Rosa chinensis heat resistance regulation gene RhNCED2 and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to a rosa chinensis heat resistance regulation gene RhNCED2 and application thereof. The invention provides application of a product for promoting expression of an RhNCED2 gene or transcriptional coding protein thereof in enhancing heat resistance of Chinese rose, a nucleotide sequence of the RhNCED2 gene is shown as SEQ ID NO.2, and a nucleotide sequence of a specific fragment of the RhNCED2 gene is shown as SEQ ID NO.1. According to expression results of the RhNCED2 at different time points of high-temperature treatment of the Chinese rose, the RhNCED2 in a Chinese rose plant is instantaneously silenced, and the heat resistance of the Chinese rose is enhanced. Abnormal expression of the RhNCED2 can influence the heat resistance of the Chinese rose plants, silence of the RhNCED2 can weaken the heat resistance of the Chinese rose plants, and spraying of the abscisic acid can enhance the heat resistance of the Chinese rose plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a rose heat tolerance regulating gene RhNCED2 and its applications. Background Technology

[0002] The rose (Rosa hybrida) is a perennial evergreen or semi-evergreen woody ornamental plant belonging to the genus Rosa in the family Rosaceae. It is valued for its year-round flowering, rich flower colors, diverse varieties, and both ornamental and medicinal uses. As one of the world's four major cut flowers, it holds an important position in the cut flower industry. However, high-temperature stress has become a key environmental factor restricting the growth, flowering, and quality formation of roses, especially in greenhouse cultivation and regions with high summer temperatures. Therefore, conducting research on the heat tolerance of roses is of great significance for ensuring year-round production and enhancing their ornamental and commercial value.

[0003] Heat tolerance is a crucial biological characteristic of plants in maintaining normal physiological functions and structural integrity under high-temperature environments. The level of a plant's heat tolerance directly affects its photosynthetic efficiency, water balance, antioxidant system, and reproductive development, thus determining its survival and productivity under high-temperature conditions. Under high-temperature stress, plants alleviate heat damage and maintain cellular homeostasis through physiological and molecular responses such as activating heat shock protein synthesis, regulating hormone signaling, and enhancing reactive oxygen species scavenging mechanisms. The final heat tolerance performance of plants is the result of the synergistic effect of multiple response mechanisms and is closely related to the expression intensity of heat-related genes and the efficiency of regulatory networks.

[0004] The heat tolerance of roses is mainly reflected in the stability of leaf structure and function, the integrity of floral organ development, and the sustained functioning of overall physiological metabolism. High temperatures often lead to leaf scorch, reduced flower color, wilting petals, and shortened flowering period, affecting the post-harvest quality and vase life of cut flowers. In cut flower production, heat tolerance is directly related to variety selection, cultivation management measures, and post-harvest treatment strategies during the hot summer months. In breeding practice, screening and cultivating heat-tolerant varieties is a key approach to improving the adaptability and market competitiveness of roses. Therefore, systematically analyzing the physiological basis and molecular mechanisms of rose heat tolerance, identifying key heat-tolerant genes, and establishing a heat tolerance evaluation and regulation technology system are of significant theoretical and practical importance for promoting the sustainable development of the rose industry in the context of climate change. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a rose heat tolerance regulating gene RhNCED2 and its application, which addresses the shortcomings of the prior art. The specific fragments of this gene and its encoded protein can be used to promote or inhibit the heat tolerance of roses.

[0006] This invention provides a rose heat tolerance regulatory gene RhNCED2, and the nucleotide sequence of a specific fragment silencing the rose heat tolerance regulatory gene RhNCED2 is shown in SEQ ID NO: 1.

[0007] The heat tolerance regulatory gene RhNCED2 for roses provided by the present invention has an amino acid sequence of the protein encoded by the gene as shown in SEQ ID NO: 3.

[0008] This invention also provides an application of the above-mentioned rose heat tolerance regulatory gene RhNCED2, the method of which is as follows:

[0009] S1. Take leaves from rose plants and extract RNA;

[0010] S2. Genomic DNA removal and reverse transcription synthesis of cDNA are performed on the extracted RNA;

[0011] S3. Using cDNA synthesized by reverse transcription as a template, RT-qPCR amplification was performed to obtain the amplification product;

[0012] S4. Vector construction: The target gene cDNA is amplified by PCR to obtain PCR product. The PCR product is recovered and purified to obtain the target gene fragment with the sequence shown in SEQ ID NO: 1. Then, according to the restriction sites of the target gene fragment inserted into the vector plasmid, the vector is double-digested. The double-digested vector is homologously recombined with the target gene fragment. Then, E. coli is transformed by heat shock method and subjected to bacterial detection and sequencing.

[0013] S5. After the sequencing results are returned, sequence alignment is performed, and samples that meet the expectations are selected for inoculation and plasmid extraction is performed using a plasmid extraction kit.

[0014] S6. Agrobacterium transformation of plasmids using the freeze-thaw method;

[0015] S7. Transient silencing: Using EcoRI and Kpn1 as restriction sites, the gene sequence of SEQ ID NO: 1 was inserted into the pTRV2 empty vector. Primers were designed using homologous recombination to construct the TRV2-RhNCED2 vector. Bacterial culture was performed, bacteria were collected and resuspended. Rose cuttings were infected by suction using a vacuum pump, and the entire plant was immersed in the bacterial solution. The treatment was repeated three times. After infection, the bacterial solution was rinsed with sterile water. After three days of equilibration, the plants were planted in the substrate to recover. One month later, the plants were transferred to a light incubator for two days of treatment. Observations were made every 24 hours, and samples were taken and photographed for record-keeping.

[0016] According to the application of the rose heat tolerance regulatory gene RhNCED2 provided by the present invention, the primer set used for reverse transcription in S2 is qRT-RhNCED2-F and qRT-RhNCED2-R, and the nucleotide sequences of the primer sets qRT-RhNCED2-F and qRT-RhNCED2-R are as shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0017] According to the application of the rose heat tolerance regulatory gene RhNCED2 provided by the present invention, the primer set used for PCR amplification in S4 is TRV2-RhNCED2-F and TRV2-RhNCED2-R, and the nucleotide sequences of the primer sets TRV2-RhNCED2-F and TRV2-RhNCED2-R are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

[0018] In the application of the rose heat tolerance regulating gene RhNCED2 provided by the present invention, the enzymes used for double digestion in S4 are EcoRI and Kpn1, and the ratio of the amount of the double-digested vector to the target gene fragment in the homologous recombination system is 1:3.

[0019] In the application of the rose heat resistance regulating gene RhNCED2 provided by the present invention, the primers for homologous recombination in S7 are TRV2-RhNCED2-F and TRV2-RhNCED2-R, the temperature for the three-day equilibration is 8°C, and the temperature of the light incubator is 42°C.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] This invention provides a heat tolerance regulatory gene RhNCED2 for roses and its application. A candidate heat tolerance gene, RhNCED2, was identified from the genome of the rose variety 'Yueyuefen'. It was found that this gene was highly expressed in leaves of roses treated with high temperature for 24 hours. Based on the expression level of RhNCED2 in leaves at different time points during high temperature treatment, transient silencing of RhNCED2 in cuttings revealed that abnormal expression of RhNCED2 affects the heat tolerance of rose plants; silencing RhNCED2 weakens the heat tolerance of rose plants. Spraying with ABA followed by high temperature treatment significantly enhances the heat tolerance of rose plants. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 The expression level of RhNCED2 in leaves at different time points after high-temperature treatment is shown in the figure.

[0024] Figure 2 Phenotypic results of the overall heat resistance of 'Luoshen' cuttings at different time points after transient silencing of RhNCED2;

[0025] Figure 3 The relative expression level of RhNCED2 was analyzed by RT-qPCR after transient silencing;

[0026] Figure 4 The ABA content in rose leaves after transient silencing of RhNCED2;

[0027] Figure 5 Phenotypic observation results of leaf heat resistance of 'Luoshen' cuttings at different time points after transient silencing of RhNCED2;

[0028] Figure 6 Figure 1 shows the phenotypic results of leaves of 'Luoshen' cuttings after DAB staining at different time points following transient silencing of RhNCED2.

[0029] Figure 7 The statistical results of leaf electrical conductivity of 'Luoshen' cuttings at different time points after transient silencing RhNCED2;

[0030] Figure 8 This study presents the statistical results of leaf electrical conductivity of 'Luoshen' cuttings at different time points after transient silencing of RhNCED2 (ABA spraying). Detailed Implementation

[0031] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Example 1

[0033] This embodiment provides a rose heat tolerance regulating gene RhNCED2. The specific nucleotide fragment sequence of the silenced gene RhNCED2 is shown in SEQ ID NO: 1, which can be used to weaken the heat tolerance of roses. The complete nucleotide sequence of the gene RhNCED2 is shown in SEQ ID NO: 2.

[0034] The amino acid sequence of the protein encoded by the rose heat tolerance regulatory gene RhNCED2 is shown in SEQ ID NO.3.

[0035] Both the encoded protein and the RhNCED2 gene specific nucleotide fragment are used to promote or inhibit the heat tolerance of roses, thereby achieving the purpose of regulating the heat tolerance of roses.

[0036] Example 2

[0037] This embodiment is based on the genome sequence of the rose variety 'Yueyuefen'. In the early stage, the transcriptome of leaves at different time points after high temperature treatment was sequenced, and the significantly differential gene RhNCED2, which is related to the heat resistance of roses, was screened out.

[0038] The gene was found to be highly expressed in the leaves of roses after 24 hours of high-temperature treatment, suggesting that RhNCED2 may be involved in the heat tolerance of roses. Transient silencing of RhNCED2 was found to indeed affect the heat tolerance of rose plants.

[0039] Therefore, this embodiment reveals the biological function of RhNCED2 in the heat resistance of roses, which is of great significance for the breeding of cut roses and the improvement of quality and efficiency in the industry.

[0040] 1. Plant materials

[0041] ①'Luoshen' is a modern cut rose variety. It was obtained from the Baofeng Base of the Flower Research Institute of Yunnan Academy of Agricultural Sciences. Cuttings were used as materials for the rose heat resistance experiment.

[0042] ② Strains and vectors

[0043] Escherichia coli: DH5α;

[0044] Agrobacterium tumefaciens strains: EHA105 and GV3101 (pSoup helper plasmid), and pSuper-1300 (Kan kanamycin resistance) were all purchased from Beijing Qingke Biotechnology Co., Ltd. VIGS (virus-induced gene silencing) vectors were pTRV1 and pTRV2 purchased from HonorGene.

[0045] ③The culture medium formulation involved

[0046] (1) The formulations of LB medium and YEB medium are shown in Table 1 below:

[0047] Table 1

[0048]

[0049] 2. Research Methods

[0050] ① RNA extraction

[0051] RNA was extracted from the leaves of 'Roselle' using the RNAprep Pure Polysaccharide and Polyphenol Plant RNA Extraction Kit (centrifuge column type).

[0052] ②cDNA synthesis

[0053] (1) The genomic DNA removal reaction system is shown in Table 2 below:

[0054] Table 2

[0055]

[0056] Mix the above components thoroughly and react at 42 °C for 2 min to obtain the reaction solution.

[0057] (2) Prepare the reverse transcription reaction system as shown in Table 3 below:

[0058] Table 3

[0059] Components volume 5×HiScript Ⅲ qRT SuperMix (Universal Reverse Transcription Premix) 4 μL reaction solution 16 μL

[0060] The reaction was carried out at 37 °C for 15 min, followed by a reaction at 85 °C for 5 s to obtain cDNA. The product was stored at -20 °C.

[0061] ③ Real-time quantitative PCR

[0062] Gene-specific RT-qPCR primers qRT-RhNCED2-F and qRT-RhNCED2-R (nucleotide sequences are shown in SEQ ID NO: 8 and SEQ ID NO: 9) were designed using Primer Premier 5 (primer design and analysis software).

[0063] The cDNA obtained by reverse transcription was diluted four times with ddH2O, and the diluted cDNA was used as a template for RT-qPCR (real-time quantitative PCR) amplification.

[0064] Using RhUBI2 (the gene encoding ubiquitin in roses) as an internal control, three biological replicates were set up.

[0065] The RT-qPCR reaction system is shown in Table 4 below:

[0066] Table 4

[0067]

[0068] In this reaction system, the control group used RhUBI2 primers qRT-RhUBI2-F and qRT-RhUBI2-R, whose nucleotide sequences are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively. The experimental group used gene-specific RT-qPCR primers qRT-RhNCED2-F and qRT-RhNCED2-R, whose nucleotide sequences are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0069] The RT-qPCR reaction procedure is shown in Table 5 below:

[0070] Table 5

[0071]

[0072] ④ Carrier Construction

[0073] 1) PCR amplification of the target gene fragment

[0074] The high-fidelity enzyme (Phusion™ Plus PCR Master Mix) was used for PCR amplification of the target gene. The PCR amplification reaction system is shown in Table 6 below:

[0075] Table 6

[0076]

[0077] The nucleotide sequences of primer sets TRV2-RhNCED2-F and TRV2-RhNCED2-R are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively;

[0078] The PCR amplification reaction procedure is shown in Table 7 below:

[0079] Table 7

[0080]

[0081] After amplification, 1% gel electrophoresis was performed, and the target band was selected for subsequent gel recovery.

[0082] 2) Glue recycling

[0083] The PCR products were recovered and purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0.

[0084] 3) Vector double enzyme digestion

[0085] Based on the restriction enzyme sites inserted according to the target fragment sequence, the vector is double-digested with the corresponding enzymes. The double-digestion system is shown in Table 8 below:

[0086] Table 8

[0087]

[0088] After adding the system reagents on ice, perform double digestion of the vector according to the specific enzyme denaturation temperature.

[0089] 4) Homologous recombination

[0090] The vector, after double enzyme digestion, is homologously recombinated with the cloned target gene fragment to construct the vector.

[0091] The homologous recombination system is shown in Table 9 below:

[0092] Table 9

[0093]

[0094] The ratio of vector to gene fragment is 1:3, and the specific amount added depends on the concentration.

[0095] Procedure: Run the PCR machine at 50 ℃ for 15 min;

[0096] The recombinant product was obtained.

[0097] 5) E. coli transformation

[0098] (1) Remove the DH5α competent states from -80 ℃ and melt them in ice;

[0099] (2) Take a 1.5 mL centrifuge tube, add 10 μL of recombinant product and 50 μL of DH5α competent cells, mix by pipetting, and let stand on ice for 30 min;

[0100] (3) Heat shock in a 42 ℃ water bath for 90 s, then quickly transfer to ice and let stand for 2 min;

[0101] (4) Add 500 μL of LB medium to the centrifuge tube and centrifuge at 37 °C and 200 rpm for 1 h;

[0102] (5) Centrifuge the cultured bacterial solution at 5000 rpm for 5 min;

[0103] (6) Discard 400 μL of supernatant in a clean bench, mix well by pipetting, spread on LB solid medium containing antibiotics, and incubate overnight at 37 °C with the medium inverted.

[0104] 6) Microbial testing and sequencing

[0105] (1) Shaking culture: Pick single colonies that have grown overnight and shake them in 500 μL of LB medium containing antibiotics for 3-4 h (at 37 ℃ and 200 rpm).

[0106] (2) Bacterial culture PCR: The bacterial culture obtained is used for PCR amplification to detect whether the target band of the constructed vector meets the expectations.

[0107] The bacterial culture PCR amplification system is shown in Table 10:

[0108] Table 10

[0109]

[0110] The nucleotide sequences of primer sets TRV2-F and TRV2-R are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively;

[0111] The bacterial culture PCR amplification reaction procedure is shown in Table 11:

[0112] Table 11

[0113]

[0114] The amplified products were used for 1% gel electrophoresis imaging, and those with band sizes that met expectations were sequenced.

[0115] ⑤ Plasmid extraction

[0116] After the test results are returned, sequence alignment is performed, and samples that meet the expectations are selected for inoculation and plasmid extraction is carried out using the plasmid extraction kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0).

[0117] ⑥ Agrobacterium-mediated transformation method:

[0118] (1) When the competent state of the straw is taken out of the -80 ℃ freezer and melted into an ice-water mixture, it is inserted into ice;

[0119] (2) Add 0.01-1 μg plasmid DNA to each 100 μL competent cells, mix well and then place on ice for 5 min, in liquid nitrogen for 5 min, in a 37 ℃ water bath for 5 min, and in an ice bath for 5 min.

[0120] (3) Add 500 μL of antibiotic-free YEB liquid medium and incubate at 28 °C with shaking for 2-3 h;

[0121] (4) Centrifuge at 5000 rpm for 2 min, discard 400 μL of supernatant in a clean bench, spread it onto YEB solid medium containing antibiotics, and incubate upside down at 28 ℃ for 2-3 days.

[0122] ⑦ Momentary silence

[0123] 1) Vector and primer design and vector construction

[0124] Using EcoRI and Kpn1 as restriction sites, the silent fragment sequence of RhNCED2 was inserted into the pTRV2 empty vector. Primers TRV2-RhNCED2-F and TRV2-RhNCED2-R were designed using homologous recombination to construct the pTRV2-RhNCED2 vector.

[0125] 2) Bacterial culture

[0126] Streak the bacterial culture on agar plates (containing 50 mg / L of Kan / Rif (kanamycin resistance / rifampicin resistance)) and incubate upside down at 28 °C for 2–3 days.

[0127] Pick a single colony and gently shake it in 500 μL of YEB medium containing antibiotics for bacterial testing; if the bands are correct, proceed with medium and large shaking (conditions: 28 ℃, 200 rpm).

[0128] 3) Collection and resuspension of bacteria

[0129] Centrifuge at 5000 rpm for 8 minutes to collect the bacteria, discard the supernatant, resuspend the bacteria in the infection solution, mix well by pipetting, and adjust to OD. 600 =1.0. When conducting transient silencing experiments, TRV1 and TRV2, TRV2-RhNCED2 bacterial cultures were mixed at a volume ratio of 1:1 and allowed to stand in the dark for 4–6 h.

[0130] 4) Vacuum suction

[0131] The stem segments with single buds were infected by suction using a vacuum pump at 0.082 MPa for 10 min, followed by holding the pressure for 10 min and releasing the pressure for 10 min, ensuring the entire stem segment was submerged in the bacterial solution. This treatment was repeated three times. After infection, the stem segments were rinsed with sterile water and incubated in an 8°C incubator for 3 days before being planted. Observations and samples were taken and photographed every two days.

[0132] The primers used in the RhNCED2 sequence experiments above are:

[0133] For RT-qPCR: qRT-UBI2-F, qRT-UBI2-R, qRT-RhNCED2-F, and qRT-RhNCED2-R;

[0134] For TRV vectors: TRV2-F, TRV2-R, TRV2-RhNCED2-F, and TRV2-RhNCED2-R;

[0135] The details are shown in Table 12 below:

[0136] Table 12

[0137]

[0138] Experimental results:

[0139] 1. Analysis of RhNCED2 expression levels in leaves treated with high temperature for different time periods

[0140] In this embodiment, the expression level of RhNCED2 in the leaves of the cut rose 'Roselle' was analyzed by real-time quantitative PCR at different time points after high-temperature treatment. The results are as follows: Figure 1 As shown in the figure, the expression of RhNCED2 increased significantly after 24 h of high temperature treatment. Therefore, it is speculated that RhNCED2 is closely related to the germination of axillary buds. The figure shows the mean ± SD (standard deviation) (n=3). This means P ≤ 0.01.

[0141] 2. RhNCED2 regulates the heat resistance of roses

[0142] This embodiment is based on the expression analysis results of RhNCED2 in leaves after different time periods of high-temperature treatment. A silenced fragment of the RhNCED2 sequence was selected, and a transient silencing vector, TRV2-RhNCED2, was constructed using homologous recombination. TRV1, TRV2, and TRV2-RhNCED2 were transformed into EHA105 straw and used to infect rose plants to silence RhNCED2. After infection, the rose plants were placed in a 25℃ constant temperature culture room for acclimatization. Subsequently, the rose plants were subjected to a 42℃ high-temperature treatment, and phenotypic observations and records were made at 0 days, 1 day, and 2 days. Figure 2 As shown.

[0143] Figure 2 In the text, 0 d, 1 d, and 2 d represent the heat tolerance of TRV2 and TRV2-RhNCED2 rose plants after high-temperature treatment on days 0, 1, and 2; "+100μmol ABA" indicates that 100μmol abscisic acid (ABA) was sprayed onto the surface of the plant leaves.

[0144] From the plant phenotype, TRV2-RhNCED2 showed worse heat resistance compared to the TRV2 control, and ABA spraying could enhance the heat resistance of rose plants.

[0145] To confirm the silencing effect, total RNA was extracted from shoots before and after RhNCED2 silencing, and its expression levels were analyzed by RT-qPCR. The results showed a highly significant decrease in RhNCED2 expression after silencing, indicating that silencing was effective. Figure 3 As shown, Figure 3The values ​​are relative RhNCED2 expression levels analyzed by RT-qPCR after transient silencing. * indicates P≤0.05.

[0146] This embodiment is based on the fact that RhNCED2 is a key gene for ABA biosynthesis, therefore the ABA content in rose leaves after transient silencing of RhNCED2 was measured, such as... Figure 4 As shown, Figure 4 The ABA content in rose leaves after transient silencing of RhNCED2 was lower than that in the TRV2 control, indicating that RhNCED2 affects the ABA content in rose leaves.

[0147] Meanwhile, phenotypic observations were conducted on the leaves of plants after high-temperature treatment, including cases with and without ABA spraying (100 μmol ABA spraying). This again demonstrated that TRV2-RhNCED2 exhibited worse heat tolerance compared to the TRV2 control, and that ABA spraying could enhance the heat tolerance of rose plants. Figure 5 As shown.

[0148] Figure 5 This is a phenotypic observation of the heat resistance of leaves of 'Luoshen' cuttings at different time points after transient silencing of RhNCED2 (including two cases: no ABA spraying and spraying with 100 μmol ABA).

[0149] Wherein 0 d represents the phenotypic observation of TRV2 and TRV2-RhNCED2 rose leaves on day 0 of high-temperature treatment; NT-24h represents the phenotypic observation of TRV2 and TRV2-RhNCED2 24h after leaf collection; Hea-24h represents the phenotypic observation of TRV2 and TRV2-RhNCED2 24h after high-temperature treatment; "+100μmol ABA" indicates that 100μmol ABA was sprayed on the surface of the plant leaves.

[0150] The leaves of the plants after high-temperature treatment were stained with DAB (3,3'-diaminobenzidine). Figure 6 This image shows the phenotypic results of leaves from 'Luoshen' rose cuttings after DAB staining at different time points following transient silencing of RhNCED2 (including cases with and without ABA spraying, and cases with 100 μmol ABA spraying). The staining results indicate that TRV2-RhNCED2 exhibits worse heat tolerance compared to the TRV2 control, and ABA spraying enhances the heat tolerance of the rose plants.

[0151] Wherein 0 d represents the phenotypic observation of rose leaves of TRV2 and TRV2-RhNCED2 after DAB staining on day 0 of high temperature treatment; NT-24h represents the phenotypic observation of TRV2 and TRV2-RhNCED2 after DAB staining 24h after leaf collection; Heat-24h represents the phenotypic observation of rose leaves of TRV2 and TRV2-RhNCED2 after DAB staining 24h after high temperature treatment; "+100μmol ABA" indicates that 100μmol ABA was sprayed on the surface of the plant leaves.

[0152] Electrical conductivity was measured in the leaves of plants treated with high temperature. Compared with the TRV2 control, TRV2-RhNCED2 showed higher electrical conductivity, more severe leaf damage, and poorer heat resistance. Figure 7 As shown.

[0153] Figure 7 This is a statistical analysis of the leaf electrical conductivity of 'Luoshen' cuttings at different time points after instantaneous silencing of RhNCED2.

[0154] 0 h represents the electrical conductivity of rose leaves of TRV2 and TRV2-RhNCED2 without high-temperature treatment; NT-24h represents the electrical conductivity of rose leaves of TRV2 and TRV2-RhNCED2 24 hours after harvesting; Heat-24h represents the electrical conductivity of rose leaves of TRV2 and TRV2-RhNCED2 24 hours after high-temperature treatment.

[0155] Meanwhile, after subjecting rose plants sprayed with ABA to high-temperature treatment, the electrical conductivity of their leaves was measured. Compared with rose plants not sprayed with ABA, the rose plants sprayed with ABA had lower leaf electrical conductivity and stronger heat resistance. Figure 7 and 8 As shown.

[0156] Figure 8 This is a statistical analysis of the leaf electrical conductivity of 'Luoshen' cuttings at different time points after instantaneous silencing of RhNCED2 (spraying ABA);

[0157] 0 h represents the electrical conductivity of rose leaves of TRV2 and TRV2-RhNCED2 without high-temperature treatment; NT-24h represents the electrical conductivity of rose leaves of TRV2 and TRV2-RhNCED2 24 hours after harvesting; Heat-24h represents the electrical conductivity of rose leaves of TRV2 and TRV2-RhNCED2 24 hours after high-temperature treatment.

[0158] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A rose heat tolerance regulatory gene RhNCED2, characterized in that, The nucleotide sequence of the specific fragment of the silenced rose heat tolerance regulatory gene RhNCED2 is shown in SEQ ID NO:

1.

2. The rose heat tolerance regulating gene RhNCED2 according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the rose heat tolerance regulatory gene RhNCED2 is shown in SEQ ID NO:

3.

3. An application of the rose heat tolerance regulating gene RhNCED2 as described in claim 1, characterized in that, The method of application is as follows: S1. Take leaf samples from rose plants and extract RNA; S2. Genomic DNA removal and reverse transcription synthesis of cDNA are performed on the extracted RNA; S3. Using cDNA synthesized by reverse transcription as a template, RT-qPCR amplification was performed to obtain the amplification product; S4. Vector construction: The target gene cDNA is amplified by PCR to obtain PCR product. The PCR product is recovered and purified to obtain the target gene fragment with the sequence shown in SEQ ID NO:

1. Then, according to the restriction sites of the target gene fragment inserted into the vector plasmid, the vector is double-digested. The double-digested vector is homologously recombined with the target gene fragment. Then, E. coli is transformed by heat shock method and subjected to bacterial detection and sequencing. S5. After the sequencing results are returned, sequence alignment is performed, and samples that meet the expectations are selected for inoculation and plasmid extraction is performed using a plasmid extraction kit. S6. Agrobacterium transformation of plasmids using the freeze-thaw method; S7. Transient silencing: Using EcoRI and Kpn1 as restriction sites, the gene sequence of SEQ ID NO: 1 was inserted into the pTRV2 empty vector. Primers were designed using homologous recombination to construct the TRV2-RhNCED2 vector. Bacterial culture was performed, bacteria were collected and resuspended. Rose cuttings were infected by suction using a vacuum pump, and the entire plant was immersed in the bacterial solution. The treatment was repeated three times. After infection, the bacterial solution was rinsed with sterile water. After three days of equilibration, the plants were planted in the substrate to recover. One month later, the plants were transferred to a light incubator for two days of treatment. Observations were made every 24 hours, and samples were taken and photographed for record-keeping.

4. The application of the rose heat tolerance regulating gene RhNCED2 according to claim 3, characterized in that, The primer sets used for reverse transcription in S2 are qRT-RhNCED2-F and qRT-RhNCED2-R, and the nucleotide sequences of the primer sets qRT-RhNCED2-F and qRT-RhNCED2-R are as shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

5. The application of the rose heat tolerance regulating gene RhNCED2 according to claim 3, characterized in that, The primer sets used for PCR amplification in S4 are TRV2-RhNCED2-F and TRV2-RhNCED2-R, and the nucleotide sequences of the primer sets TRV2-RhNCED2-F and TRV2-RhNCED2-R are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

6. The application of the rose heat tolerance regulating gene RhNCED2 according to claim 3, characterized in that, The enzymes used for double digestion in S4 are EcoRI and Kpn1, and the ratio of the amount of the double-digested vector to the target gene fragment in the homologous recombination system is 1:

3.

7. The application of the rose heat tolerance regulating gene RhNCED2 according to claim 3, characterized in that, The primers for homologous recombination described in S7 are TRV2-RhNCED2-F and TRV2-RhNCED2-R, the temperature for the three-day equilibration is 8°C, and the temperature of the light incubator is 42°C.

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