[0075] In some embodiments, the breeding kit uses the PAL2 gene as a molecular marker and comprises primers for amplifying the molecular marker, such as primers of the sequences shown in SEQ ID NO.7 and SEQ ID NO.8. Further, the breeding kit also includes: a PCR amplification enzyme, deionized water, a positive control, a negative control, or a combination of several thereof. The breeding kit can be used to realize assisted selection breeding of strawberries with pink or red fruit traits, quickly eliminate red fruit plants at the seedling stage, save subsequent maintenance and identification and evaluation costs, and significantly improve the efficiency of pink fruit breeding.

[0076] Another aspect of the present invention provides primers for amplifying the PAL2 mutant gene described above, comprising primers represented by SEQ ID NO.7 and SEQ ID NO.8.

[0077] When amplification was performed using the amplification primers described above, the procedure was as follows: pre-denaturation at 95°C for 4 min; denaturation at 95°C for 10 s, annealing at 55°C for 20 s, extension at 72°C for 60 s, and 30 cycles from denaturation to extension; full extension at 72°C for 10 min, and storage at 25°C.

[0078] In certain embodiments, the detection reagent pair can be used to assist in detecting the color of strawberry organs or to prepare a kit for detecting the color of strawberry organs or in strawberry color trait breeding.

[0079] Another aspect of the present invention provides a method for identifying pink strawberries, comprising the following steps:

[0080] 1) Identifying whether the strawberry seeds or strawberry seedlings to be tested contain the PAL2 mutant gene as described above; or,

[0081] 2) Identify whether the strawberry seeds or strawberry seedlings to be tested express the protein described above.

[0082] In certain embodiments, when the strawberry contains the PAL2 mutant gene as described above, the strawberry fruit is judged to be or is a candidate for pink, and the petiole is or is a candidate for yellow-green; when the strawberry does not contain the PAL2 mutant gene as described above, the strawberry fruit is judged to be or is a candidate for red, and the petiole is or is a candidate for purple-red.

[0083] In certain embodiments, when strawberry expresses the protein described above, the strawberry fruit is judged to be or is a candidate for pink, and the petiole is or is a candidate for yellow-green; when strawberry does not express the protein described above, the strawberry fruit is judged to be or is a candidate for red, and the petiole is or is a candidate for purple-red.

[0084] Another aspect of the present invention protects a method for inhibiting anthocyanin synthesis in strawberries, which comprises introducing the PAL2 mutant gene as described above into strawberries to obtain strawberries with inhibited anthocyanin synthesis.

[0085] Another aspect of the present invention is to protect a method for cultivating strawberries with pink fruits, which comprises cultivating strawberries containing the PAL2 mutant gene as described above to obtain strawberries with pink fruits.

[0086] In certain embodiments, the introduction of the PAL2 mutant gene can be obtained by genetic engineering or hybrid breeding.

[0087] In certain embodiments, the method comprises the following steps: hybridization is performed using the strawberry with inhibited anthocyanin synthesis and / or the strawberry with pink fruit or green petiole as parents.

[0088] In certain specific embodiments, 1) the strawberry with inhibited anthocyanin synthesis and / or the strawberry with pink fruit or green petiole is used as the male parent for hybridization with the female parent to obtain BC1F1 generation seeds;

[0089] 2) among the BC1F1 generation plants, selecting a single plant with a phenotype close to that of the maternal plant for self-pollination to obtain BC1F2 generation seeds;

[0090] 3) Among the BC1F2 generation plants, select a single plant with a phenotype close to that of the male parent and perform backcrossing to obtain BC2F2 generation seeds;

[0091] 4) among the BC2F2 generation plants, selecting a single plant with a phenotype close to that of the male parent for backcrossing to obtain BC3F2 generation seeds;

[0092] 5) Among the BC3F2 generation plants, select individual plants with phenotypes close to those of the father parent for harvest.

[0093] In certain more specific embodiments, the female parent is Fragaria ruegen.

[0094] In certain more specific embodiments, said maternal phenotype comprises red fruit and / or red petioles.

[0095] In certain more specific embodiments, the paternal phenotype comprises light red (pink) fruits and / or yellow-green petioles.

[0096] The present invention screened out two mutant plants capable of making the color of strawberry fruits lighter and the color of petioles lighter from a strawberry mutant library induced by EMS, and the anthocyanin content in the fruits and petioles of the mutants was significantly lower than that of the wild type after detection; further, the mutants were continuously backcrossed, and the genetic traits of the fruits being light red (pink) and the petioles being lighter (yellow-green) were stable.

[0097] The present invention further studies and finds that PAL2 is a key gene that controls the synthesis of strawberry anthocyanins or the color of strawberry organs. First, a hybrid F1 generation plant is created, which is hybridized by mutant rg40 and mutant rg120. The phenotype and anthocyanin content of the hybrid F1 generation plant are consistent with those of the mutant parents, indicating that the mutagenic genes controlling the two mutants are the same. Next, the mutant rg120 is located by using the MutMap strategy, and the mutagenic gene is located on the sixth chromosome. Then, the sixth chromosome is analyzed and compared, and 9 mutation sites are respectively located in the CDS regions of 9 genes. The 9 mutation sites are further analyzed and functionally annotated, and the mutant phenotype is due to the mutation of the nucleotide at position 1054 of the CDS region of the PAL2 gene from C to T, forming a stop codon, resulting in premature termination of translation and the production of non-functional protein products. At the same time, the PAL2 gene in the mutant rg40 was sequenced, and it was found that the nucleotide at position 371 of its CDS region mutated from G to A, resulting in the mutation of glycine (G) at position 124 to aspartic acid (D), producing a non-functional protein product. In addition, the present invention also uses a prokaryotic expression vector to express the mutant protein, which is used to catalyze phenylalanine in vitro. The results show that the enzyme activity of the mutant protein is much lower than that of the wild-type PAL2 protein. There are two PAL genes in forest strawberry, namely PAL1 and PAL2, and the activity of PAL2 protein in catalyzing substrate conversion is higher than that of PAL1 protein. Therefore, it is comprehensively explained that the control gene that regulates the synthesis of strawberry anthocyanins or regulates the color phenotype of strawberry organs is PAL2.

[0098] In order to confirm that the mutant phenotype is caused by the PAL2 gene mutation, a complementation verification experiment was carried out on the mutant. The phenotype, anthocyanin content and PAL2 gene expression level detection all confirmed that the mutant phenotype is caused by the PAL2 gene mutation.

[0099] In summary, the present invention has found that the PAL2 gene is closely related to the formation and accumulation of anthocyanins in strawberries, and has identified two key sites. According to the site information, the target trait can be screened at the seedling stage, effectively shortening the traditional hybrid breeding period, and achieving accurate and efficient molecular breeding of strawberry fruit color. Through auxiliary breeding, the anthocyanin content in strawberry fruits can be effectively reduced, the cultivation of pink innovative strawberry varieties can be promoted, products that meet the needs of the public and have market potential can be developed, and the innovative application of diversified strawberries can be promoted.

[0100] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0101] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0102] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0103] Wild-type red-fruited forest strawberry RG (Ruegen), with red fruits, purple-red petioles, and no runners (Ruegen, National Germplasm Repository ID PI666609).

[0104] Example 1 Creation of pink fruit, yellow-green petiole strawberry germplasm

[0105] In this Example 1, EMS was used to induce mutagenesis of strawberries to obtain pink strawberries. The process includes the following:

[0106] 1.1. EMS mutagenesis to obtain pink strawberry fruit

[0107] 1) Select 4,000 seeds of forest strawberry Ruegen (abbreviated as RG, red fruit, purple-red petiole, no runners) (the number of seeds can be more), put them into a 100 mL conical flask, add an appropriate amount of 0.1% Tween-20 (Tween 20 is a surfactant) to cover the seeds, and treat them on a shaker at room temperature for 4-6 hours. The role of Tween-20 is to increase the surface activity of the seeds so that they can fully contact with the liquid;

[0108] 2) Pour away the Tween-20 solution and wash the seeds 3-4 times with water;

[0109] 3) Add 0.4% (volume ratio) EMS solution into the bottle and shake for 6 hours;

[0110] 4) After the EMS oscillation treatment is completed, the EMS solution is poured into a container containing 7.5 times the volume of 0.5M NaOH solution. The main function of sodium hydroxide is to react with EMS and eliminate the toxicity of EMS before it can be treated as ordinary waste liquid;

[0111] 5) At the same time, wrap the seeds with white gauze and place them under running water for 12 hours to remove the remaining EMS;

[0112] 6) Sow the treated seeds (M1 generation) in a prepared flower pot containing a substrate (peat soil: vermiculite = 1:2, volume ratio), and then cover the mouth of the flower pot with plastic wrap to keep the substrate moist;

[0113] 7) Remove the plastic wrap after 5 days. If the surface is lack of water during subsequent management, you can use a watering can to spray water on the surface to keep it moist. After the seeds germinate and grow two true leaves, transplant each seedling into a small flowerpot (length, width and height are 9 cm, 9 cm, and 11 cm respectively), and then place it in a culture room to make it bloom and bear fruit. Harvest M2 generation seeds for each plant;

[0114] 8) The M2 generation seeds were sown further, and when the fruits were ripe, two mutant materials with light red fruits, reduced anthocyanin content, yellow-green petioles, and reduced anthocyanin accumulation were selected according to the fruit color. They were named mutant rg40 and mutant rg120, and the M3 generation mutant seeds were harvested;

[0115] 9) The mutant rg40 and mutant rg120 seeds in step 8) were planted for another generation to obtain M4 generation seeds. The M4 generation seeds were planted again and found to have the same phenotype as M3 and M2 without separation, indicating that the phenotypes of the rg40 and rg120 mutants were stable and could be stably inherited to the next generation.

[0116] The phenotypes of wild-type RG, mutant rg40, and mutant rg120 plants are shown in Figure 1 A, B, C, E and F.

[0117] from Figure 1 It can be seen that compared with the wild-type RG, the petioles of the mutants rg40 and rg120 changed from purple-red to yellow-green.

[0118] Fresh strawberry fruits 25 days after flowering or petioles of seedlings about 3 months old with newly unfolded leaves were collected. Ten fruits or petioles from five individual plants were collected in each group. The strawberry fruits or petioles collected in each group were ground into powder in liquid nitrogen. 0.1 g of the powder was placed in a 2.0 mL centrifuge tube, and 0.6 mL of extract (the extract was obtained by mixing hydrochloric acid and methanol in a volume ratio of 1 mL:99 mL) was added. After oscillation and mixing, the mixture was placed at 4°C overnight. On the second day, 0.4 mL of sterile water was added and mixed, and then centrifuged at room temperature and 13000 rpm for 5 min; the supernatant was placed in another clean 2.0 mL centrifuge tube, and 1 mL of chloroform was added and oscillated and mixed, and then centrifuged at room temperature and 13000 rpm for 10 min. The upper aqueous phase solution was taken to determine the absorbance values ​​at A530 and A657.

[0119] Total anthocyanin content = (A530-A657) / FW

[0120] In the formula, FW represents fresh fruit weight or petiole weight in g.

[0121] The anthocyanin contents in fruits and petioles of wild-type RG, mutant rg40 and mutant rg120 are shown in Figure 1 G. Petiole is petiole, and Fruit is fruit. Data are the mean (±SD) of three replicates and were analyzed by two-tailed t-test.

[0122] from Figure 1 As shown in Figure 3, compared with the wild-type RG, the anthocyanin content in the fruits and petioles of mutants rg40 and rg120 was significantly reduced (**P<0.01).

[0123] 1.2. Continuous backcrossing

[0124] The wild type RG was used as the female parent, and the mutants rg40 and rg120 obtained in step 8) were used as the male parent, respectively. When the female parent plant had flower buds (only the flower buds of sepals could be seen from the outside), the stamens were carefully peeled off with pointed tweezers, and the pistils were not damaged as much as possible. Then, the emasculated flowers were covered with a small paper bag made of white sulfate paper. Two days later, the stamens of the male parent's blooming flowers (the anthers of the stamens were cracked and pollen grains were attached to the surface) were used to smear the stigma of the female parent's pistil after emasculation. After pollination, the original paper bag was used to cover it, and the names of the parents and the hybridization time were marked. After 10 days of pollination, the seeds were observed to see if they were formed. If they were solid, the BC1F1 backcross seeds were harvested after the fruits were ripe.

[0125] The BC1F1 backcross seeds were planted and their phenotype was found to be the same as that of the wild-type RG. The BC1F2 seeds were harvested after the fruits matured.

[0126] After planting BC1F2 seeds, the phenotypic segregation was observed and it was found that the segregation ratio of the wild-type phenotype and the mutant phenotype was 3:1, which indicated that the mutagenic genes of the mutants rg40 and rg120 were both single gene recessive mutations.

[0127] A mutant plant was selected from BC1F2 as the male parent to perform a second backcross with the female wild-type RG to obtain BC2F1 backcross seeds.

[0128] After backcrossing, the BC2F2 seeds are harvested, and then a mutant plant is selected from the BC2F2 to continue to serve as the male parent for the third backcross with the female wild-type RG. Finally, a mutant plant with good growth is selected from the BC3F2 population to harvest seeds. The obtained germplasm is the light red (pink) strawberry germplasm.

[0129] Example 2 Location of mutagenic genes

[0130] In this Example 2, the mutagenic genes of the mutants rg40 and rg120 obtained in Example 1 were identified.

[0131] 2.1. Hybridization of mutant rg40 and mutant rg120

[0132] Considering that the two mutants have similar phenotypes, the two mutants rg40 and rg120 were hybridized to obtain hybrid F1 plants (rg40♂×rg120♀). The specific hybridization is as follows:

[0133] The mutant rg120 was used as the female parent, and the anthers were removed. The mutant rg40 was used as the male parent, and its anthers were used to smear the pistil stigma of the female parent for hybridization. The F1 generation seeds obtained by hybridization were planted to obtain hybrid F1 generation plants (rg40♂×rg120♀). The photo results are shown in Figure 1 D, E, F, anthocyanin content test results are shown in Figure 1 Middle G.

[0134] from Figure 1 As shown in D, E, F and G, the hybrid F1 plants (rg40♂×rg120♀) showed the same phenotype of yellow-green petioles and light red fruits as the hybrid parents (mutant rg40 and mutant rg120), as well as the phenomenon of reduced anthocyanin content in the fruits, indicating that the mutant rg40 and mutant rg120 had the same mutagenic (controlling) genes that caused the lighter color of fruits and petioles. Therefore, the mutant rg120 was used for subsequent experiments.

[0135] 2.2 Identification of the mutagenic gene in mutant rg120

[0136] The mutant gene of strawberry mutant rg120 was located according to the MutMap strategy.

[0137] Identification of mutagenic genes:

[0138] 1) A BC1F2 population was constructed using the mutant rg120 obtained in step 1.2 of Example 1 and the wild-type RG, 50 individual plants with mutant phenotypes were selected, and DNA was extracted from young leaves using the CTAB method.

[0139] 2) The DNA samples extracted in step 1) were sent to Shanghai Paisonno Biotechnology Co., Ltd. for equal mixing to form a mutant pool library.

[0140] Similarly, a DNA sample of wild-type RG was extracted to construct a wild-type library.

[0141] 3) Whole genome resequencing of mutant mixed pool library and wild-type library was performed using Illumina HiSeq2500.

[0142] 4) After removing the adapters and low-quality sequences from the original sequence obtained by sequencing, high-quality sequencing data is obtained.

[0143] 5) The genome sequence of diploid forest strawberry Hawaii-4 (Fragaria vesca Whole Genome v4.0.a2, https: / / www.rosaceae.org / species / fragaria_vesca / genome_v4.0.a2) was used as the reference genome, and the high-quality sequencing data obtained in step 4) were analyzed using the MutMap (https: / / github.com / YuSugihara / MutMap) method to obtain the whole genome SNP-Index distribution map. The distribution map is shown in Figure 2 .

[0144] from Figure 2 It can be seen that the Mutmap results showed an obvious peak on chromosome 6, which indicated that the mutant gene of the rg120 mutant was located on chromosome 6 (Fvb6).

[0145] Next, by screening the SNP-Index values ​​of these mutant genes on chromosome 6 (Fvb6), it was found that 84 mutation sites had a SNP-Index value of 1 (e.g. Figure 3 (A).

[0146] Then, the 84 mutation sites were analyzed and found that 75 mutation sites were located in the intergenic region, intron region or upstream untranslated region. Usually, these single point mutations do not affect the function of the related genes, while the remaining 9 mutation sites were located in the CDS region of 9 genes (such as Figure 3 (B).

[0147] Furthermore, 9 mutation sites were analyzed, among which 3 mutation sites (SNP3, SNP8 and SNP9) underwent synonymous mutations, and the other 6 mutation sites (SNP1, SNP2, SNP4, SNP5, SNP6 and SNP7) underwent non-synonymous mutations.

[0148] The functional annotation of the genes where the six non-synonymous mutation sites are located was analyzed using the online website Phytozome.

[0149] Annotation analysis showed that SNP2 was located in the coding region of phenylalanine ammonia lyase PAL2 (FvH4_6g16060) which catalyzes phenylalanine metabolism and this gene plays an important role in anthocyanin synthesis, while the functional annotations of the other five genes with non-synonymous mutations were not related to anthocyanin synthesis. Nucleotide polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. The variation of a single nucleotide can be caused by the transition or transversion of a single base.

[0150] The CDS sequence of the PAL2 gene in the wild-type RG is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0151] The nucleotide at position 1054 in the CDS region of the PAL2 gene in the mutant rg120 mutated (or converted) from C to T, resulting in a point mutation in the PAL2 gene, which caused the translation of the PAL2 protein to terminate prematurely, that is, the amino acid at position 352 of the PAL2 protein mutated from glutamine (Q) to a terminator ( Figure 3 (B) The CDS sequence of the PAL2 gene in the mutant rg120 is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.4.

[0152] Therefore, the PAL2 gene is most likely the causative gene of the light red fruit and yellow-green petiole mutant.

[0153] 2.3 Sequencing of the mutagenic gene in mutant rg40

[0154] Since the mutant rg40 and mutant rg120 have the same mutation gene, the total RNA of young leaves of mutant rg40 was extracted using an RNA extraction kit (Tiangen, Beijing, China, Cat. DP441), and then reverse transcribed to obtain cDNA using a one-step gDNA removal and cDNA synthesis kit (Transgen, Beijing, China, Cat. AE311). The experimental steps were carried out according to the manufacturer's instructions. The PAL2 gene in the rg40 mutant was amplified by PCR using the obtained cDNA as a template.

[0155] The reaction system included 12.5 μL PrimeSTAR Max Premix (2×) (TaKaRa, Dalian, China, Cat. R045), 1 μL cDNA, 1 μL forward PCR primer, 1 μL reverse PCR primer, and 9.5 μL ddH2O.

[0156] The primer sequences used for PCR amplification of the PAL2 mutant gene are as follows:

[0157] PAL2-F: 5'-ATGGAGACCGTAACCCAGAATG-3' (SEQ ID NO.7)

[0158] PAL2-R: 5'-GCATATCGGAAGAGGTTCACCG-3' (SEQ ID NO.8)

[0159] The amplification program was as follows: pre-denaturation at 95°C for 4 min; denaturation at 95°C for 10 s, annealing at 55°C for 20 s, extension at 72°C for 60 s, and 30 cycles from denaturation to extension; full extension at 72°C for 10 min, and storage at 25°C.

[0160] The PCR amplification products were detected by electrophoresis using 1.0% agarose gel. Figure 4 .

[0161] Further, sequencing analysis of the PCR amplification products revealed that the PAL2 gene in the mutant rg40 had indeed mutated, with the nucleotide at position 371 in its CDS region mutating from G to A.

[0162] The sequencing results of mutant rg40 and mutant rg120 were compared with the amino acids of wild-type RG. Figure 5 .

[0163] from Figure 5It can be seen that the nucleotide at position 371 of the CDS region of the PAL2 gene in the mutant rg40 mutated from G to A, resulting in a mutation of glycine (G) at position 124 to aspartic acid (D).

[0164] The CDS sequence of the PAL2 gene in the mutant rg40 is shown in SEQ ID NO.5, and the encoded amino acid sequence is shown in SEQ ID NO.6.

[0165] In summary, these results indicate that PAL2 is the key mutant gene that causes the strawberry fruits of the mutants rg40 and rg120 to be pink, the petioles to turn yellow-green, and the anthocyanin content in the fruits and leaf stems to be reduced.

[0166] Example 3 Verification of complementation of the mutant gene PAL2

[0167] In this Example 3, in order to verify that the candidate gene for the phenotype of light red fruit and yellow-green petiole of mutants rg40 and rg120 is PAL2, a complementation verification experiment was performed. The complementation experiment includes: constructing an expression vector containing the PAL2 gene, performing an instantaneous injection of the mutant rg120 obtained in Example 1 under the mediation of Agrobacterium, and studying the phenotype of the fruit after instantaneous injection, anthocyanin content and PAL2 gene level. Specifically, it includes the following:

[0168] 3.1. Construction of expression vector, transformation into Agrobacterium, and transient injection

[0169] A 35S::PAL2-YFP overexpression vector was constructed and then transformed into GV3101 Agrobacterium, which was then transiently injected into rg120 mutant fruits with GV3101 Agrobacterium containing 35S::PAL2-YFP. At the same time, rg120 mutant fruits were injected with GV3101 Agrobacterium containing 35S::YFP as a control group.

[0170] Construction of 35S::PAL2-YFP expression vector: First, using wild-type RG leaf cDNA as a template, the CDS sequence of the PAL2 gene was obtained by PCR amplification, and then the CDS of the PAL2 gene (excluding the stop codon) was inserted into the 35S::YFP vector by homologous recombination to obtain the 35S::PAL2-YFP expression vector.

[0171] Transient injection: The 35S::PAL2-YFP expression vector was transformed into GV3101 Agrobacterium to obtain a bacterial solution containing the 35S::PAL2-YFP expression vector; at the ginkgo stage, the bacterial solution containing 35S::PAL2-YFP was transiently injected into the fruits of the mutant rg120 obtained in Example 1, and a total of 5 fruits were injected. The culture was continued for 6 days, and the fruit color phenotype was observed; the fruits were harvested, the anthocyanin content in the fruits was detected, and the expression of the PAL2 gene was detected by fluorescent quantitative PCR.

[0172] 3.2 Investigation of fruit phenotype, anthocyanin content and PAL2 gene level

[0173] 3.2.1 Fruit phenotype detection

[0174] The phenotypes of the fruits were photographed on the 0th, 2nd, 3rd, 4th, 5th, and 6th days after transient injection in the rg120 mutant fruits. The results are shown in Figure 6 A. The scale bar in A is 1 cm.

[0175] from Figure 6 As shown in A, on the 4th day after the injection of 35S::PAL2-YFP into the fruits of the mutant rg120, the color of the fruits turned red; while in the control group where 35S::YFP was injected into the fruits of the mutant rg120, the fruits were still light red until the 6th day.

[0176] 3.2.2. Detection of anthocyanin content in fruits

[0177] After the mutant rg120 fruits were injected with 35S::PAL2-YFP and 35S::YFP, the anthocyanin content in the fruits was shown in Figure 2. Figure 6 Middle B.

[0178] from Figure 6 As shown in Figure B, the anthocyanin content in the fruits of the mutant rg120 injected with 35S::PAL2-YFP was significantly higher than that in the control group injected with 35S::YFP.

[0179] 3.2.3. Detection of PAL2 gene expression level

[0180] Fluorescence quantitative PCR experiments also confirmed that the PAL2 gene was indeed expressed in the fruit after transient injection. Figure 6 Middle C.

[0181] Total RNA was extracted from fruits injected with 35S::PAL2-YFP and 35S::YFP using RNA polysaccharide polyphenol plant total RNA extraction kit (Tiangen, Beijing, China, Cat. DP441) and one-step gDNA removal and cDNA synthesis kit (Transgen, Beijing, China, Cat. AE311), and reverse transcription was performed to obtain cDNA, which was diluted 5 times for subsequent experiments. The experimental steps were carried out according to the manufacturer's instructions. Fluorescence quantitative PCR reagents used ChamQ Blue Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China).

[0182] The reaction system included 10 μL 2×SYBR qPCR Master Mix, 1 μL cDNA, 0.4 μL forward primer, 0.4 μL reverse primer, and 8.2 μL ddH2O.

[0183] The forward and reverse primer sequences are as follows:

[0184] PAL2-QF:AACCAGGATGTCAACTCTT(SEQ ID NO.9)

[0185] PAL2-QR:TAACCGTGCTCTTCAAG(SEQ ID NO.10)

[0186] The amplification program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 20 s, and extension at 72°C for 20 s, with a total of 39 cycles from denaturation to extension.

[0187] As shown in Figure 6C, the expression level of PAL2 gene in the fruits of mutant rg120 injected with 35S::YFP was very low; however, after injection of 35S::PAL2-YFP, the expression level of PAL2 gene increased significantly, indicating that PAL2 was indeed overexpressed.

[0188] Overall, the results showed that the expression of PAL2 can indeed complement the color phenotype of the mutant rg120 fruit, which proves that the phenotype of pink strawberry fruit and yellow-green petiole is caused by the mutation of the PAL2 gene, which is the mutagenic gene of the rg120 mutant.

[0189] Example 4 Functional analysis of wild-type and mutant PAL2 proteins

[0190] In the anthocyanin synthesis pathway, PAL, as a phenylalanine ammonia lyase, can convert the reaction substrate phenylalanine (L-phenylalanine) into cinnamic acid (ninnamic acid). The mutation of PAL2 in the mutant rg120 leads to reduced anthocyanin accumulation, and it is speculated that the activity of the mutated PAL2 enzyme protein may be affected.

[0191] 4.1. Construction of expression vector

[0192] We constructed wild-type PAL2 containing RG (PAL2 RG ), rg40 mutant PAL2 (mPAL2 rg40 ), rg120 mutant PAL2 (mPAL2 rg120 ) coding sequence (wherein the rg120 mutant PAL2 only contains the first 1053bp nucleotide sequence that can encode the protein after mutation).

[0193] The construction process of the prokaryotic expression vector is as follows: first, the cDNA of wild-type RG fruit was used as a template to obtain the full-length CDS sequence of the PAL2 gene (excluding the stop codon) by PCR amplification; second, the PET28a-sumo-His vector was digested with SacⅠ and XhoⅠ, and then the CDS of the PAL2 gene (excluding the stop codon) was inserted into the PET28a-sumo-His vector after digestion by homologous recombination to obtain PET28a-sumo-PAL2 RG -His prokaryotic expression vector.

[0194] Refer to PET28a-sumo-PAL2 RG -His prokaryotic expression vector construction process Construction of PET28a-sumo-PAL2 rg40 -His and pET28a-SUMO-PAL2 rg120 -His prokaryotic expression vector, the difference is that the cDNA of rg40 and rg120 mutant plants is used as template, and PAL2 is obtained by PCR amplification. rg40 and PAL2 rg120 The full-length CDS sequence (excluding the stop codon) of

[0195] The constructed prokaryotic expression vectors were transformed into Escherichia coli DE3 strains, and wild-type PAL2 was induced by IPTG. RG , mutant mPAL2 rg40 , mutant mPAL2 rg120 Protein expression was performed and the corresponding protein expression solutions were obtained.

[0196] There are two PAL genes in forest strawberry, PAL1 and PAL2. RG In the same way, a wild-type PAL1 RG The prokaryotic expression vector was then transformed into Escherichia coli DE3 strain and wild-type PAL1 was induced by IPTG. RG protein.

[0197] 4.2 Protein induction expression and purification

[0198] Purify the wild-type PAL1 obtained in step 4.1 by nickel affinity chromatography RG Protein, wild-type PAL2 RG Protein, mutant mPAL2 rg40 Protein and mutant mPAL2 rg120 protein.

[0199] The purified target protein was stained with Coomassie Brilliant Blue. Figure 7 .

[0200] from Figure 7 It can be seen that wild-type PAL1 RG Protein, wild-type PAL2 RG Protein, mutant mPAL2 rg40 Protein and mutant mPAL2 rg120 Proteins can be expressed.

[0201] 4.3. Purified protein catalyzes the conversion of phenylalanine

[0202] The proteins purified in step 4.2 were added to 1 mL of the enzymatic reaction solution, reacted at 45°C for 1.5 h, and then 100 μL of methanol was added to terminate the reaction. The reaction solution was sterilized by filtration with a 0.22 μm filter membrane and then analyzed by HPLC. The enzymatic reaction solution was obtained by dissolving L-phenylalanine in 50 mM Tris-HCl (pH 8.5) buffer; based on the total volume of the enzymatic reaction solution, the concentration of L-phenylalanine was 10 mM.

[0203] The HPLC analysis used a Thermo Fisher C18 column, phase A was 0.1% formic acid aqueous solution, phase B was acetonitrile solution, the flow rate was 0.6 mL / min, the column temperature was 25°C, the injection volume was 10 μL, and the ratio of phase A to phase B was set as shown in Table 1:

[0204] Table 1

[0205]

[0206]

[0207] The results of HPLC are shown in Figure 8 .

[0208] from Figure 8 It can be seen that wild-type PAL1 RG 、PAL2 RG The protein can convert L-phenylalanine into trans-cinnamic acid, while the mutant mPAL2 rg40 , mutant mPAL2 rg120 It can hardly catalyze the conversion of the substrate L-phenylalanine.

[0209] This indicates that the mutation of the PAL2 gene leads to impaired catalytic function, that is, the mutant PAL2 gene almost loses the ability to convert the substrate molecule L-phenylalanine into trans-cinnamic acid.

[0210] According to wild-type PAL1 RG Protein, wild-type PAL2 RG Protein, mutant mPAL2 rg40 , mutant mPAL2 rg120 The results are shown in Fig. 9 .

[0211] The enzyme activity was determined by preparing 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.6 μg / mL and 2 μg / mL cinnamic acid standards, and constructing a standard curve of cinnamic acid by analyzing the content and peak area of ​​cinnamic acid. The cinnamic acid content was determined by the peak area of ​​cinnamic acid generated after adding different PAL proteins to the reaction system, and then the PAL enzyme activity was determined (1 U is equivalent to the generation of 1 μg / mL of cinnamic acid).

[0212] from Fig. 9 It can be seen that wild-type PAL2 RG PAL1 RG Both proteins have high catalytic activity towards the substrate L-phenylalanine, but the activity of PAL2 protein in catalyzing substrate conversion is higher than that of PAL1 protein. rg40 , mutant mPAL2 rg120 The enzyme activity is much lower than that of wild-type PAL2 RG protein.

[0213] Finally, the protein sequences of forest strawberry PAL1 and PAL2 were compared with those of octoploid strawberry, apple, Arabidopsis thaliana and other species. The analysis results are shown in Fig.10Among them, FvPAL1 represents forest strawberry PAL1 protein, XP_004307376.1; FvPAL2 represents forest strawberry PAL2 protein, XP_004304440.1; FaPAL1 represents cultivated strawberry PAL1 protein, APB87287.1; FaPAL2 represents cultivated strawberry PAL2 protein, APB87288.1; FaPAL6 represents cultivated strawberry PAL6 protein, ADQ39192.1; AtPAL1 represents Arabidopsis thaliana PAL1 protein, NP_181241.1; AtPAL2 represents Arabidopsis thaliana PAL2 protein, NP_190894.1; AtPAL3 represents Arabidopsis PAL3 protein, NP_196043.2; AtPAL4 represents Arabidopsis PAL4 protein, NP_187645.1; MdPAL1 represents apple PAL1 protein, XP_008387584.2; SlPAL3 represents tomato PAL3 protein, NP_001307538.1; NtPAL represents tobacco PAL protein, XP_016435186.1; VvPAL2 represents grape PAL2 protein ANB59163.1; PcPAL represents pear PAL protein, AGL50914.1; RcPAL1 represents rose PAL1 protein, XP_024176620.1.

[0214] from Fig.10 It can be seen that the glycine at position 124 and the aspartic acid at position 352 of the PAL1 and PAL2 proteins in forest strawberry are both conserved sites. Therefore, it is speculated that mutating the glycine at position 124 of PAL2 to aspartic acid or mutating the glutamine at position 352 to a stop codon in the octoploid strawberry can create octoploid strawberry germplasm with light red fruits and yellow-green petioles.

[0215] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the present invention should be included within the scope of the present invention.

Claims

1. A strawberry PAL2 mutant gene, characterized in that: Compared with the nucleotide sequence of the wild-type strawberry PAL2 gene, the nucleotide sequence of the PAL2 mutant gene comprises one or two of the following mutations: 1) C1054T, 2) G371A.

2. The PAL2 mutant gene according to claim 1, characterized in that: The nucleotide sequence of the wild-type strawberry PAL2 gene comprises SEQ ID NO.

1.

3. The PAL2 mutant gene according to claim 2, characterized in that: The nucleotide sequence of the PAL2 mutant gene comprises the sequence shown in SEQ ID NO.3 or SEQ ID NO.

5.

4. The biological material related to the PAL2 mutant gene according to claim 1, characterized in that: The biological material includes any of the following: a) a recombinant expression vector containing the nucleotide sequence of claim 1; b) a bioengineering bacterium containing the nucleotide as claimed in claim 1, or a bioengineering bacterium containing the recombinant expression vector as claimed in a); c) A protein encoded by the nucleotide sequence as claimed in claim 1.

5. The biomaterial according to claim 4, characterized in that The amino acid sequence of the protein comprises the sequence shown in SEQ ID NO.4 or SEQ ID NO.

6.

6. Use of the PAL2 mutant gene according to claim 1 or the biological material according to claim 4 in any of the following: A1) regulating the synthesis of strawberry anthocyanins; A2) Regulate the color of strawberry organs; A3) Develop a reagent for identifying the color of strawberry organs; A4) Strawberry breeding or germplasm improvement; A5) preparing a strawberry assisted breeding kit; A6) Preparation of transgenic strawberries.

7. The primers for amplifying the PAL2 mutant gene according to claim 1, characterized in that: Primers comprising the sequences shown in SEQ ID NO.7 and SEQ ID NO.

8.

8. A method for identifying pink strawberries, characterized in that: The following steps are involved: 1) Identifying whether the strawberry seeds or strawberry seedlings to be tested contain the PAL2 mutant gene as claimed in claim 1; or, 2) Identifying whether the strawberry seeds or strawberry seedlings to be tested express the protein as claimed in claim 5.

9. A method for inhibiting anthocyanin synthesis in strawberries, characterized in that: The PAL2 mutant gene as claimed in claim 1 is introduced into strawberries to obtain strawberries in which anthocyanin synthesis is inhibited.

10. A method for growing pink strawberries, characterized in that: Cultivate strawberries containing the PAL2 mutant gene as claimed in claim 1 to obtain strawberries with pink fruits.

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