Application of StMYB113b gene in regulation and control of potato tuber epidermal color

Abstract

The invention provides an application of an StMYB113b gene in regulating and controlling the epidermal color of a potato tuber. According to the invention, the key gene StMYB113b for regulating and controlling the formation of the red skin pigment of the potato tuber is found and cloned for the first time, and the gene can influence the skin color of the potato tuber by regulating and controlling the synthesis of an anthocyanin-related compound. Through overexpression or knockout of the gene, regulation and control of the potato tuber epidermis color can be achieved, a new way and gene resources are provided for potato variety improvement, and the gene has important theoretical value and application prospects.

Description

Application of the StMYB113b gene in regulating potato tuber epidermal color Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to the application of the StMYB113b gene in regulating the epidermal color of potato tubers. Background Technology

[0002] The potato (Solanum tuberosum L.) is a tetraploid food crop widely cultivated in 158 countries and regions worldwide, serving as a staple food source for over a billion people. It is rich in carbohydrates, protein, minerals, vitamins, polyphenols, carotenoids, flavonoids, anthocyanins, and many other essential components. The skin color of potato tubers is an important quality trait that is of concern to consumers. Furthermore, skin color and flesh color are not necessarily related; for example, the skin color of a purple-fleshed potato may be red, yellow, or white, and the flesh color of a purple-fleshed potato may also be red, yellow, or white.

[0003] Studies have shown that anthocyanin synthesis in potato tuber epidermis is mainly regulated by three key loci (D, P, and R). Specifically, P, located on chromosome 2, and R, located on chromosome 10, encode the biosynthetic enzymes flavonoid 3',5'-hydroxylase (F3'5'H) and dihydroflavonol 4-reductase (DFR), respectively. Meanwhile, the D locus on chromosome 11 encodes an R2R3-MYB transcription factor, StAN1, a homolog of petunia AN2, which plays a central regulatory role in the pigment synthesis pathway (Liu Y, Lin-Wang K, Espley RV, et al. Functional diversification of the potato R2R3 MYB anthocyanin activators AN1, MYBA1, and MYB113 and their interaction with basic helix-loop-helix cofactors[J]. J Exp Bot. 2016, 67: 2159 - 2176.).

[0004] MYB113 is present in all potato varieties, but many varieties exhibit different colors. Currently, most potato varieties with available genomic information are yellow-skinned, and the causal mechanism between specific genotypes and specific color phenotypes (especially red-skinned traits) has not been revealed.

[0005] It is worth noting that in tetraploid potatoes, due to the high complexity of the genome and gene dosage effects, even the same gene (such as StMYB113) may have functional redundancy or allele specificity in different varieties, making it difficult to predict its role in actual phenotypic regulation (Bao Z, Li C, Li G. et al. Genomearchitecture and tetrasomic inheritance of autotetraploid potato. Mol Plant. 2022;15:1211-1226.). For example, "Qingshu 9" (Q9) is a major cultivated potato variety in my country with a unique red-skinned tuber trait; however, the key genes driving the formation of this trait and its dominant alleles have not yet been identified (Wang F, Xia Z, Zou M. et al. The autotetraploid potato genome provides insights into highly heterozygous species[J]. Plant Biotechnol J. 2022, 20: 1996 - 2005.). Summary of the Invention

[0006] To address the problems in the prior art, this invention provides the application of the StMYB113b gene in regulating the skin color of potato tubers. This invention is the first to discover and clone the key gene StMYB113b that regulates the formation of red pigment in potato tubers. This gene can affect the skin color of potato tubers by regulating the synthesis of anthocyanin-related compounds.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides the application of the StMYB113b gene in regulating the epidermal color of potato tubers. The CDS sequence of the StMYB113b gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2.

[0009] Preferably, the StMYB113b gene affects the skin color of potato tubers by regulating the synthesis of anthocyanin-related compounds.

[0010] Preferably, overexpression of the StMYB113b gene can significantly increase the content of anthocyanin-related compounds; knockout of the StMYB113b gene can significantly decrease the content of anthocyanin-related compounds.

[0011] More preferably, the anthocyanin-related compounds include cyanidin, delphinidin, proanthocyanidin B4, pelargonidin, paeoniflorin, and luteolin.

[0012] Preferably, overexpression of the StMYB113b gene can change the skin color of the potato tubers of the "Atlantic" variety from yellow to pink.

[0013] Preferably, knocking out the StMYB113b gene can significantly lighten the color of the red skin of the potato tubers in the "Qingshu No. 9" variety.

[0014] The present invention also provides a method for regulating the skin color of potato tubers, including overexpression or knockout of the StMYB113b gene to regulate the skin color of potato tubers, wherein the CDS sequence of the StMYB113b gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2.

[0015] Preferably, the method includes: introducing the StMYB113b gene overexpression vector pBWA(V)KS-StMYB113b into the "Atlantic" potato variety, and introducing the StMYB113b gene knockout vector pNK2-StMYB113b into the "Qingshu No. 9" potato variety.

[0016] More preferably, the overexpression vector pBWA(V)KS-StMYB113b is constructed by the following method: primers OE-StMYB113b-F / R are designed to clone the overexpression gene, and the full-length CDS sequence of the amplified StMYB113b gene is ligated into the plant expression vector pBWA(V)KS. The sequence of primer OE-StMYB113b-F / R is as follows:

[0017] OE-StMYB113b-F: 5'-AACACGGGGGACTTTGCAACatgaataatgccaagtcattgggagtgagaaaag-3' (SEQ ID No. 5); OE-StMYB113b-R: 5'-TGAAGACAGAGCTAGTTACActaattaaatagattccataggtcaatatcaaatgaaaagt-3' (SEQ ID No. 6).

[0018] More preferably, the knockout vector pNK2-StMYB113b is constructed by the following method: a specific sgRNA is designed and synthesized targeting a specific exon region of the StMYB113b gene; a fragment containing the sgRNA expression cassette is amplified using primer D13812_0C1; and then ligated into the CRISPR / Cas9 plant knockout vector pNK2-cas9-u6 linearized with BsaI enzyme using the Golden Gate cloning method. The sequence of primer D13812_0C1 is as follows:

[0019] D13812_0C1(+):5'-cagtGGTCTCatgcaagtgctttcggtgatcttgagttttagagc-3′ (SEQID No.9);

[0020] D13812_0C1(-):5'-cagtGGTCTCaaaaccgacctcgacctcgaacagttg-3' (SEQ ID No. 10).

[0021] The beneficial effects of this invention are as follows:

[0022] This invention reveals a novel and unexpected role of the transcription factor gene StMYB113b in regulating potato tuber coloration. Experiments show that overexpression of StMYB113b can turn the tubers of the "Atlantic" variety, which originally has a yellow skin, pink; conversely, knocking out this gene using gene editing technology can significantly lighten the color of the tubers of the "Qingshu 9" variety, which has a red skin, and the flesh color also becomes paler. This demonstrates how a single gene can be manipulated to achieve bidirectional and reversible regulation of skin color in two commercially available varieties with vastly different genetic backgrounds.

[0023] Further metabolomics analysis confirmed that this phenotypic change stemmed from the key dominant regulation of the anthocyanin synthesis pathway by StMYB113b. In overexpressing plants, the levels of multiple core anthocyanin compounds, including cyanidin, delphinidin, and pelargonidin, were synergistically and significantly increased; while in knockout plants, the levels of these compounds were consistently and significantly reduced. This finding breaks through previous understanding of the mechanism of color regulation in potatoes, proving that StMYB113b does not merely modify individual components, but rather globally enhances anthocyanin synthesis and accumulation.

[0024] In summary, this invention is the first to clone and functionally validate StMYB113b, a key regulatory gene for the formation of red skin pigment in potato tubers, and further reveals its core role as a "major regulatory switch" in potato tuber color formation. Experiments demonstrate that this gene can achieve stable and controllable regulation of tuber epidermal color in cultivated varieties with different genetic backgrounds, and its regulatory range covers multiple tissue sites from the epidermis to the flesh. It can also synergistically activate the synthetic pathways of multiple anthocyanin compounds. This systematic regulatory ability exceeds the conventional understanding of the function of a single MYB transcription factor in this field. This provides a novel gene resource and breeding strategy for the targeted improvement of potato appearance quality, and has significant theoretical value and application prospects. Attached Figure Description

[0025] Figure 1 shows the basic information of the 'Qingshu 9' reference genome.

[0026] Figure 2: Observation of skin color of potato parents and F1 generation of mixed hybrids.

[0027] Figure 3: Genome-wide association analysis of potato tuber skin color. (A) Manhattan diagram of potato tuber skin color; (B) Manhattan diagram of chromosomes containing significant loci.

[0028] Figure 4: Distribution of SNP indices and differential SNP indices on chromosomes of hybrid offspring. (A) Distribution of Δ (SNP index) values ​​on chromosomes of the two offspring pools, with each point representing a SNP locus. (B) and (C) show the distribution of SNP indices on chromosomes of the two offspring pools (red-skinned potatoes and white-skinned potatoes), respectively.

[0029] Figure 5: Chromosomal localization analysis of the StMYB113 gene in the DM8.1 and Q9 genomes, where uppercase letters represent StMYB113 in the double haploid potato DM and lowercase letters represent StMYB113 in the tetraploid potato 9 genome.

[0030] Figure 6: Phylogenetic analysis of StMYB113s from the DM and Q9 genomes.

[0031] Figure 7: Phenotype of potato tubers overexpressed with the Atlantic variety StMYB113b. The white scale in the figure represents 2 cm.

[0032] Figure 8: Phenotype of knockout potato tubers of the Q9 variety StMYB113b. The white scale in the figure represents 2 cm.

[0033] Figure 9: Expression profiles of the StMYB113b gene in various organs (leaves, stems, roots, tuber pulp, and tuber epidermis) of plants overexpressing StMYB113b compared to the control (Atlantic potato variety). Significance was tested, and asterisks indicate significance. () indicates the level of statistical significance. P < 0.05; P < 0.01; NS indicates that no significant difference was determined by a two-tailed Student's t-test. The same applies below.

[0034] Figure 10: Expression profiles of the StMYB113b gene in various organs (leaves, stems, roots, tuber pulp, and tuber epidermis) of the knockout line compared to the control (Q9 potato variety).

[0035] Figure 11: Content of anthocyanin-related compounds (delphinidin, proanthocyanidin B4, cyanidin, pelargonidin, paeoniflorin and luteolin) in overexpression lines compared with controls.

[0036] Figure 12: Content of anthocyanin-related compounds (delphinidin, proanthocyanidin B4, cyanidin, geranyl oleoresin, paeoniflorin and luteolin) in the knockout lines compared with the control. Detailed Implementation

[0037] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0038] This invention isolates and clones the gene StMYB113b from the red-skinned potato variety "Qingshu 9" (Q9). This gene is a key gene that regulates the formation of red skin pigment in potato tubers.

[0039] Search for the StMYB113b gene with the ID Soltu.Q9.Chr10_A10025387.g in the published genome of the potato variety Qing薯 9 号 (http: / / bigd.big.ac.cn / ; under BioProject numbers PRJCA006096). The CDS sequence is: ATGAATAATGCCAAGTCATTGGGAGTGAGAAAAGGTTCATGGACTAAAGAAGAAGATATTCTTTTGAGGAAATGTATTGACAAGTATGGAGAAGGAAAGTGGCATCTTGTTCCTTCTAGAGCTGGTCTAAATAGATGTCGAAAGAGTTGTAGACTAAGGTGGTTGAATTATCTAAGGCCACATATCAAGAGAGGTGACTTTGCTCCGGATGAAATAGATCTCATTTTGAGACTTCATAAGCTTCTAGGCAACAGATGGTCACTTATTGCTGGGAGACTTCCAGGAAGAACAGCAAACGATGTGAAAAACTATTGGAACACACACCTACACAAGAAGTTAATAACTCCTCATCCTCAGATACAAGAGAATAAGTACAATAATACCCTCAAGATCACCGAAAGCACTATACTACGACCACGACCTCGACCTCGAATAGTCTCAAGTGCAAATAATATTTCTTGGTGCACTAACAATAGTATGATCACAAACACATTAGACAAAGATGACGAACAACGCAACAAAGAAATCATAGTAAATATTTGTGAGAAGCCAACAAGAGAAACAACGTCATCATCTATAGACGATGACGGGGTTAAATGGTGGACAGATTTATTGGAAAATTGGAAAGAATTTGAGGAAGAAGCAGCAGCAGTATTGAGTTTTGAGGAAGAAAATAAGCTGTTACCAAATTTGTTGTATGATGAACATAATTCAACAACCATGCAACAAAATGATGGTTGGGATGACTTTTCAGTTGATATTGACCTATGGAATCTATTTAATTAG (SEQ ID No.1).

[0040] The amino acid sequence encoded by StMYB113b is: MNNAKSLGVRKGSWTKEEDILLRKCIDKYGEGKWHLVPSRAGLNRCRKSCRLRWLNYLRPHIKRGDFAPDEIDLILRLHKLLGNRWSLIAGRLPGRTANDVKNYWNTHLHKKLITPHPQIQ ENKYNNTLKITESTILPRPRPRIVSSANNISWCTNNSMITNTLDKDDEQRNKEIIVNICEKPTRETTSSSIDDDGVKWWTDLLENWKEFEEEAAAVLSFEEENKLLPNLLYDEHNSTTMQQNDGWDDFSVDIDLWNLFN (SEQ ID No.2).

[0041] The specific implementation process is as follows:

[0042] Example 1

[0043] 1. Identification of tuber skin color in potato germplasm resources

[0044] After harvest, the skin color of 753 potato tubers was observed and phenotypic data were collected according to the national standard definition and standard description of potato traits (GB / T 19557.28-2018; https: / / openstd.samr.gov.cn / bzgk / gb / ) (Table 1). The observation period for tuber skin color was the harvest period (Table 2).

[0045] Table 1. Standards and Indicators for the Skin Color of Potato Tubers

[0046]

[0047] Table 2. Identification of skin color indicators in 753 tetraploid potato samples

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] 2. Genome-wide association analysis and candidate gene mining of potato tuber skin color

[0054] Based on resequencing data from 753 potato accessions, variant sites were filtered using the criteria of MAF > 0.05 and HWE > 0.001. Genome-wide association analysis (GWA) of potato tuber skin color was performed using the CMLM model (PCA+K) in GAPIT software. P < 10 was used as the cutoff value. -5 Significant SNPs were screened using a threshold, and candidate regions of 60 kb upstream and downstream of the SNP were determined based on LD decay, thereby screening candidate genes.

[0055] 3. BSA-seq sequencing analysis and candidate gene discovery

[0056] To verify the regulatory mechanism of potato tuber skin color, the red-skinned variety Qingshu 9 was crossed with the white-skinned variety Atlantic to obtain the F1 generation, whose skin color segregation ratio was approximately 1:1 (Figure 2). Extreme mixed pools of red-skinned and light yellow-skinned tubers were constructed, and resequencing was performed on both parents. The sequenced data were aligned to the reference genome, and SNP / InDel detection and filtering were performed using GATK. The SNP-index and differential values ​​of the progeny mixed pools were calculated. SNP-index was analyzed using a sliding window with a 1 Mb window and a 1 kb step size, and 99% confidence intervals were determined using 1000 permutation tests. Significant sites were screened across the entire genome (SNP-index ≥ 0.7 for pool B and ≤ 0.3 for pool A), and similar analysis was performed on InDel. Finally, ANNOVAR was used to functionally annotate candidate polymorphic sites to support the screening and validation of candidate genes.

[0057] 4. Genome-wide association analysis and BSA-seq analysis co-localized the StMYB113b gene

[0058] Genome-wide association analysis (GWAS) of potato tuber skin color using the compressed mixed linear model (CMLM) of GAPIT revealed that 61.90% of loci were primarily enriched in haplotype segments A1, A3, and A4 on chromosome 10 (Figure 3). To further validate the regulatory mechanism of tuber skin color, we constructed a population of segregating hybrid progeny with tuber skin color using the red-skinned variety Qingshu 9 and the white-skinned variety Atlantic as parents. BSA-seq analysis of 25 samples from each of the two phenotypic progeny groups revealed that significant SNP loci were also located on chromosome 10, and showed good overlap with the GWAS results (Figure 4).

[0059] To analyze the copy number and sequence variation of MYB113 in potatoes with different skin colors, we analyzed the MYB113 gene in diploid DM potato (yellow skin) and tetraploid potato variety Qingshu 9 (red skin). The results showed that MYB113 is located only on chromosome 10, with 6 copies identified in DM and 18 copies in Q9 (Figure 5). MYB113 belongs to the R2R3-MYB family. Phylogenetic analysis showed that potato StMYB113 is homologous to Arabidopsis thaliana AtMYB113, and the MYB113 gene in both species clearly clustered into four groups (Figure 6). Using the red-skinned potato genome as a reference, StMYB113b was identified through GWAS and BSA-seq co-mapping. Sequence alignment revealed that, compared to the previously identified StMYB113b protein, the StMYB113b variant contains 10 amino acid mutations and 4 residue deletions, indicating significant sequence differences between red-skinned potato StMYB113b and other StMYB113 variants (Figure 6). Based on this, we hypothesize that the StMYB113b variant, unique to red-skinned potatoes, may regulate potato skin coloration.

[0060] Example 2

[0061] Using cDNA from the potato variety "Qingshu 9" (Q9) as a template, the StMYB113b gene was amplified according to the PCR reaction system in Table 3 to obtain the CDS sequence of the StMYB113b gene. The primer sequences for specific amplification of the StMYB113b gene are as follows:

[0062] F-Primer: 5'-CGACCACGACCTCGACCTC-3' (SEQ ID No. 3);

[0063] R-Primer: 5'-TGTTGCGTTGTCGTCATCTTTG-3' (SEQ ID No. 4).

[0064] The PCR reaction procedure is shown in Table 4.

[0065] Table 3 PCR reaction system

[0066]

[0067] Table 4 PCR reaction procedure

[0068]

[0069] The Gname (630 bp) fragment was extracted by 1.5% agarose gel electrophoresis at 5 V / cm for 20 min under UV light and placed in a system for sol-gel recovery. The DNA was recovered using a gel recovery kit (Tiangen Biotech Co., Ltd.). The DNA was dissolved and recovered in 40 μL of water. After verification, the StMYB113b gene sequence was obtained.

[0070] Example 3

[0071] 1. Construction of the overexpression vector pBWA(V)KS-StMYB113b

[0072] To identify the function of StMYB113b in potato, primers OE-StMYB113b-F / R were designed and synthesized at Shanghai Sangon Biotech Co., Ltd. The full-length CDS sequence of the amplified StMYB113b gene was ligated into the plant expression vector pBWA(V)KS to construct the overexpression vector. The expression vector pBWA(V)KS was linearized by BsaI / Eco31I digestion; the 20 μL digestion system is shown in Table 5. StMYB113b and the expression vector pBWA(V)KS were ligated using homologous recombination; the ligation system is shown in Table 6.

[0073] OE-StMYB113b-F: 5'-AACACGGGGGACTTTGCAACatgaataatgccaagtcattgggagtgagaaaag-3' (SEQ ID No. 5); OE-StMYB113b-R: 5'-TGAAGACAGAGCTAGTTACActaattaaatagattccataggtcaatatcaaatgaaaagt-3' (SEQ ID No. 6).

[0074] Table 5 Enzyme digestion system and reaction conditions

[0075]

[0076] Note: The kit uses the Dicotyledonous Overexpression Vector Kit (G418), catalog number: #REC30D, manufacturer: BioRun.

[0077] Table 6. Connection system and reaction conditions

[0078]

[0079] After ligation, the strain was directly transformed into *E. coli* DH5α, and positive strains were identified by PCR, with sequencing confirming the correctness. The pBWA(V)KS-StMYB113b vector was obtained.

[0080] 2. pBWA(V)KS-StMYB113b vector was transformed into Agrobacterium GV3101.

[0081] Transform GV3101 Agrobacterium with the pBWA(V)KS-StMYB113b vector plasmid. Remove GV3101 Agrobacterium competent cells from the cryostat. Add 1 µL of plasmid to 50 µL of GV3101 Agrobacterium competent cells, mix thoroughly, and transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and transfer to a 1.5 mL centrifuge tube. Incubate at 30°C and 180 rpm for 30 min on a shaker. Inoculate 50 µL of the activated Agrobacterium culture onto LB colony solid medium and incubate in the dark at 30°C for 48 h.

[0082] 3. Agrobacterium-positive detection: Detection was performed using KAN primers. The primer sequences are as follows:

[0083] Kan-F-Primer: 5'-ACCGCTGCGTAAAAGATACGGA-3' (SEQ ID No. 7);

[0084] Kan-R-Primer: 5'-TTCTCCCAATCAGGCTTGATCC-3' (SEQ ID No. 8).

[0085] The PCR amplification system is shown in Table 7.

[0086] Table 7 Colony PCR Amplification System

[0087]

[0088] Note: 2×Taq PCR Mix (50×1 mL): Cat: P222-03; Lot: 7E2710K4; Pack: 037E4291KC2; Store at -30 ~ -15 ℃.

[0089] For gel electrophoresis detection, prepare a 1% agarose gel (weigh 1.5 g of agarose powder and dissolve it in 150 mL of 1×TAE buffer, microwave for about 3 minutes until the liquid becomes transparent. Add EB to the gel casting plate, pour the dissolved agarose liquid into the plate, mix well, insert a comb, and let stand for 40 minutes until the gel turns milky white), then spot the sample to complete the electrophoresis process.

[0090] 4. Checking PCR amplification results: If the electrophoresis bands of the positive control and the sample are clear and of the correct size, and the negative control has no band, it indicates that the sample can proceed to the next step.

[0091] Example 4

[0092] 1. Construction of the knockout vector pNK2-StMYB113b

[0093] To knock out the StMYB113b gene in potato using CRISPR / Cas9 technology, specific sgRNAs targeting specific exon regions were designed and synthesized. Fragments containing the sgRNA expression cassette were amplified using primers D13812_0C1 (system shown in Table 8, procedure shown in Table 9). D13812_0C1 primers:

[0094] D13812_0C1(+):5'-cagtGGTCTCatgcaagtgctttcggtgatcttgagttttagagc-3′ (SEQID No.9);

[0095] D13812_0C1(-):5'-cagtGGTCTCaaaaccgacctcgacctcgaacagttg-3' (SEQ ID No. 10).

[0096] Table 8 PCR amplification system for sgRNA fragments

[0097]

[0098] Table 9 PCR reaction procedure

[0099]

[0100] Electrophoresis was performed on a 1.5% agarose gel at 5 V / cm for 20 minutes. The electrophoretic fragments T1-T4 (539 bp) were excised under UV light and placed in a system for sol-gel recovery (DNA gel recovery kit: Axygen). The DNA was recovered by dissolving it in 30 μL of water (the recovered product was labeled as: rDNAT1). After verification, it was ligated into a vector.

[0101] The knockout vector pNK2-StMYB113b was constructed by ligating the BsaI-linearized CRISPR / Cas9 plant knockout vector pNK2-cas9-u6 into the Golden Gate cloning method. The 20 μL system and procedure are shown in Tables 10 and 11.

[0102] Table 10 Golden Gate Cloning System

[0103]

[0104] Note: The kit uses the Dicotyledonous Gene Editing Vector Kit (Hyg), catalog number: #REC43-I, manufacturer: BioRun.

[0105] Table 11 Golden Gate Connection Procedure

[0106]

[0107] 2. Transformation of E. coli with pNK2-StMYB113b vector and identification by plaque PCR

[0108] The 5-10 uLGolden Gate ligation product was transformed into E. coli DH5α competent cells, then transformed into kanamycin-resistant plates, and incubated at 37°C for 12 hours. Ten plaques were picked and simultaneously inoculated into 1.5 mL EP tubes for PCR identification (Tables 12 and 13). Primers for pNK2-cas9-u6 identification:

[0109] Pbw2+:5'-GCAACGCTCTGTCATCGTTACAAT-3' (SEQ ID No. 11) (9790bp);

[0110] Pbw2-:5'-gcgattaagttgggtaacgccaggg-3' (SEQ ID No. 12) (204 bp).

[0111] Table 12 Colony PCR Identification System

[0112]

[0113] Table 13 PCR Procedure

[0114]

[0115] The target band is a fragment of approximately 9606 bp. Take 100 μL of bacterial culture corresponding to 1-3 positive bands for sequencing, and inoculate the remaining 400 μL of bacterial culture into 5-10 mL of kanamycin-resistant LB broth. Shake the tubes and wait for the sequencing results. Extract the plasmid from the tube corresponding to the correctly sequenced result. Save the bacterial strain and plasmid to the database.

[0116] Example 5

[0117] 1. Genetic transformation of potatoes

[0118] Explant preparation: Select test-tube seedlings that have grown for 3-4 weeks, cut stem segments of 0.2-0.5 cm with a scalpel as explant material, inoculate them in pre-culture medium, and incubate in the dark at 23 ℃ for 2-3 days.

[0119] Agrobacterium infection and co-culture: Agrobacterium was picked into the infection solution to prepare OD. 600 Prepare a 0.2-0.5 μL Agrobacterium resuspension and inoculate the explants into the Agrobacterium suspension for 10 min. After inoculation, transfer the inoculated explants to sterile filter paper, air dry, and then inoculate them onto co-culture medium. Incubate in the dark at 23 °C for 48-72 h.

[0120] Screening: The co-cultured explants were inoculated onto the screening medium and cultured at 25°C under 16 / 8 light for 14 days.

[0121] Selection / differentiation: Explants were inoculated onto selection / differentiation medium, 30 explants per dish, and cultured at 25℃ under 16 / 8 light. The medium was changed every 20 days.

[0122] Rooting culture: Inoculate the differentiated buds into the rooting medium and culture at 25℃ under 16 / 8 light until roots are formed.

[0123] Detection: Potato genomic DNA was extracted using the CTAB method and then detected by PCR. The detection method is described in the Agrobacterium tumefaciens assay.

[0124] pNK2-StMYB113b plant detection:

[0125] After obtaining transgenic plants, genomic DNA was extracted. Fragments containing the following four sgRNA target regions were amplified using detection primers N1062-F and N1062-821R:

[0126] Detection primers:

[0127] N1062-F: 5'-GTTCGTTCGAACCCAGGAAT-3' (SEQ ID No. 13);

[0128] N1062-821R: 5'-CCCAACCATCATTTTGTTGCAT-3' (SEQ ID No. 14).

[0129] Target:

[0130] CTTGAGACTGTTCGAGGTCG AGG (SEQ ID No. 15);

[0131] AGTGCTTTCGGTGATCTTGA GGG (SEQ ID No. 16);

[0132] TTCTCTTGTATCTGAGGATG AGG (SEQ ID No. 17);

[0133] ACTGTTCGAGGTCGAGGTCG TGG (SEQ ID No. 18).

[0134] The purified PCR products were sent to a sequencing company, where the Hi-TOM high-throughput sequencing platform was used to analyze the editing efficiency of the target region and the specific mutation type.

[0135] 2. Phenotypic observation and measurement: Observe the changes in the skin color of transformed potato tubers, and measure the content of anthocyanin-related compounds and the expression level of genes.

[0136] As shown in Figure 7, overexpression of StMYB113b can turn the tubers of the "Atlantic" variety, which originally had a yellow skin, into pink; conversely, knocking out this gene using gene editing technology can significantly lighten the color of the tubers of the "Qingshu No. 9" variety, which has a red skin, and the flesh color also becomes lighter, as shown in Figure 8. This demonstrates how skin color can be reversibly regulated bidirectionally by manipulating a single gene in two commercially available varieties with vastly different genetic backgrounds.

[0137] The content of StMYB113b in the roots, stems, leaves, flesh and skin of Atlantic plants overexpressing StMYB113b was significantly increased (Figure 9), while the expression of this gene was significantly reduced in Qingshu No. 9 plants with StMYB113b knocked out (Figure 10).

[0138] The contents of anthocyanin-related compounds in the overexpression lines are shown in Figure 11, and those in the knockout lines are shown in Figure 12. The results show that in the overexpression plants, the contents of multiple core anthocyanin compounds, including cyanidin, delphinidin, and pelargonidin, were synergistically and significantly increased; while in the knockout plants, the contents of these compounds were consistently and significantly reduced. This finding breaks through previous understanding of the color regulation mechanism of potatoes, proving that StMYB113b does not only modify individual components, but can globally enhance the synthesis and accumulation of anthocyanins.

[0139] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. Application of the StMYB113b gene in regulating the epidermal color of potato tubers, wherein the CDS sequence of the StMYB113b gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that, The StMYB113b gene affects the skin color of potato tubers by regulating the synthesis of anthocyanin-related compounds.

3. The application according to claim 1, characterized in that, Overexpression of the StMYB113b gene significantly increased the content of anthocyanin-related compounds; knockout of the StMYB113b gene significantly decreased the content of anthocyanin-related compounds.

4. The application according to claim 2 or 3, characterized in that, The anthocyanin-related compounds include cyanidin, delphinidin, proanthocyanidin B4, pelargonidin, paeoniflorin, and luteolin.

5. The application according to claim 1, characterized in that, Overexpression of the StMYB113b gene can change the skin color of potato tubers in the "Atlantic" variety from yellow to pink.

6. The application according to claim 1, characterized in that, Knocking out the StMYB113b gene significantly lightens the color of the red skin on the tubers of the "Qingshu No. 9" potato variety.

7. A method for regulating the skin color of potato tubers, including overexpression or knockout of the StMYB113b gene to regulate the skin color of potato tubers, wherein the CDS sequence of the StMYB113b gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No.

2.

8. The method according to claim 7, characterized in that, The method includes: introducing the StMYB113b gene overexpression vector pBWA(V)KS-StMYB113b into the "Atlantic" potato variety, and introducing the StMYB113b gene knockout vector pNK2-StMYB113b into the "Qingshu No. 9" potato variety.

9. The method according to claim 8, characterized in that, The overexpression vector pBWA(V)KS-StMYB113b was constructed using the following method: primers OE-StMYB113b-F / R were designed to clone the overexpression gene, and the full-length CDS sequence of the amplified StMYB113b gene was ligated into the plant expression vector pBWA(V)KS. The sequences of primers OE-StMYB113b-F / R are as follows: OE-StMYB113b-F: 5'-AACACGGGGGACTTTGCAACatgaataatgccaagtcattgggagtgagaaaag-3' (SEQ ID No. 5); OE-StMYB113b-R: 5'-TGAAGACAGAGCTAGTTACActaattaaatagattccataggtcaatatcaaatgaaaagt-3' (SEQ ID No. 6).

10. The method according to claim 8, characterized in that, The knockout vector pNK2-StMYB113b was constructed using the following method: a specific sgRNA was designed and synthesized targeting a specific exon region of the StMYB113b gene; a fragment containing the sgRNA expression cassette was amplified using primer D13812_0C1; and then ligated into the CRISPR / Cas9 plant knockout vector pNK2-cas9-u6 linearized with BsaI enzyme using the Golden Gate cloning method. The sequences of the D13812_0C1 primers are: D13812_0C1(+):5'-cagtGGTCTCatgcaagtgctttcggtgatcttgagttttagagc-3′ (SEQ ID No. 9); D13812_0C1(-):5'-cagtGGTCTCaaaaccgacctcgacctcgaacagttg-3′ (SEQ ID No. 10).

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