Mango PUB29 gene and its application in regulating the content and composition of plant peel wax

Abstract

The invention discloses a mango PUB29 gene and its application in regulating the wax content and components of mango peel, belonging to the field of genetic engineering technology. The PUB29 gene encodes an amino acid sequence as shown in SEQ ID NO.2. The invention can increase the total wax content and the content of different wax components in the peel by increasing the expression of PUB29, indicating that the gene can be used to improve the storage tolerance and stress resistance of the fruit, and has important application value in cultivating storage tolerance and resistance plant varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a mango PUB29 gene and an application thereof in regulating the wax content and components of a plant peel. Background Art

[0002] Mango (Mangiferaindica L.) is an evergreen tropical fruit tree commonly grown in tropical and subtropical regions, including India, China, Thailand, Vietnam, Bangladesh and Malaysia. Mango fruit is rich in vitamins A, C and D, as well as sugar, dietary fiber and some trace elements. Mango is loved by people all over the world for its unique flavor. However, mango fruits fully ripen soon after harvest and are susceptible to adverse environmental factors, such as pathogen infection, which reduces their resistance to storage after harvest. Mangoes are processed into products such as sauces, beverages and dried mangoes to reduce the effects of their intolerance during storage. However, fresh mangoes are the first choice of consumers, so breeding durable varieties and developing good storage methods are still good means.

[0003] During their long evolutionary history, cuticular waxes have formed on the surfaces of roots, stems, leaves, flowers and fruits of plants. It has been determined that the chemical composition of cuticular waxes of fruit crops mainly includes terpenoids, sterols, alkanes, fatty acids, primary and secondary alcohols, ketones, aldehydes and phenols, which constitute the majority of the cuticular waxes. It has been reported that there are differences in wax accumulation in different fruit crops. Previous studies have shown that cuticular waxes can help plants resist ultraviolet radiation and pathogen invasion, reduce non-stomatal water loss, and maintain mechanical strength. Mango is susceptible to anthracnose during development and after harvest, which reduces economic benefits.

[0004] Studying the formation of wax from the perspective of the molecular mechanism of wax synthesis, discovering more new genes that regulate the wax content of 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 storage tolerance and resistance of fruits. Summary of the invention

[0005] One of the purposes of the present invention is to provide a gene PUB29 related to the total wax content and the content of different wax components in mango peel and its application in regulating the total wax content and / or the content of different wax components in plant peel. Further, it can be used to cultivate plant varieties with different total wax content and / or different wax component content.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] On the one hand, the present invention provides a PUB29 protein for regulating the total wax content and the content of different wax components in plant peels, and the amino acid sequence of the protein is shown as SEQ ID NO.2.

[0008] On the other hand, the present invention provides a gene PUB29 for regulating the total wax content and the content of different wax components in plant peels, wherein the gene encodes an amino acid sequence as shown in SEQ ID NO.2.

[0009] On the other hand, the present invention provides a vector for regulating the total wax content and the content of different wax components in plant peels, wherein the vector can overexpress mRNA encoding the amino acid sequence shown in SEQ ID NO.2.

[0010] 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.

[0011] 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 PUB29 gene may also include introns, promoters and various regulatory elements.

[0012] On the other hand, the present invention also provides a primer pair 1 for cloning the above gene. Preferably, the sequence of the forward primer F1 of the primer pair 1 is shown as SEQ ID NO.3, and the sequence of the reverse primer R1 is shown as SEQ ID NO.4.

[0013] On the other hand, the present invention also provides a kit for regulating the total wax content and the content of different wax components in plant peels, wherein the kit comprises the above-mentioned primer pair 1 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 total wax content and / or the content of different wax components in plant peels.

[0015] Preferably, the regulation of the total wax content of the plant peel is to increase the total wax content of the plant peel by overexpressing the aforementioned protein or mRNA.

[0016] Preferably, the regulating the content of different wax components in the plant peel is to increase the content of C16 fatty acids, C17 phenols and / or C27 esters by overexpressing the aforementioned protein or mRNA.

[0017] Optionally, the content of C19 ketones, C19 alkanes and / or C20 alkane compounds can be reduced by overexpressing the aforementioned protein or mRNA.

[0018] Preferably, the above application comprises the following steps when increasing the wax content of the plant peel:

[0019] 1) Amplify the aforementioned gene PUB29;

[0020] 2) connecting the gene PUB29 to an overexpression vector to obtain a recombinant vector;

[0021] 3) transferring the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

[0022] 4) Transforming the recombinant Agrobacterium tumefaciens into plant peels.

[0023] 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 cultivating plant varieties with storable fruits.

[0024] Preferably, the cultivated storability plant variety is a variety with a high wax content in the fruit peel.

[0025] Preferably, the high wax content has increased content of total wax, C16 fatty acids, C17 phenols and / or C27 esters.

[0026] Preferably, the above application comprises the following steps when cultivating fruit storage-resistant plant varieties:

[0027] 1) Amplify the aforementioned gene PUB29;

[0028] 2) connecting the gene PUB29 to an overexpression vector to obtain a recombinant vector;

[0029] 3) transferring the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

[0030] 4) Transforming the recombinant Agrobacterium tumefaciens into plants to achieve stable expression.

[0031] Preferably, the aforementioned plant is a dicotyledonous plant.

[0032] Furthermore, the aforementioned plant is a plant of the Anacardiaceae family.

[0033] Furthermore, the aforementioned plant is a plant of the genus Mango.

[0034] Furthermore, the aforementioned plant is mango.

[0035] Another object of the present invention is to provide a use of mango PUB29 protein and / or a gene encoding mango PUB29 protein and / or a vector encoding mango PUB29 protein in improving the stress resistance of an organism, characterized in that the amino acid sequence of the mango PUB29 protein is shown in SEQ ID NO.2.

[0036] Preferably, the organism is a plant or a bacterium.

[0037] Preferably, the plant is mango and the bacteria is Escherichia coli. The strain of Escherichia coli with improved stress resistance can be used as a positive control in scientific research.

[0038] Preferably, the stress resistance is resistance to heavy metal stress, resistance to salt stress, resistance to oxidative stress, resistance to high temperature stress and / or resistance to low temperature stress.

[0039] Compared with the prior art, the present invention has the following advantages:

[0040] 1) The present invention provides a novel protein capable of regulating the total wax content of plant peels and its encoding gene.

[0041] 2) The present invention provides a new method for regulating the total wax content of plant peels.

[0042] 3) The present invention provides a new method for increasing the total wax content and the wax content of different components.

[0043] 4) The present invention provides a new method for cultivating plant varieties with storage-resistant fruits.

[0044] 5) The present invention provides a new method for improving biological stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0046] Figure 1 Shown are the PUB29 expression levels of PUB29 transiently overexpressing samples and control samples.

[0047] Figure 2 Shown are the total wax content and the content of each component in the PUB29 transient overexpression sample and the control sample.

[0048] Figure 3 Shown are the contents of fatty acid compounds of different carbon chain lengths in PUB29 transient overexpression samples and control samples.

[0049] Figure 4 Shown are the contents of ester compounds with different carbon chain lengths in PUB29 transient overexpression samples and control samples.

[0050] Figure 5 Shown are the contents of phenolic compounds of different carbon chain lengths in PUB29 transient overexpression samples and control samples.

[0051] Figure 6 Shown are the contents of alkane compounds of different carbon chain lengths in the PUB29 transient overexpression sample and the control sample.

[0052] Figure 7 Shown are the contents of ketone compounds of different carbon chain lengths in PUB29 transient overexpression samples and control samples.

[0053] Figure 8 Shown is the prokaryotic expression analysis of PUB29 (the band in the red box is the specifically expressed target protein produced after IPTG induction).

[0054] Fig. 9 The growth of transgenic E. coli on the culture medium is shown. A: OD600 determination under heavy metal copper ion stress; B: OD600 determination under heavy metal zinc ion stress; C: OD600 determination under sodium chloride stress; D: OD600 determination under hydrogen peroxide stress; E: OD600 determination under 20℃ stress; F: OD600 determination under 45℃ stress. DETAILED DESCRIPTION

[0055] 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.

[0056] 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.

[0057] The nucleotide sequence of the mango PUB29 gene in the embodiment is shown in SEQ ID NO.1:

[0058]

[0059] The amino acid sequence of mango PUB29 protein is shown in SEQ ID NO.2:

[0060] MARNELFVTVPSLFRCPISLDVMKSPVSLCTGVTYDRSSIQHWLESGHDTCPATMQILSTKDITPNLTLHRLINLWSRQLTLSEQEIKDLIEKIGSECESEKSCVGCLAKIFEFVRGSEENRRFLSSYGGFLEAIVSVLNRKQVEIVALESVFKVLSLILNENGVKEKINHLIFLGNSNCLASILEVLEKGSLDSRIESI KILNAILLDNESKRRVVETESIFSALFDQLRTSNGDNLNDAVLSILITLSGTRSVKTQLVQFGLVQILSKILSDTSSRIPTIEKAIKLLSITIKCSEGRL AVSEDSRCAAGIVEKIIKVGKTAREEAVAVLWSMCYVYKDGRVTEAVVKSNGVTKVLLVMQSEIEGIVRKMCGDLVKVLGKKNCSSGLESYDTKTTHITPY

[0061] Example

[0062] Example 1 Cloning and prokaryotic expression of mango PUB29 gene and construction of plant transient expression vector

[0063] 1. Cloning of mango PUB29 gene

[0064] The fruit 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 FastPure Plant Total RNA Isolation Kit (purchased from Novozymes, China). After 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 RevertAid First Strand cDNA Synthesis Kit. The obtained first strand cDNA was used for amplification of the PUB29 gene. According to the PUB29 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.

[0065]

[0066] 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 ℃ pre-denaturation for 3 min; 35 amplification cycles, including 95 ℃ denaturation for 30 s, 60 ℃ annealing for 15 s, 72 ℃ extension for 2 min, 72 ℃ extension for 10 min after the cycle was completed, and then kept at 20 ℃ 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).

[0067] 2. Construction of plant overexpression vector of mango PUB29 gene

[0068] The purified PCR product was recovered and connected with the pCAMBIA1300-35S-EGFP vector, and transformed into E. coli DH5α by heat shock method. PCR detection was performed with gene-specific primers, and the positive bacterial solution was sequenced (completed by Qingke Biotechnology Co., Ltd.). The sequencing results showed that the length of the PUB29 target fragment amplified by the present invention was 1203 bp, and its nucleotide sequence was shown in SEQ ID NO.1. Through sequence comparison analysis, it was determined that the sequence was the target gene required by the present invention, and the correct recombinant target vector was named 35S-PUB29-GFP. The recombinant vector 35S-PUB29-GFP and the control empty vector 35S-EGFP were respectively transferred into Agrobacterium GV3101 by heat shock method.

[0069] Example 2 Identification of waxy traits in mango fruit by transient overexpression of mango PUB29 gene

[0070] Wax content and composition are important evaluation traits of plant cuticular wax.

[0071] 1. Transient overexpression injection of mango PUB29 gene

[0072] Take Agrobacterium tumefaciens (containing the recombinant vector of 35S-PUB29-GFP and the empty vector of 35S-GFP control) stored in a -80℃ refrigerator, streak culture on solid LB medium containing 100 mg / L kanamycin and 100 mg / L rifampicin, culture at 28℃ for 2 days, then pick up the single colony on the line with a sterilized toothpick, put it into a 50 mL centrifuge tube, add 25 mL of liquid LB medium containing 100 mg / L kanamycin and 100 mg / L rifampicin, culture at 28℃ in a shaker at 220 rpm for 12 hours, collect the bacteria in a 50 mL centrifuge tube at 5000 rpm for 5 minutes, add 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) and resuspend the bacteria, centrifuge again, add the invasion solution again, adjust the OD value of the bacterial solution to between 0.6-0.8 using a spectrophotometer, and incubate in a 28℃ shaker at 100 r / min in the dark for 4 h. Use a syringe to inject the empty 35S-GFP and 35S-PUB29-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 in each group of experiments. The experiment is repeated 3 times, and the fruits are placed in an incubator at 21℃ for dark culture for 24 h, and then placed in an incubator at 21℃, with weak light intensity and a photoperiod of 16 h light / 8 h dark, and cultured for 2 days.

[0073] 2. Detection of transient overexpression of PUB29 gene expression

[0074] Mango peel tissue samples were collected after injection. Total RNA was extracted, cDNA was synthesized, and qRT-PCR was performed. qRT-PCR was performed using the ChamQ SYBR qPCR Master Mix kit (purchased from Novozymes) and strictly followed the instructions. The 10 μL qRT-PCR reaction system included: 5 μL fluorescent dye, 3.6 μL sterilized ultrapure water, 1 μL cDNA, 0.2 μL forward primer (10 μM), and 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℃ for 10 min; 95℃ for 10s, 60℃ for 10s, 72℃ for 30s, return to the second step for 50 cycles, 72℃ for 10min, and 20℃ for 5min. Repeat 3 times and calculate the average Ct value of each cDNA sample. -ΔΔCt The relative expression level of the PUB29 gene in mango peel after transient overexpression was obtained.

[0075] 3. Detection of wax content and composition of transient overexpression samples

[0076] In a fume hood, the injected mango fruit was completely immersed in chloroform solution for 2 minutes 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 vial after filtering 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 the solution 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 it. The sample was filtered into a 1.5 mL brown gas phase vial 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 the 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; scan range, 50-650 m / z. The temperature program was as follows: 50 °C for 2 minutes, then heated to 170 °C at a rate of 20 °C / min, held for 2 minutes, then 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 increased to 320 °C at a rate of 5 °C / min and held for 20 min. The compounds were identified by comparing the retention index with the retention index information of the nist17 library and the compounds.

[0077] 4. Construction of prokaryotic recombinant vector of mango PUB29 gene

[0078] The first strand cDNA obtained in the first part of Example 1 was used to amplify the PUB29 gene, and the nucleotide sequence of the primer pair was as follows:

[0079]

[0080] The target product was amplified using the PCR reaction system in the first part of Example 1, and the specific target band was recovered. The recovered and purified PCR product was connected to the pET32a recombinant vector, transformed into E. coli BL21 (DE3) 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 is the target gene required by the present invention. The correct recombinant target vector was named pET32a-PUB29. The strains containing pET32a-PUB29 and pET32a were cultured to the same OD600, IPTG was added to a final concentration of 1mM, and the strains containing pET32a-PUB29 and pET32a were measured for heavy metal ions (Cu 2+ , 500μM; Zn 2+ The OD600 values ​​were measured after culturing under stresses of 0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h and 3.5h, respectively.

[0081] Results Figure 1-9 The expression level of PUB29 gene was significantly increased by transient overexpression ( Figure 1 The total wax and fatty acid wax content of transient expression samples were significantly higher than those of control samples ( Figure 2 Further analysis of the carbon chain length of each component revealed that the C16 fatty acid ( Figure 3 )、C27 esters( Figure 4 ) and C17 phenols ( Figure 5 ) were significantly higher than those in the control samples. In addition, the C19 and C20 alkanes ( Figure 6 ) and C19 ketones ( Figure 7 ) content was significantly lower than that of the control sample. Under IPTG induction, the PUB29 gene achieved efficient heterologous expression, and the recombinant protein contained the target protein at position 61kDa (the molecular weight of the PUB29 protein was 44kDa, and the molecular weight of the protein translated from the vector sequence was 17kDa), which was consistent with the expected size ( Figure 8 ). Under various experimental stresses, the growth rate of the strain containing pET32a-PUB29 was higher than that of the strain containing pET32a, indicating that the protein encoded by the PUB29 gene can improve the growth of the strain under heavy metal copper ion, zinc ion, sodium chloride, hydrogen peroxide stress, high temperature and low temperature stress, and also indicates that the protein encoded by the PUB29 gene may improve the tolerance of plants (such as mango, etc.) to adversity ( Fig. 9 ).

[0082] As can be seen from the above examples, the mango PUB29 provided by the present invention can positively regulate the total wax content of the fruit and can control the content of different wax components, and has potential application value in improving the storage tolerance and stress resistance of the fruit and cultivating storage tolerance and resistance plant varieties.

[0083] 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. Use of mango PUB29 protein and / or a gene encoding mango PUB29 protein and / or a vector encoding mango PUB29 protein in regulating the total wax content of mango peel and / or regulating the content of different wax components in mango peel, characterized in that: The application is achieved by overexpressing mango PUB29 protein and / or mRNA encoding mango PUB29 protein, and the amino acid sequence of the mango PUB29 protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The regulation of the total wax content of mango peel is to increase the total wax content and fatty acid content of mango peel by overexpressing mango PUB29 protein and / or mRNA encoding mango PUB29 protein.

3. The use according to claim 1, characterized in that: The method for regulating the content of different wax components in mango peel is to increase the content of C16 fatty acids, C17 phenols and / or C27 esters by overexpressing mango PUB29 protein and / or mRNA encoding mango PUB29 protein.

4. The use according to claim 1, characterized in that: The method for regulating the contents of different wax components in mango peel is to reduce the contents of C19 ketones, C19 alkanes and / or C20 alkanes by overexpressing mango PUB29 protein and / or mRNA encoding mango PUB29 protein.

5. Use of mango PUB29 protein and / or a gene encoding mango PUB29 protein and / or a vector encoding mango PUB29 protein in breeding new mango varieties, characterized in that: The application is achieved by overexpressing mango PUB29 protein and / or mRNA encoding mango PUB29 protein. The new variety is a variety with high wax content in the peel. The amino acid sequence of the mango PUB29 protein is shown in SEQ ID NO.

2.

6. The use according to claim 5, characterized in that: The high wax content refers to increased contents of total wax, C16 fatty acid, C17 phenols and / or C27 esters.

7. Use of mango PUB29 protein and / or a gene encoding mango PUB29 protein and / or a vector encoding mango PUB29 protein in improving stress resistance of an organism, characterized in that: The application is achieved by overexpressing mango PUB29 protein and / or mRNA encoding mango PUB29 protein, the amino acid sequence of the mango PUB29 protein is shown in SEQ ID NO.2, the organism is Escherichia coli, and the stress resistance is resistance to heavy metal copper ion and zinc ion stress, salt stress, hydrogen peroxide stress, high temperature stress and / or low temperature stress.

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