Potato insect-resistant yield-increasing gene StMYC1, protein encoded by the gene and application

Abstract

The application belongs to the technical field of potato molecular breeding, and particularly relates to a potato pest-resistant and yield-increasing gene StMYC1 and an encoded protein and application thereof. The application provides application of a potato StMYC1 gene or a biological material overexpressing a potato StMYC1 gene, wherein an amino acid sequence encoded by the potato StMYC1 gene is shown as SEQ ID NO:1, and the application comprises improving plant pest resistance and / or yield. The application first identifies a gene resource with pest-resistant and yield-increasing regulation functions in potatoes, and establishes a complete and repeatable breeding technical scheme based on the gene resource, thereby laying a solid foundation for breeding pest-resistant and high-yield varieties.

Description

Technical Field

[0001] This invention belongs to the field of potato molecular breeding technology, specifically involving potato insect-resistant and yield-increasing genes. StMYC1 Its encoded proteins and applications. Background Technology

[0002] Beet armyworm ( Spodoptera exigua The beet armyworm (Spodoptera litura) is a globally distributed, highly damaging, polyphagous agricultural pest with a wide host range, encompassing over 120 species of important crops across 30 families, including potatoes, tomatoes, and peppers. Its larvae are voracious eaters, causing extensive leaf damage and even plant death, resulting in significant economic losses. Currently, control of the beet armyworm primarily relies on chemical insecticides; however, this pest has developed high levels of resistance to many insecticides, including pyrethroids and abamectins. Chemical control not only suffers from significantly reduced effectiveness due to resistance but also faces serious challenges such as excessive pesticide residues, harm to natural enemies, and environmental pollution. Therefore, there is an urgent need in this field for pest control methods that can replace chemical approaches.

[0003] MYC-type transcription factors are important regulators in the jasmonic acid signaling pathway in plants, participating in the regulation of the synthesis of various secondary metabolites. In tomatoes, studies have shown... SlMYC1 By differentially regulating terpene synthesis, MYC transcription factors positively regulate monoterpene synthesis in leaf glandular trichomes and negatively regulate sesquiterpene synthesis in stem glandular trichomes. This complex regulatory mechanism influences plant resistance to spider mites. This provides a reference for studying the function of MYC transcription factors in Solanaceae crops. However, because potatoes are autotetraploid crops with a complex genetic background and high gene redundancy, directly applying the above research results to gene editing of potatoes still has significant limitations and uncertainties. Furthermore, the two pests have different damage patterns and plant defense strategies; therefore, the regulatory role of MYC transcription factors in potatoes remains unclear.

[0004] In summary, existing technologies have two major gaps: on the one hand, StMYC1 The regulatory role of genes in potatoes lacks experimental validation, and their functional predictability is low. Furthermore, in a tetraploid genetic background, achieving the synergistic expression of both insect resistance networks and yield-increasing pathways through single-gene regulation to form a stable and heritable dual superior phenotype presents significant technical challenges. Therefore, providing an experimentally validated potato variety that combines yield-increasing and insect-resistant functions is crucial. StMYC1 Genes have become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To address the lack of genes in existing technologies that effectively enhance potato insect resistance and / or yield, this invention provides a potato insect-resistant and yield-increasing gene. StMYC1Its encoded proteins and applications, specifically including the following technical solutions:

[0006] This invention provides potatoes StMYC1 Potatoes with gene or overexpression StMYC1 The application of genes in biological materials, namely potatoes StMYC1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:1, and the application includes regulating plant insect resistance and / or yield.

[0007] Preferably, the potato StMYC1 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0008] Preferably, the plant insect resistance includes plant resistance to beet armyworm.

[0009] Preferably, the yield includes the effective tuber formation rate and / or tuber yield.

[0010] Preferably, the application includes: overexpressing the potato StMYC1 Genes enhance plant insect resistance and / or yield.

[0011] Preferably, the overexpressed potato StMYC1 Biological materials for genes include potatoes StMYC1 Gene amplification primers, overexpression potatoes StMYC1 Recombinant expression vectors and overexpression potatoes StMYC1 One or more of the recombinant genes of microorganisms.

[0012] Preferably, the overexpressed potato StMYC1 The recombinant gene expression vector includes a backbone vector; the backbone vector includes the pCAMBIA1300 vector.

[0013] Preferably, the overexpressed potato StMYC1 The recombinant microorganisms of the gene include basic microorganisms; said basic microorganisms include Agrobacterium LBA4404.

[0014] Preferably, the plant includes potatoes.

[0015] This invention also provides potatoes StMYC1 Potatoes with gene or overexpression StMYC1 Application of genetically modified biological materials in potato breeding.

[0016] The beneficial effects of this invention are as follows:

[0017] 1. This invention is the first to verify and provide endogenous dual-function gene resources for insect resistance and yield increase in potatoes: This invention is the first to experimentally demonstrate in tetraploid potatoes that overexpression of endogenous... StMYC1The gene not only confers significant resistance to the beet armyworm but also increases tuber yield. Insect resistance experiment data show that... StMYC1 Transgenic lines with overexpressed genes significantly reduced the body weight of feeding larvae by 28.8%, while also significantly increasing the effective tuber-forming rate and total tuber yield per plant. These two unexpected and significant effects confirm that... StMYC1 Genes are not only excellent targets for potato insect resistance breeding, but also key genes for yield improvement, providing important gene resources and technical support for research and industrial production of simultaneous improvement of crop insect resistance and yield using transgenic technology.

[0018] 2. A complete and reproducible technical solution is provided: This invention discloses in detail the complete process and key parameters from gene cloning, vector construction, genetic transformation to insect-resistant phenotype identification and in vitro tuber formation phenotype identification, especially for the efficient transformation system of potatoes, providing a reliable basis and convenience for reproducing this invention and extending this technology to other potato varieties.

[0019] 3. Development of supporting key molecular tools: This invention provides validated tools for... StMYC1 The specific primer pairs for gene cloning, expression detection, and real-time quantitative PCR internal control provide a convenient tool for functional studies, molecular detection, and assisted breeding applications of this gene. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 Phylogenetic and evolutionary relationships between potato StMYC1 and its homologous proteins and tobacco and tomato MYC proteins;

[0022] Figure 2 For potatoes StMYC1 Agarose gel electrophoresis image of the PCR amplification product of the full-length CDS gene;

[0023] Figure 3 The recombinant plant expression vector pCAMBIA1300-35S:: StMYC1 Physical spectrum;

[0024] Figure 4 Diagram showing the key stages of the Agrobacterium-mediated potato transformation process;

[0025] Among them, A is a schematic diagram of the appearance of resistant callus formation; B is a schematic diagram of the appearance of resistant bud differentiation; C is a schematic diagram of the appearance of regenerated bud rooting; and D is a schematic diagram of the appearance of transgenic plant transplantation.

[0026] Figure 5To analyze the wild-type (Desiree) strain and transgenic strain using qRT-PCR ( StMYC1-OE )middle StMYC1 The relative expression levels of genes; among which, express P <0.0001;

[0027] Figure 6 Phenotypic diagram of beet armyworm feeding experiment;

[0028] Where A represents the wild type (Desiree) and StMYC1-OE Comparison of leaf damage in different strains; B represents the wild type (Desiree) and... StMYC1-OE Comparison of larval size in groups; C represents wild-type (Desiree) and... StMYC1-OE Statistical results of average weight of larvae in each group; among them, express P <0.05;

[0029] Figure 7 It is a wild type of potato (Desiree). StMYC1-OE Statistical chart of the content of solanine, an insect-resistant secondary metabolite, in strains 44 h after simulated insect pest treatment.

[0030] Where A represents the statistical results of α-solanine content; B represents the statistical results of α-carboxine content. express P <0.0001;

[0031] Figure 8 For wild-type potatoes (Desiree) and StMYC1 Comparison of peak areas of α-solanine quantitative ion pairs for -OE; where the mass-to-charge ratio of the parent ion and the mass-to-nucleus ratio of the daughter ion in the figure are 869.0 / 398.4;

[0032] Figure 9 For wild-type potatoes (Desiree) and StMYC1 Comparison of peak areas of α-carbohydrate quantitative ion pairs for -OE; where the mass-to-charge ratio of the parent ion and the mass-to-nucleus ratio of the daughter ion in the figure are 852.3 / 706.3;

[0033] Figure 10 For wild-type potatoes (Desiree) and StMYC1-OE In vitro tuber phenotype diagram;

[0034] In this table, A shows the tuber formation phenotype of the two groups of in vitro plants at 65 and 95 days; B shows photographs of the two groups of micro-tubers; C shows the percentage of transgenic plants with tubers in vitro; D shows the yield per plant in the two groups; E shows the number of tubers per plant in the two groups; ns indicates no significant difference. expressP <0.05. Detailed Implementation

[0035] This invention provides a potato StMYC1 Potatoes with gene or overexpression StMYC1 The application of genes in biological materials, namely potatoes StMYC1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:1, and the application includes regulating plant insect resistance and / or yield.

[0036] SEQ ID NO:1:

[0037] MTDYRLWSNSNTTNTSDDNMMMDAFLSSDPSSFWPNRTSISPTPVNGGVGETMPFFNQESLQQRLQALIDGARESWAYAIFWQSSSTSDFATPSVLGWGDGYYKGEENKNKRRASSSSANFVAEQEHRKKVLRELNSLISGVQAAGAGSGGDDAVDEEVTDT EWFFLISMTQSFANGNGLPGLAMYSSSPIWVTGTEKLAGSQCERARQAQGFGLQTIVCIPSANGVVELGSTELIFESSDLMNKVKYLFNFNIDMGSVTGSGSGSCAVHPETDPSALWLTDPSSSVVEAKDSLINSSSRDVQLVFVNENSENGTQNQQHSQQTQ GFFTKELNFSGYGFDGSSTRNKNGNSSISCKPETREILNFGDSSKRSGSLFSGQSQFGPGTGLGLMEENKNKNNNNNKKRSLASRGNNEEGMLSFVSGVILPTSTMGKSGGGGDSDHSDLEASVVKEAIVEPEKKPRKRGRKPANGREEPLNHVEAERQRRE KLNQRFYALRAVVPNVSKMDKASLLGDAIAYINELKSKVQNSDLDKEELRSQIESLRKELANKGSSNYSSSPPSNQDLKIVDMDIDVKVIGWDAMIRIQCSKKNHPAARLMAALKDLDLDVHHASVSVVNDLMIQQATVKMGSRLYAQEQLTIALTSKFAESR .

[0038] As one embodiment, the potato of the present invention StMYC1 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0039] SEQ ID NO:2:

[0040]

[0041] As one implementation, the application includes: overexpressing the potato StMYC1 Genes enhance plant insect resistance and / or yield. In one embodiment, the plant insect resistance includes resistance to the beet armyworm. In one embodiment, enhancing plant insect resistance includes increasing the solanine content in the plant, thereby enhancing insect resistance. In one embodiment, the solanine includes α-solanine and / or α-carboxine. In one embodiment, the yield includes the effective tuber-forming rate and / or tuber yield. In one embodiment, the plant includes potato.

[0042] As one implementation method, the overexpressed potato StMYC1 Biological materials for genes include potatoes StMYC1 Gene amplification primers, overexpression potatoes StMYC1 Recombinant expression vectors and overexpression potatoes StMYC1 One or more of the recombinant genes of microorganisms. As one embodiment, the potato... StMYC1 The primers for gene amplification include sequences as shown in SEQ ID NO:3 and SEQ ID NO:4.

[0043] SEQ ID NO: 3: 5′-GGGACTCTAGACGCGGATCCATGACGGACTATAGATT-3′;

[0044] SEQ ID NO: 4: 5′-TCTCCTTTACCCATGGTACCTCATCGCGATTCAGCAAA-3′;

[0045] As one implementation method, the overexpressed potato StMYC1 The recombinant gene expression vector includes a backbone vector. As one embodiment, the backbone vector includes a plasmid vector. As one embodiment, the plasmid vector includes the pCAMBIA1300 vector.

[0046] As one implementation method, the overexpressed potato StMYC1 The recombinant microorganisms of the gene include basic microorganisms. In one embodiment, the basic microorganisms include bacteria. In one embodiment, the bacteria include Agrobacterium. In one embodiment, the Agrobacterium includes Agrobacterium LBA4404.

[0047] In one embodiment, the plant described in this invention includes plants of the Solanaceae family. In another embodiment, the Solanaceae plant includes the potato.

[0048] This invention also provides potatoes StMYC1 Potatoes with gene or overexpression StMYC1 Application of genetically modified biological materials in potato breeding.

[0049] As one implementation method, the breeding method includes: overexpressing potato in potatoes. StMYC1 Genes that produce insect-resistant and / or higher-yielding potatoes.

[0050] Although existing technology has been disclosed SlMYC1 Its application in regulating tomato resistance to spider mites, and evolutionary analysis showing that potato StMYC1 and tomato SlMYC1 cluster in the same evolutionary branch (see...) Figure 1 (Phylogeny analysis of potato StMYC1 and its homologous proteins with tobacco and tomato MYC proteins), but the function of MYC transcription factors is species-specific, and their specific regulatory mechanisms are highly dependent on the species' own genetic background and metabolic network. More importantly, this invention discovered... StMYC1 Enhancing resistance to the beet armyworm by promoting solanine levels is consistent with... SlMYC1 The mechanisms by which terpenoids are regulated to combat spider mites are fundamentally different.

[0051] To further illustrate the present invention, the potato insect-resistant and yield-increasing gene provided by the present invention will be described below in conjunction with the accompanying drawings and embodiments. StMYC1 The invention describes in detail its encoded proteins and applications, but these descriptions should not be construed as limiting the scope of protection of this invention.

[0052] Example 1: Construction of the phylogenetic tree of the potato StMYC1 system

[0053] 1) Using AtMYC2~AtMYC5 (accession numbers of the TAIR (The Arabidopsis Information Resource) database: AtMYC2: AT1G32640; AtMYC3: AT5G46760; AtMYC4: AT4G17880; AtMYC5: AT1G01260) as the query sequence, in Spud Ten candidate potato MYC proteins were obtained by BLASTp alignment in the DB database, including: Soltu.DM.08G004140.1, Soltu.DM.08G022770.1, Soltu.DM.06G034400.1, Soltu.DM.01G035180.1, Soltu.DM.08G030060.1, Soltu.DM.08G005120.1, Soltu.DM.10G005300.1, Soltu.DM.01G035540.1, Soltu.DM.10G005270.1, and Soltu.DM.10G005280.1.

[0054] 2) In order to identify MYC transcription factors with conserved functions in the Solanaceae family in potatoes and to focus on the evolutionary relationships within the Solanaceae family, we used only Solanaceae sequences as references when constructing the phylogenetic tree, without including Arabidopsis sequences. This study used the amino acid sequences of SlMYC1 (Sol Genomics Network accession number: Solyc08G005050) and SlMYC2 (Sol Genomics Network accession number: Solyc08G076930) of the Solanaceae family tomato, and NtMYC2 (Sol Genomics Network accession number: Nta24G02070.1) and NtMYC3 (Sol Genomics Network accession number: Nta16G16350.1) of the tobacco MYC family as references. Using MEGA11 software, a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method. The Bootstrap test was set to 1000 replicates, and all other parameters used the software default values.

[0055] The phylogenetic tree construction results are as follows Figure 1 As shown, potato StMYC1 and tomato SlMYC1 cluster in the same evolutionary branch.

[0056] Example 2: Potato StMYC1 Gene cloning and plant expression vector construction

[0057] 2.1 Potato material cultivation:

[0058] 1) Select healthy, aseptic potato cultivar Desiree tissue culture seedlings.

[0059] 2) Inoculate stem segments with apical buds or 1-2 axillary buds into MS subculture medium; the MS subculture medium contains 4.43 g / L Murashige and Skoog (MS) basal salt (such as Phytotechnology Laboratories catalog number M519 or equivalent product), 30 g / L sucrose, and 8.4 g / L agar, and adjust the pH to 5.8±0.1 before sterilization.

[0060] 3) At (20±2)℃, photoperiod 16 h / 8 h, light intensity 50 μmol·m -2 ·s -1 Cultured under the specified conditions for 1 month.

[0061] 4) Transplant the potato plants into 16 cm diameter pots (1 plant per pot), repeat the biological process 3 times, and continue to cultivate them under the same environmental conditions for 6-8 weeks.

[0062] 5) Take healthy young leaves for RNA extraction.

[0063] 2.2 Total RNA extraction and cDNA synthesis:

[0064] All operations were performed in an RNase-free environment. Experimental equipment was treated with DEPC water overnight and then autoclaved for 15 minutes. Masks and gloves were worn throughout the operation, and operations were performed on ice whenever possible.

[0065] The specific steps are as follows:

[0066] 1) Take 200 mg of fresh potato leaf samples and quick-freeze them in liquid nitrogen.

[0067] 2) Pre-cool the mortar and pestle, and grind the young potato leaf samples into fine powder in liquid nitrogen.

[0068] 3) Quickly transfer the powder to a pre-cooled 1.5 mL centrifuge tube, immediately add 1 mL of Trizol reagent, vortex for 15 s to mix thoroughly, and let stand at room temperature for 5 min.

[0069] 4) Add 200 μL of chloroform to a 1.5 mL centrifuge tube, vortex vigorously for 15 s, let stand at room temperature for 5 min, and then incubate at 4℃ and 13000 mL. g Centrifuge for 15 min under the specified conditions.

[0070] 5) After centrifugation, carefully aspirate approximately 600 μL of the colorless aqueous phase from the top to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, gently invert to mix, let stand at room temperature for 10 min, and then incubate at 4℃ and 13000 mL. g Centrifuge again for 10 min under the same conditions.

[0071] 6) After the second centrifugation, carefully discard the supernatant, add 1 mL of pre-cooled 75% ethanol (prepared with DEPC water), gently invert to wash the precipitate, and incubate at 4°C and 13000 mL. g Centrifuge for 5 min under the specified conditions.

[0072] 7) Carefully remove the ethanol, and let it stand at room temperature for 5-10 minutes with the lid open to allow the residual ethanol to evaporate completely.

[0073] 8) Add 20-50 μL of RNase-free water to dissolve the RNA precipitate. Measure the RNA concentration and purity using a micro-volume nucleic acid quantification instrument. 260 / A 280 The ratio should be between 1.8 and 2.0. The remaining RNA sample can be stored at -80°C for a long time.

[0074] 9) cDNA synthesis was performed using the TranScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit.

[0075] The reaction system (20 μL) consisted of: 2 μg total RNA, 10 μL 2×TS Reaction Mix, and 1 μL Oligo(dT). 18 Primer (10 μM), 1 μL TransScript RT / RI Enzyme Mix, 1 μL gDNA Remover, and RNase-free water to a final volume of 20 μL.

[0076] The PCR reaction procedure was as follows: incubation at 42°C for 40 min, followed by heating at 85°C for 5 min to inactivate the reverse transcriptase. The resulting product is potato cDNA, which can be used immediately for subsequent experiments or stored at -20°C.

[0077] 2.3 StMYC1 Gene cloning:

[0078] Using the potato cDNA obtained in step 2.2 as a template, PCR amplification was performed using specific primers. StMYC1 The specific primer sequences for the gene coding region are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0079] The PCR reaction system (50 μL) consists of: 2 μL cDNA template, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 25 μL 2× Phanta Max Master Mix (or equivalent high-fidelity DNA polymerase), and double-distilled water to a final volume of 50 μL.

[0080] The PCR reaction program was as follows: 98℃ pre-denaturation for 1 min; 35 cycles (98℃ denaturation for 10 s, 55℃ annealing for 10 s, 72℃ extension for 1 kb / 5 s); 72℃ final extension for 5 min.

[0081] 2.4 Construction of plant expression vectors:

[0082] 1) The PCR amplification products were separated by 1% agarose gel electrophoresis (120 V, 20 min), and then the gel was excised and the target DNA band was recovered using a gel extraction kit (TransGold, EG101-01). The results are as follows: Figure 2 As shown, the concentration of the recovered product was determined using a micro-volume nucleic acid quantification instrument.

[0083] 2) Use restriction endonucleases Kpn I-HF and Bam The plant expression vector pCAMBIA1300 was double-digested with HI-HF at 37℃ for 3 h.

[0084] 3) The enzyme digestion product was purified using a DNA gel recovery kit to obtain a linearized vector fragment, and the concentration was determined.

[0085] 4) Purify StMYC1 The gene fragment was mixed with the linearized pCAMBIA1300 vector at a molar ratio of 3:1. 5 μL of 2×Seamless Assembly Mix (or equivalent homologous recombinase) was added to the mixture, and the volume was brought to 10 μL with double-distilled water. Ligation was completed by incubating in a 50°C water bath for 20 min.

[0086] 5) Add all the ligation products to 50 μL of *E. coli* DH5α chemocompetent cells, gently tumble to mix, and incubate on ice for 30 min. Next, heat shock at 42°C for 45 s, and then quickly transfer to ice and let stand for 2–3 min. Add 900 μL of antibiotic-free LB liquid medium to the tube and incubate at 37°C with shaking at 200 rpm for 45 min. Finally, spread an appropriate amount of the bacterial culture onto LB agar plates containing 50 mg / L kanamycin and incubate upside down at 37°C for 12–16 h.

[0087] 2.5 Screening and validation of positive clones:

[0088] 1) Select single-clone colonies and perform preliminary screening using colony PCR.

[0089] 2) Extract plasmids that initially show positive results, and use universal vector primers 35S-F and gene-specific primers. StMYC1 -F1、 StMYC1 -F2 was used for sequencing verification.

[0090] 35S-F (SEQ ID NO:5): 5′-CGACAGTGGTCCCAAAG-3′;

[0091] StMYC1 -F1 (SEQ ID NO:6):5′-AATGAAGAAGGAATGCT-3′;

[0092] StMYC1 -F2 (SEQ ID NO:7): 5′-GGGCTTCCCGGGCTTGGCGA-3′.

[0093] 3) Compare the sequencing results with the potato database in the NCBI database. StMYC1 The sequence was compared with the gene (GenBank ID: XM_006366182.2) to confirm its correctness.

[0094] It should be noted that the potato in this invention StMYC1 The gene sequence is derived from Desiree. Compared with the gene of double haploid DM in the NCBI database, there are slight differences in the nucleotide sequence, but the amino acid sequence is completely identical.

[0095] 4) Name the recombinant plasmid that has been verified to be correct through the above steps as pCAMBIA1300-35S:: StMYC1 It was then stored at -20℃ for later use. At this point, the target recombinant plant expression vector has been successfully constructed, and its physical map is as follows: Figure 3 As shown.

[0096] This invention successfully cloned potatoes. StMYC1 The gene, whose nucleotide sequence is shown in SEQ ID NO:2.

[0097] Example 3 StMYC1 Obtaining and identifying gene-overexpressing potato lines

[0098] 3.1 Agrobacterium-mediated potato genetic transformation:

[0099] 1) Select robust, sterile Desiree potato tissue culture seedlings, and inoculate stem segments with apical buds or 1-2 axillary buds onto MS subculture medium. Incubate at (20±2)℃, with a photoperiod of 16 h / 8 h and a medium of 50 μmol·m⁻²·m⁻²·cm⁻¹. -2 ·s -1 Cultured under white light. The MS subculture medium contained 4.43 g / L MS basal salt, 30 g / L sucrose, and 8.4 g / L agar.

[0100] 2) The recombinant plant expression vector pCAMBIA1300- constructed in Example 1 was subjected to a freeze-thaw method. StMYC1 Transformed into Agrobacterium tumefaciens LBA4404 competent cells.

[0101] 3) Transformants were screened on LB solid medium containing 50 mg / L kanamycin (Kan) and 50 mg / L rifampin (Rif).

[0102] 4) Take healthy potato tissue culture seedlings that have been cultured for 4 weeks, and under aseptic conditions, cut 3-5 mm stem segments without axillary buds. Place them in MS pre-culture medium for 2-3 days, with the same culture conditions as in step 1). The MS pre-culture medium formula is the same as the MS subculture medium.

[0103] 5) Use recombinant Agrobacterium (OD) 600The pre-cultured stem segments were soaked in an infection solution containing 4.43 g / L MS basal salt, 40 g / L sucrose, 10 g / L glucose, 2.5 mg / L 6-benzylaminopurine (6-BA), 0.6 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), and 50 μmol / L acetosyringone (AS) for 15 min.

[0104] 6) After blotting off excess bacterial solution from the stem segment with sterile filter paper, the explants were placed in a 2 cm × 9 cm culture dish for co-culture. The culture dish was pre-filled with two layers of sterile filter paper and 2 mL of infection culture medium, with no air bubbles between the filter papers. The culture was then incubated in the dark at 23°C for 2-3 days.

[0105] 7) Transfer the stem segments to regeneration medium and culture for 7 days (culture conditions are the same as in step 1) to induce callus formation, such as... Figure 4 As shown in Figure A. The recovery medium contains 4.43 g / L MS basal salt, 30 g / L sucrose, 8.4 g / L agar, 2.5 mg / L 6-BA, 0.6 mg / L 2,4-D, 300 mg / L cefotaxime sodium (Cef), and 100 mg / L termethin (Tmt), with a pH of 5.8±0.1.

[0106] 8) Transfer the potato stem segments after recovery culture to a resistant callus induction medium containing hygromycin. Inoculate 30 callus stem segments per dish and screen for resistant callus for two weeks. Observe and record the callus survival rate.

[0107] 9) Transfer the callus tissue to differentiation medium, changing the medium every 3 weeks until budding occurs. The differentiation medium contains 4.43 g / L MS basal salt, 30 g / L sucrose, 8.4 g / L agar, 0.01 mg / L naphthaleneacetic acid (NAA), 1 mg / L zeatin (ZT), 300 mg / L Cef, 100 mg / L Tmt, 7.5 mg / L hygromycin (Hyg), and pH 5.8±0.1.

[0108] 10) When the adventitious shoots reach 1 cm in length, transfer them to the resistant seedling rapid propagation medium (MS subculture medium containing 7.5 mg / L Hyg). When the seedlings reach 5 cm in height, perform qRT-PCR identification. The differentiation of resistant shoots should be as follows: Figure 4 As shown in Figure B, the regenerated buds take root as follows: Figure 4 As shown in C.

[0109] 11) Transplant successfully identified and well-rooted plants into sterilized nutrient soil. Transplantation of transgenic plants is as follows: Figure 4 As shown in D, the transplanted plants were numbered and cultured.

[0110] 3.2 StMYC1 Molecular identification of gene overexpression lines:

[0111] 1) After 6 weeks of growth following transplanting, young leaves of wild-type potatoes and transgenic lines were collected, and total RNA was extracted and cDNA was synthesized according to the method described in 2.2 of Example 2.

[0112] 2) Detection was performed using the ABI QuantStudio 3 real-time quantitative PCR instrument. This was for the target gene. StMYC1 Design specific primers (forward primer: SEQ ID NO:8, reverse primer: SEQ ID NO:9) to target the potato elongation factor gene. EF-1α (Accession number: NM_001288491.1, forward primer: SEQ ID NO:10, reverse primer: SEQ ID NO:11) was used as an internal reference gene, and three technical replicates were set up for each reaction.

[0113] SEQ ID NO: 8: 5′-TTCTCAATGTGAACGGGCCA-3′;

[0114] SEQ ID NO:9: 5′-CCAAGCTCGACTACACCGTT-3′;

[0115] SEQ ID NO: 10: 5′-ATTGGAAACGGATATGCTCCA-3′;

[0116] SEQ ID NO: 11: 5'-TCCTTACCTGAACGCCTGTCA-3'.

[0117] 3) qRT-PCR reaction system (20 μL): 10 μL 2× SYBR Select Master Mix (Applied Biosystems or equivalent reagents), 0.8 μL cDNA template, 0.4 μL StMYC1 Forward primer (10 μM), 0.4 μL StMYC1 Reverse primer (10 μM) (or 0.4 μL) EF-1α Forward primer + 0.4 μL EF-1α (Reverse primer), and make up to 20 μL with ddH2O.

[0118] 4) Reaction program: 50℃ for 2 min; 95℃ for 2 min (pre-denaturation); 40 cycles (95℃ for 15 s, 60℃ for 1 min); finally, perform melting curve analysis (95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s).

[0119] Use 2-ΔΔCt Method calculation StMYC1 The relative expression level of the gene in the transgenic line relative to the wild type. The identification results are as follows: Figure 5 As shown, it can be seen that among the obtained transgenic lines... StMYC1 The expression level was significantly higher than that of the wild type, successfully obtaining... StMYC1 Overexpression strains.

[0120] Example 4 StMYC1 Evaluation of resistance of overexpression lines to beet armyworm

[0121] 4.1 Insect feeding experiment:

[0122] Wild-type potatoes (Desiree) StMYC1 Overexpression lines ( StMYC1-OE ), at (20±2)℃, photoperiod 8 h / 16 h, 300 μmol·m -2 ·s -1 After culturing under white light for 70 days, leaves from each strain were randomly selected and placed at the bottom of the petri dish with moist qualitative filter paper.

[0123] Gently pick out the uniformly sized second-instar beet armyworms with a paintbrush. Spodoptera exigua Seven larvae were introduced into a petri dish containing leaves, with at least three replicates for each strain.

[0124] 4.2 Observation and data analysis of insect resistance phenotype:

[0125] Three days after the feeding treatment, the following observations were recorded:

[0126] 1) Degree of damage to plants / leaves: Photos were taken of the leaves in each petri dish, and the results are as follows: Figure 6 As shown in Figure A.

[0127] 2) Insect growth status: Carefully collect surviving larvae, photograph their size, and weigh them using a precision balance. The results are as follows: Figure 6 As shown in B and C.

[0128] Experimental results showed that compared with wild-type potatoes (Desiree), consuming... StMYC1-OE The average weight of beet armyworm larvae on the leaves of the strain decreased by 28.8%. P <0.05), clearly visible to the naked eye. StMYC1-OE The leaf damage area of ​​the strain was smaller than that of the Desiree strain.

[0129] Example 5 StMYC1 Determination of α-solanine and α-carbohydrate content in overexpression lines

[0130] 5.1 Sample preparation:

[0131] To simulate pest stress, leaves of potato tissue culture seedlings cultured under long-day conditions for 30 days were treated with 100 μM MeJA (containing 0.01% Triton X-100). After 44 h of treatment, the aboveground parts of the plants were collected and flash-frozen with liquid nitrogen.

[0132] 5.2 Solution preparation:

[0133] 1) Formic acid-ammonium formate buffer solution: Weigh 0.063 g of ammonium formate into a beaker, add an appropriate amount of water to dissolve it, and then transfer it to a 1000 mL volumetric flask. Then transfer 1 mL of formic acid into the same volumetric flask and add water to make up to the mark.

[0134] 2) Acidification of acetonitrile: Transfer 10 mL of formic acid to a 1000 mL volumetric flask and add acetonitrile to bring the volume to the mark.

[0135] 3) Methanol aqueous solution (50%, volume fraction): Transfer 500 mL of methanol into a 1000 mL volumetric flask and add water to make up to the mark.

[0136] 5.3 Preparation of standard solutions:

[0137] 1) α-Solanine Standard Stock Solution (4.00 mg / mL): Accurately weigh 40.00 mg of α-solanine standard into a beaker, dissolve it in a small amount of acetonitrile, transfer it to a 10.0 mL volumetric flask, add acetonitrile and dilute to the mark to prepare a standard working solution with a concentration of 5.00 mg / mL.

[0138] 2) α-Carbocyanine Standard Stock Solution (4.00 mg / mL): Accurately weigh 40.00 mg of α-carbocyanine standard into a beaker, dissolve in a small amount of acetonitrile, transfer to a 10.0 mL volumetric flask, add acetonitrile and dilute to the mark to prepare a standard working solution with a concentration of 5.00 mg / mL. All stock solutions should be stored at -20℃ and have a shelf life of 3 months.

[0139] 3) Accurately pipette 4.00 mg / mL of α-solanine standard stock solution and dilute it sequentially to prepare a series of standard solutions at 2.00 mg / mL, 1.00 mg / mL, 0.25 mg / mL, and 0.05 mg / mL. Accurately pipette 4.00 mg / mL of α-carboxine standard stock solution and dilute it sequentially to prepare a series of standard solutions at 2.00 mg / mL, 1.00 mg / mL, 0.50 mg / mL, 0.25 mg / mL, and 0.10 mg / mL.

[0140] 5.4 Sample extraction and purification:

[0141] Weigh 0.5 g (accurate to 0.0001 g) of the sample into a 50 mL stoppered centrifuge tube, add 5.0 mL of water and vortex for 1 min. Then accurately add 5.0 mL of acidified acetonitrile (3.2.2), vortex for 3 min, and then add 2.0 g of anhydrous magnesium sulfate and 1.0 g of anhydrous sodium acetate to the centrifuge tube. Shake vigorously manually for 30 s and then vortex for 1 min. Place the centrifuge tube in a centrifuge and centrifuge at 10,000 rpm for 8 min at 4 °C. Transfer all the supernatant to a 10 mL volumetric flask, add acidified acetonitrile to the mark, and then transfer 100 µL of the supernatant to a 5 mL volumetric flask, add methanol-water solution to the mark, and filter 1 mL through a microporous membrane (0.22 µm, polytetrafluoroethylene) for analysis.

[0142] 5.5 Instrument Measurement:

[0143] 1) Reference conditions for liquid chromatography:

[0144] a) Chromatographic column: C18 liquid chromatography column, 100 mm × 2.1 mm, particle size 1.8 µm;

[0145] b) Mobile phase: A is formic acid-ammonium formate buffer solution, B is acetonitrile, and the gradient elution program is shown in Table 1:

[0146] Table 1 Gradient elution program

[0147]

[0148] c) Flow rate: 0.4 mL / min;

[0149] d) Column temperature: 35 ℃;

[0150] e) Injection volume: 5 μL.

[0151] 2) Mass spectrometry reference conditions:

[0152] a) Ion source: Electrospray ionization (ESI);

[0153] b) Monitoring method: Multiple response monitoring (MRM);

[0154] c) Scanning mode: Positive ion mode;

[0155] d) Spray voltage: 5500 V;

[0156] e) Ion source temperature: 550 ℃;

[0157] f) Curtain gas: 35 psi;

[0158] g) Collider: 9 psi.

[0159] The qualitative and quantitative ions of the target analyte, as well as their collision energies, are shown in Table 2.

[0160] Table 2 Mass Spectrometry Parameters

[0161]

[0162] In Table 2, This indicates the quantitative ion, which is the core ion used for the quantitative analysis of the target compound (α-solanine, α-carboxine);

[0163] Experimental results are as follows Figure 7 As shown in A and B and Tables 3-4, it can be seen that... StMYC1-OE The α-solanine content in the strain was 7.46 times that of the wild type (Desiree), and the α-carbohydrate content was 4.99 times that of the wild type.

[0164] Table 3. Results of α-solanine content determination in different groups

[0165]

[0166] Table 4. Results of α-carbocyanine content determination in different groups

[0167]

[0168] In addition, the quantitative ion-pair peak area determination results for α-solanine and α-carboxine are as follows: Figures 8-9 As shown, it can be seen that StMYC1-OE The peak area of ​​the strain was significantly larger than that of the wild-type control. This result quantitatively confirms that... StMYC1 Overexpression of the gene increases the accumulation of solanine in potatoes.

[0169] Example 6 StMYC1 Analysis of the in vitro tuber formation ability of overexpression lines

[0170] 6.1 In vitro tuber formation induction experiment

[0171] Select wild-type potatoes (Desiree) and StMYC1 Overexpression lines ( StMYC1-OE Tissue culture seedlings were used as experimental materials, and the experiments were conducted at (20±2)℃, with a photoperiod of 16 h / 8 h and a concentration of 100 μmol·m⁻¹. -2 ·s -1 Cultured under white light conditions.

[0172] On day 30 of tissue culture, single stem segments were selected and transferred to a special medium for tuber induction. The tuber seedlings were then cultured under long-day conditions for another 60 days, followed by 35 days in complete darkness. To ensure reproducibility and reliability, 15 biological replicates were established for each line. The tuber induction medium contained 4.43 g / L MS basal salt, 80 g / L sucrose, and 8 g / L agar.

[0173] 6.2 Observation and Data Analysis of In Vitro Tuber Formation Phenotype

[0174] 1) Statistics on tuber formation on plants

[0175] After being transferred to dark conditions for 5 days, the tuber formation status of each plant in the test tube was observed one by one, and the number of effective tubers was accurately counted and recorded every 10 days until the induction period ended.

[0176] 2) Tuber yield determination

[0177] All tubers produced by each individual plant of each strain are harvested. First, the overall shape and size differences of the tubers are recorded by taking photos. Then, the total weight of the tubers of each individual plant is weighed using a precision balance, which is used as an indicator for yield evaluation.

[0178] Experimental results are as follows Figure 10 As shown, compared with wild-type potato (Desiree), the overexpression lines ( StMYC1-OE Under in vitro induction culture conditions, the effective tuber formation rate increased by 16.3%, and the total yield per tuber increased by 60.1%. P <0.05).

[0179] The above results collectively indicate that StMYC1 Overexpression of the gene significantly improved both potato resistance to the beet armyworm and significantly increased tuber yield. Therefore, this invention confirms that... StMYC1 The gene plays a dual crucial role in potato insect resistance and yield formation. Its encoded proteins and genes can be used for research and industrial production to simultaneously improve crop insect resistance and yield through transgenic technology, providing important genetic resources and technical support for crop breeding.

[0180] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Potatoes StMYC1 Potatoes with gene or overexpression StMYC1 The application of genes in biomaterials is characterized by, The potato StMYC1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:1, and the application is: overexpression of the potato. StMYC1 Genes that enhance potato resistance to beet armyworm and / or increase potato yield.

2. The application as described in claim 1, characterized in that, The potato StMYC1 The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. The application as described in claim 1, characterized in that, The yield includes the effective tuber formation rate and / or tuber yield.

4. The application as described in claim 1, wherein the overexpressed potato StMYC1 Biological materials for gene expression include overexpressing potatoes. StMYC1 Recombinant vectors and / or overexpression of genes in potatoes StMYC1 Microorganisms that regenerate genes.

5. The application as described in claim 4, characterized in that, The overexpressed potato StMYC1 The gene recombination vector includes a backbone vector; the backbone vector includes the pCAMBIA1300 vector.

6. The application as described in claim 4, characterized in that, The overexpressed potato StMYC1 The recombinant microorganisms of the gene include basic microorganisms; said basic microorganisms include Agrobacterium LBA4404.

7. Potatoes StMYC1 Potatoes with gene or overexpression StMYC1 The application of genetically modified biological materials in potato breeding is characterized by, The StMYC1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:1.

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