Pyriform long-chain acyl-coa synthetase, its coding gene and application

Abstract

The present application belongs to the field of plant genetic engineering, and particularly relates to a Pyrus pyrifolia long-chain acyl-CoA synthetase, a coding gene thereof and application. The Pyrus pyrifolia long-chain acyl-CoA synthetase provided by the present application has a nucleotide sequence as shown in any one of SEQ ID ID NO. 1-3 PbrLACS3 / 4 / 5 , or a nucleotide sequence as shown in any one of SEQ ID NO. 1-3, wherein one or more nucleotides are substituted, deleted and / or added, or a nucleotide sequence hybridizing with the DNA sequence defined in 1) under stringent conditions. The Pyrus pyrifolia long-chain acyl-CoA synthetase provided by the present application is verified to have an effect of promoting the synthesis of waxy substances, and the gene thereof can be transiently overexpressed or simultaneously transiently reduced in Pyrus pyrifolia fruit in sequence, so as to significantly increase or reduce the content of waxy substances in the fruit PbrLACS3 / 4 / 5 . PbrLACS3 / 4 / 5 ​

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to pear long-chain acyl-CoA synthase, its encoding gene, and its applications. Background Technology

[0002] Pear is an important economic crop in my country, ranking third in the world in terms of cultivated area, after apples and citrus. Improving pear yield and quality is one of the important research goals in the current pear research field. The waxy coating has multiple functions, including reducing water loss and mechanical damage, resisting biotic and abiotic attacks, maintaining fruit quality during storage, and extending shelf life. As one of the most important quality traits of pears, research on the composition and formation mechanism of pear fruit wax is of positive significance for improving pear fruit quality, increasing pear yield, and enhancing economic benefits.

[0003] Currently, there are no reports on the function and mechanism of LACS in pears. This invention, through gene cloning and functional research on regulating pear fruit wax synthesis, further enriches the molecular network of pear fruit wax, which will help improve pear fruit quality and enhance 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 a pear long-chain acyl-CoA synthase PbrLACS3 / 4 / 5, its encoding gene, and its applications.

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

[0006] Firstly, this invention protects a pear PbrLACS3 / 4 / 5 gene, selected from the following a), b), or c):

[0007] a) Its nucleotide sequence is as shown in any of SEQ ID NO. 1-3;

[0008] b) Any of the nucleotide sequences shown in SEQ ID NO. 1-3 are substituted, deleted, and / or have one or more nucleotides added;

[0009] c) A nucleotide sequence that hybridizes with the DNA sequence defined in 1) under strict conditions.

[0010] The pear PbrLACS3 / 4 / 5 genes refer to the pear PbrLACS3 gene, the pear PbrLACS4 gene, or the pear PbrLACS5 gene.

[0011] In a more specific embodiment, the nucleotide sequence of the pear PbrLACS3 gene is shown in SEQ ID NO.1.

[0012] In a specific implementation, the nucleotide sequence of the pear PbrLACS4 gene is shown in SEQ ID NO.2.

[0013] In a specific implementation, the nucleotide sequence of the pear PbrLACS5 gene is shown in SEQ ID NO.3.

[0014] Secondly, this invention protects the protein encoded by the pear PbrLACS3 / 4 / 5 gene described above.

[0015] In a specific implementation scheme, the protein encoded by the pear PbrLACS3 / 4 / 5 gene is selected from either A) or B):

[0016] A) Proteins with amino acid sequences as shown in any of SEQ ID NO.4-6;

[0017] B) Proteins derived from 1) with the same activity, but with one or more amino acids substituted, deleted, and / or added to any of the amino acid sequences shown in SEQ ID NO.4-6.

[0018] In a more specific embodiment, the protein encoded by the pear PbrLACS3 gene is a protein with an amino acid sequence as shown in SEQ ID NO.4.

[0019] In a more specific embodiment, the protein encoded by the pear PbrLACS4 gene is a protein with an amino acid sequence as shown in SEQ ID NO. 5.

[0020] In a more specific embodiment, the protein encoded by the pear PbrLACS5 gene is a protein with an amino acid sequence as shown in SEQ ID NO. 6.

[0021] Thirdly, the present invention also protects primer pairs for amplifying the full length or any fragment of the aforementioned pear PbrLACS3 / 4 / 5 gene.

[0022] As a preferred technical solution of this application, the primer pair is as shown in any one of SEQ ID No. 9-10, SEQ ID No. 11-12 or SEQ ID No. 13-14.

[0023] Fourthly, the present invention also protects recombinant expression vectors, expression cassettes, transgenic cell lines or genetically engineered bacteria containing the aforementioned pear PbrLACS3 / 4 / 5 genes.

[0024] Preferably, the recombinant expression vector is constructed by inserting the genes PbrLACS3 / 4 / 5 sequentially between the XbaI and BamHI sites of pCAMBIA1301 to obtain the recombinant expression vectors 35S-LACS3-GFP, 35S-LACS4-GFP, and 35S-LACS5-GFP.

[0025] Preferably, the gene PbrLACS is inserted between the BamHI and Ecor1 sites of TRV to obtain the recombinant expression vector TRV-PbrLACS.

[0026] This invention also protects the pear PbrLACS3 / 4 / 5 genes described above, and / or the proteins described above, and / or the recombinant expression vectors, expression cassettes, transgenic cell lines or genetically engineered bacteria described above in the following (1)-(4):

[0027] (1) Application in increasing or decreasing fruit wax content;

[0028] (2) Application in the preparation of products that increase or decrease the wax content of fruits;

[0029] (3) Application in improving or reducing plant breeding.

[0030] Preferably, the fruit is a pear.

[0031] The applicant constructed a plant overexpression vector for the pear long-chain acyl-CoA synthase PbrLACS3 / 4 / 5 and transiently transformed pears sequentially by injection, using pears transformed with the pCAMBIA1301-GFP vector as controls. The expression levels of the PbrLACS3 / 4 / 5 gene and the wax content in the pear peel were significantly increased.

[0032] The applicant constructed a plant gene silencing vector for the pear long-chain acyl-CoA synthase PbrLACS3 / 4 / 5 and transiently transformed pears by injection. Using pears transformed with the TRV vector as a control, the expression level of the PbrLACS3 / 4 / 5 gene and the wax content of the pear peel were significantly reduced.

[0033] The present invention also protects a method for increasing the wax content of fruit, said method being achieved by increasing the expression levels of the aforementioned genes PbrLACS3 / 4 / 5.

[0034] Increasing the expression of the aforementioned genes PbrLACS3 / 4 / 5 can promote an increase in the wax content of fruits.

[0035] Preferably, the expression level of the genes PbrLACS3 / 4 / 5 is increased by transferring a recombinant expression vector overexpressing the genes into the target plant.

[0036] Beneficial effects of the present invention

[0037] The present invention provides a pear long-chain acyl-CoA synthase PbrLACS3 / 4 / 5, its encoding gene, and its applications, which have the following advantages compared with the prior art:

[0038] (1) The pear long-chain acyl-CoA synthase PbrLACS3 / 4 / 5 provided by the present invention has been verified to promote wax synthesis through biological function testing. Transient overexpression or simultaneous transient reduction of the PbrLACS3 / 4 / 5 gene in pear fruit can significantly increase or decrease the wax content in the fruit.

[0039] (2) The PbrLACS3 / 4 / 5 gene provided by this invention promotes fruit wax synthesis and prolongs fruit storage period, laying the foundation for improving economic benefits by adjusting fruit wax to change gloss and enhance consumer preference. Attached Figure Description

[0040] Figure 1 TPM values ​​of LACS family members in different tissue transcriptomes of 'Dangshan Crisp Pear'.

[0041] Figure 2 The expression levels of PbrLACS3 / 4 / 5 genes in different tissues of 'Dangshan Crisp Pear'.

[0042] Figure 3 Analysis of the relative expression levels of PbrLACS3 / 4 / 5 in pericarp tissue during transient overexpression experiments.

[0043] Figure 4 The effects of transient overexpression of PbrLACS3 / 4 / 5 on pericarp wax content and composition were investigated. Figure 4 Figure A in the figure: Total wax content per unit area; Figure 4 Figure B in the figure shows the relative content of each component of the wax per unit area. Figure 4 Figure C in the diagram shows the content of terpenoids per unit area. Figure 4 Figure D in the diagram: Alkane content per unit area; Figure 4 Figure E in the diagram: alcohol content per unit area; Figure 4 The F-chart shows the aldehyde content per unit area.

[0044] Figure 5 The effect of transient overexpression of PbrLACS3 / 4 / 5 on the thickness of the pericarp cuticle was investigated. Figure 5 Figure A in the diagram: Oil Red O staining; Figure 5 Figure B in the diagram shows the thickness of the wax layer.

[0045] Figure 6 The effect of transient overexpression of PbrLACS3 / 4 / 5 on the waxy structure of pericarp was investigated. Figure 6 The AC graph in the figure is a blank control; Figure 6 The DF image in the image represents the PbrLACS3-GFP sample; Figure 6 The GI diagram in the image represents the PbrLACS4-GFP sample; Figure 6 The JL diagram in the image represents the PbrLACS5-GFP sample.

[0046] Figure 7 Analysis of the relative expression levels of PbrLACS3 / 4 / 5 in pericarp tissue during transient silencing experiments.

[0047] Figure 8 The effects of transient silencing of PbrLACS3 / 4 / 5 on pericarp wax content and composition were investigated. Figure 8 Figure A: Total wax content per unit area; Figure 8 Figure B in the figure shows the relative content of each component of the wax per unit area. Figure 8 Figure C in the diagram shows the content of terpenoids per unit area. Figure 8 Figure D in the diagram: Alkane content per unit area; Figure 8 Figure E in the diagram: alcohol content per unit area; Figure 8 The F-chart shows the aldehyde content per unit area.

[0048] Figure 9 The effect of transiently silencing PbrLACS3 / 4 / 5 on the thickness of the pericarp cuticle was investigated. Figure 9 Figure A in the diagram: Oil Red O staining; Figure 9 Figure B in the diagram shows the thickness of the wax layer.

[0049] Figure 10 The effect of transiently silencing PbrLACS3 / 4 / 5 on the pericarp wax structure was investigated. Figure 10 The AC graph in the figure is a blank control; Figure 10 The DF image in the image is of the PbrLACS-TRV2 sample.

[0050] Figure 11 Subcellular localization analysis of PbrLACS3 / 4 / 5. Green signals represent GFP-tagged proteins, red signals represent ER-Markers, and fused images show the overlap of green and red signals. Detailed Implementation

[0051] 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.

[0052] The applicant isolated and cloned three new genes PbrLACS3 / 4 / 5 from 'Dangshan Crisp Pear', whose nucleotide sequences are shown in the sequence listing SEQ ID NO.1-SEQ ID NO.3, with lengths of 1977bp, 1977bp, and 1716bp, respectively.

[0053] The present invention also provides the amino acid sequences encoded by the three pear long-chain acyl-CoA synthases PbrLACS3 / 4 / 5, which encode 659, 659 and 572 amino acids respectively, as shown in SEQ ID No.4-SEQ ID No.6.

[0054] Transcriptome data from different tissues showed that PbrLACS3 / 4 / 5 was highly expressed in pear pericarp tissue. Figure 1 qRT-PCR analysis also confirmed that the PbrLACS3 / 4 / 5 genes are expressed in pear peel. Figure 2 This indicates that the PbrLACS3 / 4 / 5 genes cloned in this invention may be involved in the synthesis of wax in pear fruits.

[0055] Example 1: Expression levels of pear PbrLACS3 / 4 / 5 genes in different tissues

[0056] The experimental material used was the 'Dangshan Crisp Pear' variety. Peel and pulp samples were collected 140 days after flowering at maturity, root samples from plants 70 days old in nutrient pots, and petal, anther, and style tissue samples from the large bud stage. RNA was extracted according to the operating manual of the FastPure Plant TotalRNA Isolation Kit (purchased from Novizan, China). 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 operating manual of the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. Pear PbrTUB was selected as the internal control gene, and the nucleotide sequences of the primers are as follows:

[0057] F1: 5'-TGGGCTTTGCTCCTCTTAC-3' (SEQ ID NO.7),

[0058] R1: 5'-CCTTCGTGCTCATCTTACC-3' (SEQ ID NO. 8).

[0059] Gene-specific qRT-PCR primer pairs were designed within the open reading frame of the PbrLACS3 gene using Primer Premier 5.0. The nucleotide sequences of the primers are as follows:

[0060] F2: 5'-AAGCAGCACCTCTCTTGGAC-3' (SEQ ID NO.9),

[0061] R2: 5'-GACGTGAAGCTACCACCACA-3' (SEQ ID NO. 10).

[0062] Gene-specific qRT-PCR primer pairs were designed within the open reading frame of the PbrLACS4 gene using Primer Premier 5.0. The nucleotide sequences of the primers are as follows:

[0063] F3: 5'-CTAAGGCGTTGGAGGATTGGG-3' (SEQ ID NO. 11),

[0064] R3: 5'-CCATGTCAAAGGGCTTCGGT-3' (SEQ ID NO. 12).

[0065] Gene-specific qRT-PCR primer pairs were designed within the open reading frame of the PbrLACS5 gene using Primer Premier 5.0. The nucleotide sequences of the primers are as follows:

[0066] F4: 5'-GTGGTGGTCCCTGACCCTAA-3' (SEQ ID NO. 13),

[0067] R4: 5'-GCTCAAAACCTCGGAGTTGC-3' (SEQ ID NO. 14).

[0068] qRT-PCR was performed using the ChamQ SYBR qPCR Master Mix kit (purchased from Novizan), strictly following the instructions.

[0069] The 10 μL qRT-PCR reaction system included: 5 μL fluorescent dye, 3.6 μL sterile ultrapure water, 1 μL cDNA, 0.2 μL forward primer (10 μM), and 0.2 μL reverse primer (10 μM). PCR was performed using a 96-well qRT-PCR plate (Roche) and a qRT-PCR instrument (Model: LightCycler 480, Roche). The qRT-PCR reaction program was: 95℃ for 10 min; 95℃ for 10 s, 60℃ for 10 s, 72℃ for 30 s, return to step 2 for 50 cycles, 72℃ for 10 min, 20℃ for 5 min. The reaction was repeated three times. The average Ct value for each cDNA sample was calculated using formula 2. -ΔΔCt Obtain the relative expression levels of PbrLACS3 / 4 / 5 genes ( Figure 2).

[0070] qRT-PCR results showed that the expression levels of the three genes PbrLACS3 / 4 / 5 were high in the pericarp.

[0071] Example 2: Cloning and Construction of Pear PbrLACS3 / 4 / 5 Genes and Transient Expression Vector

[0072] 1. Cloning of the pear PbrLACS3 / 4 / 5 gene

[0073] Using the peel of 'Dangshan Crisp Pear' harvested 140 days after flowering at maturity as the test material, total RNA was extracted and cDNA was synthesized using the method described in Example 1. The obtained first-strand cDNA was used for amplification of the PbrLACS3 / 4 / 5 genes. The nucleotide sequence of the PbrLACS3 primer pair is shown below:

[0074] F5: 5'-gagaacacgggggactctagaATGGATTTCATAGTGAAGGTTGAGGAAT-3' (SEQ IDNO.15),

[0075] R5: 5'-gcccttgctcaccatggatccTACCCTTGCTCCTTTTCCTTCG-3' (SEQ ID NO. 16).

[0076] The nucleotide sequences of the PbrLACS4 primer pair are shown below:

[0077] F6: 5'-gagaacacgggggactctagaATGGATTTCATAGTGAAGGTTGAGGAAT-3' (SEQ IDNO.17),

[0078] R6: 5'-gcccttgctcaccatggatccTACCCTTGCTCCTTTTCCTTCG-3' (SEQ ID NO. 18).

[0079] The nucleotide sequences of the PbrLACS5 primer pair are shown below:

[0080] F7: 5'-gagaacacgggggactctagaATGGGTTCAGCCATCAGAAGC-3' (SEQ ID NO. 19),

[0081] R7: 5'-gcccttgctcaccatggatccTACCCTTGCTCCTTTTCCTTCG-3' (SEQ ID NO. 20).

[0082] 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 a final volume of 50 μL. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 35 amplification cycles, including 95℃ denaturation for 30 s, 60℃ annealing for 15 s, 72℃ extension for 2 min, followed by a final extension at 72℃ for 10 min, and then incubation at 20℃ for 5 min. 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).

[0083] 2. Construction of plant overexpression vectors for pear PbrLACS3 / 4 / 5 genes

[0084] 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 General Biotechnology). Sequencing results showed that the amplified target fragments PbrLACS3, PbrLACS4, and PbrLACS5 were 1977 bp, 1977 bp, and 1716 bp in length, respectively, with nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.3. Sequence alignment analysis confirmed that these sequences were the target genes required in this invention, and the correct recombinant vectors were named 35S-PbrLACS3-GFP, 35S-PbrLACS4-GFP, and 35S-PbrLACS5-GFP, respectively. The recombinant vectors 35S-PbrLACS3-GFP, 35S-PbrLACS4-GFP, 35S-PbrLACS5-GFP, and the control empty vector 35S-GFP were transformed into *Agrobacterium* GV3101 using a heat shock method.

[0085] 3. Constructing plant gene silencing vectors for the pear PbrLACS3 / 4 / 5 genes.

[0086] Due to the high homology of the PbrLACS3 / 4 / 5 gene sequences, a plant gene silencing vector for PbrLACS3 / 4 / 5 was constructed using a method that simultaneously silences all three genes. The PbrLACS3 / 4 / 5 genes were amplified using the one-stranded cDNA obtained in Part 1 of Example 1, and the nucleotide sequences of the primer pairs are shown below:

[0087] F8: 5'-aaggttaccgaattctctagaTTCTGTCATGCCTTCCAAATTG-3' (SEQ ID NO. 21),

[0088] R8: 5'-cgtgagctcggtaccggatccACACTTTATCTGTTAGAAAAAGTAGTTGCT-3' (SEQ ID NO. 22).

[0089] The target product was amplified using the PCR reaction system described in Part 1 of Example 1 by adding forward primer F8 and reverse primer R8, 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 a heat shock method. PCR detection was performed using gene-specific primers, and the positive bacterial cultures were sequenced (performed by Genewiz). The sequence was confirmed to be the target gene required by this invention, and the correct recombinant target vector was named TRV-PbrLACS. The recombinant vector TRV-PbrLACS and the control empty vector TRV were transformed into *Agrobacterium* GV3101 using the heat shock method.

[0090] Example 3: Determination of wax content and components in pear fruit by transient overexpression

[0091] 1. Transient overexpression injection of PbrLACS3 / 4 / 5 genes in pear

[0092] Take Agrobacterium tumefaciens (containing '35S-PbrLACS3-GFP', '35S-PbrLACS4-GFP', or '35S-PbrLACS5-GFP' recombinant vector or 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 transferred to a 100mL Erlenmeyer flask. 30mL of a solution containing 50μg / mL kanamycin and 100mg·L⁻¹ kJ / mL kanamycin was added. -1The cells were cultured in LB liquid medium containing rifampicin at 200 rpm for 12 hours in a shaker at 28°C. The cells were then collected by centrifugation at 5000 rpm for 5 minutes in 50 mL centrifuge tubes. 10 mL of induction solution (10 mM MgCl2, 10 mM...) was added. After resuspending the bacterial cells in MES and 200mM acetylsylcholine (pH=5.5), centrifuge again, add the invasion dye again, and adjust the OD value of the bacterial solution to between 0.6 and 0.8 using a spectrophotometer. Incubate at 25℃ in a shaker at 100r / min in the dark for 4 hours. Inject the empty vector 35S-GFP, 35S-PbrLACS3-GFP, 35S-PbrLACS4-GFP, and 35S-PbrLACS5-GFP into the equatorial region of 'Dangshan Crisp Pear' fruit using a syringe. Inject 6 wells into each pear fruit, and inject at least 7 fruits into each group of experiments. Repeat the experiment 3 times. Incubate in the dark at 21℃ for 24 hours, and then incubate in a 21℃ incubator with low light intensity and a photoperiod of 16 hours light / 8 hours dark for 2 days.

[0093] 2. Detection of transient overexpression levels of PbrLACS3 / 4 / 5 genes

[0094] Collect pear peel tissue samples after injection. Extract total RNA and synthesize cDNA using the method described in Example 1.

[0095] Pear PbrACT was selected as the internal reference gene, and the nucleotide sequence of the primers is as follows:

[0096] F9: 5'-CCATCCAGGGCTGTTCTCTC-3' (SEQ ID NO. 23),

[0097] R9: 5'-GCAAGGTCCAGACGAAGG-3' (SEQ ID NO. 24).

[0098] The relative expression levels of the PbrLACS3 / 4 / 5 genes were obtained using the PbrLACS3 / 4 / 5 gene primers (SEQ ID NO. 9-SEQ ID NO. 14) and the method described in Example 1 to verify the success of transient transformation. Figure 3 ).

[0099] 3. Detection of wax content and components in samples with transient overexpression

[0100] In a fume hood, the injected pear fruit was completely immersed in chloroform solution 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 and transferred to a brown gas chromatography vial. 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. 200 μl of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma) and 200 μl of pyridine (Sigma) were added, and the mixture was derivatized in a 70°C oven for 1 hour. All reagents were dried again under chloroform, and 1 ml of chromatographic grade chloroform was added to dissolve the residue completely. The solution was then filtered through a 0.45 μm organic filter into a 1.5 ml brown gas chromatographic vial for analysis. The waxy components were detected using a gas chromatograph (TRACE 1310, Thermo Scientific) and a triple quadrupole mass spectrometer (TSQ 9000, Thermo Scientific). The compounds were separated on a TG-5MS capillary column (30m × 0.25mm ID, 0.25μm 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 port temperature 280℃; transfer line temperature 280℃; ion source temperature 250℃; quadrupole temperature 150℃; electron energy (EI) 70 eV; scan range 50–650 m / z. The temperature program was as follows: 50℃ for 2 minutes, increased to 170℃ at a rate of 20℃ / min, held for 2 minutes, increased to 200℃ at a rate of 10℃ / min, held for 3 minutes, and then increased to 220℃ at a rate of 3℃ / min, held for 2 minutes. Finally, the temperature was increased to 320°C at a rate of 5°C / min and held for 20 min. Waxy compounds were identified by matching the retention index with mass spectra in the NIST 2017 library. Alkanes were identified by comparing the mass spectra and retention times of C7-C40 saturated alkane standards (Sigma-Aldrich).

[0101] 4. Detection of wax structure in samples with transient overexpression

[0102] After injection, the pericarp of the pear fruit was taken and cut into thin slices about 0.5 cm long and 0.5 cm wide. The microstructure of the pericarp was photographed using a Hitachi 4800 field emission scanning electron microscope (Hitachi S-4800).

[0103] 5. Detection of stratum corneum thickness in samples with transient overexpression

[0104] Following the method described in Part IV of this embodiment, thin slices of pericarp were placed in a 2 ml centrifuge tube containing FAA fixative. The tube was filled with fixative, and excess air was expelled to prevent sample oxidation. The sample was first dehydrated with sucrose and then longitudinally sectioned to prepare frozen sections. The sections were then air-dried at room temperature for 15 minutes. Under light-protected conditions, the sections were stained with saturated Oil Red O staining solution for 10 minutes. Subsequently, they were differentiated with 60% isopropanol for 10 seconds, and then gently rinsed with distilled water to avoid lipid droplet migration. Excess moisture around the tissue was wiped dry, and the tissue was mounted with glycerol gelatin. Finally, the tissue was observed and photographed under a microscope (NIKON Eclipse ci).

[0105] The results showed that the expression levels of the three overexpressed genes were significantly increased, and the total wax content, alkane content, and terpene content were significantly increased by 33.19%-51.39%, 48.91%-65.96%, and 25.83%-53.52% respectively compared with the control group. The stratum corneum thickness increased significantly by 89.79%-120.44%, and the number of crystals increased significantly. Figure 3-6 ).

[0106] Example 4: Determination of wax content and components in gene-silenced pear fruit

[0107] Agrobacterium containing the 'TRV-PbrLACS' recombinant vector was activated according to the method in Part 1 of Example 3. Then, a single clone was picked up from the thread with a sterile toothpick and placed in a 100mL Erlenmeyer flask. 30mL of a solution containing 100mg / L kanamycin and 100mg / L of [unspecified ingredient] was added. -1 The cells were cultured in LB liquid medium containing rifampicin at 200 rpm for 12 hours in a shaker at 28°C. The cells were then collected by centrifugation at 5000 rpm for 5 minutes in 50 mL centrifuge tubes. 10 mL of induction solution (10 mM MgCl2, 10 mM...) was added. After resuspending the bacterial cells in MES and 200mM acetylsylcholine (pH=5.5), centrifuge again, add the inoculation solution again, and adjust the OD value of the bacterial solution to between 0.6 and 0.8 using a spectrophotometer. Prepare a mixed inoculation solution by mixing TRV1:TRV2 (PbrLACS-TRV2) in the following ratio of 1:1. Incubate the solution at 25℃ in a shaker at 100r / min in the dark for 4 hours. Inject the mixed inoculation solution of TRV1 and empty TRV2 and the mixed inoculation solution of TRV1 and PbrLACS-TRV2 into the pear fruit near the equator using a syringe. Inject 6 wells into each pear fruit, and inject at least 7 fruits in each experiment. Repeat the experiment 3 times. Incubate in the dark in an incubator at 21℃ for 24 hours, and then incubate in an incubator at 21℃ with low light intensity and a photoperiod of 16 hours light / 8 hours dark for 2 days. Finally, the expression levels of the PbrLACS3 / 4 / 5 genes and wax content of the gene-silenced and control pear fruits were determined according to the method in Example 3, and the cuticle thickness was measured using the Oil Red O staining method.

[0108] The results showed that the expression levels of PbrLACS3 / 4 / 5 genes were significantly lower in the gene silencing group compared to the control group. The total wax content and terpene content of the silenced PbrLACS3 / 4 / 5 genes were significantly lower than those in the control group by 33.47% and 39.36%, respectively. The stratum corneum thickness was significantly reduced by 30.55%, and the number of crystals was significantly decreased. Figure 7-10 ).

[0109] Example 5: Subcellular localization of PbrLACS3 / 4 / 5

[0110] Subcellular localization of PbrLACS3 / 4 / 5 was performed using the plant overexpression vector constructed in Part 2 of Example 2. Agrobacterium containing the recombinant vector '35S-PbrLACS3-GFP', '35S-PbrLACS4-GFP', or '35S-PbrLACS5-GFP', or the 35S-GFP control empty vector, and the infection solution was prepared according to the method described in Part 1 of Example 3. The vector was gently injected into the tobacco leaves from the underside, and cultured in the dark at 25°C for 1 day, followed by normal culture for 1-2 days. Fluorescence signals were observed under a laser confocal microscope (Zeiss LSM 710, Germany).

[0111] The results showed that both the red fluorescent ER-Marker and the green fluorescent fusion protein emitted fluorescent signals in the endoplasmic reticulum, and the signals overlapped to form a yellow color, indicating that PbrLACS3 / 4 / 5 is localized in the endoplasmic reticulum. Figure 11 ).

[0112] As can be seen from the above embodiments, the pear long-chain acyl-CoA synthase PbrLACS3 / 4 / 5 provided by the present invention has a positive regulatory effect on fruit wax through experiments such as spatiotemporal expression, transient overexpression in pear fruit, gene silencing, and subcellular localization.

[0113] 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. The application of a protein with the amino acid sequence shown in SEQ ID NO. 6 and / or a gene, vector, expression cassette, and / or genetically engineered bacteria expressing the protein with the amino acid sequence shown in SEQ ID NO. 6 in improving the wax content and cuticle thickness of Dangshan crisp pear peel, wherein the wax is at least one of the following waxes: a) total wax, b) total alkanes, c) total terpenes, d) docosanol, e) oleanolic acid, f) betulinic acid, g) ursolic acid, h) ursolic aldehyde, i) eicosanol.

2. The application of a protein with the amino acid sequence shown in SEQ ID NO. 6 and / or a gene, vector, expression cassette, and / or genetically engineered bacteria expressing the protein with the amino acid sequence shown in SEQ ID NO. 6 in the preparation of a product that increases the wax content and cuticle thickness of Dangshan crisp pear peel, wherein the wax is at least one of the following waxes: a) total wax, b) total alkanes, c) total terpenes, d) docosanol, e) oleanolic acid, f) betulinic acid, g) ursolic acid, h) ursolic aldehyde, i) eicosanol.

3. The application of the protein with the amino acid sequence shown in SEQ ID NO. 6 and / or the gene, vector, expression cassette and / or genetically engineered bacteria expressing the protein with the amino acid sequence shown in SEQ ID NO. 6 in the breeding of Dangshan crisp pear to improve the wax content and cuticle thickness of the pericarp, wherein the wax is at least one of the following waxes: a) total wax, b) total alkanes, c) total terpenes, d) docosanol, e) oleanolic acid, f) betulinic acid, g) ursolic acid, h) ursolic aldehyde, i) eicosanol.

4. A method for increasing the wax content and cuticle thickness of Dangshan crisp pear peel, characterized in that, The method is achieved by increasing the expression level of the gene encoding the protein with the amino acid sequence shown in SEQ ID NO.6, wherein the wax is at least one of the following waxes: a) total waxes, b) total alkanes, c) total terpenes, d) docosanol, e) oleanolic acid, f) betulinic acid, g) ursolic acid, h) ursolic aldehyde, i) eicosanol.

5. The method according to claim 4, characterized in that, The enhancement of the expression level of the gene encoding the protein with the amino acid sequence shown in SEQ ID NO.6 is achieved by transferring the recombinant expression vector of the gene into the target plant.

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