Potato DRM3 gene and application thereof in potato preservation

Abstract

The invention discloses a potato DRM3 gene and application thereof in potato preservation, the nucleotide sequence of the DRM3 gene is as shown in SEQ ID NO.1, and the amino acid sequence coded by the DRM3 gene is as shown in SEQ ID NO.2. The DRM3 gene is connected to an expression vector, agrobacterium is utilized to infect and transform potatoes, and the result shows that the germination inhibition and enzymatic browning resistance of the potatoes can be remarkably improved through overexpression of the DRM3 gene. Meanwhile, the DRM3 protein is prepared and purified, and it is proved that the DRM3 protein can remarkably inhibit enzymatic browning of potato pulp. The DRM3 gene disclosed by the invention has dual functions of inhibiting germination and resisting enzymatic browning, can provide a theoretical basis for cultivating new varieties of excellent potatoes, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to a potato DRM3 gene and an application thereof in potato preservation. Background Art

[0002] As the fourth new staple food in my country, potatoes are rich in nutritional value and play an important role in adjusting the dietary structure and human health, and are deeply favored by consumers. However, the water content of potato tubers is as high as 80% or more, and they cannot be stored dry. In addition, they are prone to problems such as germination and enzymatic browning during storage and processing, which in turn cause mold, rot, or accumulation of toxic substances such as solanine, causing harm to the health of consumers. The currently widely used low-temperature storage method is costly, and the use of chemical reagents is prone to health problems. The current control methods have limited effects. It is urgent to invent new methods to inhibit potato germination and enzymatic browning and increase the shelf life of potatoes. At present, gene editing technology has been widely used in the development of excellent new fruit and vegetable germplasm, such as the use of gene editing technology to obtain new potato varieties with corresponding functions. CN110438152A discloses a method for promoting potato tuber germination by overexpressing the potato StDWF1 gene. Overexpressing the StDWF1 gene can promote potato tuber germination, advance the germination time, and thus increase its yield, which is different from the technical problem to be solved by the present invention. CN116262922A discloses the use of potato auxin response gene StSAUR30231 in inhibiting enzymatic browning of fresh-cut potatoes. The StSAUR30231 gene is connected to an expression vector, and potatoes are transformed by Agrobacterium infection. Data show that overexpressing the StSAUR30231 gene can significantly improve the browning resistance of potatoes.

[0003] DRM3 (Dormancy-Associated protein 3) belongs to the Dormancy-Associated protein (dormancy-related gene) gene family. The dormancy-associated gene family is a highly conserved gene unique to higher plants, which 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. However, there are many members of the dormancy-associated gene family, and not all dormancy-associated genes have the effect of inhibiting germination. At present, there is a lack of DRM genes that can be used to regulate potato germination, and there is no research on using genes of this family member to solve the problem of enzymatic browning of potatoes. The present invention aims to use the dual function of the DRM3 gene to solve the two major problems of potato storage and preservation, and extend the shelf life of potatoes. Summary of the invention

[0004] The present invention discloses a potato DRM3 gene and its application in potato preservation. By overexpressing the DRM3 gene, two major problems of germination and enzymatic browning in potato storage and preservation are solved. The dual function of the DRM3 gene can provide a theoretical basis for breeding new excellent potato varieties, which is of great significance in production practice.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a DRM3 gene is provided for use in potato preservation; the DRM3 gene is a nucleic acid molecule as shown in (a) or (b) or (c):

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

[0008] (b) a nucleic acid molecule having a nucleotide sequence identity of more than 90% with that of (a) and expressing a protein with the same function;

[0009] (c) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 other than (a).

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

[0011] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated by the naked eye or by 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 is to inhibit potato sprouting and resist enzymatic browning.

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

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

[0015] ii) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in i).

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

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

[0018] Table 1 Sequences of tag proteins

[0019] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tagII 8 WSHQ c-myc 10 EQKLISEEDL

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

[0021] The recombinant expression vector can be constructed using existing plant expression vectors. The plant expression vector includes binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, such as pGreen0029, pCAMBIA3301, pCAMBIA1300 or other derived plant expression vectors. When the gene is used to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CaMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer.

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

[0023] (1) Plant breeding;

[0024] (2) regulating the ability of plants to inhibit germination;

[0025] (3) Regulate the ability of plants to resist enzymatic browning.

[0026] In the above application, the plant is preferably 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 above method, the overexpression of DRM3 gene in potato can be achieved by introducing DRM3 gene from an exogenous source; or by up-regulating the expression of DRM3 gene in potato genome. The method of up-regulating the expression of DRM3 gene in potato genome includes: introducing DNA fragments that can activate or increase the transcription level or translation level or protein activity of DRM3 gene; or controlling the synthesis of specific small RNA molecules to up-regulate the accumulation of DRM3 gene mRNA.

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

[0030] In a sixth aspect of the present invention, there is provided a method for cultivating potatoes with improved germination inhibition and enzymatic browning resistance, comprising the following steps: transferring the above-mentioned DRM3 gene into a starting potato plant to overexpress the DRM3 gene and obtain a transgenic potato plant; the transgenic potato plant has higher germination inhibition and enzymatic browning resistance than the starting potato plant.

[0031] In the above cultivation method, the method for transferring the DRM3 gene into the potato starting plant includes: Agrobacterium-mediated method, polyethylene glycol method, or gene gun bombardment method.

[0032] Beneficial effects of the present invention:

[0033] The present invention cloned the potato dormancy-related response gene DRM3 through systematic research, and transferred the gene into potatoes through Agrobacterium-mediated method. Analysis showed that the ability of potato tubers overexpressing the DRM3 gene to inhibit germination and resist enzymatic browning was significantly improved. The present invention identified for the first time that the DRM3 gene has the dual functions of inhibiting potato germination and resisting enzymatic browning, providing a research basis for cultivating new high-quality potato germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The determination of the expression level of DRM3 gene in potatoes overexpressing DRM3 gene; WT is the wild type Sichuan Yu No. 10 potato, OE16 and OE19 are Sichuan Yu No. 10 potatoes with different overexpressions of DRM3 gene;

[0035] Figure 2 Identification of germination of potatoes overexpressing the DRM3 gene; A is the determination of germination rate of potatoes overexpressing the DRM3 gene, and B is the identification of germination phenotype of potatoes overexpressing the DRM3 gene; WT is the wild-type Sichuan Yu No. 10 potato, and OE16 and OE19 are Sichuan Yu No. 10 potatoes with different overexpressions of the DRM3 gene;

[0036] Figure 3 Identification of the anti-enzymatic browning effect in potatoes overexpressing the DRM3 gene; A shows the effect of overexpressing the DRM3 gene on the L* value in potatoes, and B shows the change in the browning phenotype of potato chips overexpressing the DRM3 gene;

[0037] Figure 4 Inducible expression of DRM3-MBP fusion protein; M: 250kD protein marker; 1: IPTG induced BL21 Escherichia coli (DRM3-MBP) protein expression; 2: Blank induced BL21 Escherichia coli (DRM3-MBP) protein expression; 3: IPTG induced BL21 Escherichia coli (MBP) protein expression; 4: Blank induced BL21 Escherichia coli (MBP) protein expression;

[0038] Figure 5 The purification of DRM3-MBP fusion protein; M: 250kD protein marker; 1: MBP protein; 2: DRM3-MBP fusion protein;

[0039] Figure 6 The effect of DRM3 protein on enzymatic browning of potato pulp; A is the effect of DRM3 protein on potato pulp browning; B is the effect of DRM3 protein on potato pulp browning phenotype; MBP is MBP protein, and DRM3-MBP is DRM3-MBP fusion protein. DETAILED DESCRIPTION

[0040] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1 Discovery process of potato preservation gene DRM3

[0042] The present invention selects wild type Chuanyu No. 10 as experimental material, selects potatoes with good phenotype and complete, treats potatoes with 500nmol / L germination hormone brassinolide, and after drying in a room temperature room, stores at 20°C and 90% humidity, punches and samples the potato eye tissues at 0h and 24h (circular punching 5mm×5mm), and quickly freezes in liquid nitrogen. Subsequently, the total RNA of the sample is extracted using a plant polysaccharide polyphenol RNA extraction kit (Nanjing Novozyme Biotechnology Co., Ltd.), and sent to Hangzhou Jingjie Biotechnology Co., Ltd. for phosphorylated protein sequencing. The protein quantitative results show that after 24h of germination hormone treatment, 111 protein expressions are upregulated and 234 protein expressions are downregulated, which promotes the increase of 240 protein phosphorylation levels and downregulates 290 protein phosphorylation levels. Importantly, under the treatment of germination hormone, the expression level of DRM3 protein was downregulated by 63.3%, and the phosphorylation of 8 sites of the protein was promoted by 40%-238%. The amino acid sequence of the protein was sequenced as shown in SEQ ID NO. 2. We speculate that the DRM3 gene may be important for potato preservation.

[0043] Example 2 Cloning of DRM3 gene sequence

[0044] (1) Primer design

[0045] According to the gene number of DRM3 in phosphorylation proteomics (102581405), the cDNA sequence of the DRM3 gene was queried on NCBI (https: / / www.ncbi.nlm.nih.gov / gene / ), and primers for DRM3 amplification were designed online using primer 3. The primer sequences were:

[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] The wild-type potato tuber sample of Chuanyu No. 10 was taken and ground into powder in liquid nitrogen. The total RNA of the sample was extracted using a plant polysaccharide and polyphenol RNA extraction kit (Nanjing Novozyme Biotechnology Co., Ltd.). The specific method is as follows:

[0051] 1) Weigh 50 mg of potato tuber powder ground with liquid nitrogen, add 500 μL Buffer PRL preheated at 65°C (please add 5% β-mercaptoethanol or 5% 2M DTT solution before use), and immediately vortex vigorously for 30-60 seconds to fully lyse it. Reducing the viscosity will help increase the yield;

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

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

[0054] 4) Transfer the above mixed solution 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 Column II into new Collection Tubes 2 ml (provided in the kit), add 500 μl Buffer PRLPlus, centrifuge at 12,000 rpm (13,400 × g) for 30 s, and collect the filtrate;

[0056] 6) Add 0.5 times the volume of anhydrous ethanol to the filtrate and mix immediately by pipetting;

[0057] 7) Transfer the above mixed solution 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 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 Buffer PRW2 to FastPure RNA Column IV (please check whether 48 ml of anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the filtrate;

[0060] 10) Repeat step 9;

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

[0062] 12) Transfer FastPure RNA Column IV to a new RNase-free Collection Tubes 1.5ml centrifuge tube, and add 30-100μL of RNase-free ddH 2 O, placed at room temperature for 2 min, centrifuged at 12,000 rpm (13,400 × g) for 1 min, and the resulting RNA solution was stored at -70°C to prevent degradation.

[0063] (3) cDNA synthesis

[0064] The extracted total RNA was reverse transcribed, and the reversal system was: 3 μL RNA, 1 μL Oligo d(T)18 Primer, 8 μL RNAse-free, 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, 1 μL RT were added, at 42°C for 60 min, and then at 25°C for 5 min; finally, the obtained cDNA was diluted 10 times and stored at -20°C. The reaction was carried out according to the RNA reverse transcription kit method of Thermo Fisher Scientific.

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

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

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

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

[0069] Use 1 μL of restriction enzymes SacI and SalI to linearize 50 μL of plant expression vector pBI121, then use BM seamless Cloning Kit homologous recombination enzyme from Bomed Biotechnology to connect the PCR product obtained from the above gel recovery with the linearized vector pBI121 after restriction digestion, mix well, connect on a PCR instrument at 50℃ for 15 minutes, and cool on ice. Homologous recombination system: 100 ng of PCR product, 50 ng of linearized vector pBI121, 5 μL of 2×Seamless Cloning Mix, and make up to 10 μL with ultrapure water.

[0070] 5 μL of the ligation product was transferred to transform 100 mL of E. coli DH5α. After the strain grew, a single clone was picked and cultured overnight at 37°C and 200 rpm, and then PCR identification was performed using the primer pair DRM3-F1 and DRM3-R1. The bacterial solution of the positive clone was selected and sent to Bio-Tech for sequencing. The plasmid was extracted using the OMEGA plasmid extraction kit for the strain with the correct sequencing, and then the recombinant plasmid was heat-shocked and transformed into GV3101 Agrobacterium electrotransformation competent cells (Bomed Bio). A single clone was picked for PCR identification, using primers DRM3-F1 and DRM3-R1, and finally the GV3101 Agrobacterium with the DRM3 gene successfully transferred was obtained.

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

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

[0073] Bacterial liquid: After the GV3101 Agrobacterium containing the plant expression vector DRM3-pBI121 obtained above was sterilized by ultraviolet light on a clean bench, single colonies were picked up with a pipette and inoculated into 5 mL YEB liquid culture medium, cultured at 28°C and 200 r / min, taken out after 15-24 hours, diluted the bacterial liquid at a volume ratio of 1:100, and added to new liquid YEB culture medium. After culturing for 12-15 hours under the same conditions, the OD value of the bacterial liquid was measured, and it was taken out for use when OD600 = 0.4-0.6.

[0074] Agrobacterium infection solution (100 mL): liquid MS + 3% sucrose, pH adjusted to 5.9-6.0, sterilized at 121°C for 20 min, added with acetosyringone at a final concentration of 100 μmol / L after cooling, and set aside.

[0075] Stem regeneration medium: The basic medium is MS medium, add 0.6% agar, add antibiotics and hormones according to Table 2, prepare four stages of medium, and adjust the pH to 5.8-6.0. All mediums need to be sterilized by high pressure at 121℃ for 20min, and add filter-sterilized hormones or antibiotics when the medium drops to 60℃.

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

[0077] Cultivation Stage 6-BA TDZ 2,4-D <![CDATA[GA 3 ]]> Kan Cef Car Co-culture 0.5 1.5 1.0 0 0 0 0 Callus screening culture 0.5 1.5 0.1 0 50 100 250 Bud induction screening 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: thidiazuron, 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 bacterial solution obtained above at a speed of 5000 r / min for 15 minutes, remove the supernatant, add 10 mL of the infection solution obtained above, vortex and mix well to obtain the bacterial solution for subsequent infection. Cut the stem segments of the wild-type Sichuan Yu No. 10 potato tissue culture seedlings with vigorous growth into 1 cm long, and do not carry axillary buds. Transfer the cut stem segments to the bacterial solution obtained above and infect at room temperature for 5-8 minutes. During the infection process, the bacterial solution containing the stem end segment needs to be continuously shaken so that the stem segment can fully contact the Agrobacterium containing the expression vector. Use sterilized tweezers to transfer the infected stem segment to sterile filter paper until the moisture of the stem segment is absorbed.

[0081] (3) Co-culture

[0082] The stem segments after absorbing water were transferred to the co-cultivation medium. 20 stem segments were placed in one culture dish and co-cultivated at 24°C in the dark for 2-3 days.

[0083] (4) Bud induction screening

[0084] Take out the infected stem segments after co-cultivation from the culture dish, select the stem segments where bacteria begin to grow at the bottom edge of the stem segments, wash them 3-4 times in sterile water containing 100 mg / L Cef, absorb the moisture with sterile filter paper, and transfer them to the bud induction screening medium with tweezers. Place 20 stem segments in each culture dish and culture them at 22°C, 4000lx light intensity, 16h light / 8h dark conditions. Replace the fresh bud screening medium every 7d and culture for 30d.

[0085] (5) Callus regeneration

[0086] The stem segments that are successfully infected and have grown callus tissue are transferred to callus screening medium for culture, and the upper part of the stem segments undergoes differentiation and sprouting.

[0087] (6) Rooting culture

[0088] When the length of the bud reaches 1-2cm, cut the bud and insert it into the rooting medium. Cover the bottle cap, seal it with sealing film, and culture it under the conditions of light intensity 4000lx, light time 16h / d, temperature 22℃ for 15 days.

[0089] (7) Planting of potato seedlings overexpressing the DRM3 gene

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

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

[0092] DRM3 gene overexpressing potato tubers with uniform size, no mildew, no germination, no green change and no mechanical damage were selected. The wild-type Sichuan Yu No. 10 potato tubers were used as the control group, and the expression level of DRM3 gene in DRM3 gene overexpressing potatoes was identified.

[0093] (1) RNA extraction

[0094] Potato tubers overexpressing the DRM3 gene and wild-type potato tuber Solanum tuberosum No. 10 (WT) were ground into powder in liquid nitrogen, and total RNA was extracted using a plant polysaccharide and polyphenol RNA extraction kit (Nanjing Novozyme Biotechnology Co., Ltd.). The specific method is as follows:

[0095] 1) Weigh 50 mg of liquid nitrogen-ground DRM3 gene-overexpressing potato and wild-type Sichuan taro No. 10 potato tuber powder respectively, and perform the following steps respectively;

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

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

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

[0099] 5) Transfer the above mixed solution 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 Column II into new Collection Tubes 2 ml (provided in the kit), add 500 μl Buffer PRLPlus, centrifuge at 12,000 rpm (13,400 × g) for 30 s, and collect the filtrate;

[0101] 7) Add 0.5 times the volume of anhydrous ethanol to the filtrate and mix immediately by pipetting;

[0102] 8) Transfer the above mixed solution 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 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 Buffer PRW2 to FastPure RNA Column IV (please check whether 48 ml of anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the filtrate;

[0105] 11) Repeat step 10;

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

[0107] 13) Transfer FastPure RNA Column IV to a new RNase-free Collection Tubes 1.5ml centrifuge tube, and add 30-100μl of RNase-free ddH 2O, placed at room temperature for 2 min, centrifuged at 12,000 rpm (13,400 × g) for 1 min, and the obtained RNA solutions of potato overexpressing the DRM3 gene and wild-type Sichuan taro No. 10 potato were stored at -70°C to prevent degradation.

[0108] (2) cDNA synthesis

[0109] The total RNA extracted from the potato overexpressing the DRM3 gene and the wild-type Sichuan taro 10 potato was reverse transcribed respectively, and the reverse transcription system was: 3μL RNA, 1μL Oligo d(T)18 Primer, 8μL RNAse-free, at 65℃ for 5min; after the first step, 4μL 5×RT, 1μL Ribolock RNase Inhibitor, 2μL 10mM dNTP Mix, 1μL RT were added, at 42℃ for 60min and then at 25℃ for 5min; finally, the obtained cDNA was diluted 10 times and stored at -20℃. The reaction was carried out according to the RNA reverse transcription kit method of Thermo Fisher Scientific.

[0110] (3) Fluorescence quantitative analysis

[0111] The wild-type potato was used as the control, and the expression of DRM3 gene in the potato overexpressing DRM3 gene was analyzed by qRT-PCR. The reaction system used a 20 μL reaction system, and the reaction solution was prepared according to the following ratio: 2×Ssofast Eva Green 10 μL, cDNA volume 2.5 μL, primers 0.4 μL each, ddH 2 O to 20 μL. The qRT-PCR reaction conditions are as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 58℃ annealing and extension for 5s, 39 cycles; 95℃ denaturation for 10s, 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] Fluorescence quantitative determination revealed that Figure 1 As shown, the expression levels of DRM3 gene in potatoes overexpressing DRM3 gene OE16 and OE19 were significantly higher than that in wild type, thus finally obtaining two potato materials overexpressing DRM3 gene OE16 and OE19.

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

[0116] DRM3 gene overexpressing potatoes of groups OE16 and OE19 with uniform size, no mildew, no sprouting, no green change and no mechanical damage were selected. The wild-type tuber No. 10 of Sichuan Yu was used as the control group (WT). The potatoes were packed into fresh-keeping boxes (21.5 cm × 15.5 cm × 8 cm), with 20 potatoes in each box. The potatoes were then placed in a dark room at 20±1℃ and 90% humidity. The morphological sprouting of potato tubers in each treatment group (a tuber had at least one bud with a length ≥ 2 mm, i.e., tuber sprouting) was observed every 10 days. The sprouting of tubers at 50d, 60d, 70d, 80d, 90d and 100d was counted and photographed and recorded until a certain type of tuber was completely germinated, except for bad and rotten potatoes.

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

[0118] Example 7 Identification of the enzymatic browning effect of potatoes overexpressing the DRM3 gene

[0119] The same number of wild-type Chuanyu No. 10 potato tubers and OE19 potato tubers overexpressing the DRM3 gene (OE-DRM3) were selected, which were uniform in size, free of mildew, germination, green change, and mechanical damage, and the surface sand was washed off, washed three times with deionized water, and then immersed in a 200 μL / L sodium hypochlorite solution for 5 min for disinfection. The surface water was absorbed, the potato skin was removed, and the potato was cut into uniform 1 cm thick potato slices using a slicer. The potato was washed with deionized water to remove the surface sand. The potatoes were placed in a fresh-keeping box at 20°C and 90% humidity, and the L* value (brightness) of the surface of the fresh-cut potatoes was measured at 1d, 2d, 3d, 4d, and 5d, respectively (the detection aperture was set to 1.00mm, the VIS measurement mode was selected, and the colorimeter was calibrated with a light microscope and a standard white plate (L0=89.94a0=0.3b0=2.0)). The color of the surface of the fresh-cut potatoes was measured at 0d and 5d using an UltraScan VIS desktop colorimeter.

[0120] like Figure 3As shown in the figure, the L* values ​​of wild-type and OE-DRM3 potato slices decreased with storage time, but compared with the wild-type, overexpression of the DRM3 gene significantly inhibited the rapid decline of the L* value, and the difference in L* values ​​between the two was the largest at 5 days. In addition, the significant inhibitory effect of overexpression of the DRM3 gene on the browning of the potato slice surface can be intuitively distinguished, and overexpression of the DRM3 gene enables potatoes to have a higher ability to resist 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] Construction of DRM3-MBP (MBP, maltose-binding protein) fusion protein expression vector: pMAL-c2X (MBP tag) was double-digested with restriction endonucleases EcoR1 and BamH1 (Beijing Quanshijin Biotechnology) and the reaction system was as follows: 35 μL of pMAL-c2X vector, 2 μL of each restriction endonuclease, 5 μL of 10× Buffer, ddH 2 O50μL, react in a 37℃ water bath for 3h. The digestion product was recovered using the gel recovery kit of Sangon Biotechnology Co., Ltd.

[0124] The cDNA obtained in Example 2 was used as a template, and DRM3-F3 and DRM3-R3 were used as primers. The amplification system was as follows: 5 μL cDNA, 0.2 μL each primer, GoldPfu PCR SuperMix 25μL, ultrapure water to 50μL. Reaction conditions: 94℃, 5min; 94℃, 20s; 52℃, 20s; 72℃, 40s, 35 cycles; 72℃, 10min. 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 product was detected by 1% agarose gel electrophoresis, and then the target gene was recovered by gel extraction using the OMEGA Gel Extraction Kit.

[0128] The ligation reaction was carried out by homologous recombination, and the target gene was connected to the vector using the homologous recombination kit ClonExpress II OneStep Cloning Kit (Novagen Biotech). The reaction system was as follows: 4 μL of MBP empty load, 3 μL of target gene, 2 μL of 5×CEⅡBuffer, and 1 μL of ExnaseⅡ. The reaction was then connected in a 37°C water bath 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 as follows:

[0131] 1) Use a pipette to gently add the above 10 μL ligation product (DRM3-MBP fusion protein expression vector) into 100 μL of DH5α E. coli competent cells, tap the EP tube with your fingertips to mix, and place it on ice for 25 minutes;

[0132] 2) Then, the EP tube was placed in a 42°C water bath for heat shock treatment for 90 seconds, and then quickly placed on ice for 2 minutes. After the standing time, 0.8 mL of LB liquid culture medium was added and the tube was grown in a 37°C shaker for 60 minutes.

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

[0134] 4) Pipette the above mixed solution onto LB solid culture medium and spread it evenly using a coating rod. After the LB solid culture medium is blown dry, place the culture dish upside down in a 37°C incubator and culture overnight.

[0135] Identification of results: The monoclonal colonies were identified by PCR. The primers used were: DRM3-F2: 5'-TGGTCCCAGCCCTGATAAAG-3', DRM3-R2: 5'-GAAATGTTTGTCGGCGTCAC-3'. The single colony with the correct band detected by PCR was transferred to 5 mL of LB liquid culture medium and cultured at 37°C. The resulting bacterial liquid was sent to Biotech for sequencing, and the strains and recombinant plasmids (DRM3-MBP) with correct sequencing results were saved.

[0136] (3) Plasmid transformation of Agrobacterium

[0137] 1) Take 2 μL of the recombinant plasmid containing DRM3-MBP obtained above, gently add it to 100 μL of BL21 E. coli competent cells, tap the EP tube lightly with your fingertips to mix, and place it on ice for 25 minutes;

[0138] 2) Then, the EP tube was placed in a 42°C water bath for heat shock treatment for 90 seconds, and then quickly placed on ice for 2 minutes. After the standing time, 0.8 mL of liquid LB medium was added and the tube was grown in a shaker at 37°C for 60 minutes.

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

[0140] 4) Pipette the above mixed solution onto LB solid medium and spread it evenly with a coating rod. After the LB solid medium is blown dry, place the culture dish upside down in a 37°C incubator and culture overnight;

[0141] 5) Then, a single colony was picked and placed in 5 mL of LB liquid medium and cultured at 37°C and 200 rpm for 12 h.

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

[0143] The 5 mL LB liquid medium obtained above was cultured under shaking conditions of 37°C and 200 rpm until the bacterial solution absorbance OD600 = 0.4-0.5, and induced with a final concentration of 0.1 mmol / L IPTG (isopropyl-β-D-thiogalactoside) at 28°C for 6 h. Using DRM3-MBP Escherichia coli (without IPTG induction), empty Escherichia coli (MBP+IPTG), and empty Escherichia coli (MBP) as controls, polyacrylamide gel electrophoresis was used to screen Escherichia coli expressing DRM3-MBP:

[0144] 1) After induction, the four bacterial cultures were collected by centrifugation at 4°C and 5000 r / min for 10 min, and the precipitate was suspended in 3 mL of 0.1% phosphate buffer; the precipitate was subjected to ultrasonic disruption, and the total ultrasonic time was 5 min, including 1.5 s of working time and 6.5 s of rest time, which was repeated;

[0145] 2) Centrifuge the treated sample at 4°C, 10,000 rpm for 10 min, remove the supernatant, and suspend the precipitate with 3 mL of 0.1% PBS;

[0146] 3) Collect the supernatant, take 32 μL of protein sample, add 8 μL of 5× protein loading buffer (Shanghai Biotech Biotechnology Co., Ltd.), shake and mix, heat in a 100°C water bath for 10 min to denature the protein, cool to room temperature, and perform 10% SDS-PAGE electrophoresis (SDS-PAGE electrophoresis gel was prepared according to the Bio-rad method, with 12% separation gel and 4% concentration gel, followed by staining for 30 min and decolorization for more than 3 h).

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

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

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

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

[0151] ① Centrifuge the above bacterial solution at 5000 rpm for 15 min and discard the supernatant to obtain bacterial 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 make the concentration of each to 0.4 mmol / mL, mix thoroughly, and place on ice for 30 min;

[0153] ③Ultrasonic disruption of the bacterial solution after ice bath, setting the parameters to 85% power, 9s disruption, 5s stop, repeat for 25min; when disrupting protein, it is best to place the centrifuge tube in an ice-water mixture to maintain a low temperature environment to prevent protein denaturation;

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

[0155] ⑤ At the same time, wash the corresponding beads (Beijing Solebow Technology Co., Ltd.) with MBP washing buffer, and then add 200 μL MBP-specific beads after washing to the supernatant filtered in step ④ to incubate and bind the protein beads;

[0156] ⑥ Rotate on a rotator at 4°C for 2-3 hours, then centrifuge at 4°C, 2000 rpm for 2 minutes;

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

[0158] Table 3 MBP lysis and washing buffer ratio

[0159] Components concentration Tris-HCl (Tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4) 20mM NaCl (Sodium Chloride) 200mM EDTA (ethylenediaminetetraacetic acid) 1mM

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

[0161] Table 4 MBP elution buffer ratio

[0162] Components concentration Tris-HCl (Tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4) 20mM NaCl (Sodium Chloride) 200mM EDTA (ethylenediaminetetraacetic acid) 1mM Maltose 10mM

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

[0164] SDS-PAGE electrophoresis was used to identify the purification of the DRM3-MBP fusion protein and MBP protein obtained above. Figure 5 As shown, protein purification was performed using MBP-specific magnetic beads to obtain a 52.5 kD DRM3-MBP fusion protein and a 40 kD MBP protein, respectively.

[0165] 3) Effect of DRM3 protein on enzymatic browning of potato pulp

[0166] Wild-type Chuanyu No. 10 potato tuber powder was prepared by grinding in liquid nitrogen, 200 μL of DRM3-MBP fusion protein solution was transferred into 5 mL of potato slurry (containing 1 g of potato tuber powder, adjusted to 5 mL with sterile water), and the absorbance of the slurry at a wavelength of 410 nm at 0 h, 0.5 h, 1 h, 2 h, and 3 h of the reaction was dynamically measured by an ultraviolet spectrophotometer, and 200 μL of MBP purified protein solution was used as a control.

[0167] Enzymatic browning is the oxidation and polymerization of phenolic substances into melanin. Melanin has a good absorption peak at 410nm, so the content of melanin can be determined by absorbance, and then the degree of browning can be determined. Figure 6 It can be seen that DRM3-MBP is used to inhibit the browning of potato slurry. Compared with MBP, DRM3-MBP can significantly inhibit the enzymatic browning of slurry. In summary, the present invention prepares and purifies DRM3 protein, and uses it to inhibit the browning of potato slurry and achieves good results.

Claims

1. A potato DRM3 gene, characterized in that: The DRM3 gene is a nucleic acid molecule as shown in (a) or (b) or (c) below: (a) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; (b) a nucleic acid molecule having a nucleotide sequence identity of more than 90% with that of (a) and expressing a protein with the same function; (c) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 other than (a).

2. The use of the DRM3 gene according to claim 1 in potato preservation, characterized in that: The fresh-keeping method is to inhibit potato sprouting and resist enzymatic browning.

3. A protein encoded by the DRM3 gene according to claim 1, characterized in that: The protein is a protein shown in any one of the following i) or ii): i) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing; ii) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in i).

4. Use of the protein encoded by the DRM3 gene according to claim 3 in potato preservation.

5. Use of the recombinant expression vector, transgenic cell line or genetically engineered bacteria of the DRM3 gene according to claim 1 in potato preservation.

6. Application of DRM3 gene, protein encoded by DRM3 gene, recombinant expression vector containing DRM3 gene, transgenic cell line or genetically engineered bacteria in potato breeding.

7. A method for preserving potatoes, characterized in that: include: The DRM3 gene according to claim 1 is overexpressed in potatoes.

8. A method for cultivating potatoes with improved sprouting inhibition and anti-enzymatic browning ability, characterized in that: The following steps are involved: Transforming the DRM3 gene of claim 1 into a potato starting plant to overexpress the DRM3 gene, thereby obtaining a transgenic potato plant; The potato transgenic plants have higher sprout inhibition and enzymatic browning resistance capabilities than the original potato plants.

9. The cultivation method according to claim 8, characterized in that: Methods for transferring the DRM3 gene into potato starter plants include: Agrobacterium-mediated method, polyethylene glycol method, or gene gun bombardment method.

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