Mango E3 ubiquitin ligase PUB21 and coding gene and application thereof

Abstract

The invention discloses a mango PUB21 gene and application thereof in regulating and controlling wax content and / or structure of mango peel, and belongs to the technical field of gene engineering. The coding of the PUB21 gene is an amino acid sequence as shown in SEQ ID NO. 2. According to the invention, the wax content and / or wax structure of fruit peel can be regulated and controlled by changing the expression of PUB21, which indicates that the gene can be used for regulating and controlling the storage resistance and resistance of fruits, and has important application value in cultivation of storage-resistant and resistant plant varieties.

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 a plant peel. Background Art

[0002] Mango (Mangiferaindica) is an important economic crop in tropical and subtropical regions of my country. Its flesh has a unique aroma, is delicate and juicy, and has a moderate sweetness and sourness. In addition, it is rich in nutrients such as vitamin C, vitamin A and cellulose. It is deeply loved by people and is known as the king of tropical fruits. However, mango is susceptible to soil heavy metal pollution and biological and abiotic stresses during its growth and development stage. After harvest, it is not resistant to storage due to respiratory climacteric, which affects mango yield and quality, and seriously restricts the development of the mango industry. Plant epidermal wax is the first line of defense for plants against adverse external environments. It has multiple biological functions such as reducing water loss, resisting biological or abiotic stresses, and extending the shelf life of fruits.

[0003] Wax is one of the important quality traits of mango. Studying its composition and formation mechanism, discovering more new genes that regulate the content and structure of pericarp wax, and developing new methods to cultivate plant varieties with high wax content are of great scientific significance and application value for improving mango fruit resistance 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 application in regulating the wax content and / or wax structure of plant peel. It can further be used to cultivate plant varieties with high or low wax content in peel.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: On the one hand, the present invention provides a PUB21 protein for regulating the total wax content and / or wax structure of plant peels, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] Taking into account the degeneracy of codons, modifying the bases of the above nucleotide sequence without changing the amino acid sequence also falls within the scope of protection of the present invention.

[0010] It is well known to those skilled in the art that a gene sequence may also include introns, promoters and various regulatory elements. Therefore, the nucleotide sequence of the above-mentioned PUB21 gene may also include introns, promoters and various regulatory elements.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] Preferably, the regulating the 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 the mango peel by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein, and / or increase the content of C25 alkane compounds.

[0015] 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.

[0016] Preferably, the above application comprises the following steps when reducing the wax content of plant peel: 1) Amplify the aforementioned gene PUB21; 2) connecting the gene PUB21 with an overexpression vector to obtain a recombinant vector; 3) transferring the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens; 4) Transforming the recombinant Agrobacterium tumefaciens into plant peels.

[0017] 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.

[0018] 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.

[0019] Preferably, the above application includes the following steps when increasing the wax content of plant peel: 1) Amplifying the interference fragment SEQ ID NO.3 of the aforementioned gene PUB21; 2) connecting the SEQ ID NO.3 sequence with a gene silencing vector to obtain a recombinant vector; 3) transferring the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens; 4) Transforming the recombinant Agrobacterium tumefaciens into plant peels.

[0020] 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.

[0021] Preferably, the breeding of new plant varieties is breeding of new varieties by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein.

[0022] Preferably, the new variety has at least one of the following characteristics: The contents of total wax, total alcohol compounds, C30 alcohol compounds, C18 olefin compounds, and C25 aldehyde compounds in the peel decreased, the content of C25 alkane compounds increased, the thickness of the wax layer decreased, and the number of wax crystals decreased.

[0023] Preferably, the above application comprises the following steps when cultivating new plant varieties: 1) Amplify the aforementioned gene PUB21; 2) connecting the gene PUB21 with an overexpression vector to obtain a recombinant vector; 3) transferring the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens; 4) Transforming the recombinant Agrobacterium tumefaciens into plants for stable expression.

[0024] 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.

[0025] Preferably, the new plant variety has at least one of the following characteristics: 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.

[0026] Preferably, the above application comprises the following steps when cultivating new plant varieties: 1) Amplifying the interference fragment SEQ ID NO.3 of the aforementioned gene PUB21; 2) connecting the SEQ ID NO.3 sequence with a gene silencing vector to obtain a recombinant vector; 3) transferring the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens; 4) Transforming the recombinant Agrobacterium tumefaciens into plant peels.

[0027] Preferably, the aforementioned plant is a dicotyledonous plant.

[0028] Furthermore, the aforementioned plant is a plant of the Anacardiaceae family.

[0029] Furthermore, the aforementioned plant is a plant of the genus Mango.

[0030] Furthermore, the aforementioned plant is mango.

[0031] Compared with the prior art, the present invention has the following advantages: 1) The present invention provides a new carrier capable of regulating the wax content and wax structure of plant peels.

[0032] 2) The present invention provides a new method for regulating the wax content and wax structure of plant peels.

[0033] 3) The present invention provides a new method for regulating the total wax content and the wax content of different components.

[0034] 4) The present invention provides a method for cultivating new plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0036] 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: The expression level of PUB21 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: The expression level of PUB21 in the leaves of 'Hongxiangya' and 'Yuwen' mangoes.

[0037] Figure 2 Shown are the PUB21 expression levels in transiently expressed mango fruits and control samples (Figure A) and the effects of transiently expressed PUB21 on the total content and components of pericarp wax (Figure B).

[0038] 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.

[0039] Figure 4 Shown is the effect of transient overexpression of PUB21 on the thickness of the pericarp cuticle (wax layer).

[0040] Figure 5 The results show the effect of transient overexpression of PUB21 on the crystal structure of pericarp wax. Panel A: 35S-PUB21-GFP; Panel B: 35S-GFP.

[0041] Figure 6 Shown are the PUB21 expression levels in silenced mango fruits and control samples (A) and the effects of silencing PUB21 on the total content and components of pericarp wax (B).

[0042] Figure 7 Shown is the effect of silencing PUB21 on the carbon chain distribution of pericarp wax ester compounds.

[0043] Figure 8 Shown is the effect of transient silencing of PUB21 on the thickness of the fruit peel cuticle (wax layer).

[0044] Fig. 9 Shown is the effect of transient silencing of PUB21 on the crystal structure of pericarp wax. Panel A: TRV; Panel B: TRV-PUB21. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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 creative work are within the scope of protection of the present invention.

[0046] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0047] The nucleotide sequence of the mango PUB21 gene in the embodiment is shown in SEQ ID NO.1: The amino acid sequence of mango PUB21 protein is shown in SEQ ID NO.2: MISTWRRRRAARRAAKKLQDGEGGEMELTIPDHFRCPISLDLMKDPVTLSTGITYDRENIEKWIESGNDKCPITKQVLTSLESIPNHTIRKMIQDWCVEHRSHGIERIPT PRIPVSAMEVSEILSKITMASKEMDQVKCRDLVMKIKTLAKESERNRRCIAGNGTGSVLSATFEAFSKSFFEENAAVLGEILSALTLVFPLDEEAVSYLGSSSSLYCMVC FLKNGDLSRRRNAVLALKMLVSSEQRIVNVLSKIEGAMEALFKLIQEPICPTSTKASLIVINHMVTISATSEKIVSKFVDMGLVSVLVEILVDAQRSLCEKALCVLEVIF SFEKGREEAYTQALTVPVIVKKILRVSDIATEVSVSILWNLCKNEKREEKFALAETLQVGAFQKLLLLLQLGCAEKTKEKATELLKLLNLHRDRFECIDSMDFKDLKRPF The interference fragment of mango PUB21 gene is as shown in SEQ ID NO.3: CCCGGAGAAGAAACGCCGTGTTGGCGTTGAAAATGCTGGTTTCATCGGAGCAAAGAATAGTAAACGTATTATCGAAGATTGAAGGCGCCATGGAAGCATTATTCAAGCTGATTCAAGAGCCCATTTGTCCAACTTCCACAAAAGCTTCATTGATAGTCATCAATCACATGGTCAC CATTTCTGCCACAAGTGAGAAAATCGTTTCGAAATTTGTCGATATGGGGCTCGTCTCAGTGCTCGTAGAAATACTTGTGGATGCTCAACGAAGCCTATGTGAAAAGGCTTTATGCGTTCTGGAGGTAATTTTCAGCTTCGAAAAAGGGAGAGAAGAGGCCTACACTCAAGCCTTG Example Example 1 Determination of wax content and components in different mango varieties and different tissue parts In a fume hood, the fruits of the mango varieties 'Jinhuang' and 'Aiwen' 120 days after flowering, and the new leaves of the mango varieties 'Hong Xiangya' and 'Yuwen' after yellowing and turning green were completely immersed in chloroform solution. The fruits were immersed for 2 minutes and the leaves were immersed for 1 minute to extract the 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 filtering through a 0.45μm organic filter membrane. Then, 10μl of n-tetracosane (10μg / μl, Shanghai MacLean Biochemical Technology Co., Ltd.) was added as an internal standard, and the chloroform was blown dry with nitrogen. 200 μl N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma) and 200 μL pyridine (Sigma) were added, and the mixture was 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 the mixture. The mixture was filtered into a 1.5 mL brown gas phase bottle using a 0.45 μm organic filter membrane and then tested on the machine. The wax components were detected using a gas chromatograph (TRACE1310, ThermoScientific) and a triple quadrupole mass spectrometer (TSQ9000, ThermoScientific). The compounds were separated on a TG-5MS (30 m×0.25 mm I.D., 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; scan range, 50-650 m / z. The temperature program was as follows: 50 °C for 2 min, heated to 170 °C at a rate of 20 °C / min, held for 2 min, heated to 200 °C at a rate of 10 °C / min, held for 3 min, and then heated to 220 °C at a rate of 3 °C / min, held for 2 min. Finally, heated 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 the mass spectra in the NIST2017 library. Among them, alkanes were identified by comparing the mass spectra and retention times of C7-C40 saturated alkane standards (Sigma-Aldrich).

[0048] Example 2 Gene expression abundance of PUB21 gene in different mango varieties and different tissue parts 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.

[0049] The results showed that the expression level of PUB21 gene was negatively correlated with the wax content of mango ( Figure 1 ).

[0050] Example 3 Cloning and prokaryotic expression of mango PUB21 gene and construction of plant transient expression vector 1. Cloning of mango PUB21 gene The peel of 'Kate' mango 120 days after flowering was used as the test material, and its RNA was extracted according to the operating manual of Fast Pure Plant Total RNA Isolation Kit (purchased from Novozyme, China). After the extraction, the quality of the extracted RNA was detected by 1% agarose gel electrophoresis, and the concentration and quality of 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 Revert Aid First Strand cDNA Synthesis Kit. The obtained first strand cDNA was used for the amplification of the PUB21 gene. According to the PUB21 gene sequence obtained by transcriptome sequencing, upstream and downstream primers were designed near the start codon and the stop codon, and synthesized by Qingke Biotechnology Co., Ltd. The primer sequences are shown below.

[0051] The 50 μL PCR reaction system was as follows: 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 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 kept 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).

[0052] 2. Construction of plant overexpression vector of mango PUB21 gene The purified PCR product was recovered and ligated with the pCAMBIA1300-35S-EGFP vector, and transformed into E.coliDH5α by heat shock method. Gene-specific primers were used for PCR detection, and the positive bacterial solution was sequenced (completed by Qingke Biotechnology Co., Ltd.). The sequencing results showed that the length of the PUB21 target fragment amplified by the present invention was 1323 bp. The nucleotide sequence was compared and analyzed by NCBI to determine that the sequence was the target gene required by the present invention. The correct recombinant target vector was named 35S-PUB21-GFP. The recombinant vector 35S-PUB21-GFP and the control empty vector 35S-EGFP were respectively transferred into Agrobacterium GV3101 by heat shock method.

[0053] 3. Construction of plant gene silencing vector for mango PUB21 gene 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): The target product was amplified using the PCR reaction system in the first part of Example 3, and the specific target band was recovered. The recovered and purified PCR product was connected to the TRV vector, transformed into E. coli DH5α by heat shock method, and PCR detection was performed with gene-specific primers. The positive bacterial solution was sequenced (completed by Qingke Biotechnology Co., Ltd.) to determine that the sequence to be tested was the target gene required by the present invention, and the correct recombinant target vector was named TRV-PUB21. The recombinant vector TRV-PUB21 and the control empty vector TRV were respectively transferred into Agrobacterium GV3101 by heat shock method.

[0054] Example 4 Identification of waxy traits in mango fruits by transient overexpression of mango PUB21 gene Cuticular wax thickness, crystal structure, wax content and composition are important evaluation traits of plant cuticular wax.

[0055] 1. Mango PUB21 gene transient overexpression injection 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 of 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 in a 50mL centrifuge tube at 5000rpm for 5 minutes, add 10mL of induction solution (0.5ml200mMMgCl2, 0.5ml2 00mM MES, 20μL 200mM acetosyringone, 9ml ddH2O, pH=5.5) after resuspending the bacteria, centrifuge again, add the invasion 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 and incubate in the dark for 4h, use a syringe to inject the empty 35S-GFP and 35S-PUB21-GFP infection solutions into the equator of 'Kate' fruits, each mango fruit was injected with 6 holes, and at least 3 fruits were injected for each group of experiments. The experiment was repeated 3 times, and the fruits were 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.

[0056] 2. Detection of transient overexpression of PUB21 gene expression Mango peel tissue samples after injection were collected. Total RNA was extracted and cDNA was synthesized using the method described in the first part of Example 3, and qRT-PCR was performed. qRT-PCR was performed using the ChamQSYBRqPCRMasterMix kit (purchased from Novozymes) and strictly followed the instructions. The 10μL qRT-PCR reaction system included: 5μL fluorescent dye, 3.6μL sterilized ultrapure water, 1μL cDNA, 0.2μL forward primer (10μM), and 0.2μL reverse primer (10μM). A 96-well qRT-PCR plate (purchased from Roche) was used to perform PCR using a qRT-PCR instrument (model: LightCycler480, Roche). The qRT-PCR reaction program was: 95℃10min; 95℃10s, 60℃10s, 72℃30s, return to the second step for 50 cycles, 72℃10min, 20℃5min. 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.

[0057] 3. Detection of wax content and composition of transient overexpression samples 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 is as described in Example 1.

[0058] 4. Detection of waxy structure in transient overexpression samples The peel samples of the injected mango 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).

[0059] 5. Detection of cuticle thickness of transient overexpression samples According to the method of the fourth part of this embodiment 4, a peel slice sample was taken and placed in a 2 ml centrifuge tube containing FAA fixative. The tube was filled with fixative, and excess air was exhausted to prevent oxidation of the sample. 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 Eclipseci).

[0060] The results showed that the expression of PUB21 gene was significantly increased ( 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) The waxy compounds increased significantly and the thickness of the cuticle decreased significantly by 19.84% ( Figure 4 ), the number of wax crystals decreased significantly ( Figure 5 ).

[0061] Example 5 Identification of waxy traits in mango fruit after silencing of mango PUB21 gene According to the method in the first part of Example 4, the Agrobacterium containing the TRV-PUB21 recombinant vector was activated, and then a single clone on the line was picked up with a sterilized toothpick and placed in a 50 mL centrifuge tube. 25 mL of liquid LB medium containing 100 mg / L kanamycin and 100 mg / L rifampicin was added, and the culture was carried out at 200 rpm in a shaker at 28 ° C for 12 hours. The bacteria were collected by centrifugation at 5000 rpm for 5 minutes in a 50 mL centrifuge tube, 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, and the invasion dye was added again for use. The OD value of the bacterial solution was adjusted to between 0.6 and 0.8 by a spectrophotometer, and a mixed infection solution was prepared according to the following ratio TRV1:TRV2 (PUB21-TRV2) = 1:1, and placed in a 25°C shaker at 100 r / min in the dark 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 fruit of the control group and the experimental group near the equator with a syringe. Each mango fruit was injected with 6 holes, and at least 3 fruits were injected in each experiment. The experiment was repeated 3 times, and placed in a 21°C incubator for dark culture for 24 hours, and then placed in a 21°C incubator with weak light intensity and a light cycle of 16 hours of light / 8 hours of darkness for 2 days. Finally, the gene expression level and wax content of the PUB21 gene in the mango fruit of the gene silencing and control group were determined according to the method of Example 4, the cuticle thickness was measured by the oil red O staining method, and the wax crystal structure was observed by scanning electron microscopy.

[0062] 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 stratum corneum increased significantly by 23.76% ( Figure 8 ), the number of wax crystals increased significantly ( Fig. 9 ).

[0063] As can be seen from the above examples, the mango E3 ubiquitin ligase PUB21 provided by the present invention has been verified by transcriptome expression module analysis, transient overexpression in mango fruit, gene silencing and other experiments to negatively regulate the wax components and content of the fruit. Therefore, regulating the expression of PUB21 can have potential application value in improving fruit storage tolerance and plant resistance, and cultivating storage tolerance and resistance plant varieties.

[0064] According to the disclosure and teaching of the above description, those skilled in the art to which the present invention belongs 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 some modifications and changes to the present invention should also fall within the scope of protection of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the convenience of description and do not constitute any limitation to the present invention.

Claims

1. An overexpression vector for regulating the wax content and / or structure of mango peel, characterized in that: The overexpression vector can overexpress the mRNA encoding the amino acid sequence shown in SEQ ID NO.

2.

2. A gene silencing vector for regulating the wax content and / or structure of mango peel, characterized in that: The gene silencing vector can express the nucleotide sequence shown as SEQ ID NO.

3.

3. 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 amino acid sequence of the mango PUB21 protein is shown in SEQ ID NO.

2.

4. The use according to claim 3, characterized in that: The method for regulating the 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 the mango peel by overexpressing mango PUB21 protein and / or mRNA encoding mango PUB21 protein, and / or increase the content of C25 alkane compounds.

5. The use according to claim 3, characterized in that: 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.

6. Use of the carrier according to claim 2 in regulating the wax content and / or regulating the wax structure of mango peel.

7. The use according to claim 6, characterized in that: The regulation of mango peel wax content is to increase the content of total wax, total alcohol compounds and / or C35 ester compounds in mango peel by reducing the expression of mango PUB21 protein and / or mRNA encoding mango PUB21 protein.

8. The use according to claim 6, characterized in that: 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.

9. 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 as SEQ ID NO.2, and the breeding of the new mango variety is to breed the new mango variety by overexpressing the mango PUB21 protein and / or mRNA encoding the mango PUB21 protein.

10. Use of the vector according to claim 2 in breeding new mango varieties, characterized in that: The method for breeding a new mango variety is to reduce the expression of mango PUB21 protein and / or mRNA encoding mango PUB21 protein to breed a new mango variety. The amino acid sequence of the mango PUB21 protein is shown in SEQ ID NO.2.

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