Application of StMSSP2 gene in reducing sugar improvement of potato tuber

Abstract

The application discloses application of StMSSP2 gene in reducing sugar improvement of potato tubers, and comprises the following steps: overexpression of the StMSSP2 gene can significantly reduce the reducing sugar content of the potato tubers. Meanwhile, overexpression of the StMSSP2 gene can positively regulate growth and development of potato plants, and provides a new gene resource for molecular breeding of potato processing high-quality varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of the StMSSP2 gene in the improvement of reducing sugars in potato tubers. Background Technology

[0002] potato( Solanum tuberosum Potatoes (L.) are an important global food, vegetable, and cash crop. Reducing sugar content is one of the core indicators of potato quality, directly affecting the processing quality (such as the color and taste of fried potato chips and fries) and nutritional quality. Excessive reducing sugar content leads to the Maillard reaction during frying, producing brownish substances and harmful compounds, reducing the commercial value of processed products. Conversely, insufficient reducing sugar content affects the flavor and nutritional value of potatoes. The national standard GB / T 31784-2015, "Grading and Inspection Procedures for Commercial Potatoes," specifies detailed quality requirements for processed potatoes. The requirements for reducing sugar content differ slightly between potato chip and fries processing: less than 0.2% for chips and less than 0.25% for fries (https: / / std.samr.gov.cn / gb / search / gbDetailed). Currently, most potato varieties on the market are primarily for fresh consumption, generally having high reducing sugar content and low dry matter content, resulting in lower processing properties and making it difficult to meet the requirements of processed potato varieties. Therefore, precisely controlling the reducing sugar content of potatoes and cultivating potato varieties with low reducing sugar content is an important goal in developing processing potatoes.

[0003] Currently, the improvement of potato reducing sugar content mainly relies on traditional hybridization breeding. However, traditional breeding has shortcomings such as long cycle (usually 5-8 years), phenotypic identification is easily affected by environmental factors, and low screening efficiency, making it difficult to quickly cultivate superior varieties with low reducing sugar content. Summary of the Invention

[0004] To address the aforementioned problems, the purpose of this invention is to provide the application of the StMSSP2 gene in the improvement of reducing sugars in potato tubers, thus providing a new gene resource for the molecular breeding of high-quality potato processing varieties.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides the application of the StMSSP2 gene in the improvement of reducing sugar content in potato tubers, including: overexpression of the StMSSP2 gene can significantly reduce the reducing sugar content in potato tubers.

[0007] Furthermore, the above applications also include: overexpression of the StMSSP2 gene can significantly promote potato plant height growth.

[0008] Furthermore, the above applications also include: overexpression of the StMSSP2 gene can positively regulate the overall biomass of the plant.

[0009] The present invention also provides a method for reducing the reducing sugar content of potato tubers, comprising:

[0010] 1) Using the cDNA of potato variety Qingshu 9 as a template, the StMSSP2 gene was amplified by PCR to obtain the CDS sequence of the StMSSP2 gene, as shown in SEQ ID NO.1;

[0011] 2) The CDS sequence amplified in step 1) is ligated into the plant expression vector pBWA(V)KS to construct the overexpression vector pBWA(V)KS-StMSSP2;

[0012] 3) The pBWA(V)KS-StMSSP2 vector plasmid was transformed into Agrobacterium GV3101 to infect potato explants. After culture and screening, StMSSP2 overexpressing plants were obtained.

[0013] Furthermore, in step 1), the primers for PCR amplification are:

[0014] StMSSP2-F: 5'-atgaatggtgctgtgttagttg-3' (SEQ ID NO.3),

[0015] StMSSP2-R: 5'-ctactcatgcttcgcgatagcag-3' (SEQ ID NO. 4).

[0016] Further, in step 2), the expression vector pBWA(V)KS is linearized by digestion with BsaI / Eco31I, and the CDS sequence and the expression vector pBWA(V)KS are ligated using homologous recombination.

[0017] Further, in step 3), the potato explants are prepared by the following method: 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 materials, inoculate them in pre-culture medium, and culture them in the dark at 23°C for 2-3 days.

[0018] Further, in step 3), Agrobacterium is picked into the infection solution to prepare an Agrobacterium resuspension with OD600 = 0.2~0.5. The explants are inoculated into the Agrobacterium suspension for 10 min. The inoculated explants are dried on sterile filter paper and inoculated onto co-culture medium and cultured in the dark at 23℃ for 48~72 h. The co-cultured explants are inoculated onto selection / differentiation medium, 30 explants per plate, and cultured at 25℃ under light for 16 / 8 h, with the medium changed every 20 days. The differentiated shoots are inoculated onto rooting medium and cultured at 25℃ under light for 16 / 8 h until roots are formed.

[0019] The beneficial effects of this invention are:

[0020] This invention uses the potato variety Qingshu 9 (Q9) as research material, clones the StMSSP2 gene, and studies the function of StMSSP2 in the synthesis of reducing sugars in potatoes through overexpression of the target gene and analysis of the expression level and reducing sugar content of the StMSSP2 gene in various tissues of transgenic lines. The results show that overexpression of the StMSSP2 gene significantly promotes potato plant height growth and also has a certain positive regulatory effect on the overall plant biomass. StMSSP2 was overexpressed to varying degrees in all overexpression lines, exhibiting obvious tissue and line specificity. The gene expression level in different tissues showed a distribution pattern of stem > leaf >> root > stolon. Compared with the wild type (WT), the reducing sugar content of tubers in all StMSSP2 overexpression lines was significantly reduced (p < 0.001), with the largest reduction reaching 50%. In summary, the StMSSP2 gene in potato can significantly reduce the reducing sugar content in potato tubers and positively regulate the growth and development of potato plants, providing a new gene resource for molecular breeding of high-quality potato processing varieties. Attached Figure Description

[0021] Figure 1 Amplification of the target gene (A) Amplification of StMSSP2; (B) PCR identification of StMSSP2 recombinant vector colonies.

[0022] Figure 2 Phenotypic results of StMSSP2 overexpressing lines. (A) Phenotypic photographs of wild-type and StMSSP2 overexpressing lines at full bloom under the same conditions, scale bar = 10 cm; (B) Plant height statistics of wild-type and StMSSP2 overexpressing lines at full bloom. Data are mean ± standard deviation (n ≥ 6). This indicates that p ≤ 0.05 indicates a significant difference; p ≤ 0.01 indicates statistical significance (one-way ANOVA); (C) Total biomass per plant in wild-type and StMSSP2 overexpressing lines. Data are mean ± standard deviation (n ≥ 6). There was no significant difference between the overexpressing lines and the wild-type (P > 0.05, one-way ANOVA). WT: wild-type; StMSSP2-OE#1, OE#3, OE#9, OE#17: different lines overexpressing the StMSSP2 gene.

[0023] Figure 3 Analysis of gene expression levels in leaves, stems, roots, and stolons of StMSSP2-overexpressing plants. Figures A, B, C, and D show the gene expression levels in leaves, stems, roots, and stolons, respectively. This indicates that p ≤ 0.05 indicates a significant difference; This indicates that p ≤ 0.01 indicates a significant difference; p ≤ 0.001 indicates significant difference. WT: wild type; StMSSP2-OE#1, OE#3, OE#9, OE#17: different lines overexpressing the StMSSP2 gene.

[0024] Figure 4 The reducing sugar content in potato tubers of StMSSP2 overexpression lines. p ≤ 0.001 indicates significant difference. WT: wild type; StMSSP2-OE#1, OE#3, OE#9, OE#17: different lines overexpressing the StMSSP2 gene. Detailed Implementation

[0025] To more clearly illustrate the present invention, the invention will be further described in detail below with reference to embodiments and accompanying drawings. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0026] This invention uses the red-skinned potato variety Qingshu 9 (Q9) as the research material, clones the StMSSP2 gene, and studies the function of StMSSP2 in the process of potato reducing sugar synthesis by overexpressing the target gene and analyzing the expression level and reducing sugar content of the StMSSP2 gene in various tissues of transgenic lines.

[0027] Example

[0028] The StMSSP2 gene ID Soltu.Q9.Chr04_A20012613.g and CDS sequence were found in the published genome of potato variety Qingshu 9 (Wang, F., Xia, ZQ, Zou, ML, Zhao, L., Jiang, SR, Zhou, Y., Zhang, CJ, Ma, YZ, Bao, YT, Sun, HH, Wang, W., & Wang, J. (2022). The autotetraploid potato genome provides insights into highly heterozygous species. Plant Biotechnology Journal, 20(10), 1996–2005. https: / / doi.org / 10.1111 / pbi.13883).

[0029]

[0030] The amino acid sequence encoded by StMSSP2 is as follows:

[0031] (SEQ ID NO.2).

[0032] I. Gene Cloning

[0033] Using cDNA from the 'Qingshu 9' variety as a template, the StMSSP2 gene was amplified according to the 50 μL PCR reaction system in Table 1. The amplification primers are shown in Table 2. The CDS sequence (SEQ ID NO.1) of the StMSSP2 gene was obtained. The reaction procedure is shown in Table 3.

[0034] Table 1 PCR reaction system

[0035]

[0036] Table 2 Primer Sequences

[0037]

[0038] Table 3 PCR reaction procedure

[0039]

[0040] Electrophoresis was performed on a 1% agarose gel at 5 V / cm for 20 min. The electrophoretic fragment of StMSSP2 (2214 bp) (see...) Figure 1 (Figure A) The DNA was cut out under a UV lamp and placed in a system for sol-gel recovery. The DNA was recovered using a sol-gel recovery kit (Tiangen Biotech Co., Ltd.). The DNA was dissolved and recovered with a total volume of 40 μL of water. After the test was confirmed to be correct, the StMSSP2 gene sequence was obtained.

[0041] II. Construction of the overexpression vector pBWA(V)KS-StMSSP2

[0042] To identify the function of StMSSP2 in potato in detail, primers OE-StMSSP2-F / R were designed and synthesized by Shanghai Sangon Biotech Co., Ltd. The full-length CDS sequence of the amplified StMSSP2 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 and reaction conditions are shown in Table 4. StMSSP2 and the expression vector pBWA(V)KS were ligated using homologous recombination; the ligation system and reaction conditions are shown in Table 5.

[0043] OE-StMSSP2-F: 5'-AGAGAACACGGGGGACTTTGCAACatgaatggtgctgtgttagttg-3' (SEQID NO.5);

[0044] OE-StMSSP2-R: 5'-GTACTGAAGACACAGCTAGTTACActactcatgcttcgcgatagcag-3' (SEQ ID NO. 6).

[0045] Table 4 Enzyme digestion system and reaction conditions

[0046]

[0047] Table 5. Connection system and reaction conditions

[0048]

[0049] The ligation product was transformed into competent E. coli cells, then into Kana resistant plates, and cultured at 37°C for 12 h for plaque PCR identification.

[0050] III. Plaque PCR Identification

[0051] Ten plaques were selected and simultaneously inoculated into 1.5 mL EP tubes for PCR identification. The PCR reaction system is shown in Table 6 and the reaction procedure is shown in Table 3.

[0052] Identification primer sequences:

[0053] Seq-35S-F: 5'-ttcatttggagagaacacgggggac-3' (SEQ ID NO. 7);

[0054] StMSSP2-eGFP-R: 5'-ctctatgccacgcaatttctg-3' (SEQ ID NO. 8).

[0055] Table 6 PCR Reaction System

[0056]

[0057] The target band is a fragment of approximately 662 bp, such as... Figure 1 As shown in Figure B. Take 400 µL of bacterial culture corresponding to 2-3 PCR-positive clones and inoculate each into 5 mL of LB liquid medium containing Kan resistance. Incubate overnight at 37°C with shaking for plasmid extraction. To verify the accuracy of the recombinant plasmid construction (excluding the possibility of large fragment insertions / deletions), the extracted plasmid was verified by single-enzyme digestion. EcoRV restriction enzymes were used to digest the plasmids; the reaction system and conditions are shown in Table 7.

[0058] Table 7 Enzyme digestion reaction system

[0059]

[0060] Select 1-2 plasmid samples that have been verified to be correct by enzyme digestion and send them to a sequencing company for final sequence verification. The primers used for sequencing are:

[0061] StMSSP2-F1108: 5′-gttccatcttcaccttccctt-3′ (SEQ ID NO.9);

[0062] StMSSP2-F1662: 5'-ggattggtccatagcccg-3' (SEQ ID NO. 10).

[0063] After the sequencing results were confirmed to be 100% identical to the CDS sequence of the target gene StMSSP2, the pBWA(V)KS-StMSSP2 vector was obtained.

[0064] IV. Agrobacterium-mediated potato genetic transformation

[0065] 1. Transformation of GV3101 Agrobacterium using pBWA(V)KS-StMSSP2 vector

[0066] Transform GV3101 Agrobacterium with the pBWA(V)KS-StMSSP2 vector plasmid. Take GV3101 Agrobacterium competent cells from the ultra-low temperature freezer, 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℃ and 180 rpm for 30 min on a shaker. Take 50 µL of the activated Agrobacterium culture and inoculate it onto LB colony solid medium. Incubate in the dark at 30℃ for 48 h.

[0067] 2. PCR identification of Agrobacterium-positive clones

[0068] Single colonies that grew well on the plate were selected for colony PCR to verify successful plasmid transformation. Identification primers are shown in SEQ ID NO.11 and SEQ ID NO.12. Amplification was performed using primers for the vector resistance gene. The PCR amplification system is shown in Table 8.

[0069] Table 8 Colony PCR Amplification System

[0070]

[0071] Table 9 Primer Sequences

[0072]

[0073] PCR products were detected by 1% agarose gel electrophoresis. If a sample showed a specific band of the same size as the positive control, while the negative control showed no band, it was considered a positive clone, indicating that the sample could proceed to the next step.

[0074] 3. Genetic transformation of potatoes

[0075] 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-med medium (MS), and incubate in the dark at 23℃ for 2-3 days.

[0076] Agrobacterium infection and co-culture: Agrobacterium was picked and placed in the infection solution to prepare an Agrobacterium resuspension with an OD600 of 0.2–0.5. Explants were inoculated into the Agrobacterium suspension for 10 min. The infected explants were then air-dried on sterile filter paper and inoculated onto a co-culture medium (MS salt (M5524) 4.3 g, sucrose 30 g, 6-BAP (1 mg / mL) 1 mL, NAA (1 mg / mL) 2 mL, MSVI vitamin 1 mL, JHMS 1 mL, inositol 100 mg, agar 10 g, and the volume was adjusted to 1 L. The pH was adjusted to 5.6 using KOH; the pH was adjusted before adding the agar. Hormones were added only after cooling to below 50°C.) and incubated in the dark at 23°C for 48–72 h.

[0077] Selection / Differentiation: The co-cultured explants were inoculated onto selection / differentiation medium (MS salt (M5524) 4.3g, 3R vitamin 1ml, inositol 100mg, sucrose 30g, agar 10g, the following were added after sterilization: IAA (1mg / mL) 0.5ml, zeatin (1mg / mL) 3ml, termethin (200mg / mL) 2.5ml, selection hormone (e.g., Kana concentration of 50mg / mL) 250ul, and the volume was adjusted to 1L. The pH was adjusted to 5.9 with KOH, and the medium was autoclaved for 20min.) 30 explants per plate, cultured at 25℃ under 16 / 8h light, and the medium was changed every 20 days.

[0078] Rooting culture: Differentiated shoots were inoculated into rooting medium (MS salt (M5524) 4.3g, NaH2PO4 H2O 0.17g, inositol 0.1g, Thiamine HCl (0.4mg / ml) 1ml, sucrose 30g, plant gel 2.8g, termethin (200mg / ml) 0.5ml, selection hormone 250ul, and the volume was adjusted to 1L. The pH was adjusted to 6.0 with KOH). The culture was carried out at 25℃ under 16 / 8h light until roots were formed.

[0079] 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.

[0080] 4. Phenotypic observation and measurement

[0081] Photographs were taken to record the phenotypic differences between potato overexpression lines and wild-type lines, and the results were as follows: Figure 2 As shown in Figure A; the plant height during the full bloom period was measured, and the results are as follows. Figure 2As shown in B; the fresh weight of the whole plant was taken for sampling, and 6 biological replicates were taken for each line. The results are as follows. Figure 2 As shown in C.

[0082] Morphological observations and statistical analysis of plant height and biomass of StMSSP2-overexpressing potato plants showed that, compared with wild-type (WT), the StMSSP2-overexpressing lines exhibited significant growth advantages: plant height was significantly increased (P < 0.05), with StMSSP2-OE#3, OE#9, and OE#17 reaching highly significant levels (P < 0.01); although the total biomass per plant showed an increasing trend, it did not reach a statistically significant level (P > 0.05). These results indicate that overexpression of the StMSSP2 gene can significantly promote potato plant height growth and also has a certain positive regulatory effect on overall plant biomass.

[0083] The gene expression levels of StMSSP2 in roots, stems, leaves, and stolons of control and overexpression lines were determined by RT-qPCR. The top leaves of the 3rd and 4th functional leaves were collected, and the top 3-8 stem segments were collected as samples for gene expression determination.

[0084] qRT-PCR analysis of different tissues showed that StMSSP2 was overexpressed to varying degrees in all overexpression lines, exhibiting significant tissue and line specificity. Gene expression levels in different tissues followed a distribution pattern of stem > leaf >> root > stolon. At the line level, OE#9 showed significantly higher expression levels in leaves and stems than other lines, representing the highest overall expression level. OE#3 showed the highest expression level in roots, while OE#17 did not show significant overexpression in roots or stolons. Figure 3 ).

[0085] After the plants matured, the reducing sugar content of the tubers was determined using the 3,5-dinitrosalicylic acid (DNS) method. An appropriate amount of sample was weighed, 1 mL of sterile water was added, and the tubers were ultrasonically extracted for 10 min, followed by centrifugation at 12,000 rpm for 10 min. 0.08 mL of the extract was transferred to a new test tube, 0.06 mL of DNS was added, and the mixture was stirred. The tubes were then in a boiling water bath for 5 min, rapidly cooled to room temperature, and distilled water was added to a final volume of 1 mL. The absorbance was measured at 540 nm. Quantification was performed using the external standard method, and a fitting curve was prepared using standards of different concentrations.

[0086] Results of reducing sugar content measurements in the overexpression lines showed that, compared with the wild type (WT), the reducing sugar content in the tubers of all StMSSP2 overexpression lines was significantly reduced (p < 0.001). The reducing sugar content of wild-type plants was approximately 0.18%, while the reducing sugar content of each overexpression line decreased to 0.08%–0.10%, with a reduction of up to 50%. Figure 4 ).

[0087] 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 StMSSP2 gene in improving reducing sugar content in potato tubers, including: Overexpression of the StMSSP2 gene can significantly reduce the reducing sugar content in potato tubers.

2. The application according to claim 1, characterized in that, Also includes: Overexpression of the StMSSP2 gene can significantly promote potato plant height growth.

3. The application according to claim 1 or 2, characterized in that, Also includes: Overexpression of the StMSSP2 gene can positively regulate the overall biomass of the plant.

4. A method for reducing the reducing sugar content of potato tubers, comprising: 1) Using the cDNA of potato variety Qingshu 9 as a template, the StMSSP2 gene was amplified by PCR to obtain the CDS sequence of the StMSSP2 gene, as shown in SEQ ID NO.1; 2) The CDS sequence amplified in step 1) is ligated into the plant expression vector pBWA(V)KS to construct the overexpression vector pBWA(V)KS-StMSSP2; 3) The pBWA(V)KS-StMSSP2 vector plasmid was transformed into Agrobacterium GV3101 to infect potato explants. After culture and screening, StMSSP2 overexpressing plants were obtained.

5. The method according to claim 4, characterized in that, In step 1), the primers for PCR amplification are: StMSSP2-F: 5'-atgaatggtgctgtgttagttg-3' (SEQ ID NO.3), StMSSP2-R: 5'-ctactcatgcttcgcgatagcag-3' (SEQ ID NO. 4).

6. The method according to claim 4, characterized in that, In step 2), the expression vector pBWA(V)KS is linearized by digestion with BsaI / Eco31I, and the CDS sequence and the expression vector pBWA(V)KS are ligated using homologous recombination.

7. The method according to claim 4, characterized in that, In step 3), the potato explants are prepared by the following method: 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 materials, inoculate them in pre-culture medium, and culture them in the dark at 23°C for 2-3 days.

8. The method according to claim 4, characterized in that, In step 3), Agrobacterium is picked and placed in the infection solution to prepare an Agrobacterium resuspension with an OD600 of 0.2-0.

5. The explants are inoculated into the Agrobacterium suspension for 10 min. The inoculated explants are dried on sterile filter paper and inoculated onto co-culture medium. They are then cultured in the dark at 23°C for 48-72 h. The co-cultured explants are then inoculated onto selection / differentiation medium, with 30 explants per plate. They are cultured at 25°C under 16 / 8 h light, and the medium is changed every 20 days. The differentiated shoots are inoculated onto rooting medium and cultured at 25°C under 16 / 8 h light until roots are formed.

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