Application of StDnaj gene in improvement of salt tolerance of potatoes

Abstract

The invention relates to the technical field of biology, in particular to application of an StDnaj gene to improvement of salt tolerance of potatoes. The protein disclosed by the invention is shown in (a) or (b); (a) a protein composed of amino acids as shown in SEQ ID NO.1; and (b) a derivative protein which is obtained by substitution and / or deletion and / or addition of one or more amino acid residues on the amino acid sequence shown as SEQ ID NO.1 and has the same function. The salt tolerance function of the StDnaj gene and the StDnaj protein coded by the StDnaj gene in plants is proposed and verified for the first time, the StDnaj gene and the StDnaj protein can be applied to salt-resistant breeding and variety improvement of potatoes, and more choices are provided for potato resistance breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to StDnaj Application of genes in improving salt tolerance in potato. Background Art

[0002] Soil salinization is a phenomenon in which the salt content in the soil is too high and difficult to be directly used due to excessive accumulation or diffusion of water-soluble salt content on the surface. Soil salinization will hinder the normal growth and development of crops or other plants, which is extremely challenging to agricultural production and a global problem. The ever-expanding area of ​​salinized soil has seriously affected crop yields and restricted local agricultural production. The continuous intensification of soil salinization has changed the physical and chemical properties of the soil, making the soil easy to form compaction and poor air permeability. The increased osmotic pressure in the soil creates obstacles for plants to absorb water and nutrients. At the same time, the high concentration of salt ions in the soil affects the growth and development of plants and even withers and dies by affecting seed germination, seedling growth and vitality, and plant flowering and fruiting. In order to achieve more effective management and utilization of saline-alkali soil, it has become an important issue to be solved to reveal the mechanism of plant tolerance to salt-alkali stress and cultivate excellent salt-alkali stress-tolerant crop varieties.

[0003] potato( Solanum tuberosum L.) is an annual herbaceous plant of the genus Solanum in the Solanaceae family. Potatoes originated in the Andes Plateau in South America and are one of the most important food crops in the world. 1.3 billion people in the world rely on potatoes as their staple food, making it the third largest food crop in the world after rice and wheat. my country is the country with the largest potato production, with the planting area and total output accounting for more than a quarter of the global total for a long time. Potatoes are widely distributed in my country and are mainly divided into four planting areas: the northern single-crop area, the central plains double-crop area, the southwestern mixed cropping area of ​​one and two seasons, and the southern winter cropping area. However, potato is a crop that is sensitive to salt. The salt stress caused by soil salinization leads to a decrease in the photosynthetic rate of potato plants, a disruption of ion homeostasis, and an imbalance in osmotic regulation, which in turn affects the growth and development of tubers and causes yield losses. Due to its own high heterozygosity, potatoes have slow analysis of important traits, including unclear excellent salt-tolerant genes and lack of excellent material support in breeding. Therefore, it is urgent to analyze the excellent salt-tolerant genes of potatoes and create new potato varieties with salt tolerance through genetic improvement. Summary of the invention

[0004] The invention provides a kind of potato wild species StDnaj Coding sequence and the protein it encodes The product has been shown to significantly enhance the salt tolerance of potatoes and can be used in potato salt tolerance breeding and variety improvement, providing more options for potato resistance breeding.

[0005] To achieve the above object, the present invention provides a potato salt tolerance-related protein, wherein the protein is (a) or (b); (a) a protein consisting of the amino acids 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; Plants containing one of the proteins of (a) or (b) above have improved survival rate of potatoes under salt stress compared with wild-type plants.

[0006] In some specific embodiments, StDnaj The gene is derived from potato A056.

[0007] 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 and having enhanced salt tolerance; preferably, 85% identity, more preferably, 90% identity, more preferably, 95% identity, and most preferably, 99% identity.

[0008] The present invention also provides a protein encoding the above StDnaj A gene, wherein the nucleotide sequence of the gene is (1), (2) or (3); (1) the nucleotide sequence shown in SEQ ID NO. 2; (2) a nucleotide sequence that hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions; (3) A nucleotide sequence encoding a gene that has 80% or more homology to the nucleotide sequence shown in SEQ ID NO. 2.

[0009] The present invention also provides a kind of containing the above-mentioned protein StDnaj Gene.

[0010] It is well known to those skilled in the art that there may be multiple different codons for the same amino acid. Therefore, the nucleotide sequence encoding the above-mentioned protein is not limited to one type. It can be a nucleotide sequence that can also encode the mutant amino acid sequence of the present invention by mutating one or more nucleotides of the RNase inhibator mutant nucleotide sequence shown in SEQ ID NO. 2 to form a synonymous mutation, or a nucleotide sequence that can encode the mutant amino acid sequence of the present invention can be designed based on codon optimization.

[0011] 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.

[0012] The recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the above gene also fall within the protection scope of the present invention.

[0013] 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 salt stress also falls within the protection scope of the present invention.

[0014] The present invention also provides StDnaj Application of genes as selection markers using high salt as selection pressure.

[0015] The present invention also provides StDnaj Application of genes in breeding transgenic potatoes tolerant to high salt levels.

[0016] The present invention also provides StDnaj Application of genes in genetic engineering to adjust salt tolerance in potato.

[0017] A method for preparing transgenic plants also falls within the scope of protection of the present invention, comprising the following steps: introducing the coding gene of the above protein into a starting plant to obtain a transgenic plant; compared with wild-type plants, the salt tolerance of the transgenic plant is enhanced.

[0018] Furthermore, 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-eYGFPuv.

[0019] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

[0020] Furthermore, the plant is potato.

[0021] Beneficial effects: The present invention provides StDnaj Overexpression of the gene in potato cultivar C65 can improve the salt tolerance of transgenic potato plants, providing important theoretical support for the screening of genetic transformation-positive plants and the study of the mechanism of potato salt stress resistance, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Validation for qRT-PCR StDnaj Expression patterns in Leaf and Root; Figure 2 Fluorescent markers for genetic transformation of potato; Figure 3 for StDnaj Detection of expression levels of transgenic lines; Figure 4 for StDnaj Identification of salt tolerance of transgenic lines; Figure 5 for StDnaj Analysis of wilting rate of transgenic lines. DETAILED DESCRIPTION

[0023] 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 present invention. The present invention is described in detail. The experimental methods in the following examples without specifying specific conditions are usually 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, are 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 meanings as those familiar to those skilled in the art. In addition, any methods and materials similar to or equivalent to the recorded contents can be applied to the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0024] Example 1 StDnaj Gene and expression pattern studies The plant material used in this study was salt-tolerant wild potato material A056 (A056 was provided by the potato molecular breeding team of the Potato Science Research Institute of Yunnan Normal University. A056 was disclosed in the document "Identification of potato HAK gene family and analysis of salt tolerance expression. Journal of Yunnan Normal University (Natural Science Edition), 2023, 43(4): 22-28". The public can obtain the above biological materials from the applicant). The plants were grown on MS medium supplemented with 3% sucrose and 0.35% agar at 20±1°C for 2 weeks. After that, the 2-week-old tissue culture seedlings were transplanted into plastic pots (10×10 cm) and planted in a plant growth climate chamber (22±2°C, 14 h light / 10 h dark photoperiod). The 4-week-old plants grown under normal conditions were subjected to salt stress treatment and 200 mM NaCl aqueous solution, root and leaf tissues were taken at 0h, 6h, 12h, and 24h, and quick-frozen in liquid nitrogen; leaves were taken from the treated plants, RNA was extracted, and reverse transcribed into cDNA for qRT-PCR (qRT-PCR was performed using Takara's TB Green Premix Ex Taq II), and the amplification primers were as follows: Elongation factor 1α, EF1α ) was used as the internal reference gene, and 2–ΔΔCT Calculate the relative expression of genes (see Figure 1 ), error bars represent SE (n = 3, ∗ P <0.05, ∗∗ P <0.01, ∗∗∗ P <0.001; Student's t-test).

[0025]

[0026] The reaction system is as follows:

[0027] The reaction procedure is as follows:

[0028] from Figure 1 It can be seen that after 24 h of salt stress treatment, the leaf tissue of potato material A056 StDnaj The expression of was upregulated by 6 times. StDnaj For the research object, StDnaj To investigate whether salt tolerance in potato cultivars can be regulated.

[0029] Example 2 StDnaj1 Gene cloning and overexpression vector construction StDnaj The gene cloning used the above cDNA as a template, and according to the cDNA sequence of SEQ ID NO. 2, the homology arm sequences of the pCAMBIA 2300-YeGFPuv vector were added to both sides of the primers; StDnaj -F:5'-GAGAACACGGGGGACTCTAGAATGTTTGGAAGAGGAGGGAAAA-3' StDnaj -R: 5'-ACTAGTTCTGGATCCTCTAGACTGTTGAGCACACTGTACTCTCTGT-3' The primer combination was used to amplify the cDNA with a full length of 1260 bp. The operation was carried out according to the instructions of Phanta® Max MasterMix (Dye Plus) of Vazyme Company. The reaction system was as follows:

[0030] The reaction procedure is as follows:

[0031] Overexpression vector construction (1) BamH I and Xba I digested the vector plasmid pCAMBIA 2300-eYGFPuv, and the reaction system was as follows:

[0032] The reaction program was 37°C, 4 h; (2) Purify the PCR product a and the vector plasmid b after restriction digestion, and connect them using the instructions of Vazyme ClonE expressionII One Step Cloning Kit (C112). The specific steps are as follows: The following connection system is configured on ice:

[0033] The reaction program was 37°C, 30 min; 12°C, 5 min; (3) Conversion Use the instructions of the Weidi Biotechnology DH5α Chemically Competent Cell product to transform E. coli. The specific steps are as follows: a. Take out the DH5α competent cells from -80°C and quickly put them in ice. After 3 minutes, wait for the bacterial block to melt, add 5 μL of the above ligation product and gently stir the bottom of the EP tube by hand to mix gently, and let it stand in ice for 30 minutes; b. Heat shock in a 42°C water bath for 80 seconds, then quickly return to ice and let stand for 2 minutes; c. Add 700 μL of LB liquid culture medium without antibiotics, mix well, and place at 37°C, shake at 200 rpm for 60 minutes to recover; d. Centrifuge at 12000 rpm for 5 min to collect the cells, take about 150 μL of the supernatant, gently blow to resuspend the cells and spread on the LB plate containing Kan; e. Place the plate upside down in a 37°C incubator overnight.

[0034] (4) Identification of positive clones The specific primers p2300-sF / sR on the vector were used for amplification, and the positive clones were sequenced. The plasmid extracted from the positive clones with correct sequencing was named pCAMBIA 2300-eYGFPuv- StDnaj spare; p2300-sF: 5'-TCTCTCTCTCAAGCTTGGATCC-3' p2300-sR: 5'-AAACAGTTTTCCAATGCCAT-3' Example 3 Agrobacterium-mediated genetic transformation of potato Agrobacterium was transformed with plasmid pCAMBIA 2300-eYGFPuv- StDanj , use the instructions of Weidi Bio GV3101 Chemically Competent Cell product to carry out Agrobacterium transformation, the specific operation is as follows: (1) Thaw the GV3101 Agrobacterium competent cells stored at −80°C on ice; (2) Add 1 μL of the plasmid DNA to be transformed into every 50 μL of competent cells, mix gently, and place on ice for 30 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min; (3) Add 700 μL of LB liquid medium without antibiotics and incubate at 28°C with a shaker at 220 rpm for 2 h; (4) Pipette about 100 μL of bacterial solution and spread it on the LB plate containing Kan and Rif. Invert the plate and place it in a 28°C incubator for 2-3 days.

[0035] (5) Colony PCR identification.

[0036] 2. Agrobacterium-mediated genetic transformation of potato The recipient material for genetic transformation was C65, and the following process was involved: (1) Pre-culture: Select 20-day-old sterile potato seedlings, cut stem segments with a length of 0.5 to 1 cm without axillary buds, and place them on a plate for pre-culture for 2 days; (2) Co-cultivation: Shake 20 mL of successfully transformed Agrobacterium (LB+Kan+Rif) to OD600 = 0.5-0.6 at 8000 rpm for 10 min, resuspend the bacteria in 40 ml MS20 liquid culture medium, transfer the above stem segments to the bacterial solution, infect for 10-15 min, transfer to sterile filter paper to absorb the bacterial solution, place on a plate, cover with tin foil and culture in the dark for 2 days; (3) Regeneration culture: Transfer the co-cultivated potato stem segments to the plate of regeneration culture medium and place them in a light incubator at 22°C with alternating conditions of 16 h light (light intensity 2000 lx) / 8 h dark. Replace the culture medium every two weeks until regenerated seedlings grow out.

[0037] (4) Positive plant screening: The vector contains a separate promoter driving the eYGFPuv tag protein. When illuminated by a 365nm ultraviolet flashlight, positive plants appear fluorescent green, while negative plants appear red ( Figure 2 ), The positive plants were preliminarily identified by fluorescence detection, and the positive seedlings were further transferred to the rooting medium.

[0038] Example 4 Transgenic seedlings StDnaj Expression level detection Leaves of wild-type WT (non-transgenic) and overexpressed transgenic lines were taken respectively, and total RNA was extracted using the method in Example 1, and reverse transcribed to generate cDNA. qPCR was used to identify the transgenic lines. StDnaj expression level.

[0039] from Figure 3 It can be seen that the four transgenic lines StDnaj The expression level was upregulated 20-80 times compared with the wild type WT.

[0040] Example 5 Overexpression StDnaj Improving potato salt tolerance We selected four overexpression-positive plants for salt tolerance identification and counted the plant wilting rate (wilted leaves / total leaves). Figure 4 As can be seen from the figure, after 14 days of 200 mM NaCl stress treatment, the growth of the transgenic line was significantly better than that of the wild type, and Figure 5 As can be seen in StDnaj The wilting rate of the gene overexpression strain was significantly lower than that of the wild type, indicating StDnaj Overexpression of the gene can significantly enhance potato salt tolerance. StDnaj After the gene is overexpressed, the salt tolerance of potatoes can be significantly improved, laying the foundation for potato resistance breeding.

[0041] 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 a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the scope of protection of the present invention.

Claims

1. A protein related to potato salt tolerance, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

1.

2. A protein encoding the protein of claim 1 StDnaj A gene, wherein the nucleotide sequence of the gene is (1), (2) or (3); (1) the nucleotide sequence shown in SEQ ID NO. 2; (2) a nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO. 2 and encodes the protein shown in SEQ ID NO. 1; (3) A nucleotide sequence that has more than 80% homology with the nucleotide sequence shown in SEQ ID NO. 2 and encodes the protein shown in SEQ ID NO.

1.

3. A method comprising the method according to claim 2 StDnaj Recombinant vector, expression cassette, transgenic line or recombinant bacteria of the gene.

4. A method according to claim 2 StDnaj Application of genes as selection markers using high salt as selection pressure.

5. A method according to claim 2 StDnaj Application of genes in breeding transgenic potatoes tolerant to high salt levels.

6. Use of the gene according to claim 2 in regulating salt tolerance of potatoes by genetic engineering.

7. A protein according to claim 1, a protein according to claim 2 StDnaj Use of the gene, the recombinant vector, the expression cassette, the transgenic line or the recombinant bacteria as claimed in claim 3 in improving salt tolerance of potato cultivars.

8. A method for preparing a transgenic plant, comprising the following steps: introducing the gene encoding the protein according to claim 1 into a starting plant to obtain a transgenic plant.

9. The method according to claim 8, 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-eYGFPuv.

10. The method according to claim 8 or 9, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

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