Application of ScGAME9 gene in improving cold resistance of potatoes

Abstract

The invention relates to the technical field of biology, in particular to application of an ScGAME9 gene in improving the cold resistance of potatoes. The nucleotide sequence of the ScGAME9 gene for regulating the cold resistance of the potatoes disclosed by the invention is shown as (a), (b) or (c), and (a) is a nucleotide sequence shown as SEQ ID NO.2; (b) a nucleotide sequence which is hybridized with the nucleotide sequence as shown in SEQ ID NO.2 and is coded; and (c) a nucleotide sequence which has more than 80% of homology with the nucleotide sequence as shown in SEQ ID NO.2 and is coded. The cold resistance of the potato plant can be regulated and controlled by applying the ScGAME9 or a transgenic biological material thereof, important theoretical support is provided for screening of genetic transformation positive plants and research of a potato low-temperature stress resistance mechanism, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to ScGAME9 the application of a gene in improving the cold resistance of potatoes. Background Art

[0002] Potato ( Solanum tuberosum L.) is the most important tuber crop in the world, widely cultivated worldwide, and plays an important role in ensuring global food security. Frost damage greatly affects the growth, development, production efficiency and geographical distribution of plants. However, most potato cultivars have weak cold resistance and lack the ability of low-temperature acclimation. Low-temperature frost will significantly affect the production of potatoes in winter fallow fields and hinder the development and utilization of winter fallow fields in the south, which will seriously affect the further development of the potato industry in China.

[0003] Most potato cultivars are autotetraploids with a narrow genetic background and almost no cold resistance. There are strains with low-temperature frost tolerance among wild potato species, including S. aclaule 、 S. commersoni 、 S. malmeanum and S. boliviense etc., but there are interspecific hybridization barriers between them and cultivars, and they cannot directly perform sexual hybridization, thus preventing the exchange of cold-resistant genes. Improving the cold resistance of potatoes by genetic engineering means is an effective method. Therefore, excavating the low-temperature resistance genes of wild potato species and introducing them into cultivars is of great significance for cultivating new potato varieties with low-temperature resistance ability. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a ScGAME9 gene for regulating the cold resistance of potatoes and the protein encoded thereby, and proves that it can significantly enhance the cold resistance of potatoes, and can be applied to potato cold-resistant breeding and variety improvement, providing more choices for potato resistance breeding.

[0005] To achieve the above object, the present invention provides a protein encoded by a gene for regulating the cold resistance of potatoes, and the amino acid sequence of the protein is (a) or (b); ScGAME9 (a) a protein composed of the amino acids shown in SEQ ID NO. 1; (b) a derivative protein with the same function obtained by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1; The protein of (a) or (b) above, compared with the wild-type plant, improves the survival rate of potatoes under low-temperature stress.

[0006] ​In some specific embodiments, the present invention provides a protein 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, which has enhanced cold resistance.

[0007] The present invention also provides a gene encoding the above-mentioned protein, and the nucleotide sequence of the gene is (a), (b) or (c); (a) The nucleotide sequence shown in SEQ ID NO. 2; (b) The nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions and encodes it; (c) The nucleotide sequence having more than 80% homology with the nucleotide sequence shown in SEQ ID NO. 2 and encoding it.

[0008] In some specific embodiments, ScGAME9 the gene can regulate the cold resistance of potatoes.

[0009] Those skilled in the art are fully aware that since the same amino acid may be determined by multiple different codons, the nucleotide sequence encoding the above-mentioned protein is not limited to one kind. It can be a nucleotide sequence obtained by synonymous mutation of one or more nucleotides in the mutant nucleotide sequence shown in SEQ ID NO., which can also encode the mutant amino acid sequence of the present invention. It can also be a nucleotide sequence designed according to codon optimization that can encode the mutant amino acid sequence of the present invention.

[0010] In the present invention, the nucleic acid can be optimized or unoptimized, and the present invention does not limit this.

[0011] In the present invention, the stringent conditions refer to the conditions under which the probe hybridizes with its target sequence to a detectable degree exceeding that of hybridization with other sequences. The stringent conditions are sequence-dependent and will vary depending on the environment. By strictly controlling the hybridization or washing conditions, a target sequence that is 100% complementary to the probe can be identified. The stringent conditions can be selectively adjusted to allow for some sequence mismatches, thereby detecting a lower degree of similarity.

[0012] In some specific embodiments, the nucleotide sequence of the gene of a protein provided by the present invention has 80% identity to the sequence shown in SEQ ID NO. 2; preferably 85% identity, more preferably 90% identity, more preferably 95% identity, and most preferably 99% identity.

[0013] The recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above genes also fall within the protection scope of the present invention.

[0014] The applications of any of the above proteins, genes, recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria in the cold stress resistance of cultivated potato varieties also fall within the protection scope of the present invention.

[0015] Furthermore, by increasing ScGAME9 the expression level and / or activity of the gene in the crop, the cold resistance of the crop can be improved.

[0016] A method for preparing transgenic plants also falls within the protection scope of the present invention, including the following steps: introducing the coding gene of the above protein into a receptor plant to obtain a transgenic plant; compared with the wild-type plant, the cold resistance of the transgenic plant is improved; the plant is potato. Through the phenotypic identification of frost resistance, it is found that compared with the wild plant, the frost resistance of the transgenic plant is enhanced.

[0017] Furthermore, the coding gene is introduced into the plant through 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.

[0018] Furthermore, the nucleotide sequence of the coding gene is as shown in SEQ ID No. 2.

[0019] Beneficial effects: The present invention first reveals a ScGAME9 gene with the function of regulating the cold tolerance of potato, and the 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 research on the mechanism of potato cold stress resistance, and having broad application prospects. Description of the Drawings

[0020] Figure 1 For ScGAME9 Full-length cDNA amplification and ScGAME9 identification gel electrophoresis diagram of overexpression plants; 1A is ScGAME9 the full-length cDNA amplification gel electrophoresis diagram, 1B is ScGAME9 the identification gel electrophoresis diagram of overexpression plants; Figure 2 For ScGAME9 the expression level detection diagram of overexpression plants; Figure 3 For the effect of overexpression ScGAME9 on the cold resistance of potato; 3A is under cold stress ScGAME9Phenotypic difference diagram of overexpression plants. Figure 3B shows ScGAME9 Diagram for measuring the survival rate of overexpression plants under low temperature stress; Figure 3C shows ScGAME9 Diagram for measuring the electrolyte leakage rate of overexpression plants; Figure 4 is for overexpression ScGAME9 Effect on the antioxidant capacity of plants under low temperature stress; Figure 4A shows ScGAME9 DAB staining results of overexpression leaves under low temperature stress; Figure 4B shows ScGAME9 NBT staining results of overexpression leaves. Detailed implementation manners

[0021] To enable those skilled in the art to better understand the technical solutions of the present invention, the following combines specific implementation manners to make a detailed description of the present invention. The experimental methods without specific conditions noted in the following examples are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples are, unless otherwise specified, obtained from a conventional biochemical reagent store. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0022] Example 1 ScGAME9 Obtaining of genes The potato materials used in the present invention S. commersonii and Desiree both come from the potato planting resource library of the Potato Science Research Institute of Yunnan Normal University.

[0023] Using the sterile tissue culture seedlings of the potato wild species S. commersonii as materials, RNA was extracted using the TIANGEN polysaccharide 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. According to the potato ScGAME9 gene, specific amplification primers were designed; ScGAME9 -F: 5'-ATGAATATTTCAATTGATGAT-3' (SEQ ID NO.3) ScGAME9 -R: 5'-TTACATTTGTATCAACATTTG -3' (SEQ ID NO.4) The PCR amplification reaction was carried out according to the instructions of Vazyme's 2 × Rapid Taq Master Mix (P222). Using the prepared cDNA as a template, PCR amplification was performed with the above-designed primers. The reaction system was added according to the instructions, and the PCR reaction was carried out. The reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 1 min, for 34 cycles; extension at 72°C for 5 min.

[0024] The PCR amplification products were detected by agarose gel electrophoresis, and a clear and single target band was observed at around 678 bp. Subsequently, the target fragment was recovered to obtain ScGAME9 the cDNA fragment of the gene, and the results were as Figure 1 shown in A.

[0025] Example 2 ScGAME9 Construction of gene overexpression vector 2.1 Digestion of vector plasmid pCAMBIA2300 Use the restriction endonuclease Bam H I and Xba I to digest the vector plasmid pCAMBIA2300. The specific reaction system is as follows:

[0026] At 37°C, after overnight digestion, the vector fragment was recovered.

[0027] 2.2 Ligation reaction Ligate the digested vector plasmid in 2.1 and the ScGAME9 gene cDNA fragment purified in Example 1. The operation was carried out according to the instructions of Vazyme ClonE expression II One Step Cloning Kit (C112). The specific operation steps are as follows: Prepare the reaction system on ice:

[0028] At 37°C, ligate for 30 min.

[0029] 2.3 Transformation Transform the ligation product into Escherichia coli competent cell DH5α. The experiment was carried out according to the product instructions of Vidy Biotechnology DH5α Chemically Competent Cell for Escherichia coli transformation. The specific operation is as follows: Take out the DH5α competent cells from -80°C and quickly insert them into ice. After the bacterial mass melts, add the above ligation product to the competent cells and gently mix with a pipette. Let it stand on ice for 30 minutes; Heat shock at 42°C for 45 seconds, quickly place it back on ice and let it stand for 2 minutes (shaking will reduce the transformation efficiency); Add 700 μL of LB without antibiotics, mix well, and incubate at 37°C and 200 rpm for 60 minutes; Centrifuge at 5000 rpm for 1 min to collect the bacterial cells, leave about 100 μL of the supernatant, gently pipette to resuspend the bacterial cells and spread them on the LB plate containing kanamycin; Invert the plate and place it in an incubator at 37°C overnight.

[0030] 2.4 Identification of positive clones Use specific primers to perform colony PCR identification on single colonies on the plate, extract the plasmid of the positive clone and sequence it. Name the positive clone plasmid with correct sequencing as pCAMBIA2300- ScGAME9 Reserve for later use.

[0031] The primers for colony PCR identification are as follows: Forward primer, P2300- ScGAME9 -F (SEQ ID NO.5): 5'-ctctctctcaagcttggatccATGAATATTTCAATTGATGATGATGAAA-3'; Reverse primer, P2300- ScGAME9 -R (SEQ ID NO.6): 5'-gatacgaacgaaagctctagaTTACATTTGTATCAACATTTGTAAATTCAC-3'.

[0032] Example 3 Agrobacterium-mediated genetic transformation of potato 3.1 Agrobacterium transformation This example is based on the product manual of Weidi Biological GV3101 Chemically Competent Cell for Agrobacterium transformation. Transfer the constructed overexpression vector pCAMBIA2300- ScGAME9 into Agrobacterium tumefaciens. The specific operation is as follows: Take the GV3101 Agrobacterium tumefaciens competent cells stored at -80°C and thaw them on ice; Add 1 μL of the plasmid DNA to be transformed to every 50 μL of competent cells, gently pipette the bottom of the EP tube to mix well, and let it stand on ice for 5 min, in liquid nitrogen for 5 min, in a water bath at 37°C for 5 min, and in an ice bath for 5 min; Add 700 μL of LB liquid medium without antibiotics and incubate with shaking at 28°C for 2 hours; Collect the bacterial cells by centrifugation at 5000 rpm for 1 min, take about 100 μL of the supernatant, gently pipette to resuspend the bacterial cells, and spread them on an LB plate containing Kan and Rif. Incubate the plate upside down at 28 °C for 2 - 3 days.

[0033] Identify by PCR and select positive single colonies.

[0034] 3.2 Agrobacterium-mediated genetic transformation of potato The materials used for genetic transformation are Desiree , and Agrobacterium-mediated genetic transformation includes three processes: pre-culture, co-culture, and regeneration culture: Pre-culture: Select sterile seedlings at 28 days old, cut stem segments without axillary buds with a length of 0.5 - 1 cm and place them on plate A for pre-culture for 2 days; Co-culture: Inoculate the successfully transformed Agrobacterium into 20 mL of LB culture medium containing Kan and Rif, shake the bacteria until OD 600 = 0.6 - 0.8, then centrifuge at 8000 rpm for 10 min, resuspend the bacteria with 20 mL of MS20 liquid suspension. Transfer the stem segments pre-cultured for 2 days into the bacterial suspension, infect for 10 - 15 min, transfer the stem segments to sterile filter paper to absorb the bacterial liquid, and then place them on plate B, seal with tin foil and co-culture for 2 days; Regeneration culture: Transfer the co-cultured potato stem segments to a regeneration medium plate, place them in a light incubator at 22 °C, with a 16 h light (light intensity 2000 lx) / 8 h dark culture cycle, change the culture medium every two weeks, and regenerated seedlings can be obtained in 2 - 3 months.

[0035] 3.3 Obtaining overexpressing transgenic potato plants Identify the overexpressing ScGAME9 transgenic plants. Use the Kan resistance gene contained in the transgenic vector NptII for PCR detection. Extract the DNA of the regenerated seedlings by the CTAB method. According to the sequence of the ScGAME9 gene in the overexpression vector plasmid pCAMBIA2300 - NptII , design the following primers: NptII -F (SEQ ID NO.7): 5'-tcagaagaactcgtcaagaaggcgatagaaggcg-3'; NptII -R (SEQ ID NO.8): 5'-atggggattgaacaagatggattgcacgc-3'.

[0036] The reaction system is as follows:

[0037] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 1 min, 34 cycles; extension at 72°C for 5 min.

[0038] The obtained PCR products were detected by 1% agarose gel electrophoresis, and the results were as Figure 1 shown in B. Specific gene bands of the expected size (798 bp) were amplified in all three transgenic seedlings and the overexpression vector.

[0039] Example 4 Transgenic potato ScGAME9 Detection of gene expression level RNA of the regenerated seedlings was extracted and reverse transcribed into cDNA, and qRT-PCR was performed. The amplification primers are as follows. Using SEC as the internal reference gene, 2 –ΔΔCT was used to calculate the relative expression level of the gene, and the error bars represent SE (n = 3, ∗P <0.05, ∗∗P<0.01, ∗∗∗P<0.001; Student’s t-test).

[0040]

[0041] The reaction system is as follows:

[0042] The reaction program was: pre-denaturation at 95°C for 30 s, denaturation at 5°C for 5 s, annealing at 58°C for 15 s, extension at 72°C for 30 s, 40 cycles.

[0043] The results showed that it was not expressed in the non-transgenic material Desiree, and the gene expression levels in three independent transgenic single plants ScGAME9 were all significantly increased ( ScGAME9 ). Figure 2 ).

[0044] Example 5 Verification of overexpression ScGAME9 Cold tolerance of potato plants The positive transgenic plants and control plants were cold-acclimated (4°C) for 7 days and then treated with low temperature stress (−2°C) for 12 h. The damage degree, electrolyte leakage rate and survival rate of the overexpressed ScGAME9 transgenic plants and the wild type Desiree were statistically analyzed. The results were as Figure 3 shown.

[0045] Figure 3 As shown in A-3B, the leaf damage degree of the overexpressed transgenic lines was significantly lower than that of the wild-type plants, and the survival rate of the plants was significantly higher than that of the wild-type plants; The degree of plant damage can be judged by the electrolyte leakage rate of the plant. The higher the electrolyte leakage rate, the more serious the plant damage. From Figure 3 C, it can be seen that the electrolyte leakage rates of the transgenic plants overexpressing ScGAME9 were significantly lower than those of the wild-type plants, indicating that overexpression of ScGAME9 reduced the damage of potatoes under low-temperature conditions; All these results indicate that overexpression of ScGAME9 can significantly improve the low-temperature tolerance of potatoes.

[0046] Example 6 ScGAME9 Overexpression promotes the scavenging of reactive oxygen species under low-temperature stress Low temperature can damage cell structure and affect its key physiological functions. Low-temperature stress causes osmotic stress, which leads to loss of turgor pressure, destroys membrane stability, inactivates or denatures proteins, accumulates reactive oxygen species (ROS) and causes oxidative damage, and then leads to inhibition of photosynthesis, disorder of metabolic functions and destruction of cell structure. Therefore, maintaining the balance of reactive oxygen species plays a crucial role in effective plant stress responses. DAB and NBT can bind to hydrogen peroxide and superoxide anion respectively and form brown and blue compounds respectively. The content of ROS in plant tissues can be detected by DAB staining and NBT staining techniques.

[0047] As Figure 4 shown in A, after treatment at 4°C and -2°C, the Desiree leaves stained with DAB became significantly darker, indicating a large accumulation of hydrogen peroxide. In contrast, ScGAME9 the leaves of the

[0048] lines only showed local browning after cold treatment. Compared with Desiree, the level of hydrogen peroxide accumulation was lower under low-temperature conditions. Figure 4 NBT staining can detect the level of superoxide anion in plant tissues. Superoxide anion is a kind of reactive oxygen species that can reduce NBT to form a water-insoluble blue formaldehyde compound. As ScGAME9 shown in B, the Desiree leaves were stained darker than the leaves overexpressing

[0049] It can be seen that whether it is DAB staining or NBT staining, the wild-type leaves were stained darker than the leaves overexpressing ScGAME9 After overexpression of ScGAME9 the ROS scavenging ability of potato leaves was significantly improved, indicating that overexpression of ScGAME9 genes can reduce the damage suffered by potato materials under low-temperature conditions by increasing the ROS scavenging rate in leaves.

[0050] Finally, it should be noted that the above description is only the preferred embodiment of the present invention. Those skilled in the art can make various similar representations under the inspiration of the present invention without violating the purpose and claims of the present invention. Such transformations all fall within the protection scope 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 (a) or (b); (a) a protein having the amino acid composition shown in SEQ ID NO. 1; (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.

2. A ScGAME9 A recombinant vector, expression cassette, transgenic line or recombinant bacterium of a gene, characterized in that: The nucleotide sequence of the gene is (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 under stringent conditions; (c) a nucleotide sequence encoding a gene having 80% or more homology to the nucleotide sequence shown in SEQ ID NO.

2.

3. A ScGAME9 The application of the gene in potato cultivars to resistance to low temperature stress is characterized in that: The nucleotide sequence of the gene is (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 under stringent conditions; (c) a nucleotide sequence encoding a gene having 80% or more homology to the nucleotide sequence shown in SEQ ID NO.

2.

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

5. Use of the recombinant vector, expression cassette, transgenic line or recombinant bacteria according to claim 2 in the resistance of 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 following steps: introducing the coding gene of the protein according to claim 1 into a recipient plant to obtain a transgenic plant; compared with wild-type plants, the cold resistance of the transgenic plant is improved; and the plant is potato.

8. The method according to claim 7, characterized in that: 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.

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