Application of ScGAME9 gene in improving cold resistance of potato

Abstract

The present invention relates to the field of biotechnology, specifically to ScGAME9 The invention discloses a gene for regulating the cold resistance of potatoes. ScGAME9 A gene, the nucleotide sequence of which is as shown in (a), (b) or (c), (a) the nucleotide sequence shown in SEQ ID NO.2; (b) a nucleotide sequence that hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO.2; (c) a nucleotide sequence that has more than 80% homology with the nucleotide sequence shown in SEQ ID NO.2 and encodes. ScGAME9 Or its transgenic biological materials can regulate the cold tolerance of potato plants, providing important theoretical support for the screening of genetically transformed positive plants and the study of the mechanism of potato resistance to low temperature stress, and have broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically to ScGAME9 Application of genes in improving cold resistance of potato. Background Art

[0002] potato( Solanum tuberosum Potato (Solanum spp.) is the world's most important tuber crop, widely cultivated worldwide and playing a vital role in ensuring global food security. Frost damage significantly impacts plant growth, development, productivity, and geographical distribution. However, most potato cultivars have low cold tolerance and lack the ability to acclimate to low temperatures. Low temperatures and frosts can significantly impact potato production in fallow fields and hinder the development and utilization of fallow fields in southern China, severely impacting the further development of my country's potato industry.

[0003] Most cultivated potato varieties are autotetraploid and have a narrow genetic background, with almost no cold resistance. There are some wild potato varieties that are resistant to low temperatures and frost, including S. aclaule 、 S. commersoni 、 S. malmeanum and S. boliviense However, these species face interspecific hybridization barriers with cultivated varieties, preventing direct sexual hybridization and thus preventing the exchange of cold-resistance genes. Improving potato cold resistance through genetic engineering is an effective approach. Therefore, identifying cold-resistance genes from wild potato species and introducing them into cultivated varieties is crucial for developing new potato varieties with cold-resistance. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for regulating the cold resistance of potatoes. ScGAME9 Genes and

[0005] The protein it encodes has been shown to significantly enhance the cold resistance of potatoes and can be used in potato cold-resistant breeding and variety improvement, providing more options for potato resistance breeding.

[0006] To achieve the above object, the present invention provides a method for regulating the cold resistance of potatoes. ScGAME9 A protein encoded by a gene, wherein the amino acid sequence of the protein is (a) or (b);

[0007] (a) a protein consisting of the amino acids shown in SEQ ID NO. 1;

[0008] (b) a derivative protein having the same function as the amino acid sequence shown in SEQ ID NO. 1, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0009] The protein of one of (a) or (b) above, which increases the survival rate of potato under low temperature stress compared with wild-type plants.

[0010] In some specific embodiments, the present invention provides a protein having an amino acid sequence with enhanced cold resistance and having an amino acid sequence with 80% identity to the sequence shown in SEQ ID NO. 1; preferably, 85% identity, more preferably 90% identity, more preferably 95% identity, and most preferably, 99% identity.

[0011] The present invention also provides a gene encoding the above-mentioned protein, wherein the nucleotide sequence of the gene is (a), (b) or (c);

[0012] (a) the nucleotide sequence shown in SEQ ID NO. 2;

[0013] (b) a nucleotide sequence that hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions;

[0014] (c) a nucleotide sequence encoding a gene that has 80% or more homology to the nucleotide sequence shown in SEQ ID NO. 2.

[0015] In some specific embodiments, ScGAME9 Genes can regulate potato cold tolerance.

[0016] It is well known to those skilled in the art that there may be multiple different codons for the same amino acid.

[0017] Therefore, the nucleotide sequence encoding the above-mentioned protein is not limited to one type. It can be a nucleotide sequence encoding the mutant amino acid sequence of the present invention obtained by mutating one or more nucleotides of the mutant nucleotide sequence shown in SEQ ID NO. 2 to form a synonymous mutation. It can also be a nucleotide sequence encoding the mutant amino acid sequence of the present invention designed based on codon optimization.

[0018] In the present invention, the nucleic acid may be optimized or not optimized, and the present invention does not limit this.

[0019] As used herein, stringent conditions refer to conditions under which a probe hybridizes to its target sequence to a detectable degree, exceeding hybridization to other sequences. Stringent conditions are sequence-dependent and vary depending on the environment. By strictly controlling hybridization or washing conditions, target sequences that are 100% complementary to the probe can be identified. Stringent conditions can be selectively adjusted to allow for some sequence mismatches, thereby enabling detection of lower degrees of similarity.

[0020] In some specific embodiments, the present invention provides a protein whose gene nucleotide sequence has 80% identity with the sequence shown in SEQ ID NO. 2; preferably, it has 85% identity, more preferably, it has 90% identity, more preferably, it has 95% identity, and most preferably, it has 99% identity.

[0021] Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above genes also fall within the scope of protection of the present invention.

[0022] The use of any of the above proteins, genes, recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria in the resistance of potato cultivars to low temperature stress also falls within the scope of protection of the present invention.

[0023] Furthermore, by increasing ScGAME9 The expression level and / or activity of the gene in the crop is increased to improve the cold resistance of the crop.

[0024] A method for preparing a transgenic plant also falls within the scope of protection of the present invention, comprising the following steps: introducing the coding gene of the above-mentioned protein into a recipient plant to obtain a transgenic plant; compared with the wild-type plant, the transgenic plant has improved cold resistance; the plant is potato. Through frost resistance phenotype identification, it is found that the transgenic plant has improved cold resistance compared with the wild-type plant.

[0025] The transgenic plants showed enhanced frost resistance compared with the control plants.

[0026] Furthermore, the coding gene is introduced into the plant via a recombinant expression vector;

[0027] The expression vector is obtained by inserting the coding gene into the multiple cloning site of the original vector pCAMBIA2300.

[0028] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

[0029] Beneficial effect: This invention discloses for the first time a method for regulating the cold tolerance of potatoes. ScGAME9 Gene, application ScGAME9 Or its transgenic biological materials can regulate the cold tolerance of potato plants, providing important theoretical support for the screening of genetically transformed positive plants and the study of the mechanism of potato resistance to low temperature stress, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 for ScGAME9 Full-length cDNA amplification and ScGAME9 Gel electrophoresis diagram of overexpression plant identification;

[0031] 1A is ScGAME9Gel electrophoresis of full-length cDNA amplification, 1B is ScGAME9 Gel electrophoresis diagram of overexpression plant identification;

[0032] Figure 2 for ScGAME9 Expression level detection diagram of overexpression plants;

[0033] Figure 3 For overexpression ScGAME9 Effects on potato cold resistance;

[0034] 3A is under low temperature stress ScGAME9 Phenotypic differences of overexpression plants, 3B is under low temperature stress ScGAME9 Figure 3C shows the survival rate of overexpression plants under low temperature stress. ScGAME9 Electrolyte permeability measurement of overexpressing plants;

[0035] Figure 4 For overexpression ScGAME9 Effects on the antioxidant capacity of plants under low temperature stress;

[0036] 4A is under low temperature stress ScGAME9 DAB staining results of overexpressed leaves; 4B is under low temperature stress ScGAME9 NBT staining results of overexpressing leaves. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following is a detailed description of the embodiments of the present invention.

[0038] The present invention is described in detail. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0039] Example 1 ScGAME9 Gene acquisition

[0040] Potato material used in the present invention S. commersonii and Desiree , all come from the potato planting resource library of the Potato Science Research Institute of Yunnan Normal University.

[0041] Wild potato species S. commersoniiAseptic tissue culture seedlings of potato were used as materials, RNA was extracted using the TIANGEN Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Code No. DP441), and then the RNA was reverse transcribed into cDNA using the TaKaRa (Code No. 047A) reverse transcription kit. ScGAME9 gene, design specific amplification primers;

[0042] ScGAME9 -F: 5'-ATGAATATTTCAATTGATGAT-3' (SEQ ID NO.3)

[0043] ScGAME9 -R: 5'-TTACATTTGTATCAACATTTG -3' (SEQ ID NO.4)

[0044] PCR amplification was performed according to the instructions for Vazyme's 2× Rapid Taq Master Mix (P222). The prepared cDNA was used as a template and the designed primers were used for PCR amplification. The reaction mixture was added according to the instructions and the PCR reaction was performed. The reaction conditions were as follows: initial denaturation at 95°C for 3 min, followed by 34 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 1 min, followed by extension at 72°C for 5 min.

[0045] The PCR amplification product was detected by agarose gel electrophoresis, and a clear and single target band was found at about 678 bp. The target fragment was then recovered and obtained. ScGAME9 The cDNA fragment of the gene was Figure 1 As shown in A.

[0046] Example 2 ScGAME9 Gene overexpression vector construction

[0047] 2.1 Enzyme digestion of vector plasmid pCAMBIA2300

[0048] Use restriction endonucleases Bam HI and Xba I enzyme digested the vector plasmid pCAMBIA2300, and the specific reaction system was as follows:

[0049]

[0050] After overnight digestion at 37°C, the vector fragment was recovered.

[0051] 2.2 Ligation reaction

[0052] The enzyme-digested vector plasmid in 2.1 and the purified vector plasmid in Example 1 were ScGAME9The gene cDNA fragments were connected according to the instructions of Vazyme ClonE expression II One Step Cloning Kit (C112). The specific steps are as follows:

[0053] Set up the reaction system on ice:

[0054]

[0055] Ligate at 37°C for 30 min.

[0056] 2.3 Conversion

[0057] The ligation product was transformed into E. coli competent cells DH5α. The E. coli transformation was carried out according to the instructions of the DH5α Chemically Competent Cell product of Weidi Biotechnology. The specific steps are as follows:

[0058] Remove the DH5α competent cells from −80°C and quickly place them on ice. After the bacterial block thaws, add the above ligation product to the competent cells and gently mix with a pipette. Let it stand on ice for 30 minutes.

[0059] Heat shock at 42°C for 45 seconds, then quickly return to ice and let stand for 2 minutes (shaking will reduce transformation efficiency);

[0060] Add 700 μL of LB without antibiotics, mix well, and recover at 37°C, 200 rpm for 60 minutes;

[0061] Centrifuge at 5000 rpm for 1 min to collect the cells, take about 100 μL of the supernatant, gently pipette to resuspend the cells, and spread them on LB plates containing kanamycin;

[0062] Invert the plate and place in a 37°C incubator overnight.

[0063] 2.4 Identification of positive clones

[0064] The single colonies on the plate were identified by colony PCR using specific primers, and the positive clone plasmids were extracted and sequenced. The positive clone plasmids with correct sequencing were named pCAMBIA2300- ScGAME9 spare.

[0065] The primers for colony PCR identification are as follows:

[0066] Upstream primer, P2300- ScGAME9 -F (SEQ ID NO.5):

[0067] 5'-ctctctctcaagcttggatccATGAATATTTCAATTGATGATGATGAAA-3';

[0068] Downstream primer, P2300- ScGAME9 -R (SEQ ID NO.6):

[0069] 5'-gatacgaacgaaagctctagaTTACATTTGTATCAACATTTGTAAATTCAC-3'.

[0070] Example 3 Agrobacterium-mediated genetic transformation of potato

[0071] 3.1 Agrobacterium transformation

[0072] This example is based on the instructions of Weidi Bio GV3101 Chemically Competent Cell product.

[0073] The constructed overexpression vector pCAMBIA2300- ScGAME9 Transform into Agrobacterium tumefaciens, the specific operation is as follows:

[0074] GV3101 Agrobacterium tumefaciens competent cells stored at −80°C were thawed on ice;

[0075] Add 1 μL of the plasmid DNA to be transformed to every 50 μL of competent cells, gently stir the bottom of the EP tube to mix, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0076] Add 700 μL of LB liquid medium without antibiotics and culture at 28°C with shaking for 2 hours;

[0077] Centrifuge at 5000 rpm for 1 min to collect the cells. Take about 100 μL of the supernatant and gently pipette to resuspend the cells. Spread the cells on LB plates containing Kan and Rif and culture them upside down at 28°C for 2-3 days.

[0078] PCR identification was performed and positive single colonies were selected.

[0079] 3.2 Agrobacterium-mediated genetic transformation of potato

[0080] The materials used for genetic transformation were Desiree Agrobacterium-mediated genetic transformation includes three processes: pre-culture, co-culture, and regeneration culture:

[0081] Pre-culture: Select sterile seedlings of 28 days old, cut stem segments of 0.5-1 cm in length without axillary buds and place them on plate A for pre-culture for 2 days;

[0082] Co-cultivation: Inoculate the successfully transformed Agrobacterium into 20 mL LB culture medium containing Kan and Rif and shake the culture to OD 600 = 0.6-0.8, then centrifuged at 8000 rpm for 10 min, and used 20 mL of MS20 liquid suspension to transfer the stem segments pre-cultured for 2 days into the suspension. Infected for 10-15 min, the stem segments were transferred to sterile filter paper to absorb the suspension, and then placed on plate B, sealed with tin foil, and cultured for 2 days.

[0083] Regeneration culture: Transfer the co-cultured potato stem segments to the regeneration culture medium plate and place them in a light incubator at 22°C with 16 h light (light intensity 2000 lx) / 8 h dark. Change the culture medium every two weeks.

[0084] Cultivate the medium and regenerate seedlings can be obtained in 2 to 3 months.

[0085] 3.3 Obtaining overexpression transgenic potato plants

[0086] Overexpression ScGAME9 The Kan resistance gene contained in the transgenic vector was used to identify the transgenic plants. NptII PCR detection was performed. DNA of regenerated seedlings was extracted by CTAB method and the overexpression vector plasmid pCAMBIA2300- ScGAME9 middle NptII The following primers were designed based on the gene sequence:

[0087] NptII -F (SEQ ID NO.7):5'-tcagaagaactcgtcaagaaggcgatagaaggcg-3';

[0088] NptII -R (SEQ ID NO. 8):5'-atggggattgaacaagatggattgcacgc-3'.

[0089] The reaction system is as follows:

[0090]

[0091] Pre-denaturation at 95°C for 3 min; 34 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 1 min; and extension at 72°C for 5 min.

[0092] The PCR products were detected by 1% agarose gel electrophoresis. Figure 1 As shown in B, specific gene bands of the expected size (798 bp) were amplified from the three transgenic seedlings and the overexpression vector.

[0093] Example 4 Transgenic Potato ScGAME9 Gene expression level detection

[0094] RNA was extracted from regenerated seedlings and reverse transcribed into cDNA for qRT-PCR. The amplification primers were as follows: SEC was used as the internal reference gene, and 2 –ΔΔCT The relative expression of genes was calculated, and the error bars represent SE (n = 3, ∗P

[0095] <0.05, ∗*P<0.01, ****P<0.001; Student's t-test).

[0096]

[0097] The reaction system is as follows:

[0098]

[0099] The reaction program was as follows: pre-denaturation at 95°C for 30 s, denaturation at 5°C for 5 s, annealing at 58°C for 15 s, and extension at 72°C for 30 s, for 40 cycles.

[0100] The results showed that in the non-GMO material Desiree ScGAME9 No expression, three independent transgenic lines ScGAME9 Gene expression levels increased significantly ( Figure 2 ).

[0101] Example 5 Verification of overexpression ScGAME9 Cold resistance of potato plants

[0102] The positive transgenic plants and control plants were cold acclimated (4°C) for 7 days and then subjected to low temperature stress (−2°C) for 12 hours. ScGAME9 Transgenic plants and wild type Desiree The damage degree, electrolyte permeability and survival rate of the patients were statistically analyzed. The results are as follows Figure 3 shown.

[0103] Figure 3 As can be seen from A-3B, the leaf damage degree of the overexpressing transgenic lines was significantly lower than that of the wild-type plants, and the plant survival rate was significantly higher than that of the wild-type plants;

[0104] The degree of plant damage can be determined by the electrolyte permeability of the plant. The higher the electrolyte permeability, the more serious the plant damage. Figure 3 C can be seen that overexpression ScGAME9 The electrolyte permeability of the transgenic plants was significantly lower than that of the wild-type plants, indicating that the overexpression ScGAME9Reduces damage to potatoes under low temperature conditions;

[0105] These results indicate that overexpression ScGAME9 It can significantly improve the low temperature tolerance of potatoes.

[0106] Example 6 ​ Overexpression promotes the scavenging of reactive oxygen species under low temperature stress

[0107] Low temperatures can damage cell structure and affect key physiological functions. Low temperature stress causes osmotic stress, which leads to a loss of turgor pressure, disrupting membrane stability, inactivating or denaturing proteins, and accumulating reactive oxygen species (ROS), causing oxidative damage. This in turn leads to inhibition of photosynthesis, metabolic dysfunction, and damage to cell structure. Therefore, maintaining a balance of ROS plays a crucial role in achieving an effective plant stress response. DAB and NBT can bind to hydrogen peroxide and superoxide anions, respectively, to form brown and blue compounds. DAB and NBT staining techniques can be used to detect ROS levels in plant tissues.

[0108] like ​ As shown in A, after treatment at 4°C and −2°C, the leaves of Desiree stained with DAB became significantly darker, indicating a large accumulation of hydrogen peroxide. ​ The leaves of this line showed only partial browning after cold treatment. Compared with Desiree, the accumulation level of hydrogen peroxide was lower under low temperature conditions.

[0109] NBT staining can detect the level of superoxide anions in plant tissues. Superoxide anions are a type of reactive oxygen species that can reduce NBT to form a water-insoluble blue formaldehyde compound. ​ As shown in B, Desiree leaves were significantly different from those overexpressed ​ The leaves are darker in color.

[0110] It can be seen that no matter DAB staining or NBT staining, the wild-type leaves are significantly better than the overexpression leaves. ​ The leaves of the overexpressed ​ The ROS scavenging ability of potato leaves was significantly improved after overexpression ​ The gene can reduce the damage of potato materials under low temperature conditions by improving the ROS clearance rate of leaves.

[0111] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make various similar expressions without violating the purpose and claims of the present invention. Such changes fall within the scope of protection of the present invention.

Claims

1. A method for regulating the cold resistance of potatoes ScGAME9 The protein encoded by the gene is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

1.

2. A ScGAME9 A recombinant vector, expression cassette or recombinant bacterium of a gene, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. Overexpression ScGAME9 The application of a gene in potato cultivars to resistance to low temperature stress is characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. Use of overexpressing the protein according to claim 1 in resisting potato cultivars to low temperature stress.

5. Use of the recombinant vector, expression cassette or recombinant bacterium according to claim 2 in resisting potato cultivars to low temperature stress.

6. The use according to any one of claims 3 to 5, characterized in that: By raising ScGAME9 The expression level and / or activity of the gene in the crop is increased to improve the cold resistance of the crop.

7. A method for preparing a transgenic plant, comprising the steps of: introducing the gene encoding the protein of claim 1 into a recipient plant to obtain a transgenic plant; the transgenic plant has improved cold resistance compared to a wild-type plant; the plant is a potato.

8. The method according to claim 7, wherein: The coding gene is introduced into the plant via a recombinant expression vector; the recombinant expression vector is obtained by inserting the coding gene into the multiple cloning site of the initial vector pCAMBIA2300.

9. The method according to claim 7 or 8, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

10. The method according to claim 7 or 8, characterized in that: The primer pair for amplifying the coding gene includes an upstream primer F and a downstream primer R. The nucleotide sequence of the upstream primer F is shown in SEQ ID NO. 3, and the nucleotide sequence of the downstream primer R is shown in SEQ ID NO. 4.

Images