Use of peach ppnac2 gene in improving cold resistance of plants

Abstract

The application provides a peach PpNAC2 gene in improving plant cold resistance, belongs to the technical field of plant breeding. A peach PpNAC2 gene or a gene derivative product containing the peach PpNAC2 gene is applied in at least one of improving plant cold resistance, constructing cold-resistant transgenic plants and breeding cold-resistant plants. The application transforms the peach PpNAC2 gene into peach callus and Arabidopsis thaliana respectively under the mediation of Agrobacterium, and obtains transgenic materials. Low-temperature treatment (-4 DEG C) test finds that the fresh weight of the transgenic peach callus is obviously higher than that of the untransgenic peach callus, and the survival rate of the transgenic Arabidopsis thaliana is obviously higher than that of the wild type (WT). The peach PpNAC2 gene provides a candidate resource for plant cold-resistant breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, specifically relating to a peach... PpNAC2 The application of genes in improving plant cold resistance. Background Technology

[0002] Low temperatures occur frequently, often causing frost damage to crops and resulting in large-scale yield reductions, severely impacting agricultural development. Therefore, exploring genetic resources and molecular mechanisms that respond to low-temperature stress is of significant theoretical and practical importance for solving current agricultural problems.

[0003] NAC, as one of the largest transcription factor families in plants, is widely involved in plant growth and development and responses to abiotic stresses. Numerous studies have shown that NAC genes play an important role in plant disease responses. However, no reports have been found regarding the role of NAC genes in low-temperature stress responses. Summary of the Invention

[0004] The purpose of this invention is to provide a peach PpNAC2 A new use for genes, namely their application in improving plant cold resistance.

[0005] This invention provides a peach PpNAC2 Gene or conifer PpNAC2 The application of gene-derived products in at least one of the following: improving plant cold resistance, constructing cold-resistant transgenic plants, and breeding cold-resistant plants.

[0006] Preferably, the peach PpNAC2 Gene-derived products include at least one of the following: gene expression cassettes, recombinant vectors, recombinant strains, and recombinant plant cells.

[0007] Preferably, the peach PpNAC2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0008] Preferably, the peach PpNAC2 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0009] Preferably, the plant includes dicotyledonous plants.

[0010] Preferably, the dicotyledonous plants include at least one of the following: Rosaceae fruit trees, deciduous fruit trees, economic forest trees, and overwintering crops.

[0011] Preferably, the plant includes at least one of the following growth forms: callus, tissue culture seedling, seedling, and plant.

[0012] Preferably, the temperature range for cold resistance includes -8 to 4°C.

[0013] Preferably, improving plant cold resistance includes reducing the plant's frost mortality rate in cold environments and increasing the plant's growth rate in cold environments.

[0014] This invention provides a method for improving the cold resistance of plants, specifically peaches. PpNAC2 Gene overexpression in plants or application of growth promoters to plants PpNAC2 Gene or peach PpNAC2 Reagents for overexpressing gene-encoded proteins.

[0015] This invention provides a peach PpNAC2 Gene or conifer PpNAC2 The application of gene-derived products in at least one of the following: improving plant cold resistance, constructing cold-resistant transgenic plants, and breeding cold-resistant plants. Experiments show that the peach... PpNAC2 The expression level of the gene gradually increased with the duration of low-temperature stress treatment, suggesting that it may be involved in the regulation of cold resistance in plants. This was achieved through Agrobacterium-mediated transformation of peach... PpNAC2 The gene was overexpressed in peach callus and Arabidopsis seedlings, respectively. The results showed that, compared with the control group where the gene was not overexpressed, peach callus tissue showed improved efficacy. PpNAC2 Overexpression of genes is beneficial for biological materials to cope with the effects of low-temperature stress, not only maintaining a faster growth rate but also improving the survival rate of the biological materials. Therefore, the peach... PpNAC2 Genes enhance plant cold resistance through positive regulation, providing a new approach for constructing cold-resistant transgenic plants and breeding cold-resistant plants. Attached Figure Description

[0016] Figure 1 For peach PpNAC2 PCR amplification of the gene, gel electrophoresis image; Figure 2 For peach PpNAC2 Results of relative gene expression levels under low temperature stress; Figure 3 The spectrum of the pNC-Cam2304-35S vector; Figure 4 For peach PpNAC2 Subcellular localization map; Figure 5 The results of phenotypic analysis of peach callus under low temperature stress are shown in A, B, and C. Phenotypic diagram, B, and C respectively. This indicates a significant difference between the treatment group and the WT group. P <0.05; Figure 6 The results of Arabidopsis thaliana phenotypic analysis under low temperature stress are shown in A, B, and C. Phenotypic diagrams, B, and C respectively. This indicates a significant difference between the treatment group and the WT group. P<0.05. Detailed Implementation

[0017] This invention provides a peach PpNAC2 Gene or conifer PpNAC2 The application of gene-derived products in at least one of the following: improving plant cold resistance, constructing cold-resistant transgenic plants, and breeding cold-resistant plants.

[0018] In this invention, the peach PpNAC2 The preferred amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:2. The peach... PpNAC2 The gene includes a coding sequence derived from peach trees or a sequence optimized based on the codon bias of the host plant. The nucleotide sequence of the peach-derived coding sequence is preferably as shown in SEQ ID NO:1. Used for amplifying peach... PpNAC2 The primers for the gene preferably include a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4. In one embodiment of the invention, subcellular localization analysis revealed that the peach... PpNAC2 The proteins encoded by the gene are expressed only in the cell nucleus and cytoplasm.

[0019] In this invention, the cinnamon... PpNAC2Gene-derived products preferably include at least one of the following: gene expression cassettes, recombinant vectors, recombinant strains, and recombinant plant cells. The gene expression cassette preferably includes a promoter, an enhancer, a target gene, and a terminator. The promoter includes constitutive promoters, inducible promoters, or tissue-specific promoters, and the expression cassette can be constructed using the corresponding promoter based on the cold-resistant component. Using a constitutive promoter can drive the continuous expression of the target gene in all tissues and all growth stages of host cells (Arabidopsis, peach cells, etc.), unaffected by external environmental factors (such as low temperature and light) and tissue specificity. Constitutive promoters preferably include the CaMV 35S promoter, the Ubiquitin promoter, and the Actin promoter. Commonly used terminators for plants are the Nos terminator and the CaMV 35S terminator. The recombinant vector preferably includes at least one of the following: plant recombinant vectors, prokaryotic recombinant vectors, and shuttle recombinant vectors. The plant recombinant vector is used for plant cell transformation, such as Agrobacterium-mediated recombinant vectors (pLB-T, pNC-Cam2304-35S vector). The prokaryotic recombinant vectors are used for amplification and preliminary expression of target genes in prokaryotic cells (Escherichia coli, Agrobacterium), and are divided into clonal recombinant vectors (pUC18, pUC19) or expression-type recombinant vectors. The expression-type recombinant vectors are used for efficient expression of the target gene in prokaryotic cells, such as the pET series (pET-28a, pET-32a) and pGEX series. The shuttle recombinant vectors can replicate and express in two or more host cells (e.g., simultaneous replication in Escherichia coli and Agrobacterium, or simultaneous expression in prokaryotic cells and plant cells), enabling rapid amplification of peach genes and efficient transformation of Arabidopsis thaliana. This invention does not impose any special restrictions on the preparation method of gene-derived products; any preparation method well-known in the art can be used.

[0020] In this invention, the plant preferably includes dicotyledonous plants. The dicotyledonous plants preferably include at least one of the following: Rosaceae fruit trees, deciduous fruit trees, economic forest trees, and overwintering crops. The Rosaceae fruit trees include peach, plum, apricot, apricot, cherry, apple, pear, etc. The plant preferably includes at least one of the following growth forms: callus tissue, tissue culture seedlings, seedlings, and plants. In this embodiment of the invention, peach callus tissue and Arabidopsis thaliana plants are used as examples to illustrate the peach... PpNAC2 The cold resistance of genes.

[0021] In this invention, the preferred temperature range for cold resistance is -8 to 4°C, but it can be -5 to 0°C or -4 to -2°C. Improving plant cold resistance preferably includes reducing the rate of plant death due to cold environments and increasing the plant's growth rate in cold environments.

[0022] In this invention, the method for constructing cold-resistant transgenic plants preferably includes using peaches... PpNAC2 Gene or peach PpNAC2The gene-encoded protein is overexpressed in the plant. The preferred method for breeding cold-resistant plants is using peaches. PpNAC2 Gene expression levels were used as a screening indicator to select peaches. PpNAC2 Plants with relatively high gene expression levels are used as breeding materials, intermediate materials, or planting materials.

[0023] This invention provides a method for improving the cold resistance of plants, specifically peaches. PpNAC2 Gene overexpression in plants or application of growth promoters to plants PpNAC2 Gene or peach PpNAC2 Reagents for overexpressing gene-encoded proteins.

[0024] In this invention, detection PpNAC2 The preferred method for detecting gene overexpression in plants is qPCR. The preferred primers for detection are the forward primer shown in SEQ ID NO:5 and the reverse primer shown in SEQ ID NO:6. qPCR detection also includes internal reference gene detection primers, preferably the forward primer shown in SEQ ID NO:7 and the reverse primer shown in SEQ ID NO:8.

[0025] The following describes, in conjunction with embodiments, a peach provided by the present invention. PpNAC2 The application of genes in improving plant cold resistance is described in detail, but it should not be construed as limiting the scope of protection of this invention.

[0026] Table 1 Primer sequences involved in this application

[0027] Example 1 PpNAC2 Acquisition of genes Peach branches were used as experimental material and planted in a natural environment. A disease-free plant was selected, and young branches were cut. Total RNA was extracted from the samples using a polysaccharide and polyphenol plant total RNA extraction kit (Tiangen, Beijing, China). The integrity and purity of the samples were checked by 1% agarose gel electrophoresis, and the concentration was checked using a UV spectrophotometer. Then, the total RNA was reverse transcribed into cDNA using a FastKing cDNA first-strand synthesis kit (Tiangen, Beijing, China). A pair of specific primers (see SEQ ID NO: 3 and SEQ ID NO: 4 in Table 1) were designed for PCR amplification using peach cDNA as a template. The amplification system consisted of 25.0 μl PrimeSTARMax Premix (2×), 2.0 μl cDNA, 1.5 μl forward primer, 1.5 μl reverse primer, and 20.0 μl RNase-free ddH2O. The amplification program was 94℃ for 2 min; 94℃ for 15 s, 57℃ for 20 s, 72℃ for 30 s, for 37 cycles; and 72℃ for 10 min. The amplified fragment was detected by gel electrophoresis, and the results are shown below. Figure 1 As shown.

[0028] After purification and recovery, the PCR product was ligated into the pLB-T vector (Tiangen, Beijing, China) to obtain pLB-T:: PpNAC2 Plasmid. Sequencing revealed that the nucleic acid sequence was 879 bp in length, as shown in SEQ ID NO: 1, which encodes a protein containing 292 amino acid residues, as shown in SEQ ID NO: 2.

[0029] PpNAC2Nucleic acid sequence: ATGGGAAGTGGAAGTGAGTTGGAGTTACCGCCAGGGTTCAGATTTCACCCAACGGACGAGGAGTTGGTGAATTATTACTTGTGCCGGAAATGTGCTGGGCAGCCTCTTGCTGTTCCCATCATCAAAGAGATTGATCTTTACAAGTTTGATCCTTGGCAGCTACCTGATATGGCTCTTTATGGAGAAAAAGAGTGGTATTTCTTTTCGCCAAGGGATAGAAAATATCCGAACGGTTCAAGGCCGAACCGGGCAGCAGGAACCGGGTACTGGAAGGCGACCGGGGCAGACAAGCACATTGGAAAGCCCAAGGCACTTGGGATTAAAAAGGCACTCGTGTTCTACGCTGGCAAAGCTCCTAAAGGAGTTAAAACCAATTGGATCATGCACGAGTATCGCCTTGCAAATGTTGATAGGTCGGCTTCCAAGAAAAACAACAACAACTTGAGGCTTGATGATTGGGTGCTGTGCCGAATATACAACAAGAAAGGTAGCATAGAGAAATATAACGTTGCCATGGAGCGCAGTAAAATGACCAAATACCCAGAAATATTGCATGAGCAAAAACCAGAAATGACCCAAATGCCACCACCCCATACGGATATGTCGTCGATGGATTCAGCACCGAGGGTGCAGCAGACGACGGACTACTCTAGCTGCTCGGAGCACGTGCTGTCGCCAGAGGTCACGTGGGAGAAGGAGGTCCAAAGTGAGCTACAATGGAGCGGTGATGAATTAGAGAATTCCTTTAATACCCTTGATAATCAGTTCATCAATTACATGGATGGCTTCTCAGATATTCTTGACTCTTTTGGCGCTGCTCAGCCTCAGTACCAAATGGACCAGCAGAACATGTTTGCCTACTTACAAACCCAATTTTAA (SEQ ID NO: 1).

[0030] The PpNAC2 protein sequence is as follows: MGSGSELELPPGFRFHPTDEELVNYYLCRKCAGQPLAVPIIKEIDLYKFDPWQLPDMALYGEKEWYFFSPRDRKYPNGSRPNRAAGTGYWKATGADKHIGKPKALGIKKALVFYAGKAPKGVKTNWIMHEYRLANVDRSAS KKNNNNLRLDDWVLCRIYNKKGSIEKYNVAMERSKMTKYPEILHEQKPEMTQMPPPHTDMSSMDSAPRVQQTTDYSSCSEHVLSPEVTWEKEVQSELQWSGDELENSFNTLDNQFINYMDGFSDILDSFGAAQPQYQMDQQNMFAYLQTQF (SEQ ID NO: 2).

[0031] Example 2 PpNAC2 Analysis of gene expression patterns under low temperature stress First, peach saplings were subjected to 0℃ low-temperature stress treatment. Leaf samples were collected at 0, 1, 2, and 4 hours after treatment, and total RNA was extracted and cDNA was obtained by reverse transcription. 0 hours served as a control. [Further details on cDNA extraction are needed for accurate translation.] PpNAC2 The relative expression levels of genes under low temperature stress (see Table 1) PpNAC2 The primers used for gene qPCR amplification were SEQ ID NO: 5 and SEQ ID NO: 6, and the primers for internal control gene detection were SEQ ID NO: 7 and SEQ ID NO: 8. The amplification system consisted of 10.0 μl SYBR Green (2×), 1.0 μl cDNA, 1.0 μl forward primer, 1.0 μl reverse primer, and 7.0 μl RNase-free ddH2O. The amplification program was 95℃ for 2 min; 95℃ for 5 s, 60℃ for 10 s, and 72℃ for 15 s, for 40 cycles. Melting curve analysis was performed. Method calculation PpNAC2 The relative expression level of genes.

[0032] Depend on Figure 2 As can be seen from the qRT-PCR test results, PpNAC2 Gene expression levels were significantly upregulated under low temperature stress, reaching their peak at 4 hours, indicating that... PpNAC2 Genes are involved in the peach's response to low-temperature stress.

[0033] Example 3 PpNAC2 subcellular localization analysis pLB-T:: PpNAC2Using the plasmid as a template, primers SEQ ID NO: 9 and SEQ ID NO: 10 from Table 1 were used to clone plasmids with homologous arms. PpNAC2 Gene fragments, and then homologous recombination is used to... PpNAC2 The gene was cloned and ligated into the pNC-Cam2304-MCS35S vector (Yan P, Tuo D, Shen W. A Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants Plant Communications, 2022; 4) (see...) Figure 3 ), obtain the stop codon removed PpNAC2 -pNC-Cam2304-MCS35S recombinant vector. Extraction PpNAC2 The recombinant vector pNC-Cam2304-MCS35S and the control vector pNC-Cam2304-MCS35S were transformed into Agrobacterium GV3101. After confirming the presence of positive colonies, the bacteria were activated by shaking. Then, 100 μl of the activated bacterial solution was inoculated into a sterile glass tube containing 10 ml of LB liquid medium (containing kanamycin and rifampin) and incubated at 220 rpm for 10–14 h at 28°C in a shaker. OD was monitored in real time. 600 The bacterial cell density was controlled to approximately 1.0. The cells were efficiently collected by centrifugation at 5000 rpm for 10 min. After discarding the supernatant, the cells were gently resuspended in MMA infection buffer (containing 2.033 g / L MgCl2·6H2O, 2.132 g / L MES, and 100 μM acetylsylcholine, pH 5.5), and the suspension was adjusted to OD. 600 =1.0. The samples were placed in a dark environment at room temperature for 2–3 h. The bacterial solution was inoculated onto the dorsal side of *Nicotiana benthamiana* leaves using a sterile syringe. After transformation, the transplanted plants were transferred to a dark incubator and maintained for 3 days. Green fluorescent protein (GFP) signals were detected in the treated leaves using a laser confocal microscopy system (Leica TCSSP8 SR).

[0034] like Figure 4 As shown, the empty GFP vector is expressed in all parts of tobacco leaf cells, while the PpNAC2 protein is expressed only in the nucleus and cytoplasm, indicating that... PpNAC2 The gene-encoded protein is a protein that is localized in the cell nucleus and cytoplasm.

[0035] Example 4 PpNAC2 Phenotypic identification of peach callus after low-temperature treatment Take 100 μl containing the recombinant vector PpNAC2 Agrobacterium GV3101 (pNC-Cam2304-MCS35S) was added to 50 ml of YEP (Kana+Rif) and cultured at 220 rpm for 16 h. The bacterial cells were collected by centrifugation at 6000 rpm for 10 min, the supernatant was discarded, and 25 ml of callus infection solution (MS 4.43 g / L, sucrose 35.0 g / L) was added and the bacterial solution was thoroughly mixed. The callus tissue was placed in the bacterial infection solution mixture and cultured on a shaker at 220 rpm for 30 min. The callus tissue was then filtered through sterile gauze. The peach callus tissue was transferred to filter paper in a petri dish and allowed to stand for 30 min to absorb surface moisture. Peach callus tissue was transferred to a co-culture medium (MS 4.43 g / L, sucrose 35.0 g / L, agar 6.0 g / L, 6-BA 1.0 mg / L, IBA 0.2 mg / L, GA3 0.1 mg / L) and cultured in the dark for 2–3 days. The infected peach callus tissue was then transferred to a selection medium (MS 4.43 g / L, sucrose 35.0 g / L, agar 6.0 g / L, 6-BA 1.0 mg / L, IBA 0.2 mg / L, GA3 0.1 mg / L, Kana 80 mg / L, Cef 300 mg / L) and cultured in the dark until callus tissue showing a clear growth trend was observed. This tissue was then transferred to a new selection medium for propagation to obtain… PpNAC2 Overexpression of positive peach callus was detected in the tissue. qRT-PCR was used to detect the positive peach callus (primers were SEQ ID NO: 5 and SEQ ID NO: 6 from Table 1; primers for internal control gene detection were SEQ ID NO: 7 and SEQ ID NO: 8). Method calculation PpNAC2 The relative expression level of genes.

[0036] Figure 5 The results from qRT-PCR testing (as shown in section B) indicate that... PpNAC2 The relative expression level in positive peach callus tissue was significantly higher than that in the control, indicating that it had been acquired. PpNAC2 Overexpression of peach callus tissue.

[0037] Will PpNAC2 Overexpressing positive peach callus and non-transgenic peach callus were inoculated into the same culture flask, with one-third of the area of ​​each inoculated. They were cultured at 23°C under light for 8 days. After being treated at -4°C for 16 hours, they were transferred to normal conditions and cultured for another 7 days. The phenotype was then observed.

[0038] Depend on Figure 5 It can be seen that the volume of non-GMO peach callus tissue was significantly smaller after low-temperature treatment. PpNAC2 Overexpression of positive peach callus tissue was observed in the fresh weight analysis. PpNAC2 The fresh weight of transgenic peach callus was significantly higher than that of non-transgenic peach callus, indicating that... PpNAC2 It can improve the cold resistance of peaches.

[0039] Example 5 PpNAC2 Phenotypic identification of Arabidopsis thaliana overexpression under low temperature treatment Utilizing PpNAC2 Recombinant Agrobacterium-mediated flower-dipping method was used to infect Arabidopsis inflorescences, yielding... PpNAC2 Positive Arabidopsis thaliana plants were overexpressed. Positive plants were detected using qRT-PCR (primers were SEQ ID NO: 5 and SEQ ID NO: 6 from Table 1; primers for the internal control gene were SEQ ID NO: 7 and SEQ ID NO: 8). Method calculation PpNAC2 The relative expression level of genes.

[0040] Depend on Figure 6 As can be seen from B, the results of qRT-PCR testing show that... PpNAC2 The relative expression levels of the three positive lines were significantly higher than those of WT, indicating that three positive lines have been obtained. PpNAC2 Overexpression in Arabidopsis thaliana positive plants.

[0041] T3 generation transgenic Arabidopsis seeds and WT seeds were placed in 2 mL centrifuge tubes and surface-sterilized with 1 mL of 75% ethanol for 60 seconds, followed by removal of the ethanol solution in a laminar flow hood. Then, 1 mL of 6% sodium hypochlorite solution (prepared by dilution of the stock solution) was added for 10 min of sterilization. After removing the sodium hypochlorite in a laminar flow hood, the seeds were rinsed four times with sterile double-distilled water to thoroughly remove any ethanol and sodium hypochlorite residues. The sterilized seeds were inoculated into different sections of antibiotic-free 1 / 2 MS medium plates. After vernalization, the materials were transferred to a 23°C incubator for cultivation. Seven days after germination, the seeds were subjected to a -4°C cryopreservation treatment for 3.5 h, and phenotypic analysis was performed.

[0042] like Figure 6 As shown in Figures A and C, after low-temperature treatment, 3 PpNAC2 The survival rate of the overexpressing Arabidopsis thaliana lines was significantly higher than that of the WT lines, while the survival rate was 100% under normal temperature conditions, indicating that... PpNAC2 It can improve the cold resistance of Arabidopsis thaliana.

[0043] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of peach PpNAC2 Gene or conifer PpNAC2 The application of gene-derived products in at least one of the following: improving plant cold resistance, constructing cold-resistant transgenic plants, and breeding cold-resistant plants.

2. The application according to claim 1, characterized in that, The peach PpNAC2 Gene-derived products include at least one of the following: gene expression cassettes, recombinant vectors, recombinant strains, and recombinant plant cells.

3. The application according to claim 1, characterized in that, The peach PpNAC2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

4. The application according to claim 3, characterized in that, The peach PpNAC2 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

5. The application according to claim 1, characterized in that, The plants mentioned include dicotyledonous plants.

6. The application according to claim 5, characterized in that, The dicotyledonous plants include at least one of the following: Rosaceae fruit trees, deciduous fruit trees, economic forest trees, and overwintering crops.

7. The application according to claim 1, characterized in that, The plant includes at least one of the following growth forms: callus, tissue culture seedling, seedling, and plant.

8. The application according to claim 1, characterized in that, The temperature range for cold resistance is -8 to 4℃.

9. The application according to claim 1, characterized in that, Improving plant cold resistance includes reducing the rate of plant death due to cold environments and increasing the growth rate of plants in cold environments.

10. A method for improving the cold resistance of plants, characterized in that, peach PpNAC2 Gene overexpression in plants or application of growth promoters to plants PpNAC2 Gene or peach PpNAC2 Reagents for overexpressing gene-encoded proteins.

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