Potato gene StPACT and application thereof in regulation and control of drought resistance of potatoes

Abstract

The invention relates to the technical field of biology, and discloses a potato gene StPACT and application thereof in regulation and control of drought resistance of potatoes. According to the invention, through overexpression of the StPACT gene, an important role of the StPACT gene in regulation of drought resistance of potatoes is disclosed. The overexpression of the StPACT gene significantly reduces the drought resistance of potatoes, and the mutant shows more obvious drought sensitivity than a wild type. The invention provides a method for optimizing the drought resistance of potatoes by regulating the StPACT gene through a gene editing technology, and the method has a wide breeding application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a potato gene. StPACT And its application in regulating potato drought resistance. Background Technology

[0002] potato( Solanum tuberosum L. As the world's third largest food crop, potatoes are widely cultivated worldwide. They are not only a pillar of agricultural production and economic development, but also play a key role in maintaining global food security. However, with the increasing severity of global warming and environmental pollution, water shortages are becoming more and more severe. Drought has become an important abiotic stress factor limiting agricultural production. Because potatoes have shallow roots, they are extremely susceptible to abiotic stresses such as drought during their growth. This characteristic makes them particularly sensitive to water deficit during critical growth periods. Drought stress during this period not only inhibits the synthesis of photosynthetic products and their transport to tubers, directly limiting tuber enlargement and yield, but may also lead to tuber malformation, secondary growth, and other problems, seriously damaging their commercial quality.

[0003] As the world's largest potato producer, my country's planting areas are mainly concentrated in arid and semi-arid regions. Therefore, drought stress has become a major environmental limiting factor restricting the improvement of potato yield and quality in my country. Against this background, it is crucial to deeply analyze the molecular mechanism of potato drought resistance, which opens up a new and efficient way to cultivate highly drought-resistant potato varieties and is of great significance to ensuring food security. Summary of the Invention

[0004] To better study the drought resistance of potatoes, this invention provides a potato gene. StPACT Its application in regulating the drought resistance of potatoes, wherein the potatoes StPACT The nucleotide sequence of the gene is as shown in SEQ ID NO.1 or a sequence with more than 80% homology to the sequence shown in SEQ ID NO.1, potato. StPACT The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0005] To achieve the above objectives, the present invention provides the following technical solution: By building StPACT The gene overexpression vector was transformed into potatoes using Agrobacterium-mediated transformation to obtain the overexpressed gene. StVac14 The transgenic potato plants were verified to be diploid, and the resulting transgenic potatoes showed reduced drought resistance.

[0006] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention discloses for the first time a method for inhibiting drought resistance in potatoes. StPACT Genes, Applications StPACT Their transgenic biological materials can inhibit potato drought resistance, providing important theoretical support for the screening of genetically transformed positive plants and the study of the molecular mechanism of potato drought resistance, and have broad application prospects. Attached Figure Description

[0007] Figure 1 for StPACT Image showing the electrophoresis results of full-length cDNA amplification.

[0008] Figure 2 for StPACT Graph showing the expression level of overexpressing plants.

[0009] Figure 3 for StPACT ploidy detection in overexpression plants.

[0010] Figure 4 for StPACT The effect of overexpression on drought resistance in potato plants. Detailed Implementation

[0011] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0012] The specific embodiments are for the purpose of demonstrating StPACT The methods used in verifying the role of genes in potatoes are all routine operations familiar to those skilled in the art, such as the construction of overexpression vectors (the vectors used in the construction of overexpression vectors do not limit the present invention, and other those skilled in the art can choose any overexpression vector according to their actual situation) and the construction of transgenic plants.

[0013] Example 1 StPACT The specific steps for obtaining genes are as follows: Potato material used in this invention S. tuberosum group Stenotomum and S. Phureja All data are from the Potato Planting Resource Bank of the Potato Science Research Institute of Yunnan Normal University.

[0014] (1) Using wild potato species S. tuberosum group StenotomumUsing sterile tissue culture seedlings as material, RNA was extracted using the TIANGEN Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Code No. DP441). Subsequently, the RNA was reverse transcribed into cDNA using the TaKaRa (Code No. 047A) reverse transcription kit. Based on the potato... StPACT Gene, design specific amplification primers (underlined parts are homologous arms): StPACT- F:5'- CTCTCTCTCAAGCTTG ATGAATGTGAAAATTGAGAGTTCA-3' (SEQ ID NO.3); StPACT- R:5'- GCTCCTGCAGCTCGAG TCATTTTGCTTTCAAATCTAGAGAG-3' (SEQ ID NO. 4).

[0015] PCR was performed using cDNA as a template. The PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2.

[0016] Table 1 PCR reaction system Table 2 PCR reaction procedures (2) The PCR amplification products were analyzed by agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, a clear, single, specific band was observed at the expected position of approximately 1332 bp. This target band was then successfully recovered by gel cutting. StPACT The cDNA fragment of a gene.

[0017] Example 2 StPACT The specific steps for constructing gene overexpression vectors are as follows: (1) The pCAMBIA2300 vector was digested with enzymes at 37°C for 4 hours according to the reaction system shown in Table 3 to make it completely linearized. The linearized vector was subsequently verified and used for cloning experiments.

[0018] Table 3 Enzyme digestion system (2) The linearized pCAMBIA2300 vector obtained in step (1) was purified using the Tiangen PCR Product Kit (DP214), and then cloned with the vector obtained in Example 1 using the Vazyme ClonE expression II one-step cloning kit (C112). StPACTHomologous recombination was performed on the cDNA fragment of the gene, and the homologous recombination system was as shown in Table 4. The mixture was then incubated at 37°C for 30 minutes to obtain the recombinant vector.

[0019] Table 4 Homologous Recombination System (3) The recombinant vector obtained in step (2) was transformed into Escherichia coli DH5α competent cells (Weidi Biotechnology), and the steps are as follows: a. Thaw competent cells stored at -80°C on ice, add 5 μL of recombinant vector, gently mix, and incubate on ice for 25 minutes.

[0020] b. Heat shock in a 42°C water bath for 45 seconds, followed by an ice bath for 2 minutes.

[0021] c. Add 700 μL of antibiotic-free LB medium and incubate at 37°C and 200 rpm for 60 minutes with shaking.

[0022] d. Collect bacterial cells by centrifugation at 5000 rpm for 1 minute, resuspend in approximately 100 μL of residual supernatant, and spread onto LB plates containing Kan.

[0023] e. Invert the plate and incubate overnight in a 37°C incubator to screen for single colonies.

[0024] (4) Colony PCR verification and recombinant plasmid extraction: Single colonies grown on the plate in step (3) were picked and inoculated into LA liquid medium containing kanamycin (Kan) for expansion culture. The culture was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. By comparing the sequencing results with the expected target site and vector homologous arm sequences, the vector construction was confirmed to be correct. The bacterial culture with completely identical sequences was retained, and the plasmid was extracted from it. Finally, the required recombinant vector was obtained and named pCAMBIA2300- StPACT .

[0025] The primer sequences used for sequencing are as follows: p2300-F:5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO. 5).

[0026] Example 3 The specific steps of Agrobacterium-mediated potato genetic transformation are as follows: (1) Agrobacterium transformation: The recombinant vector pCAMBIA2300- prepared in Example 2 was transformed into Agrobacterium-200. StPACT Agrobacterium transformation was performed according to the product instructions for Weidi Bio's GV3101 chemically competent cells. The specific steps are as follows: a. Take GV3101 Agrobacterium competent cells stored at -80°C and place them on ice to thaw slowly.

[0027] b. Add 1 μL of recombinant plasmid to every 50 μL of competent cells, mix gently, and then perform the following treatments in sequence: stand on ice for 5 minutes, treat in liquid nitrogen for 5 minutes, heat shock in a 37°C water bath for 5 minutes, and finally in an ice bath for 5 minutes.

[0028] c. Add 700 μL of antibiotic-free LB liquid medium and incubate at 28°C and 200 rpm for 2 hours with shaking.

[0029] d. Take approximately 100 μL of bacterial culture and spread it on an LB agar plate containing kanamycin (Kan). Invert the plate and incubate at 28°C until single colonies appear. Pick single colonies for colony PCR identification, and then perform agarose gel electrophoresis on the colony PCR results. Agrobacterium strains with bands around 1300 bp are stored for future use.

[0030] The primers used for PCR identification are as follows: P2300- StPACT -F:5'-ATGAATGTGAAAATTGAGAGTTCA-3' (SEQ ID NO.6); P2300- StPACT -R:5'-TCATTTTGCTTTCAAATCTAGAGAG-3' (SEQ ID NO. 7).

[0031] Example 4 potatoes S.Phureja The specific steps for varieties to act as genetic transformation recipients are as follows: (1) Pre-culture: Select sterile potato seedlings with a seedling age of 28 days, cut stem segments with a length of 0.5~1cm without axillary buds, inoculate them on solid medium of MS30+2mg / L NAA+1mg / L 6-BA, and pre-culture them in a light incubator for 2 days.

[0032] (2) Co-culture: Agrobacterium, which has been identified as containing the target plasmid, was cultured in 20 mL of LB liquid medium (containing 50 μg / mL Kan and 50 μg / mL LRif) with shaking until OD. 600 The value was 0.6. Subsequently, the cells were collected by centrifugation at 6000 rpm for 10 minutes and resuspended in 20 mL of liquid MS20 medium. The pre-cultured stem segments were immersed in the resuspended bacterial solution for 15 minutes. After being removed, the cells were placed on sterile filter paper to remove excess bacterial solution and then transferred to co-culture medium (MS30 + 2 mg / L NAA + 1 mg / L 6-BA + 1 mg / L AS). The cells were wrapped in aluminum foil to protect them from light and co-cultured for 2 days.

[0033] (3) Regeneration culture: The potato stem segments after co-culture were transferred to regeneration medium (MS20+1mg / L ZT+0.5mg / L VB+100mg / L TMT+50mg / L Kan) and cultured in a light incubator at 22°C with a cycle of 16 hours of light (2000lx) / 8 hours of darkness. Fresh medium was replaced every two weeks until regenerated seedlings were induced, thus obtaining transgenic potato plants.

[0034] Example 5 Identify overexpression StPACT transgenic plants To identify overexpression StPACT Transgenic plants were used for PCR detection using the kanamycin resistance gene (NptII) carried by the vector as a molecular marker. First, genomic DNA was extracted from the regenerated plants using the CTAB method. Then, specific primers were designed based on the sequence of the NptII gene in the overexpression vector pCAMBIA2300-StPACT. The PCR system and reaction procedure were as shown in Tables 5 and 6, and the primer sequences are as follows: NptII-F:5′-TCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCG-3′ (SEQ ID NO.8); NptII-R: 5′-ATGGGGATTGAACAAGATGGATTGCACGC-3′ (SEQ ID NO. 9).

[0035] The amplified PCR products were subjected to agarose gel electrophoresis. Plants corresponding to PCR products with bands around 1300 bp were identified as overexpressing the gene. StPACT Genetically modified plants.

[0036] Table 5. Identification of overexpression StPACT PCR system of transgenic plants Table 6 Identification of Overexpression StPACT PCR program for transgenic plants Example 6 Genetically modified potatoes StPACT Gene expression level detection RNA was extracted from regenerated seedlings and reverse transcribed into cDNA. qRT-PCR was then performed using the following primers, with EF1α as the internal reference gene. –ΔΔCT The relative expression levels of genes were calculated, and the reaction system and process are shown in Tables 7 and 8. (Transgenic potatoes) StPACT The primers for gene qRT-PCR are as follows: EF1α-F:5′-ATTGGAAACGGATATGCTCCA-3′ (SEQ ID NO.10); EF1α-R:5′-TCCCTTACCTGAACGCCTGTCA-3′ (SEQ ID NO. 11); qp- StPACT -F:5′-CATCAAGCCATTGTATGAAGGAAT-3′ (SEQ ID NO. 12); qp- StPACT -R: 5′-ATAACGTCTTTCGAAGTCCCG-3′ (SEQ ID NO. 13).

[0037] Table 7. Genetically Modified Potatoes StPACT Gene expression level detection system Table 8. Genetically Modified Potatoes StPACT Gene expression level qRT-PCR program See results Figure 2 This indicates that, compared to wild-type potatoes, three independent transgenic plants showed... StPACT Gene expression levels increased significantly, indicating StPACT The gene was effectively overexpressed in transgenic potatoes, indicating that the exogenous gene has been successfully integrated and is functioning.

[0038] Example 7 Potato ploidy test ploidy analysis of the control group potato Phureja and three transgenic knockout lines was performed by flow cytometry. The specific steps are as follows: (1) Sample preparation: Take about 20 mg of fresh leaves and place them in a 2 mL centrifuge tube. Add 300 μL of pre-cooled cell lysis buffer and two steel balls.

[0039] (2) Tissue disruption: Place the sample tube in the tissue sampler and disrupt it at 30 Hz for 30 seconds at 4°C.

[0040] (3) Cell lysis: Add 1 mL of lysis buffer, mix gently, and let stand at 4°C for 1 hour to lyse. Then filter the cell suspension through a 400-mesh nylon filter to remove tissue fragments.

[0041] (4) Cell collection: Centrifuge at 1500 rpm for 5 minutes at 4°C and carefully discard the supernatant.

[0042] (5) DNA staining: Add 400 μL of propidium iodide (PI) staining solution (diluted at a ratio of 1:1000) to the precipitate and let it stand in the dark for 10 minutes for staining.

[0043] (6) Flow cytometry analysis: Gently mix the stained cell suspension and perform analysis using a flow cytometer. The excitation wavelength is 488 nm. Collect the fluorescence signal of the PE-A channel and perform ploidy analysis using the accompanying software.

[0044] The results are as follows Figure 3 As shown, overexpression StPACT All the strains are diploid.

[0045] Example 8 Validated expression StPACT To improve the drought resistance of potato plants, the specific steps are as follows: The survival rates of positive transgenic plants and control plants were counted after drought treatment for 7, 10, and 13 days. Figure 4 As shown in A-4B, the survival rate of transgenic plants was significantly lower than that of wild-type plants; the experimental results indicate that overexpression... StPACT It can significantly inhibit the drought resistance of potatoes.

[0046] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. A potato gene StPACT The nucleotide sequence of the gene is as shown in SEQ ID NO.1 or a sequence with more than 80% homology to the sequence shown in SEQ ID NO.1, the potato gene. StPACT The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

2. The potato gene of claim 1 StPACT Application in regulating drought resistance in potatoes.

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