Application of stmgd1 protein, encoding gene and mutants thereof in improving postharvest quality of potato

Abstract

This invention discloses the application of the StMGD1 protein, its encoding gene, and its mutants in improving postharvest quality of potatoes, belonging to the field of biotechnology. This invention is the first to discover the StMGD1 protein and its encoding gene... StMGD1 Closely related to the post-harvest quality of potatoes, by... StMGD1 Gene knockout reduces the chlorophyll content and starch content in the epidermis of postharvest potato tubers, effectively inhibiting greening and extending the shelf life and appearance quality of marketable potatoes. Simultaneously, gene knockout... StMGD1 The gene can also significantly reduce the accumulation of α-solanine and α-carboxine, improving the safety of potatoes for consumption.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of StMGD1 protein, its encoding gene, and its mutants in improving postharvest quality of potatoes. Background Technology

[0002] potato( Solanum tuberosum Potatoes (L.) are an important staple food and economic crop worldwide. Their tubers are rich in B vitamins (including B1, B2, B6, and pantothenic acid) and high-quality dietary fiber, giving them extremely high nutritional value. However, during field exposure, harvesting, and transportation, the starch granules in the epidermal cells of potato tubers transform into chloroplasts under light stimulation, leading to a large accumulation of chlorophyll, a phenomenon known as "greening." More seriously, the greening process is usually accompanied by the co-synthesis and excessive accumulation of toxic steroidal alkaloids (mainly α-solanine and α-carboxine), resulting in food safety risks and significant post-harvest losses.

[0003] Currently, the control of potato greening in production mainly relies on methods such as light-proof storage, low-temperature controlled atmosphere treatment, chemical preservative treatment, and conventional variety breeding. Physical storage methods are energy-intensive and have limited conditions, making it difficult to achieve long-term control throughout the entire process; chemical preservatives are prone to pesticide residues and have short-term effects, posing safety and environmental risks with long-term use; and the potato has a complex genetic background, traditional hybridization breeding has a long cycle and low efficiency in targeted improvement, making it difficult to achieve the aggregation of multiple traits such as resistance to greening and high yield, and thus failing to stably solve the tuber greening problem from its genetic roots.

[0004] Existing molecular studies have preliminarily identified key components, such as genes related to the photosynthesis pathway and alkaloid metabolism pathway, in the regulation of greening in potato tubers, a critical issue affecting postharvest potato quality. However, the identification of core functional genes regulating greening remains insufficient, and precise gene targets and targeted editing schemes for direct germplasm improvement are lacking. Therefore, identifying and elucidating the key functional genes regulating potato greening and clarifying their mechanisms of action are of great significance for breeding new greening-resistant potato varieties, effectively improving postharvest potato quality, and reducing postharvest losses. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of StMGD1 protein, its encoding gene and its mutants in improving the postharvest quality of potatoes.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of StMGD1 protein in improving postharvest quality of potatoes; the amino acid sequence of said StMGD1 protein is shown in SEQ ID NO.1.

[0007] In the above applications, the postharvest quality of potatoes is improved by inactivating the StMGD1 protein.

[0008] In the above applications, improving the postharvest quality of potatoes specifically means: reducing the chlorophyll content in the epidermis of postharvest potato tubers, reducing the starch content in the epidermis of postharvest potato tubers, and / or reducing the content of toxic steroidal alkaloids in the epidermis of postharvest potato tubers.

[0009] Furthermore, the toxic steroidal alkaloid is α-solanine or α-carboline.

[0010] As a feasible approach, one option is to modify the gene encoding the StMGD1 protein, using gene editing technology to inhibit the expression and synthesis of the StMGD1 protein at the source, thereby causing the StMGD1 protein to lose its original biological function. Another option is to use exogenous spraying of specific agents to directly act on the StMGD1 protein in plant cells, effectively inhibiting its activity by destroying the protein structure and blocking enzyme activity, thus achieving protein inactivation.

[0011] A second aspect of the present invention provides the application of the gene encoding the StMGD1 protein in improving postharvest quality of potatoes; the gene encoding the StMGD1 protein is... StMGD1 A gene is a nucleic acid molecule as shown in (i) or (ii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.1.

[0012] In the above applications, targeted knockout StMGD1 Genes improve post-harvest quality of potatoes.

[0013] As a preferred method, targeted knockout is achieved using the following substances. StMGD1 Gene: e1) Targeted knockout StMGD1 Nucleic acid molecules of genes; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3).

[0014] Furthermore, CRISPR / Cas9 editing vectors carrying the sgRNA coding region were used to target and knock out [the virus]. StMGD1Gene; the sequence of the coding region of the sgRNA is shown in SEQ ID NO.7.

[0015] A third aspect of the present invention provides StMGD1 Gene mutants, the StMGD1 The nucleotide sequence of the gene mutant is shown in SEQ ID NO.8 or SEQ ID NO.9.

[0016] A fourth aspect of the present invention provides the above. StMGD1 Applications of gene mutants in (1) or (2) below: (1) Improve post-harvest quality of potatoes; (2) Create potato germplasm resources resistant to greening. The beneficial effects of this invention are: (1) This invention is the first to discover that the StMGD1 protein and its encoding gene StMGD1 Closely related to the post-harvest quality of potatoes, by... StMGD1 Gene knockout reduces both chlorophyll and starch content in the epidermis of postharvest potato tubers. Since greening of potato tubers primarily occurs in the epidermis and near-epidermal tissues, and depends on the conversion of starch granules in this region into chloroplast-like plastids, the reduction in epidermal starch content indicates a weakening of the storage plastid base at the site of greening, thus reducing the number or developmental level of starch granules that can be converted into chloroplasts under light conditions. Simultaneously, the decreased epidermal starch reserves reduce the likelihood of starch degradation after light exposure releasing carbon sources to support chlorophyll synthesis, thereby inhibiting chlorophyll accumulation and the formation of a green phenotype in the epidermis. Therefore, reducing the epidermal starch content helps weaken the sensitivity of tubers to light-induced greening, demonstrating a beneficial effect in inhibiting potato tuber greening. By reducing chlorophyll and starch content, postharvest greening of potatoes is effectively inhibited, effectively extending the shelf life and appearance quality of commercial potatoes.

[0017] At the same time, knock out StMGD1 The gene can also significantly reduce the accumulation of α-solanine and α-carboxine, improving the safety of potatoes for consumption.

[0018] (2) with StMGD1 Genes are used as targets, and genome editing technologies such as CRISPR / Cas9 are used to target and knock them out, thereby creating... stmgd1 Mutant materials are beneficial for creating potato germplasm materials with resistance to green staining, providing important genetic resources and breeding methods for cultivating new potato varieties with excellent postharvest traits, and have significant theoretical value and application prospects. Attached Figure Description

[0019] Figure 1 A homozygous mutant line of potato stmgd1-1、stmgd1-2 Sequencing results of the mutation target.

[0020] Figure 2 For the wild type and StMGD1 Wild-type and mutant strains after 6 days of storage under simulated shelf-life conditions StMGD1 Gene mutants ( stmgd1-1、stmgd1-2 The difference in chlorophyll content.

[0021] Figure 3 For the wild type and StMGD1 Gene mutants ( stmgd1-1、stmgd1-2 The difference in concentration between α-solanine and α-carbohydrate.

[0022] Figure 4 Wild type and StMGD1 Gene mutants ( stmgd1-1、stmgd1-2 Differences in starch content after tuber epidermal analysis. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0025] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: Callus induction medium: MS (without agar and sucrose) 4.74 g / L, agar powder 8 g / L, sucrose 20 g / L, α-naphthaleneacetic acid (NAA) 0.2 mg / L, gibberellin (GA3) 0.02 mg / L, zeatin (ZT) 2.5 mg / L, termethin (Tim) 100 mg / L, kanamycin (kan) 50 mg / L.

[0026] Differentiation medium: MS (without agar and sucrose) 4.74 g / L, agar powder 8 g / L, sucrose 20 g / L, NAA 0.02 mg / L, GA3 0.02 mg / L, ZT 2.0 mg / L, Tim 100 mg / L, Kan 50 mg / L.

[0027] Rooting medium: MS (without agar and sucrose) 4.74 g / L, agar powder 8 g / L, sucrose 20 g / L, Tim 100 mg / L, Kan 50 mg / L.

[0028] Example 1: Potato StMGD1 The creation of mutants.

[0029] 1. StMGD1 Construction of gene knockout vector With potato varieties " Desiree Using potatoes as the raw material, total RNA was extracted from their tubers and reverse transcribed to synthesize cDNA. This process was based on previously reported potato... StMGD1 Primers were designed based on homologous gene sequences, and the homologous gene sequences were obtained through PCR amplification. StMGD1 The full-length cDNA sequence of the gene was obtained. Sequencing results showed that the nucleotide sequence of the gene is shown in SEQ ID NO.2, with a full length of 1599 bp, and the amino acid sequence encoding the StMGD1 protein is shown in SEQ ID NO.1.

[0030] Using the CRISPR-2.0 online tool, in StMGD1 Four target sequences were selected from the gene coding region, namely: Target 1: CCTCTGGGTTTGCCTCAGT. (SEQ ID NO.3) Target 2: TCATTGTGGGGAGAGCAATG. (SEQ ID NO.4) Target 3: GGGGAGTGTACTGGAGAATG. (SEQ ID NO.5) Target 4: TAGAGAAGAGGGGAGTGTAC. (SEQ ID NO.6) Based on the target sequences described above, their corresponding complementary sequences were synthesized as spacer sequences and assembled with the sgRNA backbone sequence to construct a complete sgRNA coding frame. After gene synthesis using the sgRNA coding frame and the AtU6-26 promoter, it was then inserted into the BasI restriction site of the CRISPR / Cas9 vector PHSN / BUN401-Cas9 (Biorun, China) to construct the complete sgRNA coding frame. StMGD1 Gene knockout vector. Sequencing confirmed that the vector was constructed correctly.

[0031] The sgRNA coding region (the core coding part of the sgRNA coding frame, which determines the sequence and function of the sgRNA) sequence is shown in SEQ ID NO.7, as follows:

[0032] 2. Potatoes StMGD1 Creation of mutants The above is correctly constructed StMGD1 The gene knockout vector was transformed into Agrobacterium tumefaciens EHA105 to obtain engineered host cells. Using potato as an example... Desiree "The cotyledonary explants of the variety are used as recipient materials, and the explants are infected through Agrobacterium-mediated genetic transformation."

[0033] Cotyledonary explants infected with Agrobacterium were inoculated into callus induction medium to induce callus formation; then transferred to differentiation medium for differentiation culture, and finally transferred to rooting medium for rooting culture to obtain transgenic plants. Extracting genomic DNA from transgenic plants, targeting StMGD1 PCR amplification and sequencing analysis were performed on the gene editing target region. The specific sequence at the target site in the mutant is shown below. Figure 1 The results showed that two homozygous mutation types were successfully obtained: stmgd-1 A 7 bp deletion occurs at the target site, resulting in a frameshift of the coding sequence, the nucleotide sequence of which is shown in SEQ ID NO. 8; stmgd-2 A 5 bp deletion occurs at the target site, which also leads to a frameshift mutation, and its nucleotide sequence is shown in SEQ ID NO. 9.

[0034] Example 2: Potato StMGD1 Study on the resistance to greening of mutants 1. Test method: The potatoes obtained in Example 1 StMGD1 mutant ( stmgd-1, stmgd-2 The shoot tips of wild-type potatoes were inoculated onto MS medium and propagated asexually; Desiree Propagate in the same way. Harvest potato tubers.

[0035] Select wild-type potatoes of similar size. Desiree "with potatoes" StMGD1 mutant ( stmgd-1、 stmgd-2 The starch content in the epidermis of the tuber was determined.

[0036] Then, the potatoes were treated for 6 days under simulated shelf-life conditions (temperature 20 ℃, humidity 80-85%, light intensity 150-200 lux, 16 h / d). The chlorophyll content, α-solanine content, and α-carboxine content in the potato tuber epidermis were measured.

[0037] The determination method is as follows: (1) Chlorophyll content determination: The ultraviolet spectrophotometer method was used. Potato peels were ground into powder using a grinder. 1.0 g of potato peel sample was taken and 25 mL of 95% ethanol was added. Extraction was carried out at 0 °C in the dark for 24 h, followed by filtration into a glass centrifuge tube. The residue, filter paper, and glass rod were washed with the same ethanol. The filtrate was transferred to a volumetric flask and the extraction was continued until no green color was observed on the residue and filter paper. The volume was then adjusted to 30 mL with 95% ethanol. Using 95% ethanol as a blank control, the absorbance of the extract was measured at wavelengths of 665 nm and 649 nm.

[0038] Chlorophyll a concentration (µg / mL) = 13.95 × OD 665 -6.88×OD 649 ; Chlorophyll b concentration (µg / mL) = 24.96 × OD 649 -7.32×OD 665 ; Total chlorophyll concentration (µg / mL): C(a+b)=Ca+Cb; the sum of the total concentrations of chlorophyll a and chlorophyll b is the total chlorophyll concentration.

[0039] The mass fraction of total chlorophyll (μg / g) = C(a+b)×50 / M, where M is the fresh weight of the sample.

[0040] Total chlorophyll concentration (mg / L): C(a+b)=Ca +Cb; the sum of the total concentrations of chlorophyll a and chlorophyll b is the total chlorophyll concentration.

[0041] The mass fraction of total chlorophyll (μg / g) = C(a+b)×50 / M, where M is the fresh weight of the sample.

[0042] (2) Determination of α-solanine and α-carboxine content: Weigh approximately 0.2 g of potato tuber epidermal sample, add 1 mL of 5% acetic acid solution, homogenize in an ice bath, sonicate in an ice-water bath for 30 min, extract overnight, centrifuge at 8000 g for 10 min, collect the supernatant, add 0.5 mL of 5% acetic acid solution to the precipitate, sonicate for 30 min for re-extraction, combine the supernatants, adjust the pH to approximately 11.0 with NaOH aqueous solution, extract three times with chloroform, combine the organic phases, dry under nitrogen, redissolve in 1 mL of methanol, filter with a syringe filter, and then proceed with analysis.

[0043] HPLC conditions: Thermo U3000 high-performance liquid chromatograph, Thermo C18 reversed-phase column (250 mm × 4 mm, 5 μm), mobile phase preparation: A: acetonitrile, B: 0.02 M potassium dihydrogen phosphate solution (A:B = 25:75), injection volume 10 μL, flow rate 1 mL / min, column temperature 30 ℃, UV wavelength 270 nm. After the baseline stabilized, the sample was added and measured.

[0044] (3) Starch content determination: A. Starch Extraction Weigh 0.05 g of potato tuber skin sample into a 15 mL centrifuge tube, add 1.5 mL of 85% ethanol and mix thoroughly. Sonicate at 25 °C for 30 min. Centrifuge the homogenate at 5000 g for 10 min and discard the supernatant.

[0045] Add 1.5 mL of 85% ethanol to the precipitate again, shake well, centrifuge at 5000 g for 10 min, and discard the supernatant. Add 500 μL of 6 mol / L HCl and 700 μL of ultrapure water, vortex, boil in a water bath for 60 min, filter, add two drops of methyl red indicator to the filtrate, adjust the pH of the filtrate to 7.0-7.5, and then make up to 10 mL.

[0046] B. Determination of starch content Take 2 mL of the above solution into a 15 mL centrifuge tube, add 1.5 mL of DNS, mix well, boil in a water bath for 5 min, cool rapidly to room temperature, make up to 10 mL, and then measure its absorbance at 540 nm.

[0047] The corresponding reducing sugar content was found in the standard curve for all test results. The percentage content of reducing sugar in the sample was calculated using the following formula: Starch content = [(m'×V×N) / (Vs×m×1000)]×0.9×100 Where: m': glucose mass obtained from the standard curve, mg; V: Total volume of sample extract, mL; N: Dilution factor of sample extract; Vs: Volume of sample extract taken during the determination, mL; m: Sample mass, g; 0.9: The coefficient for converting glucose to starch.

[0048] 2. Test Results: The results of the chlorophyll content determination in the epidermis of potato tubers are as follows: Figure 2 As shown, potatoes StMGD1 mutant ( stmgd-1, stmgd-2 The chlorophyll content in the tuber epidermis of the ) type was significantly lower than that of the wild type, indicating that StMGD1 Gene deletion can inhibit chlorophyll accumulation.

[0049] The results of the determination of α-solanine and α-carboxane content in potato tuber epidermis are as follows: Figure 3 As shown, StMGD1 The levels of α-solanine and α-carboxine in the gene mutant were significantly lower than those in the wild type, indicating that... StMGD1 Gene deletion can inhibit the accumulation of toxic steroidal alkaloids.

[0050] The results of starch content determination in potato tuber epidermis are as follows: Figure 4 As shown, StMGD1 The starch content in the tuber epidermis of the mutant was significantly lower than that of the wild type. The reduced epidermal starch reserves decrease the likelihood of starch degradation after light exposure releasing carbon sources to support chlorophyll synthesis, thereby inhibiting chlorophyll accumulation and the formation of a green epidermal phenotype. Therefore, reducing the starch content in the tuber epidermis helps to weaken the sensitivity of tubers to light-induced greening, demonstrating a beneficial effect in inhibiting greening of potato tubers.

[0051] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of StMGD1 protein in improving postharvest quality of potatoes, characterized by: The amino acid sequence of the StMGD1 protein is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, Improving postharvest quality of potatoes by inactivating the StMGD1 protein.

3. The application according to claim 1, characterized in that, The improvement of postharvest potato quality specifically includes: reducing the chlorophyll content in the epidermis of postharvest potato tubers, reducing the starch content in the epidermis of postharvest potato tubers, and / or reducing the content of toxic steroidal alkaloids in the epidermis of postharvest potato tubers.

4. The application according to claim 3, characterized in that, The toxic steroidal alkaloids are α-solanine or α-carboxine.

5. The application of the gene encoding the StMGD1 protein in improving postharvest potato quality, characterized by: The gene encoding the StMGD1 protein is StMGD1 A gene is a nucleic acid molecule as shown in (i) or (ii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.

1.

6. The application according to claim 5, characterized in that, Through targeted knockout StMGD1 Genes improve post-harvest quality of potatoes.

7. The application according to claim 5, characterized in that, Targeted knockout using the following substances StMGD1 Gene: e1) Targeted knockout StMGD1 Nucleic acid molecules of genes; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3).

8. The application according to claim 7, characterized in that, Targeted knockout using CRISPR / Cas9 editing vectors carrying sgRNA coding regions StMGD1 Gene; the sequence of the coding region of the sgRNA is shown in SEQ ID NO.

7.

9. A kind StMGD1 Gene mutants, characterized by, The StMGD1 The nucleotide sequence of the gene mutant is shown in SEQ ID NO.8 or SEQ ID NO.

9.

10. The claim 9 StMGD1 Applications of gene mutants in (1) or (2) below: (1) Improve post-harvest quality of potatoes; (2) Create potato germplasm resources resistant to greening.

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