Application of apple MdPRX37L gene in maintaining color quality of postharvest apple fruit

Abstract

The invention discloses the use of the apple MdPRX37L gene in maintaining the color quality of post-harvest apple fruits, and belongs to the technical field of plant genetic engineering. The PRX family gene MdPRX37L isolated from apples by the invention has a nucleotide sequence as shown in SEQ ID NO.1. The apple instantaneous injection experiment and the experiment on the effect of the MdPRX37L protein on anthocyanin degradation under in vitro conditions show that MdPRX37L has a significant effect on maintaining the color of post-harvest apple fruits. This provides a new and rapid approach for apple breeding. The discovery of the MdPRX37L gene provides a basis for improving the appearance quality of apple fruits.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to use of an apple MdPRX37L gene in maintaining the color quality of post-harvest apple fruits. Background Art

[0002] As one of the four major fruit trees in the world, apple is the main cultivated tree species in temperate regions. Apple is an important horticultural crop, and its fruit is widely eaten. The color of apple fruit affects consumers' first impression of purchase. Bright red apples often stimulate their desire to buy and thus increase the sales of fruit. Apple producing countries around the world have made the selection of new high-quality apple varieties with excellent coloring one of their breeding goals. The color of apple fruit mainly depends on the content of anthocyanins, which are important secondary metabolites of plants and are synthesized in the phenylalanine metabolic pathway (Tanaka et al., 2008; Jaakola et al., 2013). Anthocyanins interact with a series of modifying enzymes (chs synthase (CHS), dihydroflavonol-4-reductase (DFR), UDP-glucose flavonoid 3-oxy-glucosyltransferase (UF3GT)) through the phenylalanine pathway (Dooner et al., 1991; Tanaka et al., 2008). The biosynthetic pathway of anthocyanins is well known, but little is known about their degradation, especially in postharvest apple fruit.

[0003] As a perennial woody plant, apple has a complex genetic background, high heterosis, long juvenile period, and poor self-compatibility. These problems have brought great obstacles to genetic breeding, and conventional breeding has progressed slowly. In recent years, the continuous improvement of apple genome sequencing technology and the rapid development of modern biotechnology have opened up new ways to solve these problems. Purposefully searching for key factors that regulate fruit color through genetic engineering technology has become an important research direction for genetic improvement of fruit trees. The isolation and identification of a large number of plant genes provide target genes for genetic engineering genetic improvement.

[0004] The enzymatic degradation mechanism of anthocyanins in plants is still unclear, but three enzyme families are involved in the degradation of anthocyanins in juice: polyphenol oxidase (PPO), class III peroxidase, and β-glycosidase (Oren-Shamir et al., 2009; Luo et al., 2017; Luo et al., 2019) However, further studies have found that PPO is located in the cytoplasm, while peroxidase is mainly located in the vacuole. Therefore, peroxidase activity is more likely to lead to anthocyanin degradation in most plant species (Zipor et al., 2015; Jukanti et al., 2017). This hypothesis has been further verified in Jasmine sambac, where BcPrx01 is a vacuolar class III peroxidase isolated from Jasmine sambac, which has been shown to be involved in the degradation of plant anthocyanins (Zipor et al., 2015).

[0005] Class III peroxidase (PRX) is a plant secretory peroxidase that is widely distributed in organisms and is considered to be a potential multifunctional protein that can participate in auxin metabolism, cell wall extension and hardening, and plant resistance to pathogens (Bindschedler et al., 2006; Passardi et al., 2004). This class of enzymes catalyzes redox reactions between hydrogen peroxide (H2O2) as an electron acceptor and a variety of electron donors (such as auxins, phenolic compounds, and secondary metabolites) (Almagro et al., 2009; Passardi et al., 2005). Studies have shown that class III peroxidase is a ubiquitous enzyme in plant cell walls and vacuoles, and is also involved in the accumulation of most secondary metabolites, many of which can be used as class III peroxidase substrates (Wang et al., 2004).

[0006] At present, the research on PRX is mainly focused on model plants such as Arabidopsis, but little research is done on fruit trees. Mining apple PRX genes and conducting functional studies will provide potential effective candidate genes for regulating the growth and development of fruit trees and the formation of quality, which not only has important theoretical significance, but also has a wide range of application value. Summary of the invention

[0007] In view of the above-mentioned prior art, the purpose of the present invention is to provide the use of the apple MdPRX37L gene in maintaining the color quality of post-harvest apple fruit. The present invention shows through the instantaneous injection experiment of apple fruit and the experiment of the effect of MdPRX37L protein on anthocyanin degradation under in vitro conditions that it has a significant effect on regulating anthocyanin. It provides a new and rapid way for apple breeding. The discovery of the MdPRX37L gene provides a basis for improving the quality of apple fruit.

[0008] The first aspect of the present invention provides the use of the apple MdPRX37L gene as a negative regulatory gene in the following (1) or (2):

[0009] (1) Inhibit the degradation of anthocyanins in postharvest apple peels;

[0010] (2) Maintain the color quality of postharvest apple fruit;

[0011] The apple MdPRX37L gene is a nucleic acid molecule as shown in i) or ii) below:

[0012] i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0013] ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

[0014] The second aspect of the present invention provides the use of a protein encoded by the apple MdPRX37L gene as a negative regulatory protein in the following (1) or (2):

[0015] (1) Inhibit the degradation of anthocyanins in postharvest apple peels;

[0016] (2) Maintain the color quality of postharvest apple fruits.

[0017] In the above application, the amino acid sequence of the protein encoded by the apple MdPRX37L gene is shown in SEQ ID NO.2.

[0018] The third aspect of the present invention provides the use of (1) or (2) in inhibiting the degradation of anthocyanins in post-harvest apple peels:

[0019] (1) gRNA sequence, recombinant knockout vector or transformant for specific knockout of MdPRX37L gene;

[0020] (2) A silencing fragment, gene silencing vector or transformant for specifically silencing the MdPRX37L gene.

[0021] In the above application, preferably, the gene silencing vector for specifically silencing the MdPRX37L gene comprises the nucleotide sequence shown in SEQ ID NO.3.

[0022] More preferably, the gene silencing vector is a vector in which the nucleotide sequence shown in SEQ ID NO.3 is reversely placed under the CaMV35S promoter.

[0023] A fourth aspect of the present invention provides a method for inhibiting the degradation of anthocyanins in post-harvest apple peels, comprising:

[0024] Steps to knock out or silence the MdPRX37L gene in apple;

[0025] Alternatively, steps to render the protein encoded by the MdPRX37L gene in apple loss-of-function.

[0026] Among the above methods, VIGS technology, T-DNA insertion, CRISPR-Cas9 gene editing technology, RNA interference, introduction of gene silencing vectors and other technologies can be used to knock out or silence the MdPRX37L gene in apples.

[0027] A fifth aspect of the present invention provides a method for cultivating an apple variety that maintains stable color of post-harvest apple fruits, comprising the following steps:

[0028] The MdPRX37L gene in wild apple plants was knocked out or silenced to obtain transgenic apple plants.

[0029] In the above method, the transgenic apple plant has the following characteristics 1) or 2) compared with wild apple plants:

[0030] 1) The anthocyanin stabilization ability of the transgenic apple plant is higher than that of the wild apple plant;

[0031] 2) The apple fruit of the transgenic apple plant has a higher ability to maintain color than that of the wild apple plant.

[0032] Beneficial effects of the present invention:

[0033] The present invention has isolated a class III peroxidase PRX gene MdPRX37L from apples. Through the analysis of transgenic function in apple fruits, it is found that it has a significant effect on stabilizing the color of post-harvest apple fruits. The gene can be transformed into plants such as strawberries and peaches to stabilize their fruit coloring ability, improve their yield and quality, and has important economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 :Anthocyanin content and ROS-related indicators of Fuji apple fruits at different storage times. A. Phenotype of Fuji apple fruits at different storage times. B. Anthocyanin content of Fuji apple fruit peel at different storage times. C. H2O2 content of Fuji apple fruit peel at different storage times. D. POD activity of Fuji apple fruit peel at different storage times. E. SOD activity of Fuji apple fruit peel at different storage times.

[0035] Figure 2 : Phylogenetic tree analysis of Arabidopsis PRX family and apple PRX members.

[0036] Figure 3:The expression level of PRX gene in Fuji apple fruits at different storage times. A. The expression level of MdPRX37L gene in Fuji apple fruits at different storage times. B. The expression level of MdPRX34L gene in Fuji apple fruits at different storage times. C. The expression level of MdPRX33L gene in Fuji apple fruits at different storage times.

[0037] Figure 4 :Using VIGS transient expression technology to overexpress MdPRX37L and inject colored Fuji apple fruits, anthocyanin content and reactive oxygen related indicators were detected. AB. Phenotypic diagram of apple fruits and anthocyanin content after MdPRX37L-IL60 and IL60 were transiently injected. C. Gene expression level of MdPRX37L in apple fruits after MdPRX37L-IL60 and IL60 were transiently injected. D. Anthocyanin content of apple fruits after MdPRX37L-IL60 and IL60 were transiently injected. E. H2O2 content of apple fruits after MdPRX37L-IL60 and IL60 were transiently injected. F. POD activity of apple fruits after MdPRX37L-IL60 and IL60 were transiently injected. G. SOD activity of apple fruits after MdPRX37L-IL60 and IL60 were transiently injected.

[0038] Figure 5 :VIGS technology was used to silence MdPRX37L and inject colored Fuji apple fruits, and anthocyanin content and reactive oxygen related indicators were detected. AB. Phenotypic diagram of MdPRX37L-TRV and TRV instantaneous injection apple fruits and anthocyanin content. C. Gene expression level of MdPRX37L in MdPRX37L-TRV and TRV instantaneous injection apple fruits. D. Anthocyanin content of MdPRX37L-TRV and TRV instantaneous injection apple fruits. E. H2O2 content of MdPRX37L-TRV and TRV instantaneous injection apple fruits. F. POD activity of MdPRX37L-TRV and TRV instantaneous injection apple fruits. G. SOD activity of MdPRX37L-TRV and TRV instantaneous injection apple fruits.

[0039] Figure 6 :Inducing MdPRX37L protein in vitro promotes anthocyanin degradation. A. Obtaining MdPRX37L-His and His proteins by prokaryotic induction. B. Detecting the POD activity of MdPRX37L-His and His proteins. C-D. Anthocyanins extracted from Fuji apple fruit were incubated with MdPRX37L-His and His proteins, and the anthocyanin content was detected at different time periods. DETAILED DESCRIPTION

[0040] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0041] As mentioned above, discovering apple PRX genes and conducting functional studies provide potential effective candidate genes for regulating fruit color quality, which not only has important theoretical significance but also has broad application value.

[0042] Based on this, the present invention uses homologous cloning technology to obtain the apple class III peroxidase MdPRX37L gene, and the specific method is as follows:

[0043] Total RNA was extracted from Gala apple tissue culture seedlings and reverse transcribed to obtain cDNA. According to the conservative amino acid sequence of AtPRX37 gene in Arabidopsis thaliana published in the international gene bank, blastp was performed in the apple genome (Malus×domestica-genome.v1.0) to obtain the homologous gene MdPRX37L (MD01G1034400) of AtPRX37 in apple. Primers were designed based on the protein sequence of MD01G1034400 for amplification and ligated with BluntSimple vector, transformed into Escherichia coli DH5α competent cells, screened recombinants, and sequenced and analyzed to obtain the full-length cDNA sequence.

[0044] The full-length cDNA of the apple MdPRX37L gene is 1014 bp. It is deduced that the gene encodes 337 amino acids, with a predicted molecular weight of about 37.4 kDa and an isoelectric point PI of 5.89. Its nucleotide sequence and amino acid sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, as follows:

[0045] Nucleotide sequence:

[0046]

[0047] Description: The transparent box indicates the start codon, and the Indicates the stop codon.

[0048] The grey area indicates the specific fragment SEQ.ID.NO.3 used to construct the silencing vector.

[0049] Amino Acid Sequence:

[0050]

[0051] The present invention provides a method for applying the apple class III peroxidase gene MdPRX37L in apple callus tissue, and silencing the gene can maintain the color quality of post-harvest apple fruits. The steps are:

[0052] (a) Using the MdPRX37L gene as described above, the specific sequence (SEQ ID NO. 3) in the cDNA was reversely placed under the CaMV 35S promoter to construct a plant gene silencing vector.

[0053] (b) Using methods well known in the art, such as Agrobacterium-mediated method, the constructed expression vector and gene silencing vector are introduced into plant cells to obtain transgenic plants.

[0054] The invention relates to a plant expression vector comprising a nucleotide sequence SEQ.ID.NO.1, and is used for promoting the degradation of anthocyanin and reducing the coloring ability of apple fruit.

[0055] The invention relates to a plant gene silencing vector TRV, comprising the gray part of the nucleotide sequence SEQ.ID.NO.1, and is used for inhibiting the degradation of anthocyanin in post-harvest apple fruits and improving the color quality of post-harvest apple fruits.

[0056] The present invention involves introducing the plant expression vector and gene silencing vector into plant cells. The introduction method is the Agrobacterium-mediated transformation method well known to those skilled in the art, and a transgenic plant with strong color stabilization ability is obtained.

[0057] The present invention is described in detail using apple fruit as material, and the MdPRX37L gene and the plant gene silencing vector containing the gene in the present invention can also be used to produce other transgenic plants that maintain the color quality of the fruit.

[0058] The present invention uses VIGS technology instantaneous injection technology and in vitro conditions to induce MdPRX37L protein to promote anthocyanin degradation. The experiment found that silencing the expression of MdPRX37L in apple can stabilize the degradation of anthocyanin in apple fruit. Improving the color quality of transgenic plant fruits has significant economic benefits and social value.

[0059] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0060] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out in accordance with conventional test methods or in accordance with the operating instructions recommended by the supplier. The vectors IL60-2, IL60-1, TRV2 and TRV1 used in the present invention were purchased from Shanghai Yueyoubao Biotechnology Co., Ltd., and were propagated in large quantities in this laboratory and stored in a -80°C ultra-low temperature refrigerator in the form of glycerol bacteria for long-term use.

[0061] Example 1: Cloning of the apple MdPRX37L gene.

[0062] 1) Total RNA was extracted using the CTAB method. The specific method is as follows:

[0063] 1) Take Gala apple tissue culture seedlings, put them into a pre-cooled mortar, add liquid nitrogen to grind them, and transfer them into a pre-cooled 50 ml centrifuge tube;

[0064] 2) Quickly add 10 ml of extraction buffer preheated to 65°C (PVP and mercaptoethanol are added immediately after use), gently mix, and place in a 65°C water bath for 0.5 hours;

[0065] Extraction Buffer:

[0066]

[0067] 3) Add a mixture of water-saturated phenol / chloroform / isoamyl alcohol (25:24:1, volume ratio) equal to the volume of the centrifuge tube liquid in the previous step, shake in an ice bath for 0.5 hours, and centrifuge at 4°C, 12,000 rpm for 20 minutes; transfer the supernatant to a new 50 ml centrifuge tube;

[0068] 4) Add 1 / 3 of the volume of the supernatant with 10 mol / L pre-cooled LiCl, place at -20°C for 3 hours, centrifuge at 12,000 rpm for 30 minutes, and discard the supernatant;

[0069] 5) Add 500 μl SSTE buffer to fully suspend the precipitate, and divide evenly into two 1.5 ml centrifuge tubes;

[0070] SSTE buffer:

[0071]

[0072] 6) Add a water-saturated phenol / chloroform / isoamyl alcohol (25:24:1, volume ratio) mixture equal to the suspension volume, shake in an ice bath for 10 minutes, and centrifuge at 4°C, 12,000 rpm for 10 minutes; transfer the supernatant to a new 1.5 ml centrifuge tube,

[0073] 7) Add a mixture of chloroform / isoamyl alcohol (24:1, volume ratio) equal to the volume of the supernatant, shake in an ice bath for 10 minutes, and centrifuge at 4°C and 12,000 rpm for 10 minutes; transfer the supernatant to a new 1.5 ml centrifuge tube;

[0074] 8) Add 2.5 times the volume of the supernatant to pre-cooled anhydrous ethanol and place at -20°C for 1-2 hours;

[0075] 9) Centrifuge at 12,000 rpm for 20 min at 4°C and wash twice with 70% ethanol;

[0076] 10) Centrifuge at 4°C, 14,000 rpm for 10 minutes, air-dry the precipitate on a clean bench, and add 20 μl of DEPC water to dissolve the RNA.

[0077] 11) Store at -80℃ for future use, or proceed to the following reverse transcription experiment immediately.

[0078] 2) Synthesis of the first strand of reverse transcribed cDNA:

[0079] 1) Prepare the following mixed solution in a 0.2 ml microcentrifuge tube (wherein the RNA is extracted from step 1 of Example 1; if RNA is stored at -80°C, it must be slowly thawed on ice):

[0080]

[0081] 2) Mix gently with a pipette tip, place the microcentrifuge tube on a PCR instrument (65°C for 5 minutes) for denaturation and annealing reactions, and then cool on ice;

[0082] 3) Continue to prepare the following reverse transcription reaction solution in the above microcentrifuge tube.

[0083]

[0084] 4) Perform reverse transcription reaction on a PCR instrument under the following conditions: 30°C for 10 minutes, 42°C for 60 minutes, 70°C for 15 minutes, and store at 4°C. The synthesized reverse transcription product cDNA is used for subsequent related experiments.

[0085] 3) Obtaining the full-length cDNA sequence:

[0086] Based on the conserved amino acid sequence of AtPRX37 in Arabidopsis thaliana found by Tair, the closest homologous sequence to AtPRX37 in apple was found, and primers (5P1 and 3P1) were further designed. PCR amplification was performed using the cDNA synthesized by reverse transcription in step (ii) as a template.

[0087] 5P1: 5′-ATGTCACCTCTCTTCTTCCTCC-3′; (SEQ ID NO.4)

[0088] 3P1: 5′-GAAAGTCGGATTCATCCCCCCA-3′. (SEQ ID NO.5)

[0089] PCR amplification system (the following double-primer PCR reactions all use this system)

[0090]

[0091] PCR reaction program: pre-denaturation at 94°C for 5 minutes; cycle parameters: denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 1 minute and 40 seconds, for 35 cycles; full extension at 72°C for 10 minutes.

[0092] After the PCR reaction, 1.0% agarose gel electrophoresis was performed to detect whether there were bands of appropriate size, and the PCR product was recovered (recovery was performed according to the "Agarose Gel DNA Purification Kit" of Takara Company), vector ligation (3.0 μl of PCR recovery product was taken and connected to the BluntSimple vector, and the operation steps were performed according to the BluntSimple instruction manual), transformation (the ligation product was transformed into Escherichia coli competent cells DH5α, and inverted cultured at 37°C for 12-20 hours on the LB plate culture medium of the corresponding resistance; white colonies were picked and cultured in LB liquid culture medium overnight), and sequence determination (1 ml of the shaken bacteria was taken and placed in a 1.5 ml centrifuge tube, sealed, and sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing). The complete sequence of the MdPRX37L gene was obtained, as shown in SEQ ID NO.1.

[0093] Example 2: Anthocyanin content in apple peel at different storage periods

[0094] 1) The method for determining the anthocyanin content in the peel sample is as follows:

[0095] Weigh 0.1 g of apple peel into 10 ml of anthocyanin extract (mix 95% anhydrous ethanol and 1.5 M HCl in a volume ratio of 85:15), place in the dark for 24 hours, take the supernatant and detect its absorbance at 530 nm, 620 nm and 650 nm to detect the number of nanogram molecules of anthocyanin per unit area.

[0096] Calculation formula: [(A 530 -A 620 )-0.1(A 650 -A 620 )] / ελ*V / M

[0097] V: volume M: mass ελ: extinction coefficient of 530 nanometer light waves of anthocyanin light molecule.

[0098] The results are as follows Figure 1 As shown in B, the results show that during the storage of apple fruit, the degree of anthocyanin degradation in apple peel gradually increases with the extension of storage period.

[0099] 2) The physiological indicators of H2O2 content, POD and SOD were measured using the kits of Suzhou Keming Biotechnology Co., Ltd. Figure 1 As shown in CE, the results showed that the H2O2 content, POD and SOD of apple fruit increased to a certain extent with the extension of storage time.

[0100] Example 3: Expression levels of MdPRX37L, MdPRX34L, and MdPRX33L genes in fruit peels at different storage periods

[0101] 1) In order to explore the main effect genes affecting the degradation of anthocyanins in post-harvest apple fruits, we found through previous studies in other species that class III peroxidases located in the vacuole promote the degradation of anthocyanins. Therefore, we selected the class III peroxidases AtPRX33L, AtPRX34L, and AtPRX37L located in the vacuole in the model crop Arabidopsis thaliana and analyzed them through phylogenetic tree analysis ( Figure 2 ) and found the closely related class III peroxidases MdPRX33L (MD13G1053000), MdPRX34L (MD03G1013700), and MdPRX37L (MD01G1034400) localized in the vacuole in apple.

[0102] 2) Fuji apple peels at different storage periods were sampled and rapidly fixed in liquid nitrogen. Total RNA was extracted from the peels at different storage periods and reverse transcribed into cDNA using the same method as step 2 in Example 1.

[0103] 3) Specific primers 5P2 and 3P2, 5P3 and 3P3, 5P4 and 3P4 as well as the internal reference primers Md18S-F and Md18S-R of apple were designed in the non-conserved regions of MdPRX37L, MdPRX34L, and MdPRX33L genes.

[0104] Md18S-F: 5′-AAACGGCTACCACATCCA-3′, (SEQ.ID.NO.6);

[0105] Md18S-R: 5′-CACCAGACTTGCCCTCCA-3′, (SEQ.ID.NO.7);

[0106] 5P2: 5′-TAATCATGGCGGCTCGAGAC-3′, (SEQ.ID.NO.8);

[0107] 3P2: 5′-TCAATGAGGGAGCTTGCGTT-3′, (SEQ.ID.NO.9);

[0108] 5P3: 5′-CCAACGAAGACCTTCCAGCT-3′, (SEQ.ID.NO.10);

[0109] 3P3: 5′-TCTAGCACGCCCAAATGTGT-3′, (SEQ.ID.NO.11);

[0110] 5P4: 5′-GCCCAGGAATCGTTTCCATGC-3′, (SEQ.ID.NO.12);

[0111] 3P4: 5′-TCTGCAAGTTCAGGGCTAGC-3′, (SEQ.ID.NO.13);

[0112] 4) Using the cDNA obtained in step (2) as a template, adjust these cDNA templates using apple internal reference primers Md18S-F and Md18S-R to make the concentration of each cDNA template consistent.

[0113] 5) qRT-PCR was performed using cDNA templates of consistent concentration to detect the expression differences of MdPRX37L, MdPRX34L, and MdPRX33L genes in Fuji fruits at different storage periods. It was found that the expression of MdPRX37L increased significantly with the extension of storage period, and the expression level was highest at 120 days of storage ( Figure 3 A), therefore, MdPRX37L was further studied in the future.

[0114] Example 3: Construction of MdPRX37L gene vector

[0115] 1) Construction of MdPRX37L gene IL60 expression vector

[0116] In order to study the function of the MdPRX37L gene, the fragment containing the MdPRX37L gene coding region was correctly inserted into the expression vector IL60.

[0117] (1) Using software such as DNAMAN, a PCR reaction was performed based on the nucleotide sequence of the isolated MdPRX37L gene (SEQ ID NO. 1) by homologous recombination technology and using the Blunt simple-MdPRX37L plasmid DNA stored in step 3) of Example 1 as a template.

[0118] 5P5: 5′-TTCGTCGACAAGCTTTAATCATGGCGGCTCG-3′, (SEQ ID NO. 14);

[0119] 3P5: 5′-CACGTGTGGTCTAGATCAATGAGGGAGCTTG-3′, (SEQ ID NO. 15);

[0120] 2) After the PCR reaction is completed, 1.0% agarose gel electrophoresis, PCR product recovery, homologous recombination vector ligation, transformation, sequencing are performed, and the products are used in subsequent experiments after correct identification.

[0121] 3) The constructed recombinant vector MdPRX37L-IL60 was used to transform E. coli DH5α competent cells. PCR identification was performed, and positive colonies were selected for sequencing. The correctly sequenced recombinant monoclonal clones were used for the transient injection experiment of apple fruit.

[0122] II) Construction of TRV expression vector of MdPRX37L gene

[0123] Based on homology comparison, a 294 bp sequence fragment of the non-conserved region of MdPRX37L was inserted into the expression vector TRV.

[0124] 1) Using software such as DNAMAN, primers 5P6 and 3P6 were designed in the non-conserved region of MdPRX37L, and PCR amplification was performed using the plasmid DNA of Blunt simple-MdPRX37L stored in step 3) of Example 1 as a template.

[0125] 5P6: 5′-CCTCCATGGGGATCCGTGACCGGAGACCTG-3′, (SEQ.ID.NO.16);

[0126] 3P6: 5′-CGGGACATGCCCGGGCGGATTCATCCCCCC-3′, (SEQ.ID.NO.17);

[0127] 2) After the PCR reaction is completed, 1.0% agarose gel electrophoresis, PCR product recovery, homologous recombination vector ligation, transformation, sequencing are performed, and the products are used in subsequent experiments after correct identification.

[0128] 3) The plasmid was transformed into Agrobacterium competent cell LB4404, and PCR identification was performed, and positive colonies were selected for sequencing. The recombinant MdPRX37L-TRV single clone with correct sequencing was used for the subsequent transformation of apples.

[0129] III) Construction of prokaryotic inducible expression vector of MdPRX37 gene

[0130] In order to study the function of the MdPRX37L gene, the fragment containing the MdPRX37L gene coding region was correctly inserted into the expression vector PET32a.

[0131] (1) Using software such as DNAMAN, a PCR reaction was performed based on the nucleotide sequence of the isolated MdPRX37L gene (SEQ.ID.NO: 1) by homologous recombination technology, using the Blunt simple-MdPRX37L plasmid DNA stored in step 3) of Example 1 as a template.

[0132] 5P7: 5′-GCTGATATCGGATCCTAATCATGGCGGCTCG-3′, (SEQ.ID.NO.18);

[0133] 3P7: 5′-CGCAAGCTTGTCGACTCAATGAGGGAGCTT-3′, (SEQ.ID.NO.19);

[0134] 2) After the PCR reaction is completed, 1.0% agarose gel electrophoresis, PCR product recovery, homologous recombination vector ligation, transformation, sequencing are performed, and the products are used in subsequent experiments after correct identification.

[0135] 3) The constructed recombinant vector MdPRX37L-PET32a was used to transform E. coli BL21 competent cells. PCR identification was performed, and positive colonies were selected for sequencing. The recombinant monoclonal clones with correct sequencing were used for subsequent protein induction.

[0136] Example 4: VIGS apple fruit transient injection experiment

[0137] 1) Select fresh Fuji fruits with good size and color consistency and normal color for use.

[0138] 2) The vectors of IL60-1, IL60-2, and MdPRX37L-IL60 were extracted with plasmids. The injection combinations were IL60: IL60-1+IL60-2; MdPRX37L-IL60: IL60-1+MdPRX37L-IL60-2. IL60-1 acted as an auxiliary plasmid. The plasmid concentration was diluted to about 10. IL60-1 and IL60-2 were mixed at a ratio of 1:9 (volume ratio). The injection dose for each treatment was 200 μL, and injection experiments were carried out on apple fruits.

[0139] 3) Pick the correct Agrobacterium monoclonal colony and inoculate it into 10mL LB liquid medium (containing 50mg / L kanamycin and 100mg / L rifampicin), 28℃, 200rpm, overnight culture; take 1mL of the bacterial solution and add it into 40mL fresh LB liquid medium, 28℃, 200rpm, shake culture until OD600 is 0.6-0.8 (about 5 hours). Collect the bacteria by centrifugation, dilute and suspend the bacteria with 10mM MgCl2, adjust the bacterial solution concentration to about 0.6, add 10Mm MES and 150μM ACE after adjusting the concentration, and the injection combination is TRV:TRV1+TRV2; MdPRX37L-TRV2+TRV1, TRV1 and TRV2 are mixed according to 3:7 (volume ratio), let stand for 2-3h after mixing, and inject, each treatment injection dose is 200μL, and the injection experiment of apple fruit is carried out.

[0140] 4) After injection, the apple fruit was placed in a dark place for 12 hours, and then placed in a dark incubator for cultivation at a temperature of 19° C. The phenotype was observed for about 5 to 7 days and samples were taken for testing of relevant indicators.

[0141] Example 5: Detection of MdPRX37L gene expression, anthocyanin content and ROS-related indicators in instantaneously injected fruits.

[0142] The corresponding RNA was extracted from the peel samples after instantaneous injection in Example 4, and qRT-PCR detection was performed (see Example 2 for the specific method) to determine the expression level of the MdPRX37L gene in the injected apple fruit.

[0143] The results showed that compared with the control, the expression level of MdPRX37L gene in apple fruit injected with MdPRX37L-IL60 vector was significantly increased ( Figure 4 C), the expression level of MdPRX37L gene in apple fruit injected with MdPRX37L-TRV vector was significantly reduced ( Figure 5 C).

[0144] The method for determining anthocyanin content is the same as in Example 2.

[0145] The results showed that the anthocyanin content in apple fruits injected with MdPRX37L-IL60 vector was reduced compared with that in the control ( Figure 4 D), the anthocyanin content in apple fruit injected with MdPRX37L-TRV vector increased ( Figure 5 D).

[0146] The method for measuring the physiological indicators of H2O2 content, POD and SOD is the same as in Example 2.

[0147] The results showed that compared with the control, the H2O2 content, POD and SOD in apple fruits injected with MdPRX37L-IL60 vector were upregulated to a certain extent ( Figure 4 EG). The H2O2 content, POD and SOD in apple fruits injected with MdPRX37L-TRV vector were downregulated to a certain extent ( Figure 5 EG).

[0148] Example 6: Prokaryotic induction of MdPRX37L-His and His proteins

[0149] In order to determine the function of MdPRX37L in maintaining the degradation of anthocyanins in postharvest fruits, we analyzed its anthocyanin degradation ability. The prokaryotic protein was induced by IPTG-induced Escherichia coli, and the specific steps were as follows:

[0150] 1) Pick a single colony and culture it in LB liquid medium in a shaking incubator for 12 h at 37°C and 200 rpm;

[0151] 2) Absorb the bacterial solution to activate it into new culture medium and continue culturing until OD600 = 0.5;

[0152] 3) Add 3 mM IPTG, continue culturing at 37°C and 200 rpm for 4 hours, take 1 mL of the bacterial solution at 0 h and 4 h, collect the bacterial precipitate by centrifugation, add SDS loading buffer and perform SDS-PAGE electrophoresis and staining detection;

[0153] 4) Collect the cells after 4 hours of induction, suspend them in PBS, and disrupt them by ultrasonication on ice;

[0154] 5) Centrifuge at 4°C, 10,000 rpm for 10 min to separate the precipitate and supernatant, take 100 μL of each of the supernatant and precipitate, add SDS loading buffer and perform SDS-PAGE electrophoresis and staining detection;

[0155] 6) According to the staining test results, determine whether the protein is successfully induced. If the prokaryotic protein is expressed in the supernatant, the prokaryotic protein can be further purified for use. If the prokaryotic protein is expressed in the precipitate, urea should be used to denature and renature the precipitate to release the prokaryotic protein in the inclusion body. The successfully induced prokaryotic protein needs to be purified before use, which can effectively reduce the stray bands. According to the label of the prokaryotic protein, select the HIS protein or GST protein purification kit.

[0156] Example 7: Experiment on the promotion of anthocyanin degradation by MdPRX37L protein under in vitro conditions

[0157] Select the Fuji apple peel with red color, grind the sample with liquid nitrogen, add a small amount of water to dissolve at 37°C, centrifuge for 20 min at 12000rpm, and obtain clear anthocyanins without impurities. Take 500 μL of the prokaryotic induced MdPRX37L-His and His proteins and add them to 3 mL of extracted anthocyanins. Set up more than 3 groups of controls, and observe and detect the anthocyanin content at different time periods after the addition of protein. The method for determining the anthocyanin content is the same as in Example 2.

[0158] The results showed that when the MdPRX37L-His protein was added to anthocyanins in vitro, the degradation of anthocyanins was faster and more obvious than that of the control ( Figure 6 AD).

[0159] Based on the above technology, a class III peroxidase PRX gene MdPRX37L was isolated from apple. Through the functional verification of apple transient injection experiments, it can be seen that MdPRX37L has a significant role in anthocyanin degradation ( Figure 3 ). This gene can be transformed into plants such as strawberries and peaches to improve their coloring ability, increase their yield and quality, and have important economic and social benefits.

[0160] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of the apple MdPRX37L gene as a negative regulatory gene in the following (1) or (2): (1) Inhibit the degradation of anthocyanins in postharvest apple peels; (2) Maintain the color quality of postharvest apple fruit; The apple MdPRX37L gene is a nucleic acid molecule as shown in i) or ii) below: i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

2. Use of the protein encoded by the apple MdPRX37L gene as a negative regulatory protein in the following (1) or (2): (1) Inhibit the degradation of anthocyanins in postharvest apple peels; (2) Maintain the color quality of postharvest apple fruit; The amino acid sequence of the protein encoded by the apple MdPRX37L gene is shown in SEQ ID NO.

2.

3. Use of a gene silencing vector for specifically silencing the MdPRX37L gene in inhibiting anthocyanin degradation in post-harvest apple peel; the gene silencing vector for specifically silencing the MdPRX37L gene comprises the nucleotide sequence shown in SEQ ID NO.

3.

4. The use according to claim 3, characterized in that: The gene silencing vector is a vector in which the nucleotide sequence shown in SEQ ID NO.3 is reversely placed under the CaMV 35S promoter.

5. A method for inhibiting the degradation of anthocyanins in post-harvest apple peels, characterized in that: include: Steps to knock out or silence the MdPRX37L gene in apple; Alternatively, the steps of rendering the protein encoded by the MdPRX37L gene in apple loss-of-function; The apple MdPRX37L gene is a nucleic acid molecule as shown in i) or ii) below: i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

6. A method for cultivating an apple variety that maintains stable color of apple fruits after harvest, characterized in that: The following steps are involved: The MdPRX37L gene in wild apple plants was knocked out or silenced to obtain transgenic apple plants; The apple MdPRX37L gene is a nucleic acid molecule as shown in i) or ii) below: i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

7. The method according to claim 6, characterized in that Compared with wild apple plants, the transgenic apple plants have the following characteristics 1) or 2): 1) The anthocyanin stabilization ability of the transgenic apple plant is higher than that of the wild apple plant; 2) The apple fruit of the transgenic apple plant has a higher ability to maintain color than that of the wild apple plant.

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