Potato drm3 gene and application thereof in potato preservation

Abstract

The application discloses a potato DRM3 gene and application thereof in potato preservation, wherein a nucleotide sequence of the DRM3 gene is shown as SEQ ID NO.1, and an amino acid sequence encoded by the DRM3 gene is shown as SEQ ID NO.2. The DRM3 gene is connected to an expression vector, and the potato is transformed by using an agrobacterium infection, and the result shows that overexpression of the DRM3 gene can significantly improve the abilities of the potato in inhibiting sprouting and resisting enzymatic browning. Meanwhile, the DRM3 protein is prepared and purified, and it is confirmed that the DRM3 protein can significantly inhibit the enzymatic browning of potato serum. The DRM3 gene has the dual functions of inhibiting sprouting and resisting enzymatic browning, and can provide a theoretical basis for cultivating new potato varieties, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a potato DRM3 gene and application thereof in potato preservation. BACKGROUND

[0002] Potato is the fourth largest new staple food in China, has rich nutritional value, plays an important role in adjusting the dietary structure and human health, and is favored by consumers. However, the potato tuber has a water content of more than 80%, cannot be dried and stored, and is prone to germination, enzymatic browning and other adverse problems during storage and processing, thereby causing mold, rot or accumulation of toxic substances such as solanine, which can harm the health of consumers. The current widely used low-temperature storage method is high in cost, and the use of chemical reagents can easily cause health problems. The current control method has limited effect, and there is an urgent need to invent a new method to inhibit potato germination and enzymatic browning and to improve the potato preservation time. At present, gene editing technology has been widely used in the development of excellent new germplasm of fruits and vegetables. For example, a new potato variety with corresponding functions is obtained by using gene editing technology. CN110438152A discloses a method for promoting potato tuber germination by overexpressing potato StDWF1 gene. Overexpression of StDWF1 gene can promote potato tuber germination, advance the germination time, and thereby improve the yield. This is different from the technical problems to be solved in the present application. CN116262922A discloses application of potato auxin response gene StSAUR30231 in inhibiting enzymatic browning of fresh-cut potato. The StSAUR30231 gene is connected to an expression vector, and the potato is transformed by using agrobacterium infection. The data shows that overexpression of StSAUR30231 gene can significantly improve the anti-browning ability of potato.

[0003] DRM3 (Dormancy-Associated protein 3, dormancy-associated gene 3) belongs to the Dormancy-Associated protein gene family, which is unique to higher plants and highly conserved, and can play a role in plant growth and development, disease resistance and stress resistance. It is often used as a marker gene for meristem and axillary bud dormancy, but there are many members of the dormancy-associated gene family, and not all dormancy-associated genes have the function of inhibiting germination. At present, there is a lack of DRM gene that can be used to regulate potato germination, and there is no research on applying the gene of the family member to solve the problem of potato enzymatic browning. The present application aims to solve the two problems of potato storage and preservation by using the dual function of DRM3 gene, and prolong the preservation time of potato. SUMMARY

[0004] This invention discloses the potato DRM3 gene and its application in potato preservation. By overexpressing the DRM3 gene, two major problems in potato storage and preservation—sprouting and enzymatic browning—are solved. The dual function of the DRM3 gene can provide a theoretical basis for breeding superior new potato varieties and has significant implications for production practice.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A first aspect of the present invention provides the application of the DRM3 gene in potato preservation; said DRM3 gene is a nucleic acid molecule as shown in (a), (b), or (c) below:

[0007] (a) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0008] (b) Nucleic acid molecules that have more than 90% nucleotide sequence identity with (a) and express the same functional protein;

[0009] (c) Nucleic acid molecules other than (a) that encode the amino acid sequence shown in SEQ ID NO.2.

[0010] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA. The cDNA sequence of the DRM3 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2.

[0011] The term "identity" used here refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0012] Furthermore, the potato preservation method involves inhibiting potato sprouting and resisting enzymatic browning.

[0013] A second aspect of the present invention provides the application of a protein encoded by the DRM3 gene in potato preservation; said protein is any one of the following i) or ii):

[0014] i) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing;

[0015] ii) Fusion proteins obtained by attaching tags to the N-terminus and / or C-terminus of the protein defined in i).

[0016] The proteins described in i) and ii) can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0017] To facilitate the purification of the protein in i), a tag may be attached to the amino or carboxyl terminus of the protein in i). The tagged protein includes, but is not limited to, the tagged proteins listed in Table 1.

[0018] Table 1. Sequences of the tag proteins

[0019] Tag Residue Sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0020] A third aspect of the present invention provides the application of a recombinant expression vector containing the above-mentioned DRM3 gene, a transgenic cell line, or a genetically engineered bacterium in potato preservation.

[0021] The recombinant expression vector can be constructed using existing plant expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, or other derived plant expression vectors. When constructing the recombinant expression vector using the gene, any enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CaMV) 35S promoter, the ubiquitin gene Ubiquitin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters. Furthermore, when constructing the recombinant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used.

[0022] In a fourth aspect, the present invention provides the use of the DRM3 gene, the protein encoded by the DRM3 gene, a recombinant expression vector containing the DRM3 gene, a transgenic cell line or genetically engineered bacteria in any of the following (1) or (2) or (3):

[0023] (1) Plant breeding;

[0024] (2) Regulate the plant's ability to inhibit germination;

[0025] (3) Regulate the plant’s ability to resist enzymatic browning.

[0026] In the above applications, the plant is preferably a potato.

[0027] A fifth aspect of the present invention provides a method for preserving potatoes, comprising: a method for overexpressing the DRM3 gene in potatoes.

[0028] In the methods described above, DRM3 gene overexpression in potatoes can be achieved through exogenous transfer of the DRM3 gene; or by upregulating the expression of the DRM3 gene in the potato genome. Methods for upregulating DRM3 gene expression in the potato genome include: introducing DNA fragments that can activate or enhance the transcriptional, translational, or protein activity of the DRM3 gene; or controlling the synthesis of specific small RNA molecules to upregulate the accumulation of DRM3 gene mRNA.

[0029] The specific small RNA molecules include: microRNA (miRNA), small interfering RNA (siRNA), or artificial microRNA (amiRNA).

[0030] In a sixth aspect, the present invention provides a method for cultivating potatoes with enhanced ability to inhibit sprouting and resist enzymatic browning, comprising the following steps: transferring the above-mentioned DRM3 gene into a potato starting plant to overexpress the DRM3 gene, thereby obtaining a transgenic potato plant; wherein the transgenic potato plant has a higher ability to inhibit sprouting and resist enzymatic browning than the potato starting plant.

[0031] Among the above-mentioned cultivation methods, the methods for transferring the DRM3 gene into potato starting plants include: Agrobacterium-mediated transformation, polyethylene glycol transformation, or gene gun bombardment.

[0032] The beneficial effects of this invention are:

[0033] This invention, through systematic research, cloned the potato dormancy-related response gene DRM3 and transferred it into potatoes using Agrobacterium-mediated transformation. Analysis showed that overexpression of the DRM3 gene significantly enhanced the ability of potato tubers to inhibit sprouting and resist enzymatic browning. This invention is the first to identify the dual function of the DRM3 gene in inhibiting potato sprouting and resisting enzymatic browning, providing a research foundation for cultivating superior new potato germplasm. Attached Figure Description

[0034] Figure 1 To determine the expression level of the DRM3 gene in potatoes overexpressing the DRM3 gene; WT is wild-type Chuanyu 10 potato, and OE16 and OE19 are Chuanyu 10 potatoes with different DRM3 gene overexpressions.

[0035] Figure 2 To identify the sprouting status of potatoes overexpressing the DRM3 gene; where A is the determination of the sprouting rate of potatoes overexpressing the DRM3 gene, and B is the identification of the sprouting phenotype of potatoes overexpressing the DRM3 gene; where WT is wild-type Chuanyu 10 potato, and OE16 and OE19 are Chuanyu 10 potatoes with different DRM3 gene overexpressions.

[0036] Figure 3 To identify the anti-enzymatic browning effect of potatoes overexpressing the DRM3 gene; where A represents the effect of DRM3 gene overexpression on the L* value in potatoes, and B represents the changes in browning phenotype of potato chips overexpressing the DRM3 gene.

[0037] Figure 4 The expression of the DRM3-MBP fusion protein was induced by IPTG; where M: 250kD protein marker; 1: IPTG-induced expression of BL21 Escherichia coli (DRM3-MBP) protein; 2: blank expression of BL21 Escherichia coli (DRM3-MBP) protein; 3: IPTG-induced expression of BL21 Escherichia coli (MBP) protein; 4: blank expression of BL21 Escherichia coli (MBP) protein.

[0038] Figure 5 This section shows the purification status of the DRM3-MBP fusion protein; where M: 250kD protein marker; 1: MBP protein; 2: DRM3-MBP fusion protein;

[0039] Figure 6 The effects of DRM3 protein on enzymatic browning of potato slurry are shown in Figure A; where A represents the effect of DRM3 protein on browning of potato slurry; and B represents the effect of DRM3 protein on the browning phenotype of potato slurry. MBP is the MBP protein, and DRM3-MBP is the DRM3-MBP fusion protein. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1: Discovery of the potato preservation gene DRM3

[0042] This invention selected wild-type Sichuan potato variety 10 as experimental material. Phenotypic and intact potatoes were selected and treated with the sprouting hormone brassinolide at 500 nmol / L. After drying at room temperature, the potatoes were stored at 20℃ and 90% humidity. Potato eye tissue samples were taken at 0h and 24h (5mm × 5mm circular holes) and flash-frozen in liquid nitrogen. Subsequently, total RNA was extracted from the samples using a plant polysaccharide and polyphenol RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.) and sent to Hangzhou Jingjie Biotechnology Co., Ltd. for phosphorylated protein sequencing. Protein quantification results showed that after 24h of treatment with the sprouting hormone, the expression of 111 proteins was upregulated and the expression of 234 proteins was downregulated, promoting an increase in the phosphorylation level of 240 proteins and a decrease in the phosphorylation level of 290 proteins. Importantly, treatment with sprouting hormone downregulated the expression level of DRM3 protein by 63.3% and increased phosphorylation at eight sites of the protein by 40%–238%. The amino acid sequence of this protein is shown in SEQ ID NO.2. We hypothesize that the DRM3 gene may be significantly related to potato preservation.

[0043] Example 2: Cloning of the DRM3 gene sequence

[0044] (1) Primer design

[0045] Based on the gene number (102581405) of DRM3 in phosphorylated proteomics, the cDNA sequence of the DRM3 gene was retrieved from NCBI (https: / / www.ncbi.nlm.nih.gov / gene / ). DRM3 amplification primers were designed online using Primer 3. The primer sequences are as follows:

[0046] DRM3-F1: 5'-GAGAACACGGGGGACTCTAGACAAGCTGCCACTGAACATTC-3'

[0047] (SEQ ID NO.3);

[0048] DRM3-R1: 5'-CATAAGGGACTGACCACCCGGGGCACTTCTCATGATGCTTAG-3' (SEQ ID NO. 4).

[0049] (2) RNA extraction

[0050] Wild-type Sichuan potato tuber samples were ground into powder in liquid nitrogen. Total RNA was extracted from the samples using a plant polysaccharide and polyphenol RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.). The specific method is as follows:

[0051] 1) Weigh 50mg of potato tuber powder ground in liquid nitrogen, add 500μL of Buffer PRL preheated at 65℃ (add 5% β-mercaptoethanol or 5% 2M DTT solution before use), and immediately vortex vigorously for 30-60s to fully break it down. Reducing viscosity helps to increase yield.

[0052] 2) Incubate the lysate in a 65°C water bath for 5 min, inverting it 1-2 times during the process to aid lysis, and centrifuge at 12,000 rpm (13,400 × g) for 10 min.

[0053] 3) Transfer the supernatant to a new 1.5ml RNase-free centrifuge tube, add 0.5 times the volume of the supernatant in anhydrous ethanol, and immediately mix by pipetting.

[0054] 4) Transfer the above mixture to FastPure gDNA-Filter Column II (FastPure gDNA-Filter Column II has been placed in the collection tube), centrifuge at 12,000 rpm (13,400×g) for 2 min, and discard the filtrate;

[0055] 5) Place FastPure gDNA-Filter ColumnII into a new Collection Tube 2ml (as provided in the kit), add 500μl Buffer PRLPlus, centrifuge at 12,000rpm (13,400×g) for 30s, and collect the filtrate;

[0056] 6) Add 0.5 times the volume of anhydrous ethanol to the filtrate and immediately mix by blowing and stirring.

[0057] 7) Transfer the above mixture to FastPure RNA Column IV (FastPure RNA Column IV has been placed in the collection tube), centrifuge at 12,000 rpm (13,400×g) for 2 min, and discard the filtrate;

[0058] 8) Add 700 μl of Buffer PRW1 to FastPure RNA Column IV, incubate at room temperature for 1 min, centrifuge at 12,000 rpm (13,400 × g) for 30 s, and discard the filtrate;

[0059] 9) Add 500 μl of Buffer PRW2 to FastPure RNA Column IV (please check that 48 ml of anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400 × g) for 30 s, and discard the filtrate;

[0060] 10) Repeat step 9;

[0061] 11) Place the FastPure RNA Column IV adsorption column back into the collection tube, centrifuge at 12,000 rpm (13,400×g) for 2 min to remove any residual Buffer PRW2 in the FastPure RNA Column IV;

[0062] 12) Transfer FastPure RNA Column IV to a new RNase-free Collection Tube 1.5ml centrifuge tube, add 30-100μL of RNase-free ddH2O to the center of the adsorption column membrane, incubate at room temperature for 2min, centrifuge at 12,000rpm (13,400×g) for 1min, and store the resulting RNA solution at -70℃ to prevent degradation.

[0063] (3) cDNA synthesis

[0064] The extracted total RNA was reverse transcribed using the following reaction mixture: 3 μL RNA, 1 μL Oligo d(T)18 Primer, 8 μL RNAse-free, incubated at 65°C for 5 min. After the first step, 4 μL 5×RT, 1 μL Ribolock RNase Inhibitor, 2 μL 10 mM dNTP Mix, and 1 μL RT were added, and the mixture was incubated at 42°C for 60 min, followed by incubation at 25°C for 5 min. Finally, the obtained cDNA was diluted 10-fold and stored at -20°C. The reaction was performed according to the RNA reverse transcription kit method of Thermo Fisher Scientific.

[0065] (4) Amplification and sequence analysis of the target gene DRM3

[0066] Using the above cDNA as a template and DRM3-F1 and DRM3-R1 as primers, the amplification system was as follows: 5 μL cDNA, 0.2 μL each primer. GoldPfu PCR SuperMix 25 μL, ultrapure water added to 50 μL. Reaction conditions: 94℃, 5 min; 94℃, 20 s; 52℃, 20 s; 72℃, 40 s, 35 cycles; 72℃, 10 min.

[0067] The PCR amplification products were detected by 1% agarose gel electrophoresis, and the target band was then recovered by gel excision using the OMEGA Gel Extraction Kit. The recovered PCR products were sent to Sangon Biotech for base sequencing. The DRM3 gene sequence is shown in SEQ ID NO.1.

[0068] Example 3: Construction of the plant expression vector DRM3-pBI121

[0069] 50 μL of plant expression vector pBI121 was linearized using 1 μL each of restriction endonucleases SacI and SalI. Subsequently, the PCR product recovered from the gel was ligated to the enzyme-digested linearized vector pBI121 using the homologous recombination enzymes from BOMIDE Biotechnology's BM Seamless Cloning Kit. After mixing, the ligation was performed at 50°C for 15 min and then cooled on ice. Homologous recombination system: 100 ng PCR product, 50 ng linearized vector pBI121, 5 μL 2×Seamless Cloning Mix, and ultrapure water to a final volume of 10 μL.

[0070] 5 μL of the ligation product was transferred and transformed into 100 mL of *E. coli* DH5α. After the strain grew, single clones were picked and cultured overnight at 37°C and 200 rpm. PCR identification was then performed using primers DRM3-F1 and DRM3-R1. Positive clones were sent to Sangon Biotech for sequencing. Plasmids were extracted from correctly sequenced strains using the OMEGA plasmid extraction kit. The recombinant plasmid was then heat-shocked and transformed into *Agrobacterium* GV3101 electroporation competent cells (Bomaide Biotechnology). Single clones were picked and identified by PCR using primers DRM3-F1 and DRM3-R1, ultimately yielding *Agrobacterium* GV3101 successfully transformed with the DRM3 gene.

[0071] Example 4: Genetic transformation of potatoes overexpressing the DRM3 gene

[0072] (1) Agrobacterium culture and preparation of transgenic culture medium

[0073] Bacterial culture: After sterilizing the GV3101 Agrobacterium containing the plant expression vector DRM3-pBI121 under ultraviolet light on a clean bench, single colonies were picked up with a pipette and inoculated into 5ml YEB liquid medium. The culture was then placed at 28℃ and 200r / min for 15-24h. After that, the culture was taken out and diluted 1:100 by volume. The culture was then added to fresh liquid YEB medium and cultured under the same conditions for 12-15h. The OD value of the culture was measured. When the OD600 = 0.4-0.6, the culture was taken out for use.

[0074] Agrobacterium infection solution (100 mL): Liquid MS + 3% sucrose, pH adjusted to 5.9-6.0, sterilized at 121℃ for 20 min, cooled and then added to a final concentration of 100 μmol / L acetylsylgenone for later use.

[0075] Stem segment regeneration medium: The basal medium is MS medium with 0.6% agar added. Antibiotics and hormones are added according to Table 2 to prepare four stages of medium, with the pH adjusted to 5.8-6.0. All mediums must be autoclaved at 121℃ for 20 minutes. After the medium cools to 60℃, filter-sterilized hormones or antibiotics are added.

[0076] Table 2. Culture medium composition (mg / L)

[0077] Culture stage 6-BA TDZ 2,4-D GA3 Kan Cef Car Co-culture 0.5 1.5 1.0 0 0 0 0 Callus selection culture 0.5 1.5 0.1 0 50 100 250 Bud induction selection culture 2.0 0.25 0.03 0.1 50 100 250 Rooting culture 0 0 0 0 50 100 250

[0078] Note: 6-BA: 6-benzylaminopurine, TDZ: thiazolinone, 2,4-D: 2,4-dichlorophenoxyacetic acid, GA3: gibberellin, Kan: kanamycin, Cef: cephalosporin, Car: carbenicillin.

[0079] (2) Agrobacterium infection

[0080] Centrifuge 50 mL of the obtained bacterial solution at 5000 rpm for 15 min, remove the supernatant, and add 10 mL of the obtained infection solution. Vortex the mixture to obtain the bacterial solution for subsequent infection. Cut 1 cm long stem segments from vigorous wild-type Sichuan potato tissue culture seedlings, ensuring no axillary buds are present. Transfer the cut stem segments to the obtained bacterial solution and incubate at room temperature for 5-8 min. During incubation, continuously agitate the bacterial solution containing the stem segments to ensure thorough contact between the stem segments and the Agrobacterium tumefaciens containing the expression vector. Use sterile tweezers to transfer the infected stem segments to sterile filter paper until the moisture is absorbed.

[0081] (3) Co-cultivation

[0082] After the stem segments were dried, they were transferred to a co-culture medium. Twenty stem segments were placed in one culture dish and co-cultured at 24°C in the dark for 2-3 days.

[0083] (4) Bud induction screening

[0084] Remove the infected stem segments from the culture dish after co-culture. Select stem segments where bacteria begin to grow from the bottom edge and wash them 3-4 times in sterile water containing 100 mg / L Cef. After blotting with sterile filter paper, transfer them to the bud induction selection medium with tweezers. Place 20 stem segments in each culture dish and culture them at 22℃, 4000 lx light intensity, 16h light / 8h dark conditions. Replace the bud selection medium with fresh medium every 7 days and culture for 30 days.

[0085] (5) Callus regeneration

[0086] The stem segments that were successfully infected and grew callus were transferred to callus selection medium for culture, where the upper part differentiated and sprouted.

[0087] (6) Rooting culture

[0088] When the bud reaches 1-2 cm in length, cut it off and inoculate it into rooting medium. Cover the bottle with the cap, seal it with sealing film, and place it in a culture environment with a light intensity of 4000 lx, a light duration of 16 h / d, and a temperature of 22℃ for 15 days.

[0089] (7) Cultivation of potato plantlets overexpressing the DRM3 gene

[0090] The DRM3 gene overexpressing potato test-tube seedlings obtained above were transplanted into peat soil and cultured in a tissue culture room at a temperature of 22±2℃, humidity of 70%, light intensity of 4000 lux, and light duration of 16h / d for 4 months. The potatoes were harvested when the leaves turned yellow and withered by half.

[0091] Example 5: Identification of DRM3 gene expression levels in potato tubers overexpressing the DRM3 gene

[0092] Potato tubers with uniform size, free from mold, sprouting, greening, and mechanical damage were selected. Wild-type Chuanyu 10 potato tubers were used as the control group. The expression level of DRM3 gene in the DRM3 gene-overexpressing potatoes was identified.

[0093] (1) RNA extraction

[0094] Potato tuber samples (WT) from potatoes overexpressing the DRM3 gene and wild-type Chuanyu 10 were ground into powder in liquid nitrogen. Total RNA was extracted from the samples using a plant polysaccharide and polyphenol RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.). The specific method is as follows:

[0095] 1) Weigh out 50 mg of DRM3 gene overexpressing potato powder and wild-type Chuanyu 10 potato tuber powder ground in liquid nitrogen, and perform the following steps respectively;

[0096] 2) Add 500 μL of preheated Buffer PRL at 65°C (add 5% β-mercaptoethanol or 5% 2MDTT before use), and immediately vortex vigorously for 30-60 seconds to fully decompose it, reduce viscosity and help increase yield;

[0097] 3) Incubate the lysate in a 65°C water bath for 5 min, inverting it 1-2 times during the process to aid lysis, and centrifuge at 12,000 rpm (13,400 × g) for 10 min;

[0098] 4) Transfer the supernatant to a new 1.5ml RNase-free centrifuge tube, add 0.5 times the volume of the supernatant in anhydrous ethanol, and immediately mix by pipetting.

[0099] 5) Transfer the above mixture to FastPure gDNA-Filter Column II (FastPure gDNA-Filter Column II has been placed in the collection tube), centrifuge at 12,000 rpm (13,400×g) for 2 min, and discard the filtrate;

[0100] 6) Place FastPure gDNA-Filter ColumnII into a new Collection Tube 2ml (as provided in the kit), add 500μl Buffer PRLPlus, centrifuge at 12,000rpm (13,400×g) for 30s, and collect the filtrate;

[0101] 7) Add 0.5 times the volume of anhydrous ethanol to the filtrate and immediately mix by blowing and stirring.

[0102] 8) Transfer the above mixture to FastPure RNA Column IV (FastPure RNA Column IV has been placed in the collection tube), centrifuge at 12,000 rpm (13,400×g) for 2 min, and discard the filtrate;

[0103] 9) Add 700 μl of Buffer PRW1 to FastPure RNA Column IV, incubate at room temperature for 1 min, centrifuge at 12,000 rpm (13,400×g) for 30 s, and discard the filtrate;

[0104] 10) Add 500 μl of Buffer PRW2 to FastPure RNA Column IV (please check that 48 ml of anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400 × g) for 30 s, and discard the filtrate;

[0105] 11) Repeat step 10;

[0106] 12) Place the FastPure RNA Column IV adsorption column back into the collection tube, centrifuge at 12,000 rpm (13,400×g) for 2 min to remove any residual Buffer PRW2 in the FastPure RNA Column IV;

[0107] 13) Transfer FastPure RNA Column IV to a new RNase-free Collection Tube 1.5ml centrifuge tube, add 30-100μl of RNase-free ddH2O to the center of the adsorption column membrane, incubate at room temperature for 2 min, centrifuge at 12,000 rpm (13,400×g) for 1 min, and store the obtained DRM3 gene overexpressing potato and wild-type Chuanyu 10 potato RNA solutions at -70℃ to prevent degradation.

[0108] (2) cDNA synthesis

[0109] Total RNA extracted from potatoes overexpressing the DRM3 gene and from wild-type Sichuan potato variety 10 was reverse transcribed. The reverse transcription system consisted of 3 μL RNA, 1 μL Oligo d(T)18 Primer, and 8 μL RNase-free, incubated at 65°C for 5 min. After the first step, 4 μL of 5×RT, 1 μL of Ribolock RNase Inhibitor, 2 μL of 10 mM dNTP Mix, and 1 μL of RT were added, and the mixture was incubated at 42°C for 60 min followed by 25°C for 5 min. Finally, the resulting cDNA was diluted 10-fold and stored at -20°C. The reaction was performed according to the RNA reverse transcription kit from Thermo Fisher Scientific.

[0110] (3) Quantitative fluorescence analysis

[0111] Using wild-type potatoes as a control, the expression level of the DRM3 gene in potatoes overexpressing the DRM3 gene was analyzed by qRT-PCR. The reaction system was a 20 μL system, prepared as follows: 10 μL of 2×Ssofast Eva Green, 2.5 μL of cDNA, 0.4 μL of each primer, and ddH2O to a final volume of 20 μL. The qRT-PCR conditions were as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 58℃ annealing and extension for 5 s, 39 cycles; 95℃ denaturation for 10 s. Primer sequences are as follows:

[0112] DRM3-F2: 5'-TGGTCCCAGCCCTGATAAAG-3' (SEQ ID NO.5);

[0113] DRM3-R2: 5'-GAAATGTTTGTCGGCGTCAC-3' (SEQ ID NO. 6).

[0114] Quantitative fluorescence assay revealed that, for example Figure 1As shown, the DRM3 gene expression level in potatoes OE16 and OE19 overexpressing the DRM3 gene was significantly higher than that in wild type. Therefore, two potato materials OE16 and OE19 overexpressing the DRM3 gene were finally obtained.

[0115] Example 6: Identification of the effect of DRM3 gene overexpression on potato tuber sprouting

[0116] Potatoes with DRM3 gene overexpression from groups OE16 and OE19 that are uniform in size, free from mold, sprouting, greening, and mechanical damage were selected. Wild-type Chuanyu 10 tubers were used as the control group (WT). The tubers were placed in a 21.5cm×15.5cm×8cm container with 20 tubers per container. The container was then placed in a dark room at 20±1℃ and 90% humidity. Every 10 days, the morphology of tuber sprouting (at least one sprout with a length ≥2mm on each tuber) was observed. The sprouting status of tubers at 50d, 60d, 70d, 80d, 90d, and 100d was recorded and photographed until all tubers of a certain type had sprouted, excluding bad or rotten tubers.

[0117] like Figure 2 As shown in Figure A, the germination rate of potatoes gradually increased over time. The germination rate of potatoes overexpressing the DRM3 gene in groups OE16 and OE19 was lower than that in the control group at all time points, indicating that overexpression of the DRM3 gene can significantly inhibit potato germination and effectively extend shelf life. Figure 2 B shows the sprouting phenotype results of potatoes in different treatment groups after 80 days of storage. It can be seen that the number of sprouts in the control group was significantly higher than that in the group overexpressing the DRM3 gene.

[0118] Example 7: Identification of the enzymatic browning effect of DRM3 gene overexpression in potatoes

[0119] Select equal number of wild-type Chuanyu 10 potato tubers and OE19 potato tubers overexpressing the DRM3 gene (OE-DRM3) that are uniform in size, free from mold, sprouting, greening, and mechanical damage. Rinse off surface dirt, wash three times with deionized water, then soak in a 200 μL / L sodium hypochlorite solution for 5 minutes for disinfection. Pat dry, remove potato skin, and slice potatoes into uniform 1 cm thick slices using a slicer. Rinse potatoes with deionized water to remove surface dirt. Impurities were removed, and the potatoes were then placed in a preservation box at 20℃ and 90% humidity. The L* value (lightness) of the fresh-cut potato surface was measured at 1, 2, 3, 4, and 5 days using a colorimeter (with a detection aperture of 1.00 mm, VIS measurement mode selected, and the colorimeter calibrated with a light microscope and a standard white plate (L0 = 89.94, a0 = 0.3, b0 = 2.0) to reflect the degree of browning on the surface. The surface color of the fresh-cut potato was measured at 0 and 5 days using an UltraScanVIS benchtop colorimeter.

[0120] like Figure 3 As shown, the L* values ​​of both wild-type and OE-DRM3 potato chips decreased with storage time. However, compared with wild-type, overexpression of the DRM3 gene significantly inhibited the rapid decrease in L* value, with the greatest difference in L* values ​​observed at 5 days. Furthermore, the significant inhibitory effect of DRM3 gene overexpression on browning on the surface of potato chips was clearly evident, indicating that DRM3 gene overexpression endows potatoes with a higher resistance to enzymatic browning.

[0121] Example 8: Effect of DRM3 protein on enzymatic browning of potato slurry

[0122] (1) Construction of DRM3-MBP fusion protein expression vector

[0123] A fusion protein expression vector for DRM3-MBP (maltose-binding protein) was constructed: the pMAL-c2X (MBP tag) empty vector was double-digested with restriction endonucleases EcoR1 and BamH1 (Beijing TransGen Biotech). The reaction system was as follows: 35 μL pMAL-c2X vector, 2 μL each of restriction endonucleases, 5 μL 10× Buffer, 50 μL ddH2O, and incubated at 37℃ for 3 h. The digested products were recovered using a gel extraction kit from Sangon Biotech Co., Ltd.

[0124] Using the cDNA obtained in Example 2 as a template, and DRM3-F3 and DRM3-R3 as primers, the amplification system was as follows: 5 μL cDNA, 0.2 μL each primer. GoldPfu PCR SuperMix 25 μL, ultrapure water to 50 μL. Reaction conditions: 94℃, 5 min; 94℃, 20 s; 52℃, 20 s; 72℃, 40 s, 35 cycles; 72℃, 10 min. Primer sequences are as follows:

[0125] DRM3-F3: 5'-TAATTCGAGCTCCAGCGAATTCATGGTGTTGATTGATAAACT-3' (SEQ IDNO.7);

[0126] DRM3-R3: 5'-GCTTGCGGGTACCCAGGGATCC GCACTTCTCATGATGCTTAG-3' (SEQ ID NO. 8).

[0127] The PCR amplification products were detected by 1% agarose gel electrophoresis, and the target gene was then recovered by gel excision using the OMEGA Gel Extraction Kit.

[0128] The ligation reaction was performed using homologous recombination. The target gene and the vector were ligated using the ClonExpress II OneStep Cloning Kit (Novizan Biotechnology). The reaction system was as follows: 4 μL of MBP empty vector, 3 μL of target gene, 2 μL of 5×CEⅡBuffer, and 1 μL of ExnaseⅡ. The ligation was then carried out in a water bath at 37°C for 30 min to obtain the DRM3-MBP fusion protein expression vector.

[0129] (2) Plasmid transformation of Escherichia coli

[0130] The DRM3-MBP fusion protein expression vector was transformed into E. coli DH5α using the following steps:

[0131] 1) Using a pipette, gently add 10 μL of the above ligation product (DRM3-MBP fusion protein expression vector) to 100 μL of LDH5α E. coli competent cells, gently mix with your fingertip, and incubate on ice for 25 min.

[0132] 2) Then, the EP tubes were placed in a 42°C water bath for 90 seconds for heat shock. After heat shock, they were quickly placed on ice and allowed to stand for 2 minutes. After standing, 0.8 mL of LB liquid medium was added and the tubes were grown in a shaker at 37°C for 60 minutes.

[0133] 3) Centrifuge the incubated competent cells at 5000 rpm for 2 min, discard the supernatant, add 20 μL of sterile water, and mix well by pipetting.

[0134] 4) Pipette the above mixture onto LB solid medium and spread it evenly using a spreader. After the LB solid medium has dried, invert the petri dish and place it in a 37°C incubator overnight.

[0135] Results identification: Single colonies were identified by PCR using the following primers: DRM3-F2: 5'-TGGTCCCAGCCCTGATAAAG-3', DRM3-R2: 5'-GAAATGTTTGTCGGCGTCAC-3'. Single colonies with the correct PCR bands were transferred to 5 mL of LB liquid medium and cultured at 37°C. The resulting bacterial culture was sent to Sangon Biotech for sequencing, and the bacterial strains with the correct sequencing results and the recombinant plasmid (DRM3-MBP) were preserved.

[0136] (3) Plasmid transformation of Agrobacterium

[0137] 1) Take 2 μL of the recombinant plasmid containing DRM3-MBP obtained above and gently add it to 100 μL of BL21 Escherichia coli competent cells. Gently mix the EP tube with your fingertip and place it on ice for 25 min.

[0138] 2) Then, the EP tubes were placed in a 42℃ water bath for 90s for heat shock. After heat shock, they were quickly placed on ice and allowed to stand for 2min. After standing, 0.8mL of liquid LB medium was added and the tubes were grown in a shaker at 37℃ for 60min.

[0139] 3) Centrifuge the incubated competent cells at 5000 rpm for 2 min, discard the supernatant, add 20 μL of sterile water, and mix well by pipetting.

[0140] 4) Pipette the above mixture onto LB solid medium and spread it evenly using a spreader. After the LB solid medium has dried, invert the petri dish and place it in a 37°C incubator overnight.

[0141] 5) Subsequently, pick a single colony and incubate it in 5 mL of LB liquid medium at 37°C and 200 rpm for 12 h.

[0142] (4) Screening of Escherichia coli expressing DRM3-MBP

[0143] The obtained 5 mL LB liquid medium was cultured by shaking at 37℃ and 200 rpm until the absorbance OD600 of the bacterial culture reached 0.4-0.5. The culture was then induced with 0.1 mmol / L IPTG (isopropyl-β-D-thiogalactopyranoside) at 28℃ for 6 h. DRM3-MBP-expressing *E. coli* (without IPTG induction), empty vector *E. coli* (MBP+IPTG), and empty vector *E. coli* (MBP) were used as controls. Polyacrylamide gel electrophoresis was used to screen *E. coli* expressing DRM3-MBP.

[0144] 1) After induction, collect the four bacterial cultures by centrifugation at 4℃ and 5000r / min for 10min, and suspend the precipitate in 3mL of 0.1% phosphate buffer; the precipitate is then subjected to ultrasonic disruption for a total time of 5min, including a working time of 1.5s and an interval of 6.5s, and the process is repeated.

[0145] 2) After centrifuging the treated sample at 4℃ and 10000r / min for 10min, aspirate the supernatant and suspend the precipitate in 3mL of 0.1% PBS;

[0146] 3) Collect the supernatant, take 32 μL of protein sample, add 8 μL of 5× protein loading buffer (Shanghai Beyotime Biotechnology Co., Ltd.), vortex to mix, heat in a 100℃ water bath for 10 min to denature the protein, cool to room temperature, and then perform 10% SDS-PAGE electrophoresis (SDS-PAGE gel preparation is according to Bio-rad's method, prepare 12% separating gel and 4% stacking gel, then stain for 30 min and destain for more than 3 h).

[0147] The results show that, Figure 4 As shown, regardless of whether IPTG induction is used, E. coli containing the DRM3-MBP plasmid and E. coli containing the empty vector can both express the corresponding proteins. However, IPTG can enhance the expression of both proteins. The DRM3-MBP and MBP proteins are 52.5kD and 40kD in size, respectively. Therefore, DRM3-MBP fusion protein expression can be induced by BL21 E. coli.

[0148] (5) Effect of DRM3 protein on enzymatic browning of potato slurry

[0149] 1) Preparation of DRM3-MBP fusion protein

[0150] The BL21(DRM3-MBP) Escherichia coli culture induced by 0.1 mmol / L IPTG obtained above was disrupted by low-temperature sonication and purified using MBP-coupled magnetic beads. Using IPTG-induced empty vector Escherichia coli culture (MBP+IPTG) as a control, 50 mL of IPTG-induced BL21(DRM3-MBP) Escherichia coli culture and 50 mL of empty vector Escherichia coli culture (MBP+IPTG) were respectively placed into 50 mL centrifuge tubes, and the following operations were performed:

[0151] ① After centrifuging the above bacterial solution at 5000 rpm for 15 min, discard the supernatant to obtain bacterial cell precipitate;

[0152] ② Add 40 mL of MBP protein lysis buffer to suspend the precipitate, then add 20 mg of lysozyme, PMSFP (phenylmethylsulfonyl fluoride) and β-mercaptoethanol in sequence to a concentration of 0.4 mmol / mL. Mix thoroughly and place on ice for 30 min.

[0153] ③ The bacterial culture after the ice bath was ultrasonically disrupted. The parameters were set to 85% power, 9 seconds of disruption, 5 seconds of pause, and repeated for 25 minutes. When disrupting the protein, it is best to place the centrifuge tube in an ice-water mixture to maintain a low temperature environment and prevent protein denaturation.

[0154] ④ Add 4g Triton X-100 during the last 5 minutes of disruption. After disruption, centrifuge at 4℃ and 10000r / min for 15 minutes. Filter the centrifuged cells through a filter membrane to maintain the purity of the liquid and avoid excessive protein contamination.

[0155] ⑤ Simultaneously wash the corresponding Beads with MBP washing buffer (Beijing Solarbio Science & Technology Co., Ltd.), and then add 200 μL MBP-specific Beads after washing to the supernatant after filtration in step ④ for protein Bead incubation and binding.

[0156] ⑥ Rotate on a rotary instrument at 4℃ for 2-3 hours, then centrifuge at 4℃ and 2000 rpm for 2 minutes;

[0157] ⑦ Take the supernatant after centrifugation, add 10 mL of MBP washing buffer to resuspend the protein beads, and then centrifuge at 4℃ and 4000 rpm. Repeat this step 5-6 times to ensure that the protein beads are free from contamination by other proteins. Collect the protein beads.

[0158] Table 3 MBP lysis and washing buffer ratios

[0159] Component Concentration Tris-HCl (pH 7.4) 20 mM NaCl (sodium chloride) 200 mM EDTA (ethylenediaminetetraacetic acid) 1 mM

[0160] ⑧ Add 300 μL MBP elution buffer to elute the protein bound to the beads, and obtain purified MBP protein and DRM3-MBP fusion protein.

[0161] Table 4 MBP Elution Buffer Ratio

[0162] Component Concentration Tris-HCl (pH 7.4) 20 mM NaCl (sodium chloride) 200 mM EDTA (ethylenediaminetetraacetic acid) 1 mM Maltose 10 mM

[0163] 2) Purification and identification of DRM3-MBP protein

[0164] The purification status of the obtained DRM3-MBP fusion protein and MBP protein was identified by SDS-PAGE electrophoresis, such as... Figure 5 As shown, protein purification was performed using MBP-specific magnetic beads, yielding a 52.5 kD DRM3-MBP fusion protein and a 40 kD MBP protein, respectively.

[0165] 3) The effect of DRM3 protein on enzymatic browning of potato slurry

[0166] Wild-type Chuanyu 10 potato tuber powder was prepared by grinding in liquid nitrogen. 200 μL of DRM3-MBP fusion protein solution was transferred to 5 mL of potato slurry (containing 1 g of potato tuber powder, diluted to 5 mL with sterile water). The absorbance of the slurry at 410 nm wavelength was dynamically measured by ultraviolet spectrophotometer at 0 h, 0.5 h, 1 h, 2 h and 3 h of reaction. 200 μL of purified MBP protein solution was used as a control.

[0167] Enzymatic browning involves the oxidation and polymerization of phenolic substances into melanin. Melanin has a good absorption peak at 410 nm, so the melanin content can be determined by absorbance, and thus the degree of browning. Figure 6 It is known that DRM3-MBP can significantly inhibit the enzymatic browning of potato slurry compared with MBP. In summary, the present invention prepared and purified DRM3 protein and achieved good results in inhibiting the browning of potato slurry.

Claims

1. The application of the DRM3 gene in potato preservation, characterized by: The preservation method is to inhibit potato sprouting and resist enzymatic browning; the sequence of the DRM3 gene is shown in SEQ ID NO.

1.

2. Application of DRM3 gene-encoded protein in potato preservation; The protein is either i) or ii) as shown below: i) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing; ii) Fusion proteins obtained by attaching tags to the N-terminus and / or C-terminus of the protein defined in i).

3. Application of recombinant expression vectors, transgenic cell lines, or genetically engineered bacteria of the DRM3 gene in potato preservation; the sequence of the DRM3 gene is shown in SEQ ID NO.

1.

4. The application of the DRM3 gene, the protein encoded by the DRM3 gene, a recombinant expression vector containing the DRM3 gene, a transgenic cell line or genetically engineered bacteria in potato breeding, wherein the sequence of the DRM3 gene is shown in SEQ ID NO.1, and the sequence of the protein encoded by the DRM3 gene is shown in SEQ ID NO.2; wherein the potato breeding is for cultivating potatoes with improved ability to inhibit sprouting and resist enzymatic browning.

5. A method for preserving potatoes, characterized in that, include: The DRM3 gene was overexpressed in potatoes, and the sequence of the DRM3 gene is shown in SEQ ID NO.

1.

6. A method for cultivating potatoes with enhanced ability to inhibit sprouting and resist enzymatic browning, characterized in that, Includes the following steps: The DRM3 gene was transferred into potato plants to overexpress the DRM3 gene, resulting in transgenic potato plants. The transgenic potato plants exhibited higher inhibition of sprouting and resistance to enzymatic browning than the original potato plants. The sequence of the DRM3 gene is shown in SEQ ID NO.

1.

7. The cultivation method according to claim 6, characterized in that, Methods for transferring the DRM3 gene into potato starting plants include: Agrobacterium-mediated transformation, polyethylene glycol transformation, or gene gun bombardment.

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