Application of potato StTST1 gene in breeding for cold and drought resistance

Abstract

The application provides a potato StTST1 gene and its application in cold resistance and drought resistance, and discloses the following technical scheme: through a sequence published in a potato genome database, total RNA of a low-temperature sensitive e potato No.3 (E3) is taken as a template to prepare cDNA by reverse transcription, the cDNA is taken as a template to clone a StTST1 gene, an overexpression vector is constructed, genetic transformation is carried out on E3 to verify the gene function, and the endogenous StTST1 gene of the interference E3 is found to be able to improve the cold resistance and reduce the drought resistance by combining with the interference strain obtained in the early stage; the overexpression StTST1 gene is able to improve the drought resistance and reduce the cold resistance.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics and breeding, specifically relating to the potato StTST1 gene and its application in cold and drought resistance. Background Technology

[0002] Potato (Solanum tuberosum L.) is the fourth most important food crop after rice, corn, and wheat, playing a vital role in global economic development and food security. However, most cultivated potato varieties are sensitive to low temperatures, and because low-temperature stress is irreversible, it ultimately leads to large-scale yield reduction and quality degradation (Kou Shuang 2019). Simultaneously, most cultivated potato varieties are also sensitive to drought. Therefore, the rapid breeding of low-temperature and drought-resistant varieties is of paramount importance for increasing potato yields and improving product quality in my country.

[0003] Sugars play a vital role in plant growth and development, not only providing energy essential for cellular life activities but also acting as signaling molecules to regulate cellular metabolism. In Arabidopsis thaliana, sucrose in the cytoplasm plays a crucial role in maintaining osmotic stability and preventing cell membrane damage from low temperatures. (Yamada et al 2013). Besides sugar content, transporter-mediated subcellular compartmentalization of sugars is also an adaptive strategy for plants against biotic and abiotic stresses, but the mechanism remains unclear (Yamada and Osakabe 2018). Commonly reported transporters in plants include SWEET (Sugars Will Eventually Be Exported Transporters), SUT (Sucrose Transporters), and VGT (Vacuolar Sugar Transporter), which play important functions in plant growth and development (Aluri and Büttner 2007; Baker et al 2012; Kühn and Grof 2010). Since the StTST1 gene in this invention encodes a vacuolar membrane sugar transporter that can transport sucrose into the vacuoles, based on existing reports, we hypothesize that the StTST1 gene may have some influence on the cold and drought tolerance of potatoes. Interference lines of the StTST1 gene have been used in research on low-temperature sugar formation in potatoes (Liuet et al 2023). Therefore, we constructed an overexpression vector for StTST1 and combined it with interfering lines to verify the function of the StTST1 gene under low temperature and drought conditions.

[0004] On June 8, 2022, the applicant filed a patent application for protection regarding the application of the StTST1 gene in enhancing the low-temperature saccharification resistance of potatoes (CN202210640076.5). However, there are currently no literature reports on the application of StTST1 in potato cold and drought resistance. This application has significant scientific research value for creating high-quality cold-resistant and drought-resistant materials, and provides important theoretical basis and practical significance for ensuring yield and improving product quality. Summary of the Invention

[0005] In view of the shortcomings of existing technologies, this invention provides the potato StTST1 gene and its application in cold and drought resistance. Using sequences published in the potato genome database, and total RNA from leaves of the low-temperature sensitive E3 potato variety as a template, cDNA was prepared by reverse transcription. The cDNA was then used as a template to amplify CDS, and an overexpression vector was constructed. Genetic transformation of E3 potatoes was then performed to verify transgenic function. It was found that overexpression of the StTST1 gene can improve the drought resistance of potatoes, while interference with the expression of the StTST1 gene can improve their cold resistance. To achieve the above objectives, this invention adopts the following technical solution:

[0006] 1. Potato StTST1 gene, the transcript sequence of the potato StTST1 gene is shown in SEQ No. 1 and... Figure 6 As shown.

[0007] 2. Method for verifying the function of potato StTST1 gene, specifically including the following steps: (1) primer design, (2) RNA extraction, (3) cDNA preparation, (4) CDS fragment amplification, (5) homologous recombination, (6) transformation to GV3101, (7) potato genetic transformation, (9) verification of cold resistance function, and (10) verification of drought resistance function.

[0008] 3. Application of interfering with StTST1 gene expression to improve cold resistance in potatoes. The transcript sequence of the potato StTST1 gene is shown in SEQ No. 1 of the sequence listing.

[0009] 4. Application of interfering with StTST1 gene expression to reduce electrolyte permeability in potatoes under low temperature stress. The transcript sequence of the potato StTST1 gene is shown in SEQ No. 1 of the sequence listing.

[0010] 5. Application of overexpression of the StTST1 gene to improve drought resistance in potatoes. The transcript sequence of the potato StTST1 gene is shown in SEQ No. 1 of the sequence listing.

[0011] 6. Application of overexpression of the StTST1 gene to reduce stomatal aperture in potatoes. The transcript sequence of the potato StTST1 gene is shown in SEQ No. 1 of the sequence listing.

[0012] 7. Application of overexpression of the StTST1 gene to increase the relative water content in potato leaves. The transcript sequence of the potato StTST1 gene is shown in SEQ No. 1 of the sequence listing.

[0013] 8. Application of the StTST1 gene in the breeding of drought-resistant or cold-resistant varieties: varieties with high expression of the StTST1 gene have drought resistance, while varieties with low expression of the StTST1 gene have cold resistance.

[0014] The beneficial effects of this invention are as follows: the electrolyte permeability of transgenic lines Ri-StTST1-10, Ri-StTST1-18, and Ri-StTST1-33 was significantly lower than that of the control E3 under low-temperature treatment, and the plant condition after frost was better than that of E3, indicating that interfering with StTST1 gene expression can effectively improve the cold resistance of E3. The relative water content and plant condition of transgenic lines OE-StTST1-7, OE-StTST1-15, and OE-StTST1-18 were better than those of the control E3, and the stomatal opening state was reduced, indicating that overexpression of the StTST1 gene can effectively improve the drought resistance of E3. Attached Figure Description

[0015] Figure 1 Phenotypic diagrams of transgenic lines and E3(WT) before and after frost treatment, and after recovery to the final state.

[0016] Figure 2 Electrolyte permeability of transgenic lines and E3 at different low temperatures.

[0017] Figure 3 The plant status of transgenic lines and E3 before drought treatment, after drought treatment, and after watering.

[0018] Figure 4 The relative water content of transgenic lines and E3 before and after drought treatment.

[0019] Figure 5 Stomatal status of transgenic lines and E3 before and after drought treatment.

[0020] The data presented are the average of three biological replicates, with the error bar representing the SD of the three replicates. Significance analysis was performed using a t-test, where * represents P < 0.05 and ** represents P < 0.01.

[0021] Figure 6 : A schematic diagram of the potato StTST1 gene structure, with underlined start and stop codons. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the invention clearer, specific embodiments of the invention will be described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the drawings. The embodiments of the invention shown in and described with reference to the drawings are merely exemplary, and the invention is not limited to these embodiments. It should also be noted that, in order to avoid obscuring the technical solutions of the invention with unnecessary details, only structures and / or processing steps closely related to the solutions according to the invention are shown in the drawings, while other details that are not closely related are omitted.

[0023] Example 1

[0024] This embodiment provides the potato StTST1 gene, and the potato StTST1 gene transcript sequence is shown in sequence listing SEQ No. 1 and... Figure 6 As shown.

[0025] Example 2

[0026] This embodiment provides a method for screening the StTST1 gene associated with cold and drought resistance in potatoes. Recent studies in our laboratory have found that inhibiting StTST1 gene expression has a certain effect on improving potato cold resistance. The hypothesized mechanism is that when plants are exposed to low temperatures, the expression levels and activities of starch hydrolases and phosphorylases are upregulated, which helps catalyze the breakdown of starch stored in leaves, leading to an increase in sucrose concentration in plastids and cytoplasm, thereby protecting the cell membrane and preventing or mitigating low-temperature damage to cells. (et al 2013). However, in the presence of TST protein, sucrose in the cytoplasm is transported to the vacuoles and broken down into reducing sugars, resulting in a decrease in sucrose content in the cytoplasm, which cannot meet the normal needs of cells to resist low temperatures, thus reducing the plant's cold resistance. When the expression of the StTST1 gene was interfered with, it was found that the transgenic lines were more cold-resistant than the wild type.

[0027] Overexpression of the StTST1 gene can improve the drought resistance of potatoes. The mechanism is as follows: after overexpression of the StTST1 gene, the number of TST proteins on the vacuolar membrane increases, which leads to the transport of most of the sucrose in the cytoplasm to the vacuoles and its breakdown into glucose and fructose. This increases the soluble sugar content in the vacuoles and the water potential in the vacuoles, enabling the vacuoles to store more water to cope with drought stress.

[0028] Example 3

[0029] This embodiment provides a method for cloning potato StTST1, which specifically includes the following steps:

[0030] 1. Primer design

[0031] The StTST1 gene in this invention was amplified using primers designed based on the sequence (Soltu.DM.04G037920.1) published in the Potato Genome Database (http: / / solanaceae.plantbiology.msu.edu / pgsc_download.shtml). The primer sequences are as follows:

[0032] Table 1. CDS amplification primer information for StTST1

[0033]

[0034] 2. RNA extraction

[0035] Potato E3 leaf samples were collected, ground into powder in liquid nitrogen, and extracted using the Plant Total RNA Kit (centrifuge tube type) ultrafast plant total RNA extraction kit from Beijing Zhuangmeng International Biotechnology Co., Ltd. The specific method is as follows:

[0036] ① Take plant leaves, freeze them quickly in liquid nitrogen, grind them, and dispense them into 2.0ml screw-cap centrifuge tubes, then let them stand on ice.

[0037] ② Add 1 ml of lysis buffer R to every 20-80 mg of young plant tissue, and continuously vortex during the process.

[0038] ③ Add 0.2 ml of chloroform to each 1 ml of plant lysate, tighten the cap, vortex, and let stand at room temperature for 1 min.

[0039] ④ Centrifuge at 12000r / min for 10min in a 4℃ centrifuge. The sample can be observed to be divided into three layers: the lower organic phase, the middle and upper colorless aqueous phases, and the RNA distributed in the upper aqueous phase. Transfer 500μl of supernatant to a new 1.5ml centrifuge tube.

[0040] ⑤ Add 0.5 times (250 μl) of anhydrous ethanol and 100 μl of binding solution N, and mix by inverting. Attach the adsorption column to a 2.0 ml centrifuge tube and transfer the resulting product into the adsorption column.

[0041] ⑥ Centrifuge at 12000 r / min (4℃) for 30 seconds and discard the waste liquid.

[0042] ⑦ Add 500 μl of washing buffer RW (check whether anhydrous ethanol has been added before opening or use), centrifuge at 12000 r / min for 30 sec, and discard the waste liquid. Repeat this step.

[0043] ⑧Isolate at 13000r / min for 2min to remove rinsing solution as much as possible.

[0044] ⑨ Attach the adsorption column to a new RNase-free centrifuge tube, add 60-100 μl of RNase-free H2O to the center of the adsorption membrane, incubate at room temperature for 1 min, and centrifuge at 12000 rpm for 1 min to obtain the RNA solution. Take 4 μl of the obtained RNA solution for agarose gel electrophoresis (1% agarose gel) to detect the RNA quality and concentration. For those that meet the standards, proceed to the subsequent reverse transcription cDNA preparation experiment.

[0045] 3. cDNA preparation

[0046] ① Aspirate the RNA template into an octet (the amount aspirated depends on the concentration measured in the previous step; when the concentration is lowest, aspirate 2 μg), add 2 μl of AccuRT Reaction Mix (4×), and bring the volume to 8 μl with Nuclease-free H2O. Incubate in a 42℃ metal bath for 2 min.

[0047] ② Add 2 μl of AccuRT Reaction Stopper (5×), mix well, then add 4 μl of 5× all-in-one RTmaster mix and 6 μl of Nuclease-free H2O. Incubate at 25℃ for 10 min, then incubate at 42℃ for 15 min (qPCR) or 50 min (PCR), then inactivate at 85℃ for 5 min, and store at -20℃.

[0048] ③ cDNA detection results, the product is used for subsequent qRT-PCR, gene cloning and other experiments, or stored at -70℃.

[0049] 4. CDS fragment amplification (50 μl system as follows):

[0050] Table 2 StTST1 CDS Fragment amplification system Table 2 StTST1 CDS fragment amplification system

[0051]

[0052] Amplification program: 95℃ / 3min, 95℃ / 30sec, 55℃ / 30sec, 72℃ / 20sec (return to step 2, 35 cycles), 72℃ / 10min, 12℃ / 1h. After amplification, add 5μl of 10×DNA loading buffer, spot the sample and run on a gel. Select the correct band for gel recovery and purification PCR using the Gel Extraction Kit from Jiangsu Kangwei Century Biotechnology Co., Ltd.

[0053] 5. Homologous recombination

[0054] The product and the enzyme-digested vector (enzyme StuI) were recombined according to the system in Table 3. The reaction was carried out at 37℃ for 30 min, followed by transformation into DH5α, plated, and incubated overnight. Single colonies were picked for PCR identification, and positive bands were sent to the company for sequencing. The plasmid was then returned to obtain the target vector pH7LIC8-StTST1. CDS .

[0055] Table 3 Recombination Reaction System

[0056]

[0057] Example 4

[0058] This embodiment uses the cloning vector pH7LIC8-StTST1 obtained in Example 3. CDS The method for verifying low-temperature and drought resistance is as follows:

[0059] 1. Conversion of GV3101

[0060] ① Rinse the electric rotary cup with distilled water, then rinse it three times with 75% alcohol. Invert it onto absorbent paper on a clean bench, dry it, and then place it on ice to pre-cool.

[0061] ② Take 2 μl of plasmid and add it to Agrobacterium GV3101 competent cells. Use a yellow pipette tip to mix it evenly, transfer it to an electroporation cuvette, place it in an electroporator (Bio-RAD), and add 600 μl of LB or YEB liquid medium.

[0062] ③ Transfer all bacterial culture and culture medium to the original centrifuge tube, incubate at 220 r / min for 2 h at 28℃, centrifuge at 8000 r / min for 30 sec, and spread evenly on LB medium containing 2‰ Rif and 1‰ Kan.

[0063] ④ Incubate at 28℃ for 2-3 days. Pick single clones and shake them, then perform PCR detection on the bacterial culture. Preserve positive Agrobacterium culture.

[0064] 2. Genetic transformation of potatoes

[0065] The overexpressed Agrobacterium GV3101 was streaked onto YEB solid medium for activation. Single colonies were picked and incubated in 20 ml of YEB liquid medium at 28°C and 220 rpm for 24 h. 2 ml of the bacterial culture was then transferred to 40 ml of YEB liquid medium and incubated again at 28°C and 220 rpm until OD was reached. 600 Approximately 0.5 (5-6 h); centrifuge at 5000 r / min for 6 min, discard the supernatant, and resuspend in 10 ml of 3% MS (pH 5.8) liquid medium. Cut tubers grown for 6-8 weeks into thin slices approximately 1-2 mm thick; then inoculate in the resuspended medium for 10 min, gently shaking every 3-5 min to facilitate infection; transfer the tuber pieces to sterile filter paper to blot off the surface bacterial solution, then transfer to P1 co-culture medium (3% MS solid medium + 0.2 mg / L GA3 + 0.2 mg / L IAA + 0.5 mg / L 6-BA + 2 mg / L ZT, pH 5.8), and incubate in the dark at 23℃ for 48 h. The resistant shoots were then transferred to P2 differentiation medium (3% MS solid medium + 0.2 mg / L IAA + 0.2 mg / L GA3 + 2 mg / L ZT + 0.5 mg / L 6-BA + 75 mg / L Kan + 400 mg / L Cef) and cultured at 23°C (16 h light / 8 h dark). When the resistant shoots grew to 0.5-1 cm, they were inoculated into P3 rooting medium (3% MS + 50 mg / L Kan + 400 mg / L Cef). After the resistant shoots grew larger, they underwent secondary rooting, and then positive detection was performed by qRT-PCR. The primer information used is as follows:

[0066] Table 4 Primer information used in qRT-PCR

[0067]

[0068] Reaction program: 95℃ / 1min; 95℃ / 10s; 56℃ / 20s, with fluorescence collected, 40 cycles. After the PCR program, 95℃ / 30s; 65℃ / 1min; slowly increase the temperature to 95℃, collecting fluorescence every 0.5℃ increase, and perform melting curve analysis.

[0069] 3. Verification of cold resistance function

[0070] Two methods were used: direct frost treatment (Method 1) and electrolyte permeability testing (Method 2). Interference lines, overexpression lines, and the control E3 were simultaneously planted and cultured for 4 weeks. In Method 1, the treated and control lines were simultaneously placed in a constant temperature incubator at -2 to -3°C until the leaves hardened. They were then transferred to room temperature and allowed to recover until new leaves appeared. The plant condition was photographed and recorded before, after, and after recovery. In Method 2, leaves from healthy plants were taken, washed, and placed petiole-side down in centrifuge tubes containing 500 μl of ultrapure water. The tubes were then treated in a low-temperature water bath at a cooling rate of 1°C / h (ice pellets were added when the temperature reached 0°C), with each temperature point treated for 1 hour. The treated tubes were then placed on ice overnight. Add 9.5 ml of ultrapure water, shake at 160 r / min for 2 h, heat to room temperature, and measure the conductivity before boiling (R1) using a conductivity meter (Mellert, FE30-K, Switzerland). Treat at 99℃ for 30 min, cool to room temperature, and measure the conductivity after boiling (R2). Use R1 / R2 to approximately represent the electrolyte permeability caused by the damage to the leaf cell membrane at this temperature.

[0071] Frost test results showed that there were no significant differences between the transgenic lines and the control E3 before and after frost. After recovery, the interfering lines were in better condition than the E3 lines, while the overexpression lines were in the worst condition (see...). Figure 1 This indicates that the StTST1 gene negatively regulates potato cold resistance, and interfering with the StTST1 gene improves potato cold resistance.

[0072] Electrolyte permeability measurements showed that at four temperature points (-1℃, -3℃, -4℃, and -5℃), the electrolyte permeability of the overexpression line was higher than that of E3; while the electrolyte permeability of the interference line was lower than that of E3 at all five temperature points (see [reference needed]). Figure 2 ).

[0073] Based on the existing experimental results, it is shown that interfering with the expression of the StTST1 gene significantly improves the cold resistance of potatoes.

[0074] 4. Verification of drought resistance function

[0075] Drought resistance was determined using methods including relative water content, observation of plant condition, and stomatal opening degree. Specifically, all healthy leaves from plants before, after, and after watering were collected, weighed, and recorded as fresh weight (m1). These leaves were then quickly dried, weighed, and recorded as dry weight (m2). The relative water content of the leaves was expressed as (m1-m2) / m1*100%. Plant condition was observed during drought treatment, and the stomatal opening and closing state on the lower surface of the leaves was observed using scanning electron microscopy.

[0076] The relative water content results showed that, before and after drought treatment, the relative water content in the leaves of the overexpression lines was significantly higher than that of E3. For the interference lines, before drought treatment, #10 and #18 showed no significant difference from E3, while the relative water content of #33 was even significantly higher than that of E3. However, after irrigation, the relative water content of all three lines was significantly lower than that of E3 (see...). Figure 3 This indicates that the StTST1 gene positively regulates drought resistance in potatoes.

[0077] Before and after drought treatment, there was no significant difference in the condition of transgenic lines and E3 plants. However, after watering, the condition of overexpressing lines was better than that of E3, while the condition of interference lines was worse than that of E3 (see...). Figure 4 Meanwhile, after drought treatment, the stomatal aperture of the overexpressing lines was smaller than that of E3, while the stomatal aperture of the interference lines was larger than that of E3 (see...). Figure 5 The above results indicate that overexpression of the StTST1 gene enhances the drought resistance of potatoes.

[0078] The above description is only a specific embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

[0079] Main references:

[0080] [1] Kou Shuang. Analysis of the role and mechanism of putrescine synthesis pathway in cold resistance of potato [D]. Huazhong Agricultural University, 2019.

[0081] [2]Aluri S,Büttner M.Identification and functional expression of the Arabidopsis thaliana vacuolarglucose transporter 1and its role in seedgermination and flowering.Proceedings of theNational Academy of Sciences,2007,104(7):2537-2542

[0082] [3]Baker RF,Leach KA,Braun DM.SWEET as sugar:new sucrose effluxers inplants.MolecularPlant,2012,5(4):766-768

[0083] [4]Kühn C,Grof CPL.Sucrose transporters of higher plants.CurrentOpinion in Plant Biology,

[0084] 2010,13(3):287-297

[0085] [5]Liu TF,Kawochar MA,Begum S,Wang ES,Zhou TT,Jing SL,Liu TT,Nie BH,Song BT.

[0086] Potato tonoplast sugar transporter 1 controls tuber sugaraccumulation during postharvest coldstorage.Horticulture Research.2023,10(4)

[0087] [6] T,Heyer A G.Approximating subcellular organisation ofcarbohydrate metabolismduring cold acclimation in different naturalaccessions of Arabidopsis thaliana[J].NewPhytologist,2013,198(3):777-787.

[0088] [7]Yamada K,Osakabe Y.Sugar compartmentation as an environmentalstress adaptation strategyin plants.Seminars in Cell&Developmental Biology,2018,83:106-114.

Claims

1. The application of interfering with StTST1 gene expression to improve the cold resistance of potatoes, characterized by... The potato StTST1 gene transcript sequence is shown in SEQ No. 1, and the application is achieved by reducing the electrolyte permeability of potatoes under low temperature stress.

2. The application of overexpression of the StTST1 gene to improve the drought resistance of potatoes, characterized by... The potato StTST1 gene transcript sequence is shown in SEQ No.

1. The application is achieved by reducing the stomatal aperture of the potato and increasing the relative water content of the potato leaves.

Images