Pear long-chain acyl coenzyme A synthetase as well as coding gene and application thereof

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to pear long-chain acyl coenzyme A synthetase as well as a coding gene and application thereof. The pear PbrLACS3 / 4 / 5 gene is selected from the following a), b) or c): a) the nucleotide sequence of the pear PbrLACS3 / 4 / 5 gene is shown as any one of SEQ ID NO.1-3; b) a nucleotide sequence shown as any one of SEQ ID NO.1-3 is subjected to substitution, deletion and / or addition of one or more nucleotides; and c) a nucleotide sequence hybridized with the DNA sequence limited by 1) under strict conditions.Biological function verification shows that the pear long-chain acyl coenzyme A synthetase PbrLACS3 / 4 / 5 provided by the invention has the effect of promoting wax synthesis, and the PbrLACS3 / 4 / 5 gene is sequentially and instantaneously overexpressed or simultaneously and instantaneously reduced in pear fruits, so that the wax content in the fruits can be remarkably increased or reduced.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and in particular relates to pear long-chain acyl-CoA synthetase and a coding gene and application thereof. Background Art

[0002] Pear is an important economic crop in my country. Its cultivated area is second only to apples and citrus, and it is the third largest fruit tree in the world. Improving the yield and quality of pear fruit is one of the important research goals in the current field of pear research. Wax has many functions, such as reducing water loss and mechanical damage, resisting biological or non-biological damage, 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 to improving the quality of pear fruit, increasing pear yield and economic benefits. .

[0003] At present, there are no relevant reports on the functions and mechanisms of LACS in pears. The present invention further enriches the molecular network of pear fruit wax by carrying out gene cloning and functional research on regulating the synthesis of pear fruit wax, which is helpful to improve the quality of pear fruit and enhance the competitiveness of my country's pear industry. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a pear long-chain acyl-CoA synthetase PbrLACS3 / 4 / 5 and its encoding gene and application.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] In the first aspect, the present invention first protects a pear PbrLACS3 / 4 / 5 gene selected from the following a), b) or c):

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

[0008] b) the nucleotide sequence shown in any one of SEQ ID NO.1-3 is substituted, deleted and / or added with one or more nucleotides;

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

[0010] The pear PbrLACS3 / 4 / 5 gene refers 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 as SEQ ID NO.1.

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

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

[0014] In a second aspect, the present invention protects the protein encoded by the pear PbrLACS3 / 4 / 5 gene described above.

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

[0016] A) a protein with an amino acid sequence as shown in any one of SEQ ID NOs. 4-6;

[0017] B) A protein derived from 1) having the same activity as the amino acid sequence shown in any one of SEQ ID NOs. 4-6, with one or more amino acids substituted, deleted and / or added.

[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] In a third aspect, the present invention also protects a primer pair for amplifying the full length or any fragment of the aforementioned pear PbrLACS3 / 4 / 5 gene.

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

[0023] In a fourth aspect, the present invention also protects a recombinant expression vector, an expression cassette, a transgenic cell line or a genetically engineered bacterium containing the aforementioned pear PbrLACS3 / 4 / 5 gene.

[0024] Preferably, the recombinant expression vector is constructed by the following method: the gene PbrLACS3 / 4 / 5 is sequentially inserted 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 EcoRI sites of TRV to obtain the recombinant expression vector TRV-PbrLACS.

[0026] The present invention also protects the use of the pear PbrLACS3 / 4 / 5 gene described above, and / or the protein described above, and / or the recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria described above in the following (1)-(4):

[0027] (1) Application in increasing or decreasing the wax content of fruits;

[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 fruit.

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

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

[0033] The present invention also protects a method for increasing the wax content of fruits, which is achieved by increasing the expression level of the gene PbrLACS3 / 4 / 5 mentioned above.

[0034] Increasing the expression of the gene PbrLACS3 / 4 / 5 can promote the increase of the wax content of the fruit.

[0035] Preferably, increasing the expression level of the gene PbrLACS3 / 4 / 5 is achieved by transferring a recombinant expression vector that overexpresses the gene into the target plant.

[0036] Beneficial effects of the present invention

[0037] The pear long-chain acyl-CoA synthetase PbrLACS3 / 4 / 5 and its encoding gene and application provided by the present invention have the following beneficial effects compared with the prior art:

[0038] (1) The pear long-chain acyl-CoA synthetase PbrLACS3 / 4 / 5 provided by the present invention has the effect of promoting wax synthesis through biological function verification. Sequentially overexpressing or simultaneously reducing the PbrLACS3 / 4 / 5 genes in pear fruit can significantly increase or reduce the wax content in the fruit.

[0039] (2) The PbrLACS3 / 4 / 5 gene provided by the present invention promotes the synthesis of fruit wax and prolongs the storage period of the fruit, laying a foundation for improving consumer preference by adjusting the fruit wax and further improving economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the TPM value of LACS family members in the transcriptome of different tissues of 'Dangshan Pear'.

[0041] Figure 2 is the expression level of PbrLACS3 / 4 / 5 genes in different tissues of 'Dangshan Pear'.

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

[0043] Figure 4 The effects of transient overexpression of PbrLACS3 / 4 / 5 on the wax content and composition of the fruit peel. Figure 4 Figure A: total amount of wax per unit area; Figure 4 Figure B: Relative content of wax components per unit area; Figure 4 Figure C: Terpene content per unit area; Figure 4 Figure D: alkane content per unit area; Figure 4 Figure E: alcohol content per unit area; Figure 4 Figure F: content of aldehydes per unit area.

[0044] Figure 5 The effect of transient overexpression of PbrLACS3 / 4 / 5 on the thickness of the cuticle of the fruit peel. Figure 5 Panel A: Oil Red O staining; Figure 5 Figure B: Wax layer thickness.

[0045] Figure 6 The effect of transient overexpression of PbrLACS3 / 4 / 5 on the wax structure of the fruit peel. Figure 6 Figures AC in the figure are blank controls; Figure 6 Figures DF in the figure are PbrLACS3-GFP samples; Figure 6 The GI figure in the figure is the PbrLACS4-GFP sample; Figure 6 Figures JL in the figure are PbrLACS5-GFP samples.

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

[0047] Figure 8 The effect of transient silencing of PbrLACS3 / 4 / 5 on the wax content and composition of the fruit peel. Figure 8 Figure A in the middle: total amount of wax per unit area; Figure 8 Figure B: Relative content of wax components per unit area; Figure 8 Figure C: Terpene content per unit area; Figure 8 Figure D: alkane content per unit area; Figure 8 Figure E: alcohol content per unit area; Figure 8 Figure F: content of aldehydes per unit area.

[0048] Fig. 9 The effect of transient silencing of PbrLACS3 / 4 / 5 on the thickness of the cuticle of the fruit peel. Fig. 9 Panel A: Oil Red O staining; Fig. 9 Figure B: Wax layer thickness.

[0049] Fig.10 The effect of transient silencing of PbrLACS3 / 4 / 5 on the wax structure of the fruit peel. Fig.10 Figures AC in the figure are blank controls; Fig.10 The DF diagram in the figure is the PbrLACS-TRV2 sample.

[0050] Fig.11 Subcellular localization analysis of PbrLACS3 / 4 / 5. The green signal represents GFP-tagged protein, the red signal represents ER-Marker, and the fused image represents the overlap of green and red signals. DETAILED DESCRIPTION

[0051] The present invention is described in detail below in conjunction with specific embodiments. Based on the following description and embodiments, those skilled in the art can determine the essential features of the present invention, and can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention to make it suitable for various uses and conditions.

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

[0053] The present invention also provides amino acid sequences encoded by the three pear long-chain acyl-CoA synthetases PbrLACS3 / 4 / 5, encoding 659, 659 and 572 amino acids respectively. The amino acid sequences are 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 peel tissue ( Figure 1 ), qRT-PCR analysis also demonstrated that PbrLACS3 / 4 / 5 genes were expressed in pear peel ( Figure 2 ), indicating that the PbrLACS3 / 4 / 5 genes cloned in the present invention may be involved in the synthesis of wax in pear fruit.

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

[0056] The experimental material selected was the 'Dangshan Pear' variety. The peel and pulp of pears 140 days after flowering at maturity, the root system of plants 70 days in nutrient pots, and the petals, anthers, and style tissue samples at the large bud stage were collected. The RNA was extracted according to the operating manual of FastPure Plant Total RNA Isolation Kit (purchased from Novartis, China). After the extraction was completed, the quality of the extracted RNA was detected by 1% agarose gel electrophoresis, and the concentration and quality of the RNA were detected by NanoDrop 2000 spectrophotometer. The synthesis of the first strand of cDNA was carried out according to the operating manual of Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. Pear PbrTUB was selected as the internal reference gene, and the nucleotide sequence of the primer is as follows:

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

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

[0059] Primer Premier 5.0 was used to design gene-specific qRT-PCR primer pairs within the open reading frame of the PbrLACS3 gene. 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] Primer Premier 5.0 was used to design gene-specific qRT-PCR primer pairs within the open reading frame of the PbrLACS4 gene. 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] Primer Premier 5.0 was used to design gene-specific qRT-PCR primer pairs within the open reading frame of the PbrLACS5 gene. 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 Novartis) in strict accordance with the instructions.

[0069] The 10 μL qRT-PCR reaction system includes: 5 μL fluorescent dye, 3.6 μL sterilized ultrapure water, 1 μL cDNA, 0.2 μL forward primer (10 μM), 0.2 μL reverse primer (10 μM). A 96-well qRT-PCR plate (purchased from Roche) was used to perform PCR using a qRT-PCR instrument (model: LightCycler 480, Roche). The qRT-PCR reaction program was: 95°C for 10 min; 95°C for 10 s, 60°C for 10 s, 72°C for 30 s, return to the second step for 50 cycles, 72°C for 10 min, 20°C for 5 min. Repeat 3 times, and calculate the average Ct value of each cDNA sample, using the calculation formula 2 -ΔΔCt Obtain the relative expression levels of PbrLACS3 / 4 / 5 genes ( Figure 2).

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

[0071] Example 2 Cloning of pear PbrLACS3 / 4 / 5 genes and construction of plant transient expression vectors

[0072] 1. Cloning of Pear PbrLACS3 / 4 / 5 Genes

[0073] The peel of 'Dangshan Pear' pear at maturity 140 days after flowering was used as the test material, and 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 PbrLACS3 / 4 / 5 genes. The nucleotide sequence of the PbrLACS3 primer pair is as follows:

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

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

[0076] The nucleotide sequence of the PbrLACS4 primer pair is shown below:

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

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

[0079] The nucleotide sequence of the PbrLACS5 primer pair is 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 was added to make up to 50 μL. The PCR program was as follows: 95°C pre-denaturation for 3 min; 35 amplification cycles, including 95°C denaturation for 30 s, 60°C annealing for 15 s, 72°C extension for 2 min, 72°C extension for 10 min after the cycle was completed, and then incubated at 20°C for 5 min. After amplification, the PCR product of a single target band was detected by 1.5% agarose gel electrophoresis, and the specific target band was recovered according to the extraction steps of the gel recovery kit (purchased from Novozyme, China).

[0083] 2. Construction of plant overexpression vector of pear PbrLACS3 / 4 / 5 gene

[0084] The purified PCR product was recovered and connected with the pCAMBIA1301-GFP vector, and transformed into Escherichia coli DH5α by heat shock method. PCR detection was performed with gene-specific primers, and the positive bacterial solution was sequenced (completed by General Biotechnology). The sequencing results showed that the lengths of the PbrLACS3, PbrLACS4, and PbrLACS5 target fragments amplified by the present invention were 1977 bp, 1977 bp, and 1716 bp, respectively, and their nucleotide sequences were shown in SEQ ID NO.1-SEQ ID NO.3. Through sequence comparison analysis, it was determined that the sequence was the target gene required by the present invention, and the correct recombinant target 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 respectively transferred into Agrobacterium GV3101 by heat shock method.

[0085] 3. Construction of plant gene silencing vector for pear PbrLACS3 / 4 / 5 genes

[0086] Since the sequence homology of the three genes PbrLACS3 / 4 / 5 is too high, the method of silencing the three genes simultaneously was used to construct a plant gene silencing vector of the PbrLACS3 / 4 / 5 genes. The first part of Example 1 was used to obtain a strand of cDNA to amplify the PbrLACS3 / 4 / 5 genes, and the nucleotide sequence of the primer pair is as follows:

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

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

[0089] The target product was amplified by adding forward primer F8 and reverse primer R8 using the PCR reaction system of the first part of Example 1, and the specific target band was recovered. The recovered purified PCR product was ligated with the TRV vector, transformed into Escherichia coli DH5α by heat shock method, and PCR detection was performed with gene-specific primers. The positive bacterial solution was sequenced (completed by Jin Weizhi Company) to determine that the sequence to be tested is the target gene required by the present invention, and the correct recombinant target vector was named TRV-PbrLACS. The recombinant vector TRV-PbrLACS and the control empty TRV were respectively transferred into Agrobacterium GV3101 by heat shock method.

[0090] Example 3 Determination of wax content and components in transiently overexpressed pear fruit

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

[0092] Take Agrobacterium tumefaciens (containing '35S-PbrLACS3-GFP' or '35S-PbrLACS4-GFP' or '35S-PbrLACS5-GFP' recombinant vector or 35S-GFP control empty vector) stored in ultra-low temperature refrigerator and add 100 mg·l -1 Kanamycin and 100 mg L -1 The solid LB medium of rifampicin was streaked and cultured at 28℃ for 2 days. Then a single colony on the line was picked with a sterilized toothpick and placed in a 100mL conical flask. 30mL of LB medium containing 50μg / mL kanamycin and 100mg·L -1 The liquid LB medium containing rifampicin was cultured in a shaker at 200 rpm at 28°C for 12 hours, and the cells were collected by centrifugation at 5000 rpm for 5 minutes in a 50 mL centrifuge tube. 10 mL of induction solution (10 mM MgCl 2, 10 mM MES, 200 mM acetosyringone, pH = 5.5) after resuspending the bacteria, centrifuge again, add the invasion solution again, use a spectrophotometer to adjust the bacterial solution OD value to between 0.6-0.8, place it in a 25°C shaker at 100 r / min and incubate in the dark for 4 h, use a syringe to inject the empty 35S-GFP, 35S-PbrLACS3-GFP, 35S-PbrLACS4-GFP, and 35S-PbrLACS5-GFP infection solution into the equator of the 'Dangshan Pear' fruit, each pear fruit was injected with 6 holes, and at least 7 fruits were injected in each group of experiments. The experiment was repeated 3 times, and the fruits were placed in a 21°C incubator for dark culture for 24 h, and then placed in an incubator at 21°C, with weak light intensity and a photoperiod of 16 h light / 8 h dark, and cultured for 2 days.

[0093] 2. Detection of transient overexpression of PbrLACS3 / 4 / 5 gene expression

[0094] The pear peel tissue samples after injection were collected, and total RNA was extracted and cDNA was synthesized using the method described in Example 1.

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

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

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

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

[0099] 3. Detection of wax content and composition of transient overexpression samples

[0100] In a fume hood, the injected pear fruit was completely immersed in chloroform solution for 1 minute to extract epidermal wax. The extraction solution containing epidermal wax was then transferred to a test tube, and the solution was evaporated and concentrated to a volume of about 1.5 ml using a nitrogen blower (JHD-001S, Shanghai Jiheng Industrial Co., Ltd.) at 40°C. The sample was transferred to a brown gas chromatography-mass spectrometry bottle after filtration through a 0.45 μm organic filter membrane. 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 blown dry with nitrogen. 200 μl of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma) and 200 μl of pyridine (Sigma) were added, and derivatized in an oven at 70°C for 1 hour. All reagents were blown dry with chloroform again, and 1 ml of chromatographic grade chloroform was added to fully dissolve, and filtered into a 1.5 ml brown gas phase bottle using a 0.45 μm organic filter membrane for detection. The wax 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 (30 m × 0.25 mm ID, 0.25 μm film, Thermo Scientific) capillary column. Helium was used as a carrier gas with a flow rate of 1.2 mL / min. The following parameters were used: inlet temperature, 280 °C; transfer line temperature, 280 °C; ion source temperature, 250 °C; quadrupole temperature, 150 °C; electron energy (EI), 70 eV; scanning range, 50-650 m / z. The heating program was as follows: 50 °C for 2 minutes, heated to 170 °C at a rate of 20 °C / min, held for 2 minutes, heated to 200 °C at a rate of 10 °C / min, held for 3 minutes, and then heated to 220 °C at a rate of 3 °C / min, held for 2 minutes. Finally, the temperature was raised to 320°C at a rate of 5°C / min and maintained for 20 min. Waxy compounds were identified by matching the retention index with the mass spectra in the NIST 2017 library. Among them, alkanes were identified by comparing the mass spectra and retention times of C7-C40 saturated alkane standards (Sigma-Aldrich).

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

[0102] The peel samples of the injected pear fruits were taken and cut into thin slices of about 0.5 cm in length and 0.5 cm in width. The microstructure of the peel was photographed using a Hitachi 4800 field emission scanning electron microscope (Hitachi S-4800).

[0103] 5. Detection of cuticle thickness of transient overexpression samples

[0104] According to the method of the fourth part of this embodiment, the peel slice sample was placed in a 2ml centrifuge tube containing FAA fixative, the tube was filled with fixative, and the excess air was exhausted to prevent the sample from oxidizing. The sample was first dehydrated with sucrose, and prepared into frozen sections by longitudinal cutting, and dried at room temperature for 15 minutes. The slices were placed in saturated Oil Red O staining solution for 10 minutes under light-proof conditions. Subsequently, 60% isopropanol was used for differentiation for 10 seconds, and then gently rinsed with distilled water to avoid displacement of lipid droplets. Excess water around the tissue was wiped dry and sealed with glycerol gelatin. Finally, it was observed and photographed under a microscope (NIKON Eclipse ci).

[0105] The results showed that the expression of the three genes was significantly increased after overexpression, and the total amount of wax, alkane and terpenoid contents were significantly increased by 33.19%-51.39%, 48.91%-65.96% and 25.83%-53.52% respectively compared with the control group. The thickness of the cuticle 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] According to the method in the first part of Example 3, the Agrobacterium containing the 'TRV-PbrLACS' recombinant vector was activated, and then a single clone on the line was picked up with a sterilized toothpick and placed in a 100 mL conical flask, and 30 mL of 100 mg / L kanamycin and 100 mg / L -1 The liquid LB medium containing rifampicin was cultured in a shaker at 200 rpm at 28°C for 12 hours, and the cells were collected by centrifugation at 5000 rpm for 5 minutes in a 50 mL centrifuge tube. 10 mL of induction solution (10 mM MgCl 2 , 10mM MES, 200mM acetosyringone, pH=5.5) after resuspending the bacteria, centrifuge again, add the infection solution again, use a spectrophotometer to adjust the OD value of the bacterial solution to between 0.6-0.8, and prepare a mixed infection solution according to the following ratio TRV1:TRV2(PbrLACS-TRV2)=1:1, place it in a 25℃ shaker at 100r / min and incubate in the dark for 4h, use a syringe to inject the mixed infection solution of TRV1 and empty TRV2 and the mixed infection solution of TRV1 and PbrLACS-TRV2 into the pear fruit near the equator, each pear fruit is injected with 6 holes, at least 7 fruits are injected each time, the experiment is repeated 3 times, and the fruits are placed in an incubator at 21℃ for dark culture for 24h, and then placed in an incubator at 21℃, with weak light intensity and a photoperiod of 16h light / 8h dark, and cultured for 2 days. Finally, the expression level of PbrLACS3 / 4 / 5 genes and the wax content of pear fruits in the gene silencing group and the control group were determined according to the method of Example 3, and the thickness of the cuticle was measured using the Oil Red O staining method.

[0108] The results showed that the expression of PbrLACS3 / 4 / 5 genes in the gene silencing group was significantly lower than that in the control group. The total wax content and terpenoid content of PbrLACS3 / 4 / 5 gene silencing group were significantly reduced by 33.47% and 39.36% respectively compared with the control group. The thickness of the cuticle was significantly reduced by 30.55%, and the number of crystals was significantly reduced ( Figure 7-10 ).

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

[0110] Subcellular localization of PbrLACS3 / 4 / 5 The plant overexpression vector constructed in the second part of Example 2 was used to activate the Agrobacterium containing the '35S-PbrLACS3-GFP' or '35S-PbrLACS4-GFP' or '35S-PbrLACS5-GFP' recombinant vector or the 35S-GFP control empty vector and prepare the infection solution according to the method in the first part of Example 3. The vector was gently injected into the tobacco leaf from the back of the tobacco leaf, cultured at 25°C in the dark for 1 day, and cultured normally for 1-2 days; the fluorescence signal was 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 yellow, indicating that PbrLACS3 / 4 / 5 was localized in the endoplasmic reticulum ( Fig.11 ).

[0112] It can be seen from the above embodiments that the pear long-chain acyl-CoA synthetase PbrLACS3 / 4 / 5 provided by the present invention has the effect of positively regulating fruit wax through experimental verification such as spatiotemporal expression, transient overexpression in pear fruit, gene silencing and subcellular localization.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene selected from the following a1), a2) or a3): a1) The nucleotide sequence thereof is shown in any one of SEQ ID NO.1-3; a2) The nucleotide sequence shown in any one of SEQ ID NO.1-3 is substituted, deleted and / or added with one or more nucleotides; a3) A nucleotide sequence that hybridizes to the DNA sequence defined in 1) under stringent conditions.

2. The protein encoded by the gene according to claim 1, characterized in that It is selected from the following b1) or b2): b1) a protein having an amino acid sequence as shown in any one of SEQ ID NOs. 4-6; b2) A protein derived from 1) with the same activity as that of any one of SEQ ID NOs. 4-6, wherein one or more amino acids are substituted, deleted and / or added.

3. Recombinant expression vectors, expression cassettes, transgenic cell lines or genetically engineered bacteria containing the aforementioned genes.

4. The recombinant expression vector according to claim 3, characterized in that The construction of the recombinant expression vector is selected from the following c1) or c2): c1) inserting the gene according to claim 1 into the XbaI and BamHI sites of pCAMBIA1301 to obtain a recombinant expression vector; c2) inserting the gene described in claim 1 between the BamHI and EcoRI sites of TRV to obtain a recombinant expression vector.

5. Use of the gene according to claim 1 and / or the protein according to claim 2 and / or the recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria according to claim 3 in increasing or decreasing the wax content of pear fruit.

6. Use of the gene according to claim 1 and / or the protein according to claim 2 and / or the recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria according to claim 3 in the preparation of a product for increasing or decreasing the wax content of pear fruit.

7. Use of the gene according to claim 1 and / or the protein according to claim 2 and / or the recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria according to claim 3 in pear breeding for increasing or decreasing fruit wax.

8. A method for increasing the wax content of pear fruit, characterized in that: The method is achieved by increasing the expression level of the gene described in claim 1.

9. The method according to claim 8, characterized in that The increasing the expression level of the gene described in claim 1 is achieved by transferring a recombinant expression vector that overexpresses the gene described in claim 1 into the target plant.

10. A method for reducing the wax content of pear fruit, characterized in that: The method is achieved by reducing the expression level of the gene described in claim 1.

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