Mango E3 ubiquitin ligase PUB21 and its encoding gene and applications

Abstract

The present invention discloses a mango PUB21 gene and its application in regulating the wax content and / or structure of mango peel, belonging to the technical field of genetic engineering. The PUB21 gene encodes an amino acid sequence shown in SEQ ID NO.2. By altering the expression of PUB21, the present invention can regulate the wax content and / or wax structure of the peel, indicating that this gene can be used to regulate the storage tolerance and resistance of fruits and has important application value in cultivating plant varieties with storage tolerance and resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a mango PUB21 gene and an application thereof in regulating the wax content and structure of plant peels. Background Art

[0002] Mango (Mangiferaindica) is an important cash crop in my country's tropical and subtropical regions. Its unique aroma, delicate juiciness, and balanced sweetness and sourness are its rich content of nutrients, including vitamin C, vitamin A, and fiber. It is a beloved fruit, earning it the title of "King of Tropical Fruits." However, mangoes are susceptible to heavy metal contamination in the soil and biotic and abiotic stresses during their growth and development. Postharvest, mangoes suffer from respiratory catastrophic changes and poor storage tolerance, impacting yield and quality, severely limiting the development of the mango industry. Cuticular wax is a plant's first line of defense against adverse environmental conditions, performing multiple biological functions, including reducing water loss, protecting against biotic and abiotic stresses, and extending the shelf life of the fruit.

[0003] Wax is one of the important quality traits of mango. Studying its components and formation mechanism, discovering more new genes that regulate the content and structure of wax in the peel, and developing new methods to cultivate plant varieties with high wax content are of great scientific significance and application value for improving the resistance of mango fruit and increasing mango quality and yield. Summary of the Invention

[0004] The present invention aims to provide a gene PUB21 related to the total wax content and / or wax structure of mango peel and its use in regulating the wax content and / or wax structure of plant peels. Furthermore, the gene can be used to cultivate plant varieties with high or low wax content in the peel.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides a PUB21 protein for regulating the total wax content and / or wax structure of plant peels, wherein the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0007] On the other hand, the present invention provides a gene PUB21 for regulating the wax content and / or wax structure of plant peels, wherein the nucleotide sequence of the gene encodes a nucleotide sequence of the amino acid sequence shown in SEQ ID NO.2.

[0008] On the other hand, the present invention provides a vector for regulating the wax content and / or wax structure of plant peels, wherein the vector can overexpress mRNA encoding the amino acid sequence shown in SEQ ID NO.2.

[0009] On the other hand, the present invention provides a gene silencing vector for regulating the wax content and / or wax structure of mango peel, characterized in that the gene silencing vector can express the nucleotide sequence shown in SEQ ID NO.3.

[0010] Taking into account the degeneracy of codons, modifications to the bases of the above nucleotide sequence without changing the amino acid sequence also fall within the scope of protection of the present invention.

[0011] It is well known to those skilled in the art that a gene sequence may also contain introns, promoters and various regulatory elements. Therefore, the nucleotide sequence of the above-mentioned PUB21 gene may also contain introns, promoters and various regulatory elements.

[0012] On the other hand, the present invention also provides a primer pair for cloning SEQ ID NO. 3. Preferably, the sequence of the forward primer F1 of the primer pair is shown as SEQ ID NO. 4, and the sequence of the reverse primer R1 is shown as SEQ ID NO. 5.

[0013] On the other hand, the present invention also provides a kit for regulating the wax content and / or wax structure of plant peels, wherein the kit comprises the above-mentioned primer pair and / or the above-mentioned vector.

[0014] On the other hand, the present invention also provides the use of at least one of the aforementioned proteins, genes, primers, vectors or kits in regulating the wax content and / or wax structure of plant peels.

[0015] Preferably, the regulation of mango peel wax content is to reduce the content of total wax, total alcohol compounds, C30 alcohol compounds, C18 olefin compounds and / or C25 aldehyde compounds in mango peel by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein, and / or increase the content of C25 alkane compounds.

[0016] Preferably, the regulating the mango peel wax structure is to reduce the thickness of the mango peel wax layer and the number of wax crystals by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein.

[0017] Preferably, the above application comprises the following steps when reducing the wax content of plant peel:

[0018] 1) Amplify the aforementioned gene PUB21;

[0019] 2) connecting the gene PUB21 with an overexpression vector to obtain a recombinant vector;

[0020] 3) Transforming the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

[0021] 4) Transforming the recombinant Agrobacterium tumefaciens into plant peels.

[0022] Preferably, the regulating the mango peel wax content is to increase the content of total wax, total alcohol compounds and / or C35 ester compounds in the mango peel by reducing the expression of mango PUB21 protein and / or mRNA encoding mango PUB21 protein.

[0023] Preferably, the regulating the mango peel wax structure is to increase the thickness of the mango peel wax layer and the number of wax crystals by reducing the expression of mango PUB21 protein and / or mRNA encoding mango PUB21 protein.

[0024] Preferably, the above application includes the following steps when increasing the wax content of plant peel:

[0025] 1) Amplifying the interference fragment SEQ ID NO. 3 of the aforementioned gene PUB21;

[0026] 2) connecting the SEQ ID NO. 3 sequence with a gene silencing vector to obtain a recombinant vector;

[0027] 3) Transforming the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

[0028] 4) Transforming the recombinant Agrobacterium tumefaciens into plant peels.

[0029] On the other hand, the present invention also provides the use of the aforementioned protein, gene, primer, vector or kit in breeding new plant varieties.

[0030] Preferably, the cultivating of new plant varieties is cultivating new varieties by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein.

[0031] Preferably, the new variety has at least one of the following characteristics:

[0032] The contents of total wax, total alcohol compounds, C30 alcohol compounds, C18 olefin compounds, and C25 aldehyde compounds in the peel decreased, while the content of C25 alkane compounds increased, the thickness of the wax layer decreased, and the number of wax crystals decreased.

[0033] Preferably, the above application comprises the following steps when cultivating new plant varieties:

[0034] 1) Amplify the aforementioned gene PUB21;

[0035] 2) connecting the gene PUB21 with an overexpression vector to obtain a recombinant vector;

[0036] 3) Transforming the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

[0037] 4) Transforming the recombinant Agrobacterium tumefaciens into plants for stable expression.

[0038] Preferably, the breeding of new plant varieties is breeding of new plant varieties by reducing the expression of mango PUB21 protein and / or mRNA encoding mango PUB21 protein.

[0039] Preferably, the new plant variety has at least one of the following characteristics:

[0040] The contents of total wax, total alcohol compounds, and C35 ester compounds in the peel increased, the thickness of the wax layer increased, and the number of wax crystals increased.

[0041] Preferably, the above application comprises the following steps when cultivating new plant varieties:

[0042] 1) Amplifying the interference fragment SEQ ID NO. 3 of the aforementioned gene PUB21;

[0043] 2) connecting the SEQ ID NO. 3 sequence with a gene silencing vector to obtain a recombinant vector;

[0044] 3) Transforming the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

[0045] 4) Transforming the recombinant Agrobacterium tumefaciens into plant peels.

[0046] Preferably, the aforementioned plant is a dicotyledonous plant.

[0047] Furthermore, the aforementioned plant is a plant of the Anacardiaceae family.

[0048] Furthermore, the aforementioned plant is a plant of the genus Mango.

[0049] Furthermore, the aforementioned plant is mango.

[0050] Compared with the prior art, the present invention has the following advantages:

[0051] 1) The present invention provides a new carrier capable of regulating the wax content and wax structure of plant peels.

[0052] 2) The present invention provides a new method for regulating the wax content and wax structure of plant peels.

[0053] 3) The present invention provides a new method for regulating the total wax content and the wax content of different components.

[0054] 4) The present invention provides a method for cultivating new plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The beneficial effects of the present invention are described in detail below with reference to the accompanying drawings and specific embodiments.

[0056] Figure 1 Shown are the gene expression levels of PUB21 in the peel and leaves of high- and low-wax mango varieties; Figure A: Wax content in the peel of 'Jinhuang' mango (MGDL) and 'Aiwen' mango (MGGL); Figure B: PUB21 expression level in the peel of 'Jinhuang' and 'Aiwen' mangoes; Figure C: Wax content in the leaves of 'Hongxiangya' mango (MGYDL) and 'Yuwen' mango (MGYGL); Figure D: PUB21 expression level in the leaves of 'Hongxiangya' and 'Yuwen' mangoes.

[0057] Figure 2 Shown are the PUB21 expression levels in transiently expressed mango fruits and control samples (Figure A) and the effects of transient expression of PUB21 on the total content and contents of various components of pericarp wax (Figure B).

[0058] Figure 3 Shown are the effects of transient overexpression of PUB21 on the carbon chain distribution of peel wax components; Figure A: Carbon chain distribution of peel wax alcohols; Figure B: Carbon chain distribution of peel wax olefins; Figure C: Carbon chain distribution of peel wax aldehydes; Figure D: Carbon chain distribution of peel wax alkanes.

[0059] Figure 4 Shown is the effect of transient overexpression of PUB21 on the thickness of the cuticle (wax layer) of the fruit peel.

[0060] Figure 5 Shown are the effects of transient overexpression of PUB21 on the wax crystal structure of the fruit peel. Panel A: 35S-PUB21-GFP; Panel B: 35S-GFP.

[0061] Figure 6 Shown are the PUB21 expression levels in silenced mango fruits and control samples (A) and the effects of PUB21 silencing on the total content and components of pericarp wax (B).

[0062] Figure 7 Shown is the effect of silencing PUB21 on the carbon chain distribution of wax ester compounds in the peel.

[0063] Figure 8 Shown is the effect of transient silencing of PUB21 on the thickness of the fruit peel cuticle (wax layer).

[0064] Figure 9 Shown are the effects of transiently silencing PUB21 on the crystal structure of pericarp wax. Panel A: TRV; Panel B: TRV-PUB21. DETAILED DESCRIPTION

[0065] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0066] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0067] The nucleotide sequence of the mango PUB21 gene in the embodiment is shown in SEQ ID NO.1:

[0068]

[0069] The amino acid sequence of mango PUB21 protein is shown in SEQ ID NO.2:

[0070] MISTWRRRRAARRAAKKLQDGEGGEMELTIPDHFRCPISLDLMKDPVTLSTGITYDRENIEKWIESGNDKCPITKQVLTSLESIPNHTIRKMIQDWCVEHRSHGIERIPT PRIPVSAMEVSEILSKITMASKEMDQVKCRDLVMKIKTLAKESERNRRCIAGNGTGSVLSATFEAFSKSFFEENAAVLGEILSALTLVFPLDEEAVSYLGSSSSLYCMVC FLKNGDLSRRRNAVLALKMLVSSEQRIVNVLSKIEGAMEALFKLIQEPICPTSTKASLIVINHMVTISATSEKIVSKFVDMGLVSVLVEILVDAQRSLCEKALCVLEVIF SFEKGREEAYTQALTVPVIVKKILRVSDIATEVSVSILWNLCKNEKREEKFALAETLQVGAFQKLLLLLQLGCAEKTKEKATELLKLLNLHRDRFECIDSMDFKDLKRPF

[0071] The interference fragment of the mango PUB21 gene is as shown in SEQ ID NO. 3:

[0072] CCCGGAGAAGAAACGCCGTGTTGGCGTTGAAAATGCTGGTTTCATCGGAGCAAAGAATAGTAAACGTATTATCGAAGATTGAAGGCGCCATGGAAGCATTATTCAAGCTGATTCAAGAGCCCATTTGTCCAACTTCCACAAAAGCTTCATTGATAGTCATCAATCACATGGTCAC CATTTCTGCCACAAGTGAGAAAATCGTTTCGAAAATTTGTCGATATGGGGCTCGTCTCAGTGCTCGTAGAAATACTTGTGGATGCTCAACGAAGCCTATGTGAAAAGGCTTTATGCGTTCTGGAGGTAATTTTCAGCTTCGAAAAAGGGAGAGAAGAGGCCTACACTCAAGCCTTG

[0073] Example

[0074] Example 1 Determination of wax content and components in different mango varieties and tissue parts

[0075] In a fume hood, fruits of the 'Jinhuang' and 'Aiwen' mango varieties, 120 days after anthesis, and new leaves of the 'Hong Xiangya' and 'Yuwen' mango varieties, after turning from yellow to green, were completely immersed in a chloroform solution for 2 minutes for the fruits and 1 minute for the leaves to extract epidermal waxes. The extract containing epidermal waxes 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 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 with nitrogen. 200 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma) and 200 μL of pyridine (Sigma) were added, and the mixture was oven-derivatived at 70°C for 1 hour. All reagents were then dried again with chloroform, and 1 mL of chromatographic-grade chloroform was added for complete dissolution. The mixture was filtered through a 0.45 μm organic filter into a 1.5 mL brown gas phase vial for analysis. The waxy components were analyzed using a gas chromatograph (TRACE1310, ThermoScientific) coupled with a triple quadrupole mass spectrometer (TSQ9000, ThermoScientific). Compounds were separated on a TG-5MS (30 m × 0.25 mm ID, 0.25 μm film, ThermoScientific) capillary column. Helium was used as the carrier gas at 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; and scan range, 50–650 m / z. The temperature program was as follows: 50°C for 2 minutes, then ramped to 170°C at a rate of 20°C / min, held for 2 minutes, then ramped to 200°C at a rate of 10°C / min, held for 3 minutes, then ramped to 220°C at a rate of 3°C / min, held for 2 minutes. Finally, ramped to 320°C at a rate of 5°C / min, held for 20 minutes. Waxy compounds were identified by matching retention indices with mass spectra from the NIST 2017 database. Alkanes were identified by comparing the mass spectra and retention times of C7–C40 saturated alkane standards (Sigma-Aldrich).

[0076] Example 2 Gene expression abundance of PUB21 gene in different mango varieties and different tissue parts

[0077] High-throughput sequencing technology was used to obtain the changes in the expression pattern of the fruit peel of the 'Jinhuang' and 'Aiwen' mango varieties 120 days after flowering, and the new leaves of the 'Hongyi' and 'Yuwen' mango varieties after yellow to green. Using transcriptome data, the TPM values ​​of PUB21 in different mango varieties, different tissue parts and different developmental stages of the fruit were obtained, and its expression pattern was analyzed.

[0078] The results showed that the expression level of PUB21 gene was negatively correlated with the wax content of mango ( Figure 1 ).

[0079] Example 3 Cloning and prokaryotic expression of mango PUB21 gene and construction of plant transient expression vector

[0080] 1. Cloning of the mango PUB21 gene

[0081] RNA was extracted from the peel of 'Kate' mango fruit, 120 days after anthesis, according to the FastPure Plant Total RNA Isolation Kit (purchased from Novozymes, China). After extraction, the quality of the extracted RNA was assessed by 1% agarose gel electrophoresis and the concentration and quality were determined using a NanoDrop 2000 spectrophotometer. First-strand cDNA synthesis was performed according to the ThermoScientific RevertAid First Strand cDNA Synthesis Kit. The resulting first-strand cDNA was used to amplify the PUB21 gene. Based on the PUB21 gene sequence obtained from transcriptome sequencing, upstream and downstream primers were designed near the start and stop codons and synthesized by Qingke Biotechnology Co., Ltd. The primer sequences are shown below.

[0082]

[0083] A 50 μL PCR reaction system consisted of 25 μL PrimeSTAR Max Premix (2×) (purchased from TakaRa), 0.6 μM forward primer, 0.6 μM reverse primer, 200 ng cDNA, and sterile distilled water to a final volume of 50 μL. The PCR protocol was as follows: initial denaturation at 95°C for 3 minutes; 35 cycles of amplification, consisting of denaturation at 95°C for 30 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 2 minutes. Following this cycle, the reaction was extended at 72°C for 10 minutes, followed by a 5-minute incubation at 20°C. After amplification, a single target band of the PCR product was detected by electrophoresis on a 1.5% agarose gel. The specific target band was recovered according to the instructions of a gel extraction kit (purchased from Novozymes, China).

[0084] 2. Construction of a plant overexpression vector for the mango PUB21 gene

[0085] The purified PCR product was recovered and ligated with the pCAMBIA1300-35S-EGFP vector. The product was then transformed into E. coli DH5α using the heat shock method. PCR detection was performed using gene-specific primers, and positive bacterial cultures were sequenced (performed by Qingke Biotechnology Co., Ltd.). Sequencing results indicated that the amplified PUB21 target fragment was 1323 bp in length. Nucleotide sequence analysis using NCBI confirmed that the sequence was the target gene of the present invention. The resulting recombinant target vectors were designated 35S-PUB21-GFP. The recombinant vector 35S-PUB21-GFP and a control empty vector 35S-EGFP were then transformed into Agrobacterium tumefaciens GV3101 using the heat shock method.

[0086] 3. Construction of a plant gene silencing vector for the mango PUB21 gene

[0087] The first-strand cDNA obtained in the first part of Example 3 was used to amplify the PUB21 gene. The nucleotide sequence of the primer pair is as follows (the underlined parts are SEQ ID NO. 4 and SEQ ID NO. 5):

[0088]

[0089] The target product was amplified using the PCR reaction system described in the first section of Example 3, and the specific target band was recovered. The recovered and purified PCR product was ligated with 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 suspension was sequenced (performed by Qingke Biotechnology Co., Ltd.) to confirm that the sequence under test was the target gene required by the present invention. The resulting recombinant target vector was named TRV-PUB21. The recombinant vector TRV-PUB21 and the control empty vector TRV were separately transformed into Agrobacterium tumefaciens GV3101 using the heat shock method.

[0090] Example 4 Identification of waxy traits in mango fruits by transient overexpression of the mango PUB21 gene

[0091] Cuticular wax thickness, crystal structure, wax content and composition are important evaluation traits of plant epidermal wax.

[0092] 1. Mango PUB21 gene transient overexpression injection

[0093] Take Agrobacterium tumefaciens (containing the recombinant vector of 35S-PUB21-GFP and the empty vector of 35S-GFP control) stored in a -80℃ refrigerator, streak culture on solid LB medium containing 100mg / L kanamycin and 100mg / L rifampicin, culture at 28℃ for 2d, then pick the single colony on the line with a sterilized toothpick, put it into a 50mL centrifuge tube, add 25mL liquid LB medium containing 100mg / L kanamycin and 100mg / L rifampicin, culture at 28℃ in a shaker at 220rpm for 12 hours, collect the bacteria by centrifugation at 5000rpm for 5 minutes in a 50mL centrifuge tube, add 10mL induction solution (0.5ml200mMMgCl2, 0.5ml2 00mM MES, 20μL 200mM acetosyringone, 9ml ddH2O, 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, place it in a 28℃ shaker at 100r / min in the dark and incubate for 4h, use a syringe to inject the empty 35S-GFP and 35S-PUB21-GFP infection solutions into the equator of 'Kate' fruits, respectively. Each mango fruit is injected with 6 holes, and at least 3 fruits are injected for each group of experiments. The experiment is repeated 3 times, and the fruits are placed in a 21℃ incubator for dark incubation for 24h, and then placed in a 21℃ incubator with weak light intensity and a photoperiod of 16h light / 8h dark for culturing for 2 days.

[0094] 2. Detection of transient overexpression of PUB21 gene expression

[0095] Mango peel tissue samples were collected after injection. Total RNA was extracted and cDNA synthesized using the method described in the first section of Example 3 for qRT-PCR. qRT-PCR was performed using the ChamQ SYBR qPCR MasterMix Kit (purchased from Novozymes) according to the manufacturer's instructions. A 10 μL qRT-PCR reaction system consisted of: 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 (purchased from Roche) and a qRT-PCR instrument (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, followed by 50 cycles of return to the second step, 72°C for 10 min, and 20°C for 5 min. Repeat 3 times and calculate the average Ct value of each cDNA sample. -ΔΔCt The relative expression level of the PUB21 gene in mango peel after transient overexpression was obtained.

[0096] 3. Detection of wax content and composition in transient overexpression samples

[0097] In a fume hood, the injected mango fruit was completely immersed in a chloroform solution for 2 minutes to extract epidermal wax. The specific method was similar to that in Example 1.

[0098] 4. Detection of waxy structures in transiently overexpressed samples

[0099] The peel samples of the injected mango fruits were taken and cut into thin slices of approximately 0.5 cm long and 0.5 cm wide. The microstructure of the peel was photographed using a Hitachi 4800 field emission scanning electron microscope (Hitachi S-4800).

[0100] 5. Detection of cuticle thickness of transient overexpression samples

[0101] Following the method described in Part 4 of this Example 4, thin fruit peel slices were placed in 2 ml centrifuge tubes containing FAA fixative. The tubes were filled with fixative and excess air was expelled to prevent oxidation. The samples were first dehydrated with sucrose and prepared by longitudinal sectioning into frozen sections. The sections were then air-dried at room temperature for 15 minutes. The sections were then stained in saturated Oil Red O staining solution for 10 minutes in the dark. Subsequently, the sections were differentiated in 60% isopropanol for 10 seconds and gently rinsed in distilled water to prevent lipid droplet displacement. Excess moisture was wiped away from the surrounding tissue and the sections were mounted with glycerol gelatin. Finally, the sections were observed and photographed under a microscope (NIKON Eclipse).

[0102] The results showed that overexpression of PUB21 gene significantly increased the expression level ( Figure 2 A), the total amount of wax was significantly reduced by 24.93% compared with the control group ( Figure 2 B), among which alcohol compounds were significantly reduced by 27.55% ( Figure 2 B). C30 alcohols ( Figure 3 A), C18 olefins ( Figure 3 B) and C25 aldehydes ( Figure 3 C) Waxy compounds were significantly reduced, C25 alkanes ( Figure 3 D) Wax compounds increased significantly, and the thickness of the cuticle decreased significantly by 19.84% ( Figure 4 ), the number of waxy crystals was significantly reduced ( Figure 5 ).

[0103] Example 5 Identification of waxy traits in mango fruit after silencing of the mango PUB21 gene

[0104] According to the method of the first part of Example 4, Agrobacterium containing the TRV-PUB21 recombinant vector was activated, and then a single clone on the line was picked with a sterilized toothpick and placed in a 50 mL centrifuge tube. 25 mL of liquid LB culture medium containing 100 mg / L kanamycin and 100 mg / L rifampicin was added, and the culture was shaken at 200 rpm at 28 ° C for 12 hours. The bacteria were collected by centrifugation at 5000 rpm in a 50 mL centrifuge tube for 5 minutes, and 10 mL of induction solution (0.5 ml 200 mM MgCl2, 0.5 ml 200 mM MES, 20 μL 200 mM acetosyringone, 9 ml ddH2O, pH = 5.5) was added to resuspend the bacteria, and then centrifuged again. The invasion dye was added again for use. The bacterial solution was adjusted to an OD value between 0.6 and 0.8 using a spectrophotometer. A mixed infection solution was prepared using the following ratio: TRV1:TRV2 (PUB21-TRV2) = 1:1. The solution was incubated in a shaker at 25°C, 100 rpm, and protected from light for 4 hours. The mixed infection solution of TRV1 and unloaded TRV2, and the mixed infection solution of TRV1 and PUB21-TRV2 were injected into the mango fruits of the control and experimental groups near the equator using a syringe. Six wells were injected into each mango fruit, and at least three fruits were injected in each experiment. The experiment was repeated three times. The fruit was placed in an incubator at 21°C and incubated in the dark for 24 hours. The fruit was then placed in an incubator at 21°C, with low light intensity and a photoperiod of 16 hours light / 8 hours dark, and incubated for 2 days. Finally, the expression level of the PUB21 gene and the wax content of the mango fruits in the gene-silenced and control groups were determined according to the method of Example 4. The cuticle thickness was measured using Oil Red O staining, and the wax crystal structure was observed by scanning electron microscopy.

[0105] The results showed that silencing the PUB21 gene expression level was significantly reduced ( Figure 6 A), the total amount of wax significantly increased by 10.58% compared with the control group ( Figure 6 B), among which the content of alcohol compounds increased significantly by 47.30% ( Figure 6 B). C35 esters ( Figure 7 ) Wax compounds increased significantly, and the thickness of the cuticle increased significantly by 23.76% ( Figure 8 ), the number of wax crystals increased significantly ( Figure 9 ).

[0106] As demonstrated in the above examples, the mango E3 ubiquitin ligase PUB21 provided by the present invention has been shown to negatively regulate the composition and content of mango wax through transcriptome expression module analysis, transient overexpression in mango fruit, and gene silencing experiments. Therefore, regulating PUB21 expression has potential applications in improving fruit storage tolerance and plant resistance, and in cultivating storage-tolerant and resistant plant varieties.

[0107] Based on the disclosure and teachings of the above description, those skilled in the art may also make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and any modifications and variations of the present invention should also fall within the scope of protection of the claims of the present invention. In addition, although certain specific terms are used in this description, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. Use of mango PUB21 protein and / or a gene encoding mango PUB21 protein and / or a vector encoding mango PUB21 protein in regulating the wax content and / or regulating the wax structure of mango peel, characterized in that: The application is achieved by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein, and the amino acid sequence of the mango PUB21 protein is shown in SEQ ID NO.2; The regulation of the mango peel wax content is to reduce the content of C30 alcohol compounds, C18 olefin compounds and / or C25 aldehyde compounds in the mango peel, and / or increase the content of C25 alkane compounds in the mango peel by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein, Or the regulation of mango peel wax content is to reduce the total wax content of mango peel by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein, Or the regulation of mango peel wax content is to reduce the total alcohol compound content of mango peel by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein; The regulation of the mango peel wax structure is to reduce the thickness of the mango peel wax layer and the number of wax crystals by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein.

2. Use of mango PUB21 protein and / or a gene encoding mango PUB21 protein and / or a vector encoding mango PUB21 protein in regulating the wax content and / or regulating the wax structure of mango peel, characterized in that: The application is achieved by reducing the expression of mango PUB21 protein and / or mRNA encoding mango PUB21 protein, and the amino acid sequence of the mango PUB21 protein is shown in SEQ ID NO.2; The regulation of the mango peel wax content is to increase the content of total alcohol compounds and / or C35 ester compounds in the mango peel by reducing the expression of mango PUB21 protein and / or mRNA encoding mango PUB21 protein, Or the regulation of mango peel wax content is to increase the total wax content of mango peel by reducing the expression of mango PUB21 protein and / or mRNA encoding mango PUB21 protein; The regulation of the mango peel wax structure is to increase the thickness of the mango peel wax layer and the number of wax crystals by reducing the expression of mango PUB21 protein and / or mRNA encoding mango PUB21 protein.

3. Use of mango PUB21 protein and / or a gene encoding mango PUB21 protein and / or a vector encoding mango PUB21 protein in breeding new mango varieties, characterized in that: The amino acid sequence of the mango PUB21 protein is shown in SEQ ID NO. 2, and the breeding of the new mango variety is breeding of the new mango variety by overexpressing the mango PUB21 protein and / or mRNA encoding the mango PUB21 protein.

4. Use of mango PUB21 protein and / or a gene encoding mango PUB21 protein and / or a vector encoding mango PUB21 protein in breeding new mango varieties, characterized in that: The method of cultivating a new mango variety is to cultivate a new mango variety by reducing the expression of mango PUB21 protein and / or mRNA encoding mango PUB21 protein. The amino acid sequence of the mango PUB21 protein is shown in SEQ ID NO.2.

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