Application of DcNAC78 gene in improving heat resistance of dianthus caryophyllus

Abstract

The invention relates to the technical field of gene engineering, and discloses an application of a DcNAC78 gene in improvement of heat resistance of carnation, the nucleotide sequence of the DcNAC78 gene is shown as SEQ ID NO.1, and the amino acid sequence coded by the DcNAC78 gene is shown as SEQ ID NO.2. The invention further discloses a preparation method of the DcNAC78 gene. According to the application of the DcNAC78 gene in improving the heat resistance of the dianthus caryophyllus, the DcNAC78 gene is overexpressed in a dianthus caryophyllus plant, so that the heat resistance of the dianthus caryophyllus can be obviously improved, and the DcNAC78 gene can be used for cultivating a new variety of heat-resistant dianthus caryophyllus; meanwhile, the invention discloses that the DcNAC78 gene positively regulates and controls the heat resistance of carnation by enhancing the membrane stability, regulating the infiltration capacity and enhancing the active oxygen scavenging capacity.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to DcNAC78 Application of genes in improving heat tolerance of carnation. Background Art

[0002] Carnation ( Dianthus caryophyllus Dianthus spicata is a perennial herbaceous plant of the Caryophyllaceae family, genus Dianthus. Widely cultivated in China for its ornamental value, it is well-suited for vase placement and is often used as a cut flower. As one of the world's four major cut flowers, carnations are popular with consumers for their rich, vibrant colors, and diverse flower shapes. They possess high economic, practical, and ornamental value.

[0003] Carnations are native to the Mediterranean region, prefer cool climates and cannot tolerate heat. Currently, the occurrence of extreme high temperatures caused by global warming has had an adverse impact on their cultivation and preservation. Long-term high temperatures will cause their growth to slow, the plants to wilt, the petals to shrink, and their susceptibility to diseases and pests to increase, thereby reducing their quality. Therefore, it is very necessary to improve the tolerance of carnations to high temperature environments through genetic engineering technology.

[0004] NAC is a family of plant-specific transcription factors that play an important role in plant responses to various abiotic stresses, including temperature, salinity, and drought. Studies on NAC transcription factors under high-temperature stress have been reported in Arabidopsis thaliana and other plants such as wheat and rice, as well as in ornamental plants such as wintersweet and lily. Currently, research on NAC transcription factors has primarily focused on model plants, with limited research on the functions of NAC family genes in non-model plants. Furthermore, no studies have been conducted on the role of NAC transcription factors in response to high-temperature stress in carnation. Therefore, screening and identifying NAC family genes in the carnation genome is of great significance in providing a genetic reserve and theoretical basis for heat-tolerant breeding and germplasm innovation in carnation. Summary of the Invention

[0005] The object of the present invention is to provide DcNAC78 Application of genes in improving heat tolerance of carnation, overexpression in carnation DcNAC78 Gene, can significantly improve the heat resistance of carnation, can be used to cultivate new varieties of heat-resistant carnation; at the same time, the present invention discloses DcNAC78 The gene regulates the heat tolerance of carnation by enhancing membrane stability, regulating permeability, and enhancing the ability to scavenge reactive oxygen species.

[0006] To achieve the above object, the present invention provides DcNAC78 Application of genes in improving heat tolerance of carnation, DcNAC78 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Furthermore, when applied, overexpression in Carnation DcNAC78 Genes to improve the heat tolerance of carnation.

[0008] The present invention also provides a DcNAC78 protein for improving the heat resistance of carnation. The amino acid sequence of the DcNAC78 protein is shown in SEQ ID NO.2. The coding gene of the DcNAC78 protein is DcNAC78 Gene, DcNAC78 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] The present invention also provides the use of the DcNAC78 protein in improving the heat resistance of carnation.

[0010] The present invention also provides a recombinant vector comprising the recombinant vector shown in SEQ ID NO.1 DcNAC78 Gene.

[0011] Furthermore, the recombinant vector is a plant overexpression vector.

[0012] The present invention also provides application of the recombinant vector in improving the heat resistance of carnation.

[0013] The present invention also provides a method for cultivating high temperature resistant carnation, wherein the DcNAC78 Genes were overexpressed in carnations to screen and cultivate high-temperature-resistant carnations.

[0014] The present invention DcNAC78 The advantages and positive effects of using genes to improve the heat tolerance of carnations are: 1. The present invention discloses DcNAC78 Gene, overexpressed in Carnation DcNAC78 This gene can significantly improve the heat resistance of carnation, can be used for genetic engineering improvement of plants to adapt to high temperature stress, effectively alleviate the damage caused by high temperature to plants, and has a broad application prospect.

[0015] 2. The present invention discloses DcNAC78 The molecular mechanism by which genes enhance heat tolerance, DcNAC78 The gene positively regulates the heat tolerance of carnation by enhancing membrane stability, regulating permeability, and enhancing the ability to scavenge reactive oxygen species.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Carnation in the embodiment of the present invention DcNAC78 Electropherogram of gene coding region sequence verification; Figure 2The subcellular localization of DcNAC78 protein in the embodiment of the present invention; Figure 3 In the embodiment of the present invention DcNAC78 Verification of transcriptional activation activity in yeast cells, Figure 3 A in the middle is the identification and segmented structure of the DcNAC78 NAC domain, the N-terminus is 1-450bp, and the C-terminus is 451-1641bp. Figure 3 Middle B is the verification of the transcriptional activation activity of DcNAC78, the pGBKT7-lam vector is used as a negative control, pGBKT7-53 is used as a positive control, Trp is tryptophan, and His is histidine; Figure 4 The pSuper and pSuper- DcNAC78 Phenotype and character of detached petals of Carnation DcNAC78 The expression level of Figure 4 A in the middle is the phenotype of the detached petals of Carnation. Figure 4 B is DcNAC78 In pSuper and pSuper- DcNAC78 The expression level in Figure 5 The pSuper and pSuper- DcNAC78 DAB staining and H2O2 content of isolated petals of Carnation, Figure 5 A in the middle is DAB staining of isolated carnation petals. Figure 5 B is the H2O2 content; Figure 6 The pSuper and pSuper- DcNAC78 NBT staining and O2 staining of isolated petals of Carnation . - Content, among which, Figure 6 Middle A is NBT staining of isolated carnation petals. Figure 6 B is O2 . - content; Figure 7 The pSuper and pSuper- DcNAC78 Relative electrical conductivity of isolated petals of Carnation; Figure 8 The pSuper and pSuper- DcNAC78 Phenotype, DAB and NBT staining of Dianthus caryophyllus seedlings; Figure 9 The pSuper and pSuper- DcNAC78 Carnation seedlings DcNAC78 Relative expression, O2 . - Content, H2O2 content and relative conductivity, among which, Figure 9 A in the middle DcNAC78 Relative expression, Figure 9 B is O2 . - content, Figure 9 C is the H2O2 content, Figure 9 Where D is relative conductivity; Figure 10 The pSuper and pSuper- DcNAC78 Chlorophyll content in carnation seedlings, including: Figure 10 A is the total chlorophyll content, Figure 10 B is the chlorophyll a content, Figure 10 C in the middle is the chlorophyll b content; Figure 11 The TRV and TRV- DcNAC78 Phenotype of detached petals of Carnation; Figure 12 The TRV and TRV- DcNAC78 NBT staining of isolated petals of Carnation; Figure 13 The TRV and TRV- DcNAC78 DAB staining of isolated petals of Carnation; Figure 14 Silence in the embodiment of the present invention DcNAC78 In the isolated petals of carnation, DcNAC78 In TRV and TRV- DcNAC78 Relative expression, relative conductivity determination, H2O2 content determination and O2 . - Content determination, among which, Figure 14A in the middle DcNAC78 The relative expression level of Figure 14 B is the relative conductivity measurement, Figure 14 C in the figure is the determination of H2O2 content. Figure 14 D in the equation is O2 . - Content determination. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0019] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0020] All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally determined in accordance with national standards. Experimental instruments, equipment, and reagents in the following examples where the sources are not specified are all commercially available raw materials.

[0021] Unless otherwise defined or indicated, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present method. It should be noted that, unless conflicting, the embodiments and features of the embodiments of the present invention may be combined with each other. Carnation was purchased from Yunnan Yingmao Flower Industry Co., Ltd.

[0022] Example 1.1 Carnation RNA extraction: Total RNA was extracted from dianthus using the FastPure Universal Plant Total RNA Isolation Kit.

[0023] 1.2 cDNA synthesis: (1) RNA template denaturation (Table 1): Table 1 RNA template denaturation reaction system ;

[0024] Heat at 65°C for 5 min, quickly cool on ice, and let stand on ice for 2 min.

[0025] (2) Genomic DNA removal (Table 2): Table 2 Genomic DNA removal reaction system ;

[0026] Mix by gently pipetting. Incubate at 42°C for 2 min.

[0027] (3) Prepare the first-strand cDNA synthesis reaction solution (Table 3): Table 3 Preparation of first-strand cDNA synthesis reaction solution reaction system ;

[0028] Mix thoroughly by gently pipetting. Perform the first-strand cDNA synthesis reaction under the following conditions: 37°C for 45 min, 85°C for 5 sec, and store the product at -20°C.

[0029] 1.3 Real-time fluorescence quantitative PCR: Gene-specific primers were designed using Primer premier5 (Table 4). cDNA was diluted 10-fold with ddH2O and used as a template. The experiment was performed on a Step One plus quantifier using a 96-well plate. Fluorescence quantitative PCR amplification was performed. DcGAPDH As an internal control, three biological replicates were set.

[0030] Table 4 RT-qPCR primer sequences ;

[0031] The qRT-PCR reaction system is shown in Table 5, and the reaction program is set according to the conditions in Table 6.

[0032] Table 5 RT-qPCR reaction system ;

[0033] Table 6 RT-qPCR reaction procedure ;

[0034] 1.4 Amplification and purification of target gene fragments: 1.4.1 PCR amplification of target gene: The PCR amplification reaction system for the target gene fragment is shown in Table 7, and the reaction program is set according to the conditions in Table 6.

[0035] Table 7 PCR amplification reaction system ;

[0036] Table 8 PCR amplification reaction program ;

[0037] After amplification, 1% gel electrophoresis was performed and the target band was selected for subsequent gel recovery.

[0038] 1.4.2 Rubber recycling: Purification of target product: DNA gel recovery kit (Vazyme FastPure Gel DNA Extraction Mini Kit) was used to recover and purify the PCR product.

[0039] 1.5 Vector Construction: 1.5.1 Double enzyme digestion of vector: Digest the circular vector plasmid using a double enzyme digestion method to obtain a linear vector. Mix the reagents according to the amounts in Table 9. Place the reaction mixture in a 37°C dry-bed incubator for 30 minutes. Then run gel electrophoresis and select the correct length band for gel recovery.

[0040] Table 9 Double enzyme digestion reaction system ;

[0041] 1.5.2 Homologous Recombination Set up the reaction system on ice, perform homologous recombination between the double-enzyme-digested vector and the cloned target gene fragment, and react in a PCR instrument at 50°C for 5 minutes to obtain the recombinant plasmid. The homologous recombination system is shown in Table 10: Table 10 Homologous recombination reaction system ;

[0042] 1.5.3 E. coli transformation: After removing DH5α competent cells from a -80°C freezer, immediately place them on ice and thaw for 5 minutes. Transform the ligation product into the DH5α competent cells and shake the tube to mix thoroughly. Incubate on ice for 25 minutes. Heat shock the tube in a 42°C water bath for 45 seconds, remove the tube immediately, and incubate on ice for 2 minutes. Add 700 μL of antibiotic-free LB liquid medium to the tube and incubate at 37°C in a shaker at 200 rpm for 60 minutes. After removal, centrifuge at 5000 rpm for 1 minute to collect the cells. Discard the excess liquid and remove 100 μL from the bottom of the tube. Plate the cells on LB solid medium supplemented with 100 mg / L Kan and incubate overnight at 37°C.

[0043] 1.5.4 Identification of recombinant clones: Six single colonies were picked from the above screening medium and placed in LB liquid medium containing 100 mg / L Kan. The culture was shaken at 37°C and 200 rpm for 5 h. The bacterial solution was then used for PCR detection. The PCR system is shown in Table 11, and the reaction procedure is shown in Table 12: Table 11 Bacteria detection reaction system ;

[0044] Table 12 Bacteria test reaction procedure ;

[0045] The bacterial solution with the correct PCR detection band was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0046] 1.5.5 Plasmid extraction: Compare the sequencing results with the target sequence, select the sample with the correct sequence, shake the bacteria, and use the FastPureEndoFree Plasmid Mini Kit to extract the plasmid.

[0047] 1.6 Agrobacterium transformation: Remove the competent culture from the -80°C freezer and place it in the palm of your hand or at room temperature for a while to partially thaw. When it becomes an ice-water mixture, insert it into ice; add 0.01-1 μg of recombinant plasmid DNA per 100 μL of competent culture, mix thoroughly, and then stand on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes; add 700 μL of antibiotic-free LB liquid medium and culture with shaking at 28°C for 3 hours; centrifuge at 6000 rpm for 1 minute to harvest the bacteria, retain about 100 μL of supernatant, gently resuspend the bacterial block, spread it on LB medium containing 100 mg / L Kan and 50 mg / L Rif, and culture inverted at 28°C for 2-3 days.

[0048] 1.7 Subcellular localization Using homologous recombination method, specific primers pSuper- DcNAC78 -F、pSuper- DcNAC78 -R, insert the stop codon between Hind III and Sal I sites in the pSuper vector DcNAC78 CDS region to obtain pSuper- DcNAC78 Then empty pSuper vector and pSuper- DcNAC78 The cells were transformed into Agrobacterium tumefaciens GV3101 and infected into Nicotiana benthamiana leaves. The leaves were placed in the dark for 2 days, then exposed to light for 1 day before observation and photography under a laser scanning confocal microscope.

[0049] 1.8 Transcriptional Activation Analysis Design specific primers BD- DcNAC78 -F, BD- DcNAC78 -R、BD- DcNAC78 -R 1-150 ,BD- DcNAC78 -F 151-546 , select Nde I and EcoR I as restriction sites, DcNAC78The full-length CDS, N-terminus and C-terminus of the α-terminal β-actin were amplified and inserted into the pGBKT7 vector using homologous recombination to generate pGBKT7- DcNAC78 、pGBKT7- DcNAC78 -N and pGBKT7- DcNAC78 -C. The recombinant plasmid was transformed into Y2HGold yeast cells along with pGBKT7-53 (positive control) and pGBKT7-lam (negative control). All transformed yeast cells were diluted with sterile water and plated onto SD / -Trp and SD / -Trp-His deficient media, respectively. The cells were incubated upside down in a dark incubator at 28°C for 2-3 days.

[0050] 1.9 Transient overexpression: pSuper- DcNAC78 The recombinant plasmid and the empty pSuper vector were transformed into Agrobacterium tumefaciens GV3101 and cultured. The culture suspension was collected after low-temperature centrifugation and resuspended in infection solution (2M MgCl2, 1M MES, 200mM AS). After 3 hours in the dark, the petals and seedlings of carnations were vacuum-treated (0.7MPa, followed by slow release after 10 minutes, repeated 2-3 times until the petals and plants showed signs of waterlogging). The empty pSuper vector suspension served as a control.

[0051] 1.10 Momentary Silence: Will DcNAC78 The specific silent coding fragment was PCR-PCR-PCR-PCR-PCR-assisted with the specific primer TRV- DcNAC78 -F, TRV- DcNAC78 -R, and inserted into TRV2 vector using homologous recombination. DcNAC78 , TRV1, and TRV2 were transformed into Agrobacterium tumefaciens cells and cultured. The cells were resuspended in infiltration buffer (2M MgCl2, 1M MES, 200mM AS). The OD 600 Adjust the pressure to 1 and mix in equal volumes. After incubating the infecting solution at 8°C in the dark for 3 days, use it to transform carnation petals and apply vacuum at 0.7 MPa. After 10 minutes, slowly deflate the solution. Repeat 2-3 times until the petals appear water-soaked. Then, place the petals in a Petri dish lined with filter paper soaked in deionized water and incubate at 8°C in the dark for 3 days.

[0052] The above primer sequences are shown in Table 13: Table 13 Primer list ;

[0053] 2 Experimental results: 2.1 DcNAC78 Gene cloning: The gene cloning primers were DcNAC78 -F, DcNAC78 -R, DcNAC78 Gene cloning electrophoresis diagram Figure 1 As shown, the CDS length is 1641 bp, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2.

[0054] DcNAC78 Gene (SEQ ID NO.1):

[0055] Amino acid sequence (SEQ ID NO.2): .

[0056] 2.2 Subcellular localization analysis of DcNAC78 In order to study the subcellular distribution of DcNAC78 protein, the present invention constructed pSuper- DcNAC78 Expression vector, using transient overexpression of pSuper-DcNAC78 fusion protein in tobacco for detection DcNAC78 The recombinant plasmid was transferred into Agrobacterium and injected into Nicotiana benthamiana. After 2 days of dark treatment and 1 day of light treatment, the expression was observed under a laser confocal microscope. Figure 2 As shown, the empty vector of pSuper was expressed in the nucleus, cell membrane and cytoplasm, while the empty vector of pSuper was expressed in the cell nucleus, cell membrane and cytoplasm. DcNAC78 Agrobacterium-transfected tobacco leaves expressed DcNAC78 only in the nucleus. The above results indicated that DcNAC78 was a nuclear protein.

[0057] 2.3 Analysis of transcriptional activation of DcNAC78 All transformed fragments grew well on SD medium lacking Trp (SD / -Trp), while DcNAC78 and DcNAC78-C grew well on SD medium lacking Trp, His, and (SD / -Trp-His) Figure 3 ), indicating that DcNAC78 has transcriptional activation activity and the transcriptional activation domain is located at the C-terminus.

[0058] 2.4 DcNAC78 Positive regulation of carnation heat tolerance: 2.4.1 Overexpression DcNAC78 Improved the resistance of carnation petals to high temperature stress: Transient overexpression in carnation petals DcNAC78 . Will pSuper- DcNAC78 The recombinant plasmid was transformed into Agrobacterium GV3101 and vacuum-infected into detached petals of Carnation. Phenotypic observation of transiently transformed plant tissues was performed ( Figure 4 Middle A) and RT-qPCR expression ( Figure 4 B) was measured and found that DcNAC78 The expression of α-glucose was significantly increased compared with the control group ( Figure 4 B). After 2 days of heat stress at 45℃, pSuper and pSuper- DcNAC78 The relative conductivity was measured. Compared with the control group, after heat stress, the overexpression DcNAC78 The petals showed less ion penetration ( Figure 7 DAB and NBT staining were performed to analyze the accumulation of reactive oxygen species, and the petals showed less brown and blue than the control group ( Figure 5 、 Figure 6 shown).

[0059] 2.4.2 Overexpression DcNAC78 Improved carnation's resistance to high temperature stress: The transgenic plants were treated with 45℃ high temperature. The results showed that compared with the control group, the overexpression DcNAC78 The plants suffered significantly less damage from high temperatures than the control group and showed better growth conditions ( Figure 8 Physiological index analysis showed that the content of photosynthetic pigments in carnation leaves decreased after high temperature, while overexpression DcNAC78 After some relief ( Figure 10 As shown); ROS accumulation analysis showed that high temperature caused H2O2, O2 .- The content increased, and DcNAC78 Alleviating this accumulation ( Figure 9 B, C), and the membrane stability is increased ( Figure 9 Overall, overexpression DcNAC78 Enhanced the heat resistance of carnation seedlings.

[0060] 2.4.3 Silencing DcNAC78 reduces the resistance of carnation to high temperature stress: To further explore DcNAC78 The effect of heat tolerance on carnation was studied by silencing the gene in petals using virus-induced gene silencing (VIGS). DcNAC78 .Silent Petals (TRV- DcNAC78 ) and control petals (TRV) were treated under high temperature stress (45℃) for 48h. The phenotype of the petals showed that TRV- DcNAC78 Showed more severe wilting than TRV, with more rust coloration on the petals and more wilting ( Figure 11 shown). DcNAC78 The expression level of TRV was higher than that of TRV- DcNAC78 About 0.5 times higher ( Figure 14 Middle A). The osmotic capacity regulation ability of the silenced material after heat stress was significantly weakened compared with the control group ( Figure 14 In order to clarify the effect of high temperature on the oxidase activity of carnation, the petals of carnation under normal conditions and after high temperature were stained with DAB and NBT, and the H2O2 and O2 .- Under normal circumstances, there is no significant difference in the degree of staining between the two, but the high temperature treated carnation petals are lighter in staining than the silenced plants, and the staining area is also significantly smaller than the silenced strain ( Figure 12 、 Figure 13 The oxidase activity of carnation changed significantly under high temperature stress and increased with the severity of damage. . 2 - The content increases significantly with the increase of temperature. DcNAC78 After that, H2O2, O2 .- The amount increased significantly ( Figure 14 (C, D).

[0061] Therefore, the present invention adopts the above-mentioned DcNAC78 Application of genes in improving heat tolerance of carnation, overexpression in carnation plants DcNAC78 Gene, can significantly improve the heat resistance of carnation, can be used to cultivate new varieties of heat-resistant carnation; at the same time, the present invention discloses DcNAC78 The gene regulates the heat tolerance of carnation by enhancing membrane stability, regulating permeability, and enhancing the ability to scavenge reactive oxygen species.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. DcNAC7 The application of 8 genes in improving the heat tolerance of carnation is characterized by: DcNAC78 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Overexpression in Carnation DcNAC78 Genes to improve the heat tolerance of carnation.

3. DcNAC78 protein for improving the heat tolerance of carnation, characterized by: The amino acid sequence of DcNAC78 protein is shown in SEQ ID NO.

2. The gene encoding DcNAC78 protein is DcNAC78 Gene, DcNAC78 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. Use of the DcNAC78 protein as claimed in claim 3 in improving the heat resistance of carnation.

5. A recombinant vector, characterized in that: The recombinant vector comprises the DcNAC78 Gene.

6. The recombinant vector according to claim 5, characterized in that: The recombinant vector is a plant overexpression vector.

7. Use of the recombinant vector as claimed in claim 5 or 6 in improving the heat resistance of carnation.

8. A method for cultivating high-temperature resistant carnation, characterized by: The claim 1 DcNAC78 Genes were overexpressed in carnations to screen and cultivate high-temperature-resistant carnations.

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