PbrASR1, a transcription factor that induces ripening and senescence in Guanyang pear fruit, and its application.

Abstract

This invention discloses the application of the PbrASR1 gene in regulating fruit ripening and senescence in Guanyang snow pear, belonging to the field of genetic engineering technology. The nucleotide sequence of the PbrASR1 gene is shown in SEQ ID NO.1, or is completely complementary to the sequence shown in SEQ ID NO.1, or is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2. This invention can improve fruit firmness and storage tolerance by reducing PbrASR1 expression, and can be applied to plant genetic engineering breeding, providing a new method for the creation or improvement of new germplasm with improved storage tolerance.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and relates to the construction of PbrASR1, a transcription factor that induces ripening and senescence in Guanyang pear fruit, and its recombinant expression vector, and its application in regulating the ripening and senescence of plant fruits. Background Art

[0002] Fruit preservation and storage have always been a key focus in agricultural science, especially in the context of modern agriculture that strives to extend shelf life and maintain superior quality. While traditional refrigeration techniques have alleviated fruit spoilage to some extent, a deeper exploration of the genetic mechanisms underlying fruit is crucial for developing more efficient and durable preservation strategies. In recent years, with the rapid development of genomics and molecular biology, an increasing number of genes have been discovered to participate in fruit ripening regulation, stress response, and storability formation, providing new perspectives for understanding the intrinsic mechanisms of fruit preservation. Abscisic acid (ABA), as a key plant hormone, plays a central role in regulating fruit ripening and responding to environmental stresses. It not only promotes ripening processes such as color change and softening but also participates in regulating physiological and biochemical changes within the fruit, such as sugar accumulation and ethylene production. These changes directly affect the final quality and storability of the fruit. Ethylene, as a marker hormone of fruit ripening, is a key factor determining postharvest lifespan. By regulating the activity of key enzymes in the ethylene synthesis pathway, such as ACC synthase and ACC oxidase, the ripening rate of the fruit can be effectively controlled, thereby affecting its storability. Besides hormonal regulation, the fruit's internal antioxidant system also plays a crucial role in preservation. Antioxidant enzymes, such as superoxide dismutase, catalase, and ascorbate peroxidase, can scavenge reactive oxygen species produced in the fruit after harvest, reducing oxidative damage and thus delaying fruit senescence. Simultaneously, changes in cell wall modifying enzymes and water-retaining substances are closely related to the fruit's storability. For example, the regulation of polysaccharide-degrading enzyme activity can affect fruit firmness and water loss rate, while the accumulation of soluble sugars, organic acids, and osmotic regulators helps maintain the fruit's water balance and stress resistance. Furthermore, the storability of fruit is also closely related to the expression patterns of specific gene families. For instance, differences in the expression of gene families involved in ABA signal transduction, stress response, and cell protection can directly correlate with the fruit's adaptability to the post-harvest environment. Through genetic engineering techniques, such as CRISPR-Cas9 gene editing technology, targeted modification of the function of these genes holds promise for cultivating new varieties with greater storability and achieving precise regulation of post-harvest fruit physiology. In conclusion, in-depth exploration of the intrinsic genetic mechanisms of fruits, particularly the genes and networks related to hormone regulation, antioxidant defense, cell wall modification, and water management, is of great significance for improving the storage and preservation effects of fruits. Future research should focus on revealing more potential molecular targets and applying them to practical variety improvement and preservation technology development to meet market demand for high-quality, long-shelf-life fruits.

[0003] 'Guanyang Snow Pear' is a superior variety within my country's sand pear system. It received national agricultural product geographical indication registration from the Ministry of Agriculture and Rural Affairs in 2009. The fruit can be stored in low-temperature cold storage for more than three months without significant decline in quality or flavor, exhibiting good storability and a long shelf life. However, the mechanism of its storability is still poorly understood. Therefore, elucidating the molecular mechanism of 'Guanyang Snow Pear's' storability is of great significance for improving the storability of pear fruit. Summary of the Invention

[0004] The purpose of this invention is to provide the fruit ripening and senescence-inducing transcription factor PbrASR1 in Guanyang snow pear and its application in regulating fruit ripening and senescence in plants, and / or in cultivating storable plant varieties.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] On the one hand, the present invention provides a gene PbrASR1 that regulates the maturation and senescence of plant tissues or organs. The nucleotide sequence of the PbrASR1 gene encodes a nucleotide sequence of the amino acid sequence shown in SEQ ID NO.2, or encodes a nucleotide sequence that has more than 97% homology with the amino acid sequence shown in SEQ ID NO.2.

[0007] Considering the degeneracy of codons, modifying the bases of the above nucleotide sequence without changing the amino acid sequence also falls within the scope of protection of this invention.

[0008] As is known to those skilled in the art, gene sequences can also contain introns, promoters, and various regulatory elements. Therefore, the nucleotide sequence of the above-mentioned transcription factor PbrASR1 gene can also contain introns, promoters, and various regulatory elements.

[0009] On the other hand, the present invention provides a protein related to the induction of maturation and senescence of plant tissues or organs, characterized in that the protein sequence is as shown in SEQ ID NO.2, or a protein having more than 97% homology with the amino acid sequence shown in SEQ ID No.2.

[0010] On the other hand, the present invention also provides primer pair 1 for cloning the above-mentioned gene. Preferably, the sequence of the forward primer F1 of primer pair 1 is shown in SEQ ID No. 3, and the sequence of the reverse primer R1 is shown in SEQ ID No. 4.

[0011] On the other hand, the present invention also provides primer pair 2 for regulating fruit ripening and senescence. Preferably, the sequence of the forward primer F1 of the primer pair 2 is shown in SEQ ID No. 9, and the sequence of the reverse primer R1 is shown in SEQ ID No. 10.

[0012] On the other hand, the present invention also provides gene silencing target sequences for regulating the maturation and senescence of plant tissues or organs, wherein the sequence is SEQ ID No. 11 or its complete complementary sequence.

[0013] On the other hand, the present invention also provides a kit for regulating the maturation and senescence of plant tissues or organs, the kit comprising the aforementioned primer pair 1 and / or primer pair 2.

[0014] On the other hand, the present invention also provides an overexpression recombinant expression vector or kit containing the above-mentioned PbrASR1 transcription factor that induces maturation and senescence in the tissues or organs of Guanyang pear.

[0015] Preferably, a recombinant expression vector containing the gene can be constructed using existing plant expression vectors or viral expression vectors.

[0016] On the other hand, the present invention also provides a gene loss-of-function recombinant expression vector or kit containing the above-mentioned PbrASR1 transcription factor that induces maturation and senescence in the tissues or organs of Guanyang snow pear.

[0017] Preferably, the gene loss-of-function recombinant expression vector can be a T-DNA insertion mutation vector, a CRISPR targeting mutation vector, a CRISPRi repression vector, an RNAi vector, or a VIGS vector, etc.

[0018] Preferably, a functional loss-of-function recombinant expression vector containing the gene can be constructed using existing plant expression vectors or viral expression vectors.

[0019] On the other hand, the present invention also provides the application of at least one of the aforementioned proteins, genes, primers, target sequences, vectors or kits in regulating the maturation and senescence of plant tissues or organs, and / or in cultivating storable plant varieties.

[0020] The regulation described in this invention can include either promotion or inhibition. When applied to promote the maturation and senescence of plant tissues or organs, it can be achieved by overexpressing the transcription factor PbrASR1 or its encoded protein in the target material. When inhibiting the maturation and senescence of plant tissues or organs, it can be achieved by inhibiting the expression of the gene or its encoded protein in the target material.

[0021] Preferably, the above-described application, when promoting the maturation and senescence of plant tissues or organs, includes the following steps:

[0022] 1) Amplify the plant tissue or organ maturation and senescence-inducing transcription factor PbrASR1;

[0023] 2) The plant tissue or organ maturation and senescence-inducing transcription factor PbrASR1 was ligated to an overexpression vector to obtain a recombinant vector;

[0024] 3) The recombinant vector was transferred into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

[0025] 4) Transform the recombinant Agrobacterium tumefaciens into plant tissues.

[0026] Preferably, the process of amplifying the plant tissue or organ maturation and senescence-inducing transcription factor PbrASR1 in step 1) is as follows: using plant tissue or organ cDNA as a template, PCR amplification is performed to obtain the plant tissue or organ maturation and senescence-inducing transcription factor PbrASR1; the amplification-specific primer pair includes a forward primer F1 and a reverse primer R1; the sequence of the forward primer F1 is shown in SEQ ID No. 3; the sequence of the reverse primer R1 is shown in SEQ ID No. 4.

[0027] Preferably, the aforementioned application in inhibiting fruit ripening and senescence includes the following steps:

[0028] 1) Amplify the gene silencing target sequence of the plant tissue or organ maturation and senescence-inducing transcription factor PbrASR1;

[0029] 2) The gene silencing target sequence of the plant tissue or organ maturation and senescence-inducing transcription factor PbrASR1 is ligated to a gene silencing vector to obtain a recombinant vector;

[0030] 3) The recombinant vector was transferred into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

[0031] 4) Transform the recombinant Agrobacterium tumefaciens into plant tissues or organs.

[0032] The process of amplifying the plant tissue or organ maturation and senescence-inducing transcription factor PbrASR1 in step 1) is as follows: using plant tissue or organ cDNA as a template, PCR amplification is performed to obtain the plant tissue or organ maturation and senescence-inducing transcription factor PbrASR1; the amplification-specific primer pair includes forward primer F1 and reverse primer R1; the sequence of the forward primer F1 is shown in SEQ ID No. 3; the sequence of the reverse primer R1 is shown in SEQ ID No. 4.

[0033] Preferably, the aforementioned promotion of plant tissue or organ maturation and senescence includes at least one of the following phenotypic or physiological changes: decreased tissue or organ stiffness, increased ABA content, increased ethylene content, and decreased titratable acid content.

[0034] Preferably, the aforementioned inhibition of plant tissue or organ maturation and senescence includes at least one of the following phenotypic or physiological changes: increased tissue or organ stiffness, decreased ABA content, decreased ethylene content, increased titratable acid content, and decreased soluble solids content.

[0035] On the other hand, the present invention also provides the application of the aforementioned proteins, genes, primers, target sequences, vectors or kits in plant varieties of early-maturing or storage-resistant fruits.

[0036] Preferably, the plant variety for cultivating early-maturing fruits is obtained by overexpressing the aforementioned protein or gene, and the plant variety for cultivating storable fruits is obtained by reducing the expression of the aforementioned protein or gene.

[0037] Preferably, the aforementioned plant tissue or organ is a plant fruit.

[0038] Preferably, the aforementioned plant is a dicotyledonous plant.

[0039] Furthermore, the aforementioned plants belong to the Rosaceae family.

[0040] Furthermore, the aforementioned plants belong to the Maloideae subfamily.

[0041] Furthermore, the aforementioned plants are all of the genus Pyrus.

[0042] Furthermore, the aforementioned plant is *Pyrus pyrifolia* guanyangensis.

[0043] Compared with the prior art, the present invention has the following advantages:

[0044] 1) This invention provides a novel protein that regulates the maturation and senescence of plant tissues or organs.

[0045] 2) This invention provides a new gene that regulates the maturation and senescence of plant tissues or organs.

[0046] 3) This invention provides a novel method for regulating the maturation and senescence of plant tissues or organs.

[0047] 4) This invention provides a new method for cultivating plant varieties with early-maturing or storage-resistant fruits. Attached Figure Description

[0048] The beneficial effects of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] Figure 1 This is an electrophoresis diagram of the amplification of the transcription factor PbrASR1, which induces ripening and senescence in Guanyang pear fruit according to the present invention.

[0050] Figure 2 This is a schematic diagram illustrating the cloning, localization, and functional verification process of PbrASR1, the fruit ripening and senescence-inducing transcription factor of Guanyang snow pear, according to the present invention.

[0051] Figure 3 This invention illustrates the construction process and structural diagram of the recombinant vector;

[0052] in, Figure 3 A represents the construction process of the recombinant expression vector. Figure 3 B is the structural diagram of the BS-35S-GFP vector. Figure 3 C is a structural diagram of the pTRV2 vector; Figure 3 D is a structural diagram of the pSAK277 carrier.

[0053] Figure 4 The images show the expression patterns and subcellular localization of the PbrASR1 transcription factor, which induces ripening and senescence in Guanyang snow pear fruit according to the present invention, as the fruit develops, in Examples 2 and 3.

[0054] Figure 5 This is a phenotypic diagram of the fruit after inhibition of the ripening and senescence-inducing transcription factor PbrASR1 in Guanyang snow pear fruit according to the present invention.

[0055] in, Figure 5 A shows the development of fruits from the control group and PbrASR1-TRV2. Figure 5 B shows the expression of the PbrASR1 gene in the control and PbrASR1-TRV2 fruits. Figure 5 C represents the fruit firmness of the control and PbrASR1-TRV2.

[0056] Figure 6 This diagram illustrates the effect of inhibiting the ripening and senescence-inducing transcription factor PbrASR1 on the ethylene and ABA content of Guanyang snow pear fruit according to the present invention.

[0057] Figure 7 This figure shows the effect of inhibiting the ripening and senescence-inducing transcription factor PbrASR1 in Guanyang snow pear fruit on the content of titratable acid and soluble solids in the fruit.

[0058] Figure 8 This is a phenotypic diagram of the fruit after overexpression of the transcription factor PbrASR1 induced by ripening and senescence of Guanyang snow pear fruit according to the present invention.

[0059] in, Figure 8 A shows the development of fruits from the control group and PbrASR1-SAK277. Figure 8 B shows the expression of the PbrASR1 gene in the control and PbrASR1-SAK277 fruits. Figure 8 C represents the fruit firmness of the control and PbrASR1-SAK277.

[0060] Figure 9 This diagram illustrates the effect of overexpression of the transcription factor PbrASR1 induced by the ripening and senescence of Guanyang snow pear fruit on the ethylene and ABA content of the fruit.

[0061] Figure 10This figure shows the effect of overexpression of the transcription factor PbrASR1 induced by the ripening and senescence of Guanyang snow pear fruit on the content of titratable acid and soluble solids in the fruit. Detailed Implementation

[0062] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0064] The nucleotide sequence of the PbrASR1 gene in Guanyang snow pear is shown in SEQ ID NO.1 in the example:

[0065] ATGTCTGAAGAGAAGCACCACCGCGGTCTCTTCCACCACCACAAGGAGGAAGACAGACCCTCAGACTACCCTCAGTCTGGCTACTCTGATGAAGGACGTCCCGGCGGCCTTGGTGGTGGTTACGGCGACACCAATGATTATTCTGATGAAGGACGTACCGGTGGCCTAGGTGGTGGCTACGGCGACACCAATGCTTATTCTGGTGAAGGACGTCCCGGCGGCTATGGTGGCTATAACGAGACCACTGCTTACTCTGAGGAAAGAGTTGAAAGACCCGACGGTGGCCGGTACAGTGAGACCACTGCGGCATATGGCACCACCACCACCACTCATGAATCTGAAATTGATTACAAGAAGGAGGAGAAGCACCACAAGCATCTCGAGCACCTCGGCGAGGCCGGTGTGGCTGCTGCCGGCGCTTTTGCCTTGCATGAGAAGCAGAAGGAAAAGAAAGATCCAGAGCATGCCCACAGGCACAAGATTGAAGAGGAGATTGCTGCAGCAGCTGCAGTTGGTTCGGGTGGATTTGCCTTCCATGAACATCATGAGAAGAAAGAGACAAAGGAAGAAGAGGAAGAGGCTCATGGAAAGAAGAAGCACCACCTCTTCTAG

[0066] The amino acid sequence of the PbrASR1 protein of Guanyang snow pear is shown in SEQ ID NO.2:

[0067] MSEEKHHRGLFHHHKEEDRPSDYPQSGYSDEGRPGGLGGGYGDTNDYSDEGRTGGLGGGYGDTNAYSGEGRPGGYGGYNETTAYSEERVERPDGGRYSETTAAYGTTTTTHESEIDYKKEEKHHKHLEHLGEAGVAAAGAFALHEKQKEKKDPEHAHRHKIEEEIAAAAAVGSGGFAFHEHHEKKETKEEEEEAHGKKKHHLF

[0068] Example 1: Cloning of the full-length cDNA of the PbrASR1 gene from Guanyang snow pear

[0069] A pear whole-genome cDNA library was screened to identify PbrASR1, a transcription factor that regulates the ripening and senescence of Guanyang pear fruit. Based on the characteristics of homologous recombination expression vectors and the PbrASR1 gene sequence, primers (SEQ ID No. 3: ATGTCTGAAGAGAAGCACCACCG; SEQ ID No. 4: GAAGAGGTGGTGCTTCTTCTTTCC) were designed using Primerpremier 5.0, and full-length PCR amplification was performed using Guanyang pear fruit cDNA as a template. Detailed steps are as follows:

[0070] The research material, Guanyang snow pear, was grown at the experimental base of the Guangxi Academy of Special Crops, with a tree age of 12 years. Well-developed, disease-free Guanyang snow pear fruits, 150 days after flowering, were selected, and 500 mg samples were randomly weighed and immediately flash-frozen in liquid nitrogen. Total RNA was extracted using the CTAB method. Preparation before the experiment included RNA-free blue, yellow, and white pipette tips and 1.5 ml centrifuge tubes; mortars, pestles, and small keys were sterilized with alcohol at high temperature beforehand and then flash-frozen in liquid nitrogen after cooling. CTAB extraction buffer consisted of: 2% CTAB (W / V, g / 100 ml), 2% PVP K-30 (W / V, g / 100 ml), 10 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2 M NaCl, and 0.5 g / L spermidine.

[0071] After extraction, the quality of the extracted RNA was detected by 1% agarose gel electrophoresis, and the concentration and quality of the RNA were detected by NanoDrop2000 spectrophotometer.

[0072] The synthesis of the first strand of cDNA was performed according to the operating manual of the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. The resulting first-strand cDNA was used for the amplification of the PbASR1 gene. PCR was performed according to the following program: pre-denaturation at 98°C for 2 min; 35 amplification cycles, including denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 3 min, and a final extension at 72°C for 5 min, followed by incubation at 15°C. After amplification, the PCR product, consisting of a single target band, was detected by 1% agarose gel electrophoresis. The specific target band was recovered according to the extraction procedure of the gel extraction kit (purchased from Kangwei Century, China).

[0073] Example 2: qRT-PCR analysis of PbrASR1, a transcription factor induced by ripening and senescence in Guanyang pear fruit.

[0074] To analyze the expression pattern of the PbrASR1 gene in Guanyang pear fruit during fruit development, real-time PCR was used to analyze the expression levels of the PbrASR1 gene at 10, 45, 90, 120, 150, 180, and 210 days after flowering. Based on the coding region sequence of the PbrASR1 gene, upstream and downstream PCR primers for amplifying the entire coding region were designed using Primer Primer 5.0 software, following general primer design principles. RNA was extracted using a kit, and the synthesis of the first strand of cDNA was performed according to the operating manual of the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. The 20 μL reaction system included: 10 μL SYBR Green, 5 μL sterile ultrapure water, 1 μL cDNA, 2 μL forward primer F3: 5'-AGTTGAAAGACCCGACGGTG-3' (SEQ ID No. 5), and 2 μL reverse primer R3: 5'-GCAAATCCACCCGAACCAAC-3' (SEQ ID No. 6). (Actin was used as an internal control, with the following sequences: Actin-F4: 5'-CAATGTGCCTGCCATGTATG-3' (SEQ ID No. 7); Actin-R4: 5'-CCAGCAGCTTCCATTCCAAT-3' (SEQ ID No. 8).

[0075] The qRT-PCR program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 3 s, 60℃ annealing for 10 s, 72℃ extension for 30 s, 45 cycles; 72℃ extension for 3 min, 20℃ incubation for 30 s.

[0076] Using pear fruits from Guanyang at different post-flowering days as material, the relative expression levels of the PbrASR1 gene showed certain expression characteristics, such as... Figure 4 As shown, the expression level of the PbrASR1 gene reaches its peak 150 days after flowering.

[0077] Example 3: Gene silencing and overexpression of transcription factor PbrASR1 induced by fruit ripening and senescence in Guanyang pear.

[0078] 1. Amplification of the PbrASR gene fragment

[0079] Total RNA was extracted from Guanyang pear fruits according to the instructions of the RNA extraction kit (Tiangen, DP432). Using 1 μg of total RNA as a template, cDNA was synthesized using the Thermo reverse transcription kit (USA).

[0080] For gene silencing, a 231 bp PbrASR1-specific fragment was selected using the online website https: / / www.ebi.ac.uk / interpro / . Gene cloning primers F: TTACAAGAAGGAGGAGAAGCAC (SEQ ID No. 9), R: TCTTCCTCTTCTTCCTTTGTCTC (SEQ ID No. 10). The PbrASR1-specific fragment amplified was: TTACAAGAAGGAGGAGAAGCACCACAAGCATCTCGAGCACCTCGGCGAGGCCGGTGTGGCTGCTGCCGGCGCTTTTGCCTTGCATGAGAAGCAGAAGGAAAAGAAAGATCCAGAGCATGCCCACAGGCACAAGATTGAAGAGGAGATTGCTGCAGCAGCTGCAGTTGGTTCGGGTGGATTTGCCTTCCATGAACATCATGAGAAGAAAGAGACAAAGGAAGAAGAGGAAGA (SEQ ID No. 11).

[0081] For overexpression, full-length CDS cloning was performed using the cloning primers in Example 1 (SEQ ID No. 3: ATGTCTGAAGAGAAGCACCACCG; SEQ ID No. 4: GAAGAGGTGGTGCTTCTTCTTTCC).

[0082] PCR reactions were performed using I-5TM 2×High-Fidelity Master Mix, following the instructions. After detection by agarose gel electrophoresis, the correct bands were cut out and recovered from the gel.

[0083] 2. Construction of plant transformation vectors

[0084] After PCR products were subjected to 1% agarose gel electrophoresis, the target band was recovered using a gel electrophoresis kit. The recovered and purified amplified fragment was recombined with expression vectors (silencing vector: pTRV2 vector plasmid; overexpression vector: pSAK277 vector plasmid), and transformed into E. coli competent cells DH5α using the heat shock method. The transformed bacterial culture was tested by PCR, and the PCR-positive culture was sent for sequencing. The correct recombinant target vector was named PbrASR1-pTRV2 or PbrASR1-pSAK277. The recombinant vector PbrASR1-pTRV2 or PbrASR1-pSAK277 was introduced into Agrobacterium GV3101 using the freeze-thaw method (referring to Sambrook, Huang Peitang, translated, Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002).

[0085] 3. The steps of Agrobacterium-mediated genetic transformation of Guanyang pear fruit are as follows:

[0086] (1) Agrobacterium culture: Take Agrobacterium tumefaciens culture stored in an ultra-low temperature freezer (-80℃), streak it on LB agar plates with 50 mg / L kanamycin and 50 mg / L rifampicin, pick single colonies, and culture them in LB liquid medium with 50 mg / L kanamycin and 50 mg / L rifampicin at 28-30℃ and 220-240 rpm / min for 16-24 h.

[0087] (2) OD 600 When the bacterial growth reaches 0.6-0.8, collect the bacterial cells and centrifuge at 5000 rpm for 10 min.

[0088] (3) Prepare the instantaneous conversion induction solution as shown in Table 1 below:

[0089] Table 1. Preparation method of induction solution for instantaneous transformation of Guanyang snow pear fruit.

[0090]

[0091] (4) Select well-developed, disease-free, and 150 days after flowering Guanyang pears.

[0092] (5) Infecting Guanyang snow pear fruits by injection.

[0093] (6) The preferred post-infection culture is dark culture for 12 hours, followed by normal light culture for 16 hours and then 8 hours. The culture temperature after infection is 22-25℃, and the culture is managed normally until physiological and biochemical indicators are measured.

[0094] (7) After 0 days and 15 days of cultivation, the expression of the PbrASR1 gene was detected by real-time quantitative PCR, and the firmness of the fruit, the content of ABA and ethylene in the fruit, and the content of titratable acid and soluble solids in the fruit were determined.

[0095] By injecting plasmids of PbrASR1-TRV2 or PbrASR1-SAK277 and control empty vectors TRV2 or SAK277 into Guanyang pear fruits, and culturing the fruits for 15 days, the results showed that, compared with the control, the expression level of the PbrASR1 gene in fruits injected with PbrASR1-TRV2 was significantly inhibited. Figure 5 B), while compared with the control, the expression level of the PbrASR1 gene in fruits injected with PbrASR1-SAK277 was significantly induced ( Figure 8B). After 0 days of fruit culture, the fruits with the PbrASR1 gene silenced showed no significant differences in firmness, ABA, ethylene, titratable acid, and soluble solids content compared to the control; after 15 days of fruit culture, the fruits with the PbrASR1 gene silenced showed a significant change in firmness compared to the control. Figure 5 C), ABA and ethylene contents decreased significantly ( Figure 6 The titratable acid content increased significantly. Figure 7 A) The content of soluble solids decreased significantly. Figure 7 B). After 0 days of fruit culture, the fruits overexpressing the PbrASR1 gene showed no significant differences in firmness, ABA, ethylene, titratable acid, and soluble solids content compared to the control; after 15 days of fruit culture, the fruits overexpressing the PbrASR1 gene showed a significant change in firmness compared to the control. Figure 8 C), ABA and ethylene contents increased significantly ( Figure 9 The titratable acid content is significantly reduced. Figure 10 A) The content of soluble solids did not change significantly. Figure 10 B). In summary, these results indicate that the PbrASR1 gene can regulate fruit ripening and senescence by influencing the content of ABA and ethylene in Guanyang pears, thereby affecting fruit firmness, titratable acid, and soluble solids content.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of a protein with the amino acid sequence shown in SEQ ID NO.2 in regulating the ripening and senescence of Guanyang snow pear fruit, characterized in that, The regulation of ripening and senescence of Guanyang snow pear fruit is achieved by increasing the expression of the protein to promote ripening and senescence, or by inhibiting the expression of the protein to inhibit ripening and senescence.

2. The application of a gene encoding the amino acid sequence shown in SEQ ID NO.2 in regulating the ripening and senescence of Guanyang snow pear fruit, characterized in that, The regulation of ripening and senescence of Guanyang snow pear fruit is achieved by increasing the expression of the gene to promote ripening and senescence, or by inhibiting the expression of the gene to inhibit ripening and senescence.

3. Application of gene silencing target sequence in inhibiting the ripening and senescence of Guanyang pear fruit, wherein the target sequence is SEQ ID NO.11 or its complete complementary sequence.

4. The application of overexpression vectors or kits containing overexpression vectors in promoting the ripening and senescence of Guanyang pear fruits, characterized in that... The overexpression vector can overexpress a protein with an amino acid sequence as shown in SEQ ID NO.

2.

5. The application of gene function deletion vectors or kits containing gene function deletion vectors in inhibiting the ripening and senescence of Guanyang pear fruits, characterized in that, The gene loss-of-function vector can inhibit the expression of proteins with amino acid sequences as shown in SEQ ID NO.

2.

6. The application of a protein with the amino acid sequence shown in SEQ ID NO.2 in the breeding of early-maturing or storage-resistant Guanyang pear varieties, characterized in that, The cultivation of early-maturing Guanyang snow pear varieties is achieved by increasing the expression of the protein, while the cultivation of storage-resistant Guanyang snow pear varieties is achieved by inhibiting the expression of the protein.

7. The application of a gene encoding the amino acid sequence shown in SEQ ID NO.2 in the breeding of early-maturing or storage-resistant Guanyang Xue pear varieties, characterized in that, The cultivation of early-maturing Guanyang snow pear varieties is achieved by increasing the expression of the aforementioned gene, while the cultivation of storage-resistant Guanyang snow pear varieties is achieved by inhibiting the expression of the aforementioned gene.

8. Application of gene silencing target sequence in breeding storage-resistant Guanyang snow pear varieties, wherein the target sequence is SEQ ID NO.11 or its complete complementary sequence.

9. The application of gene deletion vectors or kits containing gene deletion vectors in the cultivation of storage-resistant Guanyang snow pear varieties, characterized in that, The gene loss-of-function vector can inhibit the expression of proteins with amino acid sequences as shown in SEQ ID NO.2.

Images