Application of overexpression of the StBBX17 gene in improving the cold resistance of potatoes

Abstract

This invention provides the application of overexpression of the StBBX17 gene in improving the cold resistance of potatoes, belonging to the field of genetic engineering technology. The StBBX17 gene of this invention is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the StBBX17 gene is shown in SEQ ID NO.2. The StBBX17 gene of this invention can significantly enhance the cold resistance of potatoes and can be applied to potato low-temperature resistance breeding and variety improvement, providing technical support for potato resistance breeding and having significant importance and wide application value for promoting the development of the potato industry.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of overexpression of the StBBX17 gene in improving the cold resistance of potatoes. Background Technology

[0002] Potato (Solanum tuberosum L.) is the world's fourth largest food crop and the most important tuber crop, widely cultivated and consumed worldwide. It plays a vital role in ensuring global food security and meeting the increasing food demands of future population growth. Frost damage is one of the major environmental stressors, significantly impacting plant growth, development, productivity, and geographical distribution. With the implementation of the potato staple food strategy, fallow winter fields in the south will be the main contributor to the increase in potato acreage; however, frost damage is the most significant meteorological disaster in this region. Common potato cultivars are almost entirely intolerant of frost; once a cold snap occurs, agricultural growth is severely affected, seriously hindering the development and utilization of fallow winter fields in the south and significantly impacting the potato industry. Therefore, it is essential to explore potato resistance genes and cultivate new potato varieties with cold resistance capabilities. Summary of the Invention

[0003] In view of this, the present invention provides the application of overexpression of the StBBX17 gene in improving the cold resistance of potatoes, so as to solve the above problems.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides the application of biological materials overexpressing the StBBX17 gene or overexpressing the StBBX17 gene in any of the following:

[0006] (1) Application in improving the cold resistance of potatoes;

[0007] (2) Application in the cultivation of cold-resistant potato germplasm;

[0008] The nucleotide sequence of the StBBX17 gene is shown in SEQ ID NO.1.

[0009] This invention also provides the application of the StBBX17 gene-encoded protein in improving the cold resistance of potatoes. The amino acid sequence of the StBBX17 gene-encoded protein is shown in SEQ ID NO.2.

[0010] The present invention also provides a method for improving the cold resistance of potatoes, comprising the following steps:

[0011] S1. PCR amplification of the StBBX17 gene;

[0012] S2. After digesting the PFGC1008 cloning vector with enzymes, the linearized PFGC1008 vector was obtained; the linearized PFGC1008 vector was ligated with the StBBX17 gene fragment to obtain the overexpression vector of the StBBX17 gene.

[0013] S3. The overexpression vector of the StBBX17 gene was transformed into Agrobacterium GV3101 to obtain recombinant Agrobacterium;

[0014] S4. Recombinant Agrobacterium tumefaciens was used to infect low-temperature sensitive potato tubers, and the tubers were cultured to obtain potato lines overexpressing the StBBX17 gene.

[0015] Preferably, the primers for amplifying the StBBX17 gene are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0016] Preferably, the PFGC1008 cloning vector is double-digested with restriction endonucleases KpnI and AscI.

[0017] Preferably, when culturing potato tubers, the tubers are first desiccated and then screened for resistance. When the differentiated regenerated plants grow to 2-3 cm in length, the regenerated seedlings are cut from the callus tissue and inserted into the rooting medium for culture.

[0018] The preferred culture medium for sterilization is: MS + 4 mg / L ZT + 0.2 mg / L IAA + 250 mg / L TIM.

[0019] The preferred culture medium for resistance screening is: MS + 4 mg / L ZT + 0.2 mg / L IAA + 250 mg / L TIM + 50 mg / L Kan.

[0020] Preferably, the rooting medium is: MS + 0.4 mg / L IAA + 250 mg / L TIM + 50 mg / L Kan.

[0021] By adopting the above technical solution, the present invention has the following beneficial effects: The StBBX17 gene of the present invention is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the StBBX17 gene is shown in SEQ ID NO.2. The StBBX17 gene of the present invention can significantly enhance the cold resistance of potatoes, and can be applied to potato low-temperature resistance breeding and variety improvement, providing technical support for potato resistance breeding, and has important significance and wide application value for promoting the development of the potato industry. Attached Figure Description

[0022] Figure 1To compare the cold resistance of potato lines overexpressing StBBX17 with wild potatoes; where A represents the expression of the StBBX17 gene in WT, OE#3, and OE#23 plants; B represents the electrolyte osmotic rate of WT, OE#3, and OE#23 plants at 0℃ and -1℃; C represents the phenotype of WT, OE#3, and OE#23 plants at room temperature; D represents the phenotype of WT, OE#3, and OE#23 plants after treatment at -1℃ for 14 h and recovery to normal conditions for 3 days; different letters indicate significant differences of P < 0.05. Detailed Implementation

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1. Cloning of the potato StBBX17 gene and construction of an overexpression vector

[0025] The StBBX17 gene is derived from potato, and its nucleotide sequence is shown in SEQ ID NO.1, while the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0026] The coding sequence of the StBBX17 gene (SEQ ID NO.1):

[0027] ATGTGTAGTGGAAGAAGAGAGGGTGAAGAAGAAACTACCTCAATTTCGTATTGCAAAGGGCCATTGAAAGAGGGAGAATCCATAATTAGGTCTACAATTTCTTGTGCGCTCTGTAGTTCAGAGGCCTCTGTTTATTGTGAAGCTGATAATGCTTTTCTTTGCCGGAAATGCGATAGATCGGTTCACGGAGCTAATTT CTTGGCTCAGAGACATATAAGGTGCCTTCTTTGCAGCGTTTGCCGGAAAACAACGTGGCGGTTTCTGATCGGAACTTCGTCGGAGCTGATTTTGCCGACGATTGCCGGTTTGGAACAGAGAAACAGAAGGAGAAGTGCAGATTCTGAGACGATGGACTACAGAACGACGCCGCAAGAGCTTTTTCCTGTTTATTTGA.

[0028] The amino acid sequence of the protein encoded by the StBBX17 gene (SEQ ID NO.2):

[0029] MCSGRREGEEETTSISYCKGPLKEGESIIRSTISCALCSSEASVYCEADNAFLCRKCDRSVHGANFLAQRHIRCLLCSVCRKTTWRFLIGTSSELILPTIAGLEQRNRRRSADSETMDYRTTPQELFLFI.

[0030] To investigate the response function of StBBX17 to cold stress in potatoes, the StBBX17 gene was first cloned from the potato genome. Based on the coding region sequence analysis, specific primers StBBX17-F and StBBX17-R were designed, with restriction enzyme sites (KpnI and AscI) on the primers.

[0031] The sequence of the upstream primer StBBX17-F is as follows:

[0032] 5'-ttacaattaccatggggcgcgccATGTGTAGTGGAAGAAGAGAGG-3' (SEQ ID NO. 3);

[0033] The sequence of the downstream primer StBBX17-R is as follows:

[0034] 5'-aacatcgtatgggtaggtacc AATAAACAGGAAAAGCTCTTGC-3' (SEQ ID NO.4). The PCR amplification system is shown in Table 1; the PCR amplification program is shown in Table 2.

[0035] Table 1 PCR amplification system

[0036]

[0037] Table 2 PCR amplification program

[0038]

[0039] After amplification, agarose gel electrophoresis was performed. After confirming that the bands were correct, the gel was cut, recovered, and purified.

[0040] Extraction and double enzyme digestion of PFGC1008 plasmid DNA: The pFGC1008 vector bacterial culture stored in the laboratory was removed from -80℃ and allowed to thaw completely. 200 µL of the culture was then added to LB liquid medium containing 50 mg / L chloramphenicol. The culture was incubated overnight at 37℃ and 220 rpm for 16 hours before plasmid extraction. The extracted plasmid was then double-digested with restriction endonucleases KpnI and AscI. After adding the system shown in Table 3 to ice, the digestion was carried out at 37℃ for 3 hours. Immediately afterward, 1 / 10 of gelloading Dye (6×) was added to terminate the reaction. Electrophoresis was then performed on a 1% agarose gel, and the desired target band was recovered.

[0041] Table 3. Double enzyme digestion reaction system

[0042]

[0043] The StBBX17 gene amplification product was ligated with the linearized product of the pFGC1008 empty vector using homologous recombination to form a new plasmid containing the target gene. This step was performed using a one-step recombination kit (ClonExpress® II OneStep Cloning Kit, Vazyme). The reaction system is shown in Table 4. The recombination reaction was carried out at 37°C for 30 min, followed by an ice-water bath for 5 min to ligate the enzyme digestion products.

[0044] Table 4 ClonExpress® II Recombinant Reaction System

[0045]

[0046] The ligation product was then transformed into competent E. coli DH5α cells. The DH5α competent cells were removed from -80℃ and quickly placed on ice. After 5 min, when the bacterial block thawed, 5 μL of the above ligation product was added and the mixture was gently stirred by tapping the bottom of the EP tube. The mixture was then placed on ice for 25 min. The cells were then heat-shocked in a 42℃ water bath for 45 seconds, quickly returned to ice, and placed on ice for 2 min. 700 μL of antibiotic-free LB medium was added, mixed, and the cells were incubated at 37℃, 200 rpm for 60 min. The cells were then collected by centrifugation at 5000 rpm for 1 min. Approximately 100 μL of the supernatant was collected, and the cells were gently resuspended by pipetting and spread onto LB agar plates containing antibiotics (20 μg / mL Rif + 50 μg / mL Chl). The plates were then inverted and incubated overnight at 37℃.

[0047] Positive clone identification: Single colonies were picked for colony PCR, and positive clones were sequenced. The ligation vector with correct sequencing was named pMD-StBBX17. Subsequently, the plasmid was extracted, and the recombinant plasmid and the vector pFGC1008 containing the CaMV35S promoter were digested with Kpn I and Sac I. Homologous recombination ligation was performed in one step using the ClonExpress® II One Step Cloning Kit and Vazyme kit. The recombination reaction system was the same as in Table 2. The colonies were then transformed into E. coli and screened with chloramphenicol to obtain single colonies. The plasmid with correct sequencing was the StBBX17 overexpression vector.

[0048] Example 2. Agrobacterium-mediated potato genetic transformation

[0049] The StBBX17 overexpression vector was transformed into Agrobacterium GV3101. The procedure was as follows: GV3101 Agrobacterium competent cells stored at -80℃ were thawed on ice. 1 μL of plasmid DNA to be transformed was added to every 50 μL of competent cells. The mixture was gently mixed and incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and in an ice bath for 5 min. 700 μL of antibiotic-free LB liquid medium was added and cultured at 28℃ with shaking for 2 h. Approximately 100 μL of the bacterial culture was then plated onto LB plates containing chloramphenicol and ciprofloxacin. The plates were inverted and cultured at 28℃ for 3 days. Positive Agrobacterium clones were obtained by colony PCR identification.

[0050] The steps for infecting the low-temperature-sensitive potato variety "Desiree" with recombinant Agrobacterium are as follows:

[0051] Soak miniature potatoes in 1% sodium hypochlorite solution overnight, peel and cut into 1-2 mm thick slices, then soak in prepared Agrobacterium EH105 bacterial suspension for 10 minutes. Remove with sterile forceps, place on sterilized filter paper, blot off excess bacterial suspension, and transfer to MS co-medium containing 4 mg / L ZT and 0.2 mg / L IAA. Incubate in the dark at 28°C for 2 days, or until a faint ring of bacterial spots appears around the potato slices.

[0052] Wash the co-cultured potato pieces three times with sterile distilled water, then soak them in sterile distilled water containing 250 mg / L TIM for 15 min. Place them on sterilized filter paper and wait for the surface of the potato pieces to dry. Then transfer them to MS medium containing 4 mg / L ZT + 0.2 mg / L IIAAA + 250 mg / L TIM for 7 days of desterilization culture. Cover the culture dish with 6 layers of gauze, removing one layer of gauze each day. Gradually increase the light intensity. The culture conditions are 24℃, 1500 Lux, and a photoperiod of 16 h light / 8 h dark.

[0053] The tubers were transferred to MS medium containing 4 mg / L ZT + 0.2 mg / L IAA + 250 mg / L TIM + 50 mg / L Kan for resistance selection culture. When the differentiated regenerated plants grew to 2-3 cm in length, the regenerated seedlings were cut from the callus tissue and inserted into rooting medium containing 0.4 mg / L IAA + 250 mg / L TIM + 50 mg / L Kan for rooting culture.

[0054] After successful growth, secondary rooting was performed, and the rooted resistant seedlings were then transferred to an antibiotic-free culture medium for further cultivation. The resulting resistant seedlings had robust stems and strong roots. The resistant seedlings were propagated for two generations in the antibiotic-free medium. After approximately four weeks of growth, the seedlings were hardened off for three days before being transferred to a greenhouse for further cultivation, yielding 18 transgenic potato lines. Verification using 35S primers and gene expression levels showed that the StBBX17 gene expression was significantly upregulated in seven lines, indicating the acquisition of seven positive transgenic lines.

[0055] Example 3. Expression of the StBBX17 gene in transgenic potatoes

[0056] To investigate the expression of the StBBX17 gene in wild-type and transgenic potatoes, quantitative real-time PCR (qRT-PCR) analysis was performed. Total RNA was isolated from leaves of wild-type potatoes and two StBBX17 transgenic lines using the MiNiBEST Universal RNA Extraction Kit (TaKaRa). Reverse transcription was performed using StarScript II RT Mix (GeneStar). Real-time quantitative PCR was conducted on a CFX96 detection system (Bio-Rad, Hercules). Each qRT-PCR reaction contained 1 µL of cDNA, 0.5 µL each of forward and reverse primers, 10 µL of qPCR Master Mix, and 8 µL of ddH2O. β-tubulin was selected as an internal control gene.

[0057] Table 5 Primers for Real-Time PCR

[0058]

[0059] Using Ct(2) -ΔΔCt The relative gene expression levels were calculated using the method shown in the figure. Figure 1 As shown in A in the figure. The results showed that the expression of the StBBX17 gene in the StBBX17 transgenic potato line was higher than that in wild potatoes.

[0060] Example 4. Verification of cold resistance in transgenic potatoes

[0061] To investigate the StBBX17 gene's response to cold stress in potatoes, two transgenic lines with the highest StBBX17 gene expression levels, StBBX17-OE-3 and StBBX17-OE-23, were selected for cold resistance phenotypic identification (electrolyte osmotic pressure measurement). The steps were as follows: transgenic potatoes were planted in a greenhouse, with wild-type potatoes as a control. All plants underwent frost tolerance testing 30 days after planting, and phenotypic surveys were conducted.

[0062] Electrolyte permeability determination method: Take mature leaves, rinse them thoroughly with distilled water, and place them at the bottom of a capped test tube (25 × 150 mm), one leaf per tube, with the petiole facing upwards and the leaf surface facing outwards. Place the test tubes in a water bath (the medium in the water bath is a 1:1 mixture of ethylene glycol and distilled water). Set the initial temperature of the water bath to 0℃, maintain it for 30 min, then lower it to -0.5℃ and maintain it for another 30 min, with a cooling rate of 0.5℃ for 30 min. –1 The temperature was lowered from -0.5℃ to -1℃ and held for 30 minutes. A small piece of ice was added to each tube, and the temperature was held for another 30 minutes. The temperature was then lowered to -1.5℃ and held for 1 hour. This was repeated until the temperature reached the target temperature, and samples were taken at each temperature. Immediately after reaching each temperature point, the test tubes were removed and placed in ice, then thawed overnight in a 4℃ freezer. The next day, the thawed leaves were cut into 5 mm strips with a sharp blade, placed in glass test tubes, and 25 mL of distilled water was added. The tubes were evacuated to a vacuum of 0.1 MPa for 6 minutes, and then shaken at 220 rpm. –1 The test tubes were vibrated at a certain speed for 1 hour, and the conductivity R1 was measured after standing. The tubes were then capped and placed in an autoclave at 121℃ for 15 minutes, followed by cooling to room temperature to measure the conductivity R2. Electrolyte leakage rate (%) = R1 / R2 × ​​100%. The average of the three electrolyte leakage rates corresponding to each temperature was substituted into the Logistic equation for nonlinear fitting. The inflection point of the equation is the half-lethal temperature (LT50) of the material, used to represent the material's freeze resistance. Freeze resistance was assessed at 0 days and 12 days of cold acclimation. The cold acclimation capacity was calculated as the LT50 after acclimation minus the LT50 before acclimation. All samples were assessed in triplicate.

[0063] The results showed that at 0℃ and -1℃, the electrolyte osmotic rates of the three transgenic lines were significantly lower than those of the wild-type (WT) plants. Figure 1 (B) Compared to wild-type plants, under normal growth conditions, neither transgenic line showed morphological changes (B). Figure 1(C) Two overexpression lines and WT plants were subjected to low-temperature treatment (-1℃ for 14 h, followed by a 3-day recovery period). Compared with WT, the cold tolerance of the StBBX17 overexpression line was significantly improved. After cold treatment, the leaf damage area of ​​the overexpression line (StBBX17-OE) was lower than that of WT. Figure 1 (D in the text). These results indicate that StBBX17 plays a positive regulatory role in potato cold resistance.

[0064] As can be seen from the above embodiments, the present invention provides the application of overexpression of the StBBX17 gene in improving the cold resistance of potatoes. Overexpression of the StBBX17 gene can improve the cold resistance of potatoes and can be applied to potato low-temperature resistance breeding and variety improvement.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Overexpression StBBX17 Gene or overexpression StBBX17 Application of genetic biomaterials in any of the following: (1) Application in improving the cold resistance of potatoes; (2) Application in the cultivation of cold-resistant potato germplasm; The StBBX17 The nucleotide sequence of the gene is shown in SEQ ID NO. 1; The biomaterial is a carrier or Agrobacterium.

2. StBBX17 Application of gene-encoded proteins in improving the cold resistance of potatoes StBBX17 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

3. A method for improving the cold resistance of potatoes, characterized in that, Includes the following steps: S1. PCR amplification StBBX17 Gene; S2. After double digestion of the PFGC1008 cloning vector, the linearized PFGC1008 vector was obtained; the linearized PFGC1008 vector was then combined with... StBBX17 Gene segment linking, to obtain StBBX17 Gene overexpression vectors; S3. Will StBBX17 The gene overexpression vector was transformed into Agrobacterium GV3101 to obtain recombinant Agrobacterium; S4. Recombinant Agrobacterium was used to infect low-temperature sensitive potato tubers, and the tubers were cultured to obtain overexpressed [products]. StBBX17 Potato strains with specific genes; StBBX17 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The method according to claim 3, characterized in that, Amplification StBBX17 The primers for the gene are shown in SEQ ID NO.3 and SEQ ID NO.

4.

5. The method according to claim 3, characterized in that, The PFGC1008 cloning vector was double-digested with restriction endonucleases KpnI and AscI.

6. The method according to claim 3, characterized in that, When culturing potato tubers, first perform sterilization culture, then conduct resistance screening. When the differentiated regenerated plants grow to 2-3 cm in length, cut off the regenerated seedlings from the callus tissue and insert them into the rooting medium for culture.

7. The method according to claim 6, characterized in that, The culture medium for sterilization was: MS + 4 mg / L ZT + 0.2 mg / L IAA + 250 mg / L TIM.

8. The method according to claim 6, characterized in that, The culture medium for resistance selection was: MS + 4 mg / L ZT + 0.2 mg / L IAA + 250 mg / L TIM + 50 mg / L Kan.

9. The method according to claim 6, characterized in that, The rooting medium was: MS + 0.4 mg / L IAA + 250 mg / L TIM + 50 mg / L Kan.

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