Application of pear cytochrome P450 enzyme PbrCYP94B

Abstract

The application discloses application of a pear cytochrome P450 enzyme PbrCYP94B, and the PbrCYP94B has the application in any one of the following (A1)-(A3): (A1) application in improving or reducing wax in pear fruits; (A2) application in preparing products for improving or reducing regulation of wax in pear fruits; (A3) application in improving or reducing wax in pear breeding. The pear cytochrome P450 enzyme PbrCYP94B provided by the application has the effect of promoting wax synthesis through biological function verification, and can be used for improving or reducing the content of wax in pear fruits. PbrCYP94B The gene can be transiently overexpressed or transiently reduced in pear fruits, and the content of wax in the fruits can be significantly improved or reduced.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to the application of a pear cytochrome P450 enzyme, PbrCYP94B. Background Technology

[0002] Pears are a major fruit crop worldwide and the third largest fruit crop in my country, ranking first globally in both area and yield. However, pears are susceptible to diseases such as black spot during storage, causing significant losses to fruit growers annually. The aboveground parts of terrestrial plants are covered with a layer of hydrophobic wax. This epidermal wax is a complex mixture of aliphatic compounds, terpenes, and sterols, playing crucial roles in reducing non-stomatal water loss, enhancing the plant's ability to cope with biotic and abiotic stresses, maintaining plant surface cleanliness, and preventing organ fusion. Studies have shown that epidermal wax can effectively inhibit the germination of conidia from plant pathogens and extend the storage life of pears. For example, higher epidermal wax content effectively inhibits gray mold in roses and tomatoes. 1-MCP treatment can increase the wax content of apple pears, alter the composition and microstructure of the wax, prevent oily peel and pathogen invasion, and extend storage life. Compared to Korla fragrant pears and snowflake pears, the Yulu fragrant pear has a denser epidermal wax structure and the lowest incidence of black spot disease. Furthermore, the alkane components in pear wax are closely related to plant water loss. These studies all indicate that increasing the wax content of pear fruits is of great significance for extending their storage period. Alkanes are the main component of pear fruit wax. However, current research on pear fruit wax mainly focuses on the analysis of its metabolites, with very little research on the molecular mechanisms of pear fruit wax synthesis.

[0003] Members of the cytochrome P450 family play different functions in the decarbonylation pathway of plant cuticle waxes, but no reports have been found on the involvement of these family members in pear fruit wax synthesis. Given the limited research on the metabolic mechanisms of pear fruit waxes in my country, this invention aims to enrich the molecular network of pear fruit waxes by cloning and functionally studying genes that regulate pear fruit wax synthesis. This will contribute to improving pear fruit quality and enhancing the competitiveness of my country's pear industry. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an application of pear cytochrome P450 enzyme PbrCYP94B.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] In a first aspect, the present invention protects the use of pear cytochrome P450 enzyme PbrCYP94B in any of the following (A1)-(A3):

[0007] (A1) Application in increasing or decreasing the wax content of pear fruits;

[0008] (A2) Application in the preparation of products that increase or decrease the wax content of pear fruits;

[0009] (A3) Application in pear breeding to increase or decrease fruit wax content.

[0010] The pear cytochrome P450 enzyme PbrCYP94B is any one of the following (B1)-(B3):

[0011] (B1) A protein with the amino acid sequence shown in SEQ ID NO.2;

[0012] (B2) A protein derived from (B1) with the same activity, but with one or more amino acids substituted, deleted, and / or added to the amino acid sequence shown in SEQ ID NO.2;

[0013] (B3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of (B1) or (B2).

[0014] Secondly, this invention protects the gene encoding the pear cytochrome P450 enzyme PbrCYP94B described above. PbrCYP94B Application in any of the following (A1)-(A3):

[0015] (A1) Application in increasing or decreasing the wax content of pear fruits;

[0016] (A2) Application in the preparation of products that increase or decrease the wax content of pear fruits;

[0017] (A3) Application in pear breeding to increase or decrease fruit wax content.

[0018] In a specific implementation plan, the gene PbrCYP94B, It is selected from any one of the following (C1)-(C3):

[0019] (C1) Its nucleotide sequence is shown in SEQ ID NO.1;

[0020] (C2) The nucleotide sequence shown in SEQ ID NO.1 has been substituted, deleted, and / or have one or more nucleotides added;

[0021] (C3) A nucleotide sequence that hybridizes with a DNA sequence defined by (C1) under strict conditions.

[0022] Thirdly, this invention protects genes containing the aforementioned gene. PbrCYP94B The expression box is used in any of the following (A1)-(A3):

[0023] (A1) Application in increasing or decreasing the wax content of pear fruits;

[0024] (A2) Application in the preparation of products that increase or decrease the wax content of pear fruits;

[0025] (A3) Application in pear breeding to increase or decrease fruit wax content.

[0026] Fourthly, this invention protects genes containing the aforementioned gene. PbrCYP94B Or the recombinant expression vector containing the expression cassette described above may be used in any of the following (A1)-(A3):

[0027] (A1) Application in increasing or decreasing the wax content of pear fruits;

[0028] (A2) Application in the preparation of products that increase or decrease the wax content of pear fruits;

[0029] (A3) Application in pear breeding to increase or decrease fruit wax content.

[0030] In specific implementation schemes, the recombinant expression vector can be constructed using methods disclosed in the prior art. This application provides the following two construction methods for reference:

[0031] (D1) The gene PbrCYP94B was inserted between the XbaI and BamHI sites of pCAMBIA1301 to obtain the recombinant expression vector 35S-PbrCYP94B-GFP;

[0032] (D2) The gene PbrCYP94B was inserted between the BamHI and Ecor1 sites of TRV to obtain the recombinant expression vector TRV-PbrCYP94B.

[0033] Fifthly, the present invention protects the use of transgenic cell lines containing the gene PbrCYP94B described above or containing the recombinant expression vector described above in any of the following (A1)-(A3):

[0034] (A1) Application in increasing or decreasing the wax content of pear fruits;

[0035] (A2) Application in the preparation of products that increase or decrease the wax content of pear fruits;

[0036] (A3) Application in pear breeding to increase or decrease fruit wax content.

[0037] Sixthly, the present invention protects genes containing the foregoing. PbrCYP94B Or the use of genetically engineered bacteria containing the recombinant expression vector described above in any of the following (A1)-(A3):

[0038] (A1) Application in increasing or decreasing the wax content of pear fruits;

[0039] (A2) Application in the preparation of products that increase or decrease the wax content of pear fruits;

[0040] (A3) Application in pear breeding to increase or decrease fruit wax content.

[0041] Seventhly, the present invention protects a method for increasing the wax content of pear fruit, said method being achieved by increasing the expression level of the gene encoding the pear cytochrome P450 enzyme PbrCYP94B in pear.

[0042] In a specific implementation plan, the expression level of the gene encoding the pear cytochrome P450 enzyme PbrCYP94B in pear is increased by introducing the gene encoding the pear cytochrome P450 enzyme PbrCYP94B as described above into the target pear.

[0043] The applicant constructed a plant overexpression vector for the pear cytochrome P450 enzyme PbrCYP94B and transiently transformed pears by injection, using pears transformed with the pCAMBIA1301-GFP vector as controls. The resulting pear peel... PbrCYP94B Gene expression levels and wax content were significantly increased.

[0044] Eighthly, the present invention protects a method for reducing the wax content of pear fruit, said method being achieved by reducing the expression level of the gene encoding the pear cytochrome P450 enzyme PbrCYP94B in pear.

[0045] In a specific implementation, the reduction of the expression level of the gene encoding the pear cytochrome P450 enzyme PbrCYP94B in pear is achieved by silencing the gene encoding the pear cytochrome P450 enzyme PbrCYP94B as described above.

[0046] The applicant constructed a plant gene silencing vector for the pear cytochrome P450 enzyme PbrCYP94B and transiently transformed pears by injection, using pears transformed with the TRV vector as a control. The resulting pear peel... PbrCYP94B Gene expression levels and wax content decreased significantly.

[0047] Beneficial effects of the present invention

[0048] The application of the pear cytochrome P450 enzyme PbrCYP94B provided by this invention has the following beneficial effects compared with the prior art:

[0049] (1) The pear cytochrome P450 enzyme PbrCYP94B provided by this invention has been verified by biological function to promote wax synthesis. PbrCYP94B Transient overexpression or transient reduction of the gene in pear fruit can significantly increase or decrease the wax content in the fruit.

[0050] (2) The present invention provides PbrCYP94B Genes promote the synthesis of fruit wax and extend the storage period of fruit, laying the foundation for improving economic benefits by adjusting the gloss of fruit through wax regulation to enhance consumer preference. Attached Figure Description

[0051] Figure 1 The pear cloned by this invention PbrCYP94B Agarose gel electrophoresis image of the gene; where the left band is the marker; the right band is the pear PbrCYP94B gene cloned in this invention.

[0052] Figure 2 for PbrCYP94B Features of gene structure (A) and conserved domains (B).

[0053] Figure 3 Phylogenetic tree of PbrCRYP94B and other CYP family members in Arabidopsis.

[0054] Figure 4 The total wax content of 'Yuluxiang' pear peel after bagging treatment and PbrCRYP94B Gene expression levels and correlation analysis ( R =0.87).

[0055] Figure 5 The content of alkane in the peel of 'Yuluxiang' pear after bagging treatment and PbrCRYP94B Gene expression levels and correlation analysis ( R =0.80).

[0056] Figure 6 The pear cloned by this invention PbrCRYP94B The effect of transient overexpression of the gene in 'Yuluxiang' pear fruit on the fruit waxy texture; among which, Figure 6 Figure A in the diagram represents gene expression levels. Figure 6 Figure B in the diagram: Wax content; Figure 6 Figure C in the diagram: Alkane content; Figure 6 Figure D in the diagram shows the content of alkanes with different carbon chain lengths. Figure 6 Figure E in the diagram: Oil Red O staining in the control group; Figure 6 F-graph in the middle: PbrCRYP94B Oil Red O staining of the gene overexpression group; Figure 6 G diagram: stratum corneum thickness.

[0057] Figure 7 The pear cloned by this invention PbrCRYP94B The effect of gene silencing on the waxy texture of 'Yuluxiang' pear fruit; among which... Figure 7 Figure A in the diagram represents gene expression levels. Figure 7 Figure B in the diagram: Wax content; Figure 7 Figure C in the diagram: Alkane content; Figure 7 Figure D in the diagram shows the content of alkanes with different carbon chain lengths. Figure 7 Figure E in the diagram: Oil Red O staining in the control group; Figure 7 F-graph in the middle: PbrCRYP94B Oil Red O staining of gene silencing groups; Figure 7 G diagram: stratum corneum thickness. Detailed Implementation

[0058] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and embodiments, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention without departing from its spirit and scope to make it suitable for various uses and conditions.

[0059] The applicant isolated and cloned a new gene, PbrCRYP94B, from the 'Yuluxiang' pear. Its nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing and has a length of 1518 bp.

[0060] The present invention also provides the amino acid sequence encoded by the pear cytochrome P450 enzyme PbrCYP94B, which encodes 506 amino acids, as shown in SEQ ID No. 2.

[0061] The effects of qRT-PCR on the fruit peel before and after bagging treatment were analyzed. PbrCYP94B Gene expression patterns, and respectively on PbrCYP94B Correlation analysis of relative expression levels, total wax content, and alkane content showed that... PbrCYP94B The relative expression level was significantly positively correlated with the total wax content and alkane content, indicating that the pear PbrCYP94B gene cloned in this invention is a candidate gene involved in wax synthesis.

[0062] Example 1: Pear PbrCYP94B Gene cloning and construction of plant overexpression vectors

[0063] 1. Pear PbrCYP94B Cloning of genes

[0064] The pericarp of the 'Yuluxiang' pear variety, harvested 140 days after flowering at maturity, was used as the test material. RNA was extracted according to the instructions of the Plant Polysaccharide and Polyphenol Total RNA Extraction Kit. After extraction, the quality of the extracted RNA was assessed by 1% agarose gel electrophoresis, and the concentration and quality of RNA were determined using a NanoDrop 2000 spectrophotometer. The synthesis of the first strand of cDNA was performed according to the instructions of the ThermoScientific RevertAid First Strand cDNA Synthesis Kit. The obtained first-strand cDNA was used for... PbrCYP94BThe nucleotide sequences of the primer pairs for gene amplification are shown below:

[0065] F1: 5'-gagaacacgggggactctagaATGTTTCTTGAGCTCTTAACCTTCGCCTTC-3' (SEQ IDNO.3),

[0066] R1: 5'-gcccttgctcaccatggatccAGCCCTCTCCAAAACGGTGACCGGAAGTCC-3' (SEQ ID NO. 4).

[0067] The 50 μL PCR reaction system was as follows: 25 μL PrimeSTAR Max Premix (2×) (purchased from TakaRa), 0.6 μM forward primer F1, 0.6 μM reverse primer R1, 200 ng cDNA, and sterile distilled water to bring the total volume to 50 μL. The PCR program was as follows: 98 ℃ pre-denaturation for 3 min; 32 amplification cycles, including 98 ℃ denaturation for 10 s, 58 ℃ annealing for 5 s, 72 ℃ extension for 3 min, followed by a 72 ℃ extension for 10 min, and then incubation at 10 ℃. After amplification, the PCR product, consisting of a single target band, was detected by 1.5% agarose gel electrophoresis. The specific target band was recovered according to the instructions of the gel extraction kit (purchased from Novizan, China). Figure 1 ).

[0068] 2. Constructing a pear PbrCYP94B Plant overexpression vectors for genes

[0069] The purified PCR product was ligated into the pCAMBIA1301-GFP vector and transformed into *E. coli* DH5α using a heat shock method. PCR detection was performed using gene-specific primers, and positive bacterial cultures were sequenced (performed by Genewiz). Sequencing results showed that the target fragment amplified in this invention was 1515 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1. Sequence alignment analysis confirmed that this sequence is the target gene required in this invention. Figure 2 The phylogenetic results indicate that this gene belongs to the same group as members of the Arabidopsis CYP94B subfamily, and it is named... PbrCYP94B ( Figure 3 The correct recombinant target vector was named 35S- PbrCYP94B -GFP. The recombinant vector 35S- was synthesized using a heat shock method. PbrCYP94B -GFP and the control empty vector 35S-GFP were transformed into Agrobacterium GV3101, respectively.

[0070] 3. Constructing a pear PbrCYP94BPlant gene silencing vector

[0071] Using the one-stranded cDNA obtained in Part 1 of Example 1 for... PbrCYP94B The nucleotide sequences of the primer pairs for gene amplification are shown below:

[0072] F2: 5'-gtgagtaaggttaccgaattcCCGACCTCCTCGGCGACGGCATTCT-3' (SEQ ID NO.5),

[0073] R2: 5'-cgtgagctcggtaccggatccAAGCCACTGCGAATTTGGCTTCGGG-3' (SEQ ID NO. 6).

[0074] The target product was amplified using the PCR reaction system described in Part 1 of Example 1 by adding forward primer F2 and reverse primer R2, and the specific target band was recovered. The recovered and purified PCR product was ligated into the TRV vector and transformed into *E. coli* DH5α using the heat shock method. PCR detection was performed using gene-specific primers, and the positive bacterial cultures were sequenced (performed by Genewiz). The sequencing results showed that the target fragment amplified in this invention was 300 bp in length, and its nucleotide sequence is shown in SEQ ID NO. 7. Sequence alignment analysis confirmed that this sequence is the target gene required in this invention. Figure 2 The correct recombinant target vector was named TRV- PbrCYP94B The recombinant vector TRV- was synthesized using a thermal shock method. PbrCYP94B The empty TRV vector and the control vector were transformed into Agrobacterium GV3101, respectively.

[0075] Example 2: Pears after bagging treatment PbrCYP94B Correlation analysis between gene expression levels and waxes

[0076] 1. Pears after bagging PbrCYP94B qRT-PCR analysis of genes

[0077] Forty days after flowering, 'Yuluxiang' pears were treated with white paper bags, yellow outer bags with white inner bags, and yellow outer bags with black inner bags, respectively, with no bagging as a control. Natural field management was maintained until 80 days after flowering, when peel tissue samples were collected from both treatment and control groups. Total RNA was extracted and cDNA synthesized using the method described in Example 1. Pears were selected... PbrTUB The primer nucleotide sequence, used as an internal reference gene, is as follows:

[0078] F2: 5'-TGGGCTTTGCTCCTCTTAC-3' (SEQ ID NO.8),

[0079] R2: 5'-CCTTCGTGCTCATCTTACC-3' (SEQ ID NO. 9).

[0080] Using Primer Premier 5.0 PbrCYP94B Gene-specific qRT-PCR primer pairs were designed within the open reading frame of the gene. The nucleotide sequences of the primers are as follows:

[0081] F3: 5'- GGGGATGACTGTGACGTACC-3' (SEQ ID NO. 10),

[0082] R3: 5'-CCGCTTGAAAAACGGGATAGC-3' (SEQ ID NO. 11).

[0083] qRT-PCR was performed using the ChamQ SYBR qPCR Master Mix kit (purchased from Novizan), strictly following the instructions.

[0084] The 20 μL qRT-PCR reaction system included: 10 µL 2×ChamQ SYBR qPCR Master Mix, 7.8 µL sterile ultrapure water, 1 µL cDNA, 0.4 µL forward primer (10 µM), 0.4 µL reverse primer (10 µM), and 0.4 µL 50×ROX Reference Dye 1. PCR was performed using a 36-well qRT-PCR plate (Roche) and a qRT-PCR instrument (LightCycler 480, Roche). The qRT-PCR reaction program was: 95℃ pre-denaturation for 30 s; 95℃ for 10 s, 60℃ for 30 s, 40 cycles; the melting curve was 95℃ for 15 s, 60℃ for 60 s, 95℃ for 15 s. The reaction was repeated three times, and the average Ct value for each cDNA sample was calculated using formula 2. -ΔΔCt get PbrCYP94B Relative expression level of genes ( Figure 4 , Figure 5 ).

[0085] 2. Wax content of pear fruit after bagging treatment

[0086] The bagging treatment method for 'Yuluxiang' pear fruits is the same as in Part 1 of Example 2. The pear fruits were washed thoroughly with water and allowed to air dry. Then, the fruits were completely immersed in chloroform solution twice in a fume hood, each time for 1 minute, to extract the epidermal wax. The extract containing the epidermal wax was then transferred to a test tube and concentrated to approximately 1.5 ml using a nitrogen evaporator (JHD-001S, Shanghai Jiheng Industrial Co., Ltd.) at 40 °C. The sample was then filtered through a 0.45 μm organic filter membrane and transferred to a brown gas chromatography-mass spectrometry (GC-MS) vial. Then, 10 μl of n-tetracosane (10 μg / μl, Shanghai Maclean Biochemical Technology Co., Ltd.) was added as an internal standard, and the chloroform was dried under nitrogen. 40 μl of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma) and 40 μl of pyridine (Sigma) were added, and the mixture was incubated in a water bath at 70 °C for 1 hour. All reagents were dried again under chloroform, and the mixture was dissolved in 1.2 ml of chromatographic grade chloroform. The waxy components were detected using a gas chromatograph (TRACE 1310, Thermo Scientific) and a triple quadrupole mass spectrometer (TSQ 9000, Thermo Scientific). Compounds were separated on a TG-5MS capillary column (30m × 0.25mm ID, 0.25 μm film, Thermo Scientific). Helium was used as the carrier gas at a flow rate of 1.2 mL / min. The following parameters were used: injection temperature 280 °C; transfer line temperature 280 °C; ion source temperature 250 °C; quadrupole temperature 150 °C; electron energy (EI) 70 eV; scan range 50–650 m / z. The temperature program was as follows: 50 °C for 2 minutes. Then, the temperature was increased to 200 °C at a rate of 40 °C / min and held for 2 minutes. Finally, the temperature was increased to 320 °C at a rate of 3 °C / min and held for 30 minutes. Waxy compounds were identified by matching retention indices with mass spectra in the NIST 2017 library. Alkanes were identified by comparing mass spectra and retention times of C7-C40 saturated alkane standards (Sigma-Aldrich).

[0087] By integrating the results of qRT-PCR and GC-MS assays, it was found that bagging treatment of pears... PbrCYP94B The relative expression levels of the gene were highly correlated with both total wax content and alkane content, with correlation coefficients reaching 0.8 and 0.87, respectively. Figure 4 , Figure 5 This indicates that the pear cloned in this invention... PbrCYP94B The gene is a candidate gene involved in wax synthesis.

[0088] Example 3: Determination of wax content and components in pear fruit by transient overexpression

[0089] Take Agrobacterium tumefaciens (containing the '35S-PbrCYP94B-GFP' recombinant vector or the 35S-GFP control empty vector) stored in an ultra-low temperature freezer, and in a solution containing 100 mg·L⁻¹ -1 Kanamycin and 100 mg∙L -1 Rifampicin was streaked onto solid LB medium and incubated at 28 °C for 2 days. Then, single colonies were picked off the streaks using a sterile toothpick and placed in a 100 mL Erlenmeyer flask. 30 mL of a solution containing 100 mg / L kanamycin and 100 mg·L⁻¹ kanamycin was added. -1 The cells were cultured in LB liquid medium containing rifampicin at 200 rpm for 12 hours in a shaker at 28 ℃. The cells were then collected by centrifugation at 5000 rpm for 5 minutes in 50 mL centrifuge tubes. After resuspending the cells in 10 mL of induction solution (10 mM MgCl2, 10 mM MES, 200 mM acetylsylcholine, pH=5.5), the cells were centrifuged again. Infection solution was added again, and the OD value of the bacterial solution was adjusted to between 0.6 and 0.8 using a spectrophotometer. The cells were then incubated in a shaker at 100 rpm in the dark for 4 hours at 25 ℃. Empty vectors of 35S-GFP and 35S-PbrCYP94B-GFP were injected into pear fruits near the equator using a syringe. Six wells were injected into each pear fruit, and at least seven fruits were injected in each experiment. The experiment was repeated three times. The fruits were then incubated in the dark at 21 ℃ for 24 hours, followed by incubation at 21 ℃ with low light intensity and a photoperiod of 16 hours light / 8 hours darkness for 2 days. Finally, the method of Example 2 was used to determine... PbrCYP94B Overexpression and control group pear fruits PbrCYP94B Gene expression levels and wax content were measured using Oil Red O staining to determine stratum corneum thickness.

[0090] The results showed that the overexpression group was significantly different from the control group. PbrCYP94B Gene expression levels, total wax and alkane content, and stratum corneum thickness increased significantly, by 6-fold, 43%, 28%, and 1-fold, respectively. Figure 6 Furthermore, the content of ultra-long chain 23, 25, 27, and 29 alkanes increased by 21-39% compared to the control group. Figure 6 ).

[0091] Example 4: Determination of wax content and components in gene-silenced pear fruit

[0092] Agrobacterium containing the 'TRV-PbrCYP94B' recombinant vector was activated according to the method in Example 3. Then, a single colony was picked up from the thread with a sterile toothpick and placed in a 100 mL Erlenmeyer flask. 30 mL of a solution containing 100 mg / L kanamycin and 100 mg·L⁻¹ kanamycin was added. -1Rifampicin liquid LB medium was cultured at 200 rpm for 12 hours in a shaker at 28 ℃. The cells were collected by centrifugation at 5000 rpm for 5 minutes in 50 mL centrifuge tubes. The cells were resuspended in 10 mL of induction solution (10 mM MgCl2, 10 mM MES, 200 mM acetylsylcholine, pH=5.5), centrifuged again, and then the OD value of the bacterial suspension was adjusted to between 0.6 and 0.8 using a spectrophotometer. A mixed infection solution was prepared by mixing TRV1:TRV2 (PbrCYP94B-TRV2) at a ratio of 1:1 and incubated at 100 rpm in a shaker at 25 ℃ in the dark for 4 hours. h, using a syringe, a mixed infection solution of TRV1 and empty TRV2, and a mixed infection solution of TRV1 and PbrCYP94B-TRV2, were injected into pear fruits near the equator. Six wells were injected into each pear fruit, and at least seven fruits were injected in each experiment. The experiment was repeated three times. The fruits were then incubated in the dark at 21°C for 24 hours, followed by incubation at 21°C with low light intensity and a photoperiod of 16 hours light / 8 hours dark for 2 days. Finally, the results were measured according to the method in Example 2. PbrCYP94B Gene silencing and control group pear fruits PbrCYP94B Gene expression levels and wax content were measured using Oil Red O staining to determine stratum corneum thickness.

[0093] The results showed that the gene silencing group was significantly different from the control group. PbrCYP94B Gene expression levels, total wax and alkane content, and stratum corneum thickness were significantly reduced, decreasing by 53.7%, 22%, 22%, and 18.6%, respectively. Figure 7 Furthermore, the content of ultra-long chain 23, 25, 27, and 29 alkanes increased by 24-34% compared to the control group. Figure 7 ).

[0094] As can be seen from the above embodiments, the pear cytochrome P450 enzyme provided by the present invention... PbrCYP94B Association analysis and experiments involving transient overexpression and gene silencing in pear fruit have shown that it has a positive effect on regulating the accumulation of fruit wax, especially alkane components.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B in any of (Al)-(A6) below: (A1) in increasing wax in Pyrus bretschneideri fruit by overexpressing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A2) in decreasing wax in Pyrus bretschneideri fruit by silencing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A3) in preparing a product for increasing wax in Pyrus bretschneideri fruit by overexpressing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A4) in preparing a product for decreasing wax in Pyrus bretschneideri fruit by silencing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A5) in breeding Pyrus bretschneideri for increasing fruit wax by overexpressing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A6) in breeding Pyrus bretschneideri for decreasing fruit wax by silencing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; said Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B is a protein with an amino acid sequence as shown in SEQ ID NO.

2.

2. A gene encoding the cytochrome P450 enzyme PbrCYP94B of claim 1 PbrCYP94B Use in any one of (Al) - (A6) below: (A1) in increasing wax in Pyrus bretschneideri fruit by overexpressing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A2) in decreasing wax in Pyrus bretschneideri fruit by silencing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A3) in preparing a product for increasing wax in Pyrus bretschneideri fruit by overexpressing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A4) in preparing a product for decreasing wax in Pyrus bretschneideri fruit by silencing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A5) in breeding Pyrus bretschneideri for increasing fruit wax by overexpressing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A6) in breeding Pyrus bretschneideri for decreasing fruit wax by silencing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B.

3. Use according to claim 2, characterized in that, The nucleotide sequence of the gene PbrCYP94B is shown as SEQ ID NO.

1.

4. A composition comprising the gene of claim 2 PbrCYP94B use of the expression cassette of the gene of claim 2 in any one of (Al) - (A6): (A1) in increasing wax in Pyrus bretschneideri fruit by overexpressing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A2) in decreasing wax in Pyrus bretschneideri fruit by silencing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A3) in preparing a product for increasing wax in Pyrus bretschneideri fruit by overexpressing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A4) in preparing a product for decreasing wax in Pyrus bretschneideri fruit by silencing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A5) in breeding Pyrus bretschneideri for increasing fruit wax by overexpressing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A6) in breeding Pyrus bretschneideri for decreasing fruit wax by silencing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B.

5. A recombinant expression vector comprising the gene of claim 2 PbrCYP94B or a recombinant expression vector comprising the expression cassette of claim 4 for use in any one of (Al) - (A6) below: (A1) in increasing wax in Pyrus bretschneideri fruit by overexpressing a gene encoding Pyrus bretschneideri cytochrome P450 enzyme PbrCYP94B; (A1) use of over-expressing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to increase the wax of Pyrus fruit; (A2) use of silencing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to decrease the wax of Pyrus fruit; (A3) use of over-expressing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to prepare products for increasing the wax of Pyrus fruit; (A4) use of silencing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to prepare products for decreasing the wax of Pyrus fruit; (A5) use of over-expressing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to breed Pyrus for increasing the wax of fruit; 6. Use according to claim 5, characterized in that, (A6) use of silencing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to breed Pyrus for decreasing the wax of fruit. (D1) The gene The recombinant expression vector is constructed by any of the following methods: was inserted between Xbal and BamHI sites of pCAMBIA1301 to get the recombinant expression vector 35S- PbrCYP94B -GFP; (D2) inserting the gene PbrCYP94B into the BamHI and EcoRI sites of TRV to obtain a recombinant expression vector TRV- PbrCYP94B .

7. A transgenic cell line containing the gene of claim 2 PbrCYP94B or the recombinant expression vector of claim 5 for use in any one of (Al) - (A6) below: PbrCYP94B (A1) use of over-expressing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to increase the wax of Pyrus fruit; (A2) use of silencing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to decrease the wax of Pyrus fruit; (A3) use of over-expressing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to prepare products for increasing the wax of Pyrus fruit; (A4) use of silencing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to prepare products for decreasing the wax of Pyrus fruit; (A5) use of over-expressing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to breed Pyrus for increasing the wax of fruit; 8. A genetically engineered bacterium comprising the gene of claim 2 (A6) use of silencing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to breed Pyrus for decreasing the wax of fruit. or the recombinant expression vector of claim 5 for use in any one of (Al) - (A6) below: PbrCYP94B (A1) use of over-expressing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to increase the wax of Pyrus fruit; (A2) use of silencing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to decrease the wax of Pyrus fruit; (A3) use of over-expressing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to prepare products for increasing the wax of Pyrus fruit; (A4) use of silencing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to prepare products for decreasing the wax of Pyrus fruit; (A5) use of over-expressing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to breed Pyrus for increasing the wax of fruit; 9. A method for increasing the wax content of a pear fruit, characterized in that, (A6) use of silencing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme to breed Pyrus for decreasing the wax of fruit. The method is achieved by increasing the expression amount of the gene encoding PbrCYP94B in Pyrus; the increase of the expression amount of the gene encoding PbrCYP94B in Pyrus is achieved by introducing the gene encoding PbrCYP94B in Pyrus cell P450 enzyme into the target Pyrus.

10. A method of reducing the waxy content of a pear fruit, characterized in that, The method is achieved by reducing the expression of the gene encoding the PbrCYP94B cytochrome P450 enzyme in the pear; the reduction of the expression of the gene encoding the PbrCYP94B cytochrome P450 enzyme in the pear is achieved by silencing the gene encoding the PbrCYP94B cytochrome P450 enzyme according to claim 1.

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