Application of potato StTST1 gene in cold-resistant and drought-resistant breeding

Abstract

The invention provides a potato StTST1 gene and application thereof in cold resistance and drought resistance, through a sequence published in a potato genome database, a total RNA (Ribonucleic Acid) of a leaf of a low-temperature sensitive type Hubei potato No. 3 (E3) is taken as a template, cDNA (Complementary Deoxyribose Nucleic Acid) is prepared through reverse recording, then the cDNA is taken as a template to clone the StTST1 gene, an overexpression vector is constructed, genetic transformation of E3 is carried out for gene function verification, and the potato StTST1 gene is obtained. In combination with an interference strain obtained in the earlier stage, it is found that the interference E3 endogenous StTST1 gene can improve the cold resistance and reduce the drought resistance; the overexpression of the StTST1 gene can improve the drought resistance and reduce the cold resistance.

Description

Technical Field

[0001] The invention belongs to the field of molecular genetic breeding, and specifically relates to a potato StTST1 gene and its application in cold resistance and drought resistance. Background Art

[0002] Potato (Solanum tuberosum L.) is the fourth most important food crop after rice, corn and wheat, and plays an important role in global economic development and food security. However, most cultivated potato varieties are low-temperature sensitive materials. Every year, my country's cultivation areas are threatened by low temperature and frost to varying degrees. For example, late frost and sudden cold currents in early spring in the northern primary cropping area are common; the central plains secondary cropping area suffers from low temperature and cold damage caused by early frost in spring and late frost in autumn; late spring cold often occurs in the southwestern mixed cropping area in early spring; and in the southern winter cropping area, the potato planting period is mainly in winter or early spring, and the growth after germination is extremely susceptible to low temperature. Since low temperature stress is irreversible, it will eventually lead to large-scale yield reduction and quality reduction (Kou Shuang 2019). At the same time, most cultivated potato varieties are drought-sensitive materials, and drought stress is also a major natural factor restricting the healthy development of my country's potato industry. Therefore, it is extremely important to breed low-temperature and drought-resistant varieties as soon as possible to increase potato production and income and improve product quality in my country.

[0003] Sugar plays an important role in plant growth and development. It not only provides cells with the energy necessary for life activities, but also acts as a signal molecule and participates in the regulation of cell metabolism. In Arabidopsis, sucrose in the cytoplasm plays an important role in maintaining cell osmotic stability and preventing cell membranes from being damaged by low temperatures ( et al 2013). In addition to sugar content, transporter-mediated subcellular compartmentalization of sugars is also an adaptive strategy for plants to resist biotic and abiotic stresses, but the mechanism is still unclear (Yamada and Osakabe 2018). Common transporters reported in plants include SWEET (Sugars Will Eventually be Exported Transporters), SUT (Sucrose Transporters) and VGT (Vacuolar Glucose Transporter), etc. These transporters 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 the present invention encodes a vacuole sugar transporter that can transport sucrose into the vacuole, based on existing reports, we speculate that the StTST1 gene may have a certain effect on potato cold and drought resistance. The interference strain of the StTST1 gene has been used in potato low-temperature saccharification research (Liuet al 2023). Therefore, a StTST1 overexpression vector was constructed and combined with an interference strain to verify the function of the StTST1 gene in low temperature and drought.

[0004] On June 8, 2022, the applicant requested protection for the application of the StTST1 gene in enhancing the low temperature saccharification resistance of potatoes (CN202210640076.5), but there is no relevant literature report on the application of StTST1 in potato cold and drought resistance. This application has important 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 deficiencies of the prior art, the present invention provides a potato StTST1 gene and its application in cold and drought resistance; using the sequence published in the potato genome database, using the total RNA of the leaves of the low-temperature sensitive E-potato No. 3 (E3) as a template, reverse transcription to prepare cDNA, then using the cDNA as a template to amplify CDS, and construct an overexpression vector, genetically transform E3 to verify the transgenic function, and find that overexpression of the StTST1 gene can improve the drought resistance of potatoes, while interference expression of the StTST1 gene can improve its cold resistance. In order to achieve the above purpose, the present invention adopts the following technical scheme:

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

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

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

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

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

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

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

[0013] 8. The application of StTST1 gene in breeding drought-resistant or cold-resistant varieties. Varieties with high expression of StTST1 gene are drought-resistant, and varieties with low expression of StTST1 gene are cold-resistant.

[0014] The beneficial effects of the present invention are as follows: the electrolyte permeability of the transgenic lines Ri-StTST1-10, Ri-StTST1-18 and Ri-StTST1-33 is significantly lower than that of the control E3 when treated at low temperature, and the plant state after frost is better than that of E3, indicating that interfering with the expression of the StTST1 gene can effectively improve the cold resistance of E3. The relative water content and plant state of the transgenic lines OE-StTST1-7, OE-StTST1-15 and OE-StTST1-18 are better than those of the control E3, and the stomatal opening state is reduced, indicating that overexpressing the StTST1 gene can effectively improve the drought resistance of E3. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0018] Figure 4 : Relative water content of transgenic lines and E3 before drought treatment and after watering.

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

[0020] The data shown are the average values ​​of three biological replicates, the error bars are the SD of three replicates, and the significance analysis was performed using t-test, with * representing P < 0.05 and ** representing P < 0.01.

[0021] Figure 6 : Schematic diagram of the potato StTST1 gene structure, with the start and stop codons underlined. DETAILED DESCRIPTION

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

[0023] Example 1

[0024] This example provides a potato StTST1 gene, and the potato StTST1 gene transcript sequence is shown in the sequence table SEQ No. 1 and Figure 6 shown.

[0025] Example 2

[0026] This example provides a method for screening the StTST1 gene related to potato cold and drought resistance. Recent studies in our laboratory have found that inhibiting the expression of the StTST1 gene has a certain effect on improving potato cold resistance. The mechanism is speculated to be: when plants are exposed to low temperatures, the expression of starch hydrolases and phosphorylases is upregulated, and the activity is enhanced, which helps to catalyze the decomposition of starch stored in the leaves, resulting in an increase in the concentration of sucrose in the plastids and cytoplasm, thereby protecting the cell membrane and preventing or slowing down the damage caused by low temperatures ( et al 2013). However, in the presence of TST protein, sucrose in the cytoplasm is transported to the vacuole and decomposed into reducing sugars, resulting in a decrease in the sucrose content in the cytoplasm, which cannot meet the normal needs of cells to resist low temperatures, and the cold resistance of plants is reduced. After interfering with the expression of the StTST1 gene, it was found that the transgenic strains were more cold-resistant than the wild type.

[0027] Overexpression of the StTST1 gene can improve potato's drought resistance. The mechanism is: after overexpression of the StTST1 gene, the number of TST proteins on the vacuole membrane increases, causing most of the sucrose in the cytoplasm to be transported to the vacuole and decomposed into glucose and fructose, increasing the soluble sugar content in the vacuole and increasing the water potential in the vacuole, so that the vacuole can store more water to cope with drought stress.

[0028] Example 3

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

[0030] 1. Primer Design

[0031] The StTST1 gene in the present invention is amplified by designing primers based on the sequence (Soltu.DM.04G037920.1) published in the potato genome database (http: / / solanaceae.plantbiology.msu.edu / pgsc_download.shtml), and the primer sequences are as follows:

[0032] Table 1 StTST1 CDS amplification primer information

[0033]

[0034] 2. RNA Extraction

[0035] Potato E3 leaf samples were taken and ground into powder in liquid nitrogen. The extracts were performed using the Plant Total RNA Kit (centrifuge tube type) ultra-fast plant total RNA extraction kit of Beijing Zhuangmeng International Biogene Technology 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 2.0 ml screw-cap centrifuge tubes, and place them on ice.

[0037] ② Add 1 ml of Lysis Buffer R to every 20-80 mg of young plant tissue, vortexing continuously during the process

[0038] ③ Add 0.2 ml of chloroform to every 1 ml of plant lysate, cover tightly with the screw 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 is distributed in the upper aqueous phase. Pipette 500μl of the supernatant and transfer it to a new 1.5ml centrifuge tube.

[0040] ⑤ Add 0.5 times (250 μl) volume of anhydrous ethanol and 100 μl of binding solution N, and mix thoroughly by inversion. Put the adsorption column on a 2.0 ml centrifuge tube and transfer the obtained products into the adsorption column.

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

[0042] ⑦ Add 500 μl of rinsing solution RW (before opening or using, please make sure to add anhydrous ethanol), centrifuge at 12000 r / min for 30 seconds, discard the waste liquid, and repeat this step.

[0043] ⑧13000r / min, idling for 2min, removing the rinse liquid as much as possible.

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

[0045] 3. cDNA Preparation

[0046] ① Pipette RNA template into eight strips (the amount of aspiration depends on the concentration measured in the previous step; when the concentration is the lowest, aspirate 2 μg), add 2 μl of AccuRT Reaction Mix (4×), make up to 8 μl with Nuclease-free H2O, and incubate in a 42°C metal bath for 2 min.

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

[0048] ③cDNA detection results: the product was 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 Table 2StTST1 fragment amplification system 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 10× DNA loading Buffer and then spot the sample and run on the gel. Select the correct band for gel recovery and purify PCR using the Gel Extraction Kit rapid agarose gel DNA recovery kit of Jiangsu Kangwei Century Biotechnology Co., Ltd.

[0053] 5. Homologous Recombination

[0054] The product was recombined with the digested vector (enzyme StuI) according to the system in Table 3, reacted at 37°C for 30 minutes, and then transformed into DH5α, plated, cultured overnight, single clone colonies were selected for PCR identification, positive bands were selected and sent to the company for sequencing, and then the plasmid was returned to obtain the target vector pH7LIC8-StTST1 CDS .

[0055] Table 3 Recombination reaction system

[0056]

[0057] Example 4

[0058] This example is to clone the vector pH7LIC8-StTST1 obtained in Example 3 CDS The low temperature and drought resistance function verification method is carried out, and the specific verification steps are as follows:

[0059] 1. Transformation of GV3101

[0060] ① Wash the electric transfer cup with distilled water, then wash it three times with 75% alcohol, invert it on absorbent paper on the clean bench, dry it and place it on ice to precool it.

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

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

[0063] ④Cultivate at 28℃ for 2-3 days. Pick out single clones and perform PCR test on the culture solution, and save the positive Agrobacterium culture solution.

[0064] 2. Genetic transformation of potato

[0065] The overexpressed Agrobacterium GV3101 was streaked on YEB solid medium for activation, and a single clone was picked and placed in 20 ml YEB liquid medium, and cultured at 28°C 220 r / min for 24 h; 2 ml of the bacterial solution was placed in 40 ml YEB liquid medium, and cultured again at 28°C 220 r / min until OD 600 About 0.5 (5-6h); 5000r / min, centrifuge for 6min, discard the supernatant, and resuspend in 10ml 3% MS (PH5.8) liquid culture medium. Cut the tuber grown for 6-8 weeks into thin slices with a thickness of about 1-2mm; then infect in the above resuspension for 10min, and shake gently every 3-5min to facilitate infection; transfer the tuber to sterile filter paper to absorb the surface bacterial liquid, and then transfer to P1 co-culture medium (3% MS solid culture medium + 0.2mg / L GA3 + 0.2mg / LIAA + 0.5mg / L 6-BA + 2mg / L ZT, pH 5.8), and culture in a dark incubator at 23℃ for 48h. Then transfer to P2 differentiation medium (3% MS solid medium + 0.2mg / L IAA + 0.2mg / L GA3 + 2mg / L ZT + 0.5mg / L 6-BA + 75mg / L Kan + 400mg / L Cef), and culture in a 23°C incubator (16h light / 8h dark). When the resistant buds grow to 0.5-1cm, they are inoculated into P3 rooting medium (3% MS + 50mg / L Kan + 400mg / L Cef). After the resistant buds grow up, secondary rooting is performed, and then qRT-PCR is used to detect the positive results. 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, and collect fluorescence, 40 cycles. After the PCR program is completed, 95℃ / 30s; 65℃ / 1min; slowly increase the temperature to 95℃, collect fluorescence every 0.5℃ rise, and perform melting curve analysis.

[0069] 3. Cold resistance verification

[0070] Two methods were used: direct frost (method 1) and electrolyte permeability (method 2). The interference strain, overexpression strain and control E3 were planted at the same time and cultured for 4 weeks. Method 1: Put the treatment and control into a constant temperature incubator at the same time, treat at -2 to -3℃ until the leaves become hard, transfer to room temperature, wait for them to recover until new leaves appear, and take pictures to record the status of the plants before, after and after low temperature treatment. Method 2: Take the leaves of a healthy plant, wash them, and place them in a centrifuge tube with 500μl ultrapure water with the petiole facing down. Treat them in a low-temperature water bath with a cooling rate of 1℃ / h (add ice particles when it drops to 0℃), treat each temperature point for 1h, take them out and place them on ice overnight. Add 9.5 ml of ultrapure water, shake at 160 r / min for 2 hours, heat to room temperature, and measure the conductivity R1 before cooking with a conductivity meter (Mellet, FE30-K, Switzerland). Treat at 99°C for 30 minutes, cool to room temperature, and measure the conductivity R2 after cooking. R1 / R2 is used to approximately represent the electrolyte permeability caused by leaf cell membrane damage at this temperature point.

[0071] The frost results showed that there was no significant difference between the transgenic lines and the control E3 before and after frost. After recovery, the interference lines were in a better state than E3, while the overexpression lines were the worst (see Figure 1 ). This indicates that the StTST1 gene negatively regulates potato cold resistance, and interfering with the StTST1 gene can improve potato cold resistance.

[0072] The results of the electrolyte permeability test showed that at the four temperature points of -1°C, -3°C, -4°C, and -5°C, the electrolyte permeability of the overexpression strain was higher than that of E3; while at the five temperature points of -1°C, -2°C, -3°C, -4°C, and -5°C, the electrolyte permeability of the interference strain was lower than that of E3 (see Figure 2 ).

[0073] Comprehensive existing experimental results show that interfering with the expression of StTST1 gene significantly improves the cold resistance of potato.

[0074] 4. Drought resistance verification

[0075] The drought resistance was determined by relative water content, observation of plant status and stomatal opening. The details are as follows: take all leaves in good condition from the plants before, after and after drought treatment, weigh and record the fresh weight m1, quickly dry them, weigh and record the dry weight m2, and use (m1-m2) / m1*100% to represent the relative water content of the leaves. Observe the state of the plants during the drought treatment and use a scanning electron microscope to observe the stomatal opening and closing state on the lower surface of the leaves during the drought treatment.

[0076] The results of relative water content showed that the relative water content of leaves of overexpression lines was significantly higher than that of E3 before and after drought treatment. For the interference lines, before drought treatment, the relative water content of #10 and #18 was not significantly different from that of E3, and the relative water content of #33 was even significantly higher than that of E3. However, after watering, the relative water content of the three lines was significantly lower than that of E3 (see Figure 3 ). This indicates that the StTST1 gene positively regulates potato drought resistance.

[0077] Before and after drought treatment, there was no significant difference in the status of transgenic lines and E3 plants, but after watering, the status of the overexpression line was better than E3, while the status of the interference line was worse than E3 (see Figure 4 ). At the same time, after drought treatment, the stomatal opening of the overexpression line was smaller than that of E3, while the stomatal opening of the interference line was larger than that of E3 (see Figure 5 ). The above results show that overexpression of StTST1 gene improves potato drought resistance.

[0078] The above is only a specific implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

[0079] Main references:

[0080] [1] Kou Shuang. Role and mechanism of putrescine biosynthesis pathway in potato cold resistance[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,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.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. Application of interfering with StTST1 gene expression to improve potato cold resistance, characterized in that The potato StTST1 gene transcript sequence is shown in the sequence table SEQ No.

1.

2. Application of interfering with StTST1 gene expression to reduce electrolyte permeability of potato under low temperature stress, characterized in that The potato StTST1 gene transcript sequence is shown in the sequence table SEQ No.

1.

3. Application of overexpressing StTST1 gene to improve drought resistance of potato, characterized in that The potato StTST1 gene transcript sequence is shown in the sequence table SEQ No.

1.

4. Application of overexpressing StTST1 gene to reduce stomatal aperture in potato, characterized in that The potato StTST1 gene transcript sequence is shown in the sequence table SEQ No.

1.

5. Application of overexpressing StTST1 gene to increase relative water content of potato leaves, characterized in that The potato StTST1 gene transcript sequence is shown in the sequence table SEQ No.

1.

6. Potato StTST1 gene, characterized in that The potato StTST1 gene transcript sequence is shown in the sequence table SEQ No.

1.

7. A method for verifying the function of potato StTST1 gene, characterized in that The method specifically comprises the following steps: (1) primer design, (2) RNA extraction, (3) cDNA preparation, (4) CDS fragment amplification, (5) homologous recombination, (6) transformation of GV3101, (7) potato genetic transformation, (9) cold resistance function verification, and (10) drought resistance function verification.

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