LdHSFC1 gene and its application in improving the heat resistance of lilies

Abstract

This invention discloses an LdHSFC1 gene and its application in improving the heat tolerance of lilies. The CDS sequence of the LdHSFC1 gene is 864 bp long, as shown in SEQ ID NO:1, encoding 287 amino acids, as shown in SEQ ID NO:2. Under high-temperature stress, the gene expression level is significantly upregulated, and silencing LdHSFC1 can improve the heat tolerance of lilies. This invention provides important theoretical significance and application value for cultivating new heat-tolerant lily germplasm and for molecular breeding of heat-tolerant lilies.

Description

Technical Field

[0001] This invention belongs to the field of plant gene breeding technology, specifically relating to an LdHSFC1 gene and its application in improving the heat resistance of lilies. Background Technology

[0002] High-temperature stress is one of the most significant environmental limiting factors restricting plant growth, development, reproduction, and geographical distribution. With the continued increase in the frequency and intensity of global warming events, heat stress has become a prominent challenge in agricultural and horticultural production. High-temperature exposure disrupts the stability of plant membrane structures, interferes with protein folding processes, impairs photosynthesis, and accelerates the accumulation of reactive oxygen species. These adverse effects ultimately inhibit the normal growth and reproductive capacity of plants. For ornamental crops, the impact of heat stress is particularly severe, not only leading to reduced biomass and lower survival rates but also directly reducing the quality, ornamental value, and market performance of flowers.

[0003] Heat shock transcription factors (HSFs) are widely distributed in plants and are important factors regulating responses to various environmental stresses, playing a crucial role, especially in high-temperature adaptation mechanisms. Under high-temperature stress, HSFs can rapidly recognize and bind to heat shock elements (HSEs) in the promoters of heat shock proteins (HSPs) genes, thereby activating HSP gene expression. Simultaneously, the rapidly accumulating heat shock proteins can effectively enhance plant heat tolerance by forming functional complexes in synergy with other proteins.

[0004] Lilies (Lilium spp.) are among the world's most valuable ornamental crops, widely used in cut flowers, potted plants, and landscaping. However, many lily species and varieties are sensitive to high-temperature stress, especially during the vegetative growth and flower development stages. High temperatures not only reduce plant vigor but also cause leaf burn, flower deformities, blind flower bud drop, and shortened vase life, severely impacting yield and quality. These adverse effects are particularly pronounced under summer production conditions. Therefore, in-depth research into key genes that can enhance the heat tolerance of lilies, and the subsequent development of heat-resistant new varieties, is a critical scientific problem and technological bottleneck that the lily industry urgently needs to address. Summary of the Invention

[0005] The purpose of this invention is to provide an LdHSFC1 gene and its application in improving the heat resistance of lilies.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention seeks protection for the use of the LdHSFC1 gene as a negative regulatory target in at least one of the following a1) and a2):

[0008] a1) Its application in improving the heat resistance of lilies;

[0009] a2) Application in cultivating new lily germplasm with improved heat resistance.

[0010] Secondly, the present invention seeks protection for the use of the LdHSFC1 protein encoded by the LdHSFC1 gene as a negative regulatory target in at least one of the following a1) and a2):

[0011] a1) Its application in improving the heat resistance of lilies;

[0012] a2) Application in cultivating new lily germplasm with improved heat resistance.

[0013] Thirdly, the present invention seeks protection for the use of biological materials that inhibit LdHSFC1 gene expression in at least one of the following a1) and a2):

[0014] a1) Its application in improving the heat resistance of lilies;

[0015] a2) Application in cultivating new lily germplasm with improved heat resistance;

[0016] The biomaterial is at least one of the following: d1) - d10):

[0017] d1) DNA molecules used to suppress LdHSFC1 gene expression;

[0018] d2) An expression cassette containing the DNA molecule described in d1);

[0019] d3) A recombinant vector containing the DNA molecule described in d1) or the expression cassette described in d2);

[0020] d4) Recombinant microorganisms containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3);

[0021] d5) A transgenic plant cell line containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3);

[0022] d6) Transgenic plant tissue containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3);

[0023] d7) A transgenic plant organ containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3);

[0024] d8) A transgenic plant containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3);

[0025] d9) Regenerative cells, tissue cultures, or protoplasts produced from cells or tissue cultures of the transgenic plants described in d8);

[0026] d10) Propagation material of the transgenic plants as described in d8).

[0027] Furthermore, the above applications include: improving the heat resistance of lilies or cultivating new lily germplasm with improved heat resistance by inhibiting the expression of the LdHSFC1 gene in lilies or reducing the activity and / or content of the LdHSFC1 protein.

[0028] Fourthly, the present invention claims protection for a method for improving the heat resistance of lilies by inhibiting the expression of the LdHSFC1 gene in lilies or reducing the activity and / or content of the LdHSFC1 protein, thereby improving the heat resistance of lilies.

[0029] Fifthly, the present invention seeks protection of a method for cultivating new lily germplasm with improved heat resistance, which involves inhibiting the expression of the LdHSFC1 gene in lilies or reducing the activity or content of the LdHSFC1 protein, and screening to obtain plants with improved heat resistance.

[0030] Sixthly, the present invention seeks protection for an LdHSFC1 gene.

[0031] In a seventh aspect, the present invention seeks protection for the LdHSFC1 protein encoded by the aforementioned LdHSFC1 gene.

[0032] Eighthly, the present invention claims protection for biological materials used to silence the aforementioned LdHSFC1 gene, said biological material being at least one of the following d1)-d5):

[0033] d1) DNA molecules used to suppress LdHSFC1 gene expression;

[0034] d2) An expression cassette containing the DNA molecule described in d1);

[0035] d3) A recombinant vector containing the DNA molecule described in d1) or the expression cassette described in d2);

[0036] d4) Recombinant microorganisms containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3);

[0037] d5) A transgenic plant cell line containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3).

[0038] In this invention, the expression of the LdHSFC1 gene is suppressed using VIGS virus-induced gene silencing, RNA interference, gene editing, or antisense RNA technology. In a specific embodiment of this invention, VIGS virus-induced gene silencing technology is preferably used. The LdHSFC1 gene silencing fragment is cloned into the pTRV2 vector and infected into lily plants via Agrobacterium-mediated infection. Based on this technology, the DNA molecule used to suppress LdHSFC1 gene expression is the LdHSFC1 gene silencing fragment.

[0039] In the technical solution of this invention, the LdHSFC1 gene is a DNA molecule as shown in b1) or b2) below:

[0040] b1) A DNA molecule having the nucleotide sequence shown in SEQ ID NO:1;

[0041] b2) A DNA molecule that has more than 70% identity with the DNA molecule described in b1) and encodes a protein with the same function; preferably, a DNA molecule that has more than 80% identity with the DNA molecule described in b1) and encodes a protein with the same function; more preferably, a DNA molecule that has more than 90% identity with the DNA molecule described in b1) and encodes a protein with the same function.

[0042] In the technical solution of this invention, the LdHSFC1 protein is the protein shown in c1), c2), c3), or c4) as follows:

[0043] c1) A protein having the amino acid sequence shown in SEQ ID NO:2;

[0044] c2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in c1);

[0045] c3) A derivative protein that has undergone substitution and / or deletion and / or addition of one or more (e.g., 1-50, preferably 1-20, more preferably 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO:2, while retaining the function of the protein in negatively regulating the heat resistance of lilies.

[0046] c4) is a derivative protein that has more than 80% identity with the protein shown in c1), c2), or c3) and retains the function of the protein in negatively regulating the heat resistance of lilies.

[0047] In the technical solution of this invention, the primer pair used to amplify the LdHSFC1 gene includes upstream primer F and downstream primer R:

[0048] Primer sequence F: ATGGCCTTCTCTGGATCTTCAAAAGA (SEQ ID NO:3);

[0049] Primer sequence R: CTAGAAGAATCCCTCGCCAAGAATACA (SEQ ID NO:4).

[0050] In a specific embodiment of the present invention, the process of studying the LdHSFC1 gene and its function of improving the heat resistance of lily includes the following steps: (1) cloning and sequence analysis of the LdHSFC1 gene; (2) analysis of the subcellular localization and transcriptional activity of LdHSFC1; (3) analysis of the expression pattern of the LdHSFC1 gene; (4) silencing LdHSFC1 with VIGS to improve the heat resistance of lily.

[0051] The cloning and sequence analysis process of the LdHSFC1 gene in step (1) is as follows: First, RNA was extracted from lily leaves using the TRizol method. Then, the mRNA was reverse transcribed into cDNA using a reverse transcription kit. High-fidelity PCR amplification was then used to clone the open reading frame (ORF) of LdHSFC1 using the cDNA as a template. The correctly cloned sequence was then analyzed. Phylogenetic tree construction: Arabidopsis HSF family members were obtained from the TAIR website. Lily genome sequences were obtained from the LGD (https: / / lgd.njau.edu.cn / lily / ) Liliales multi-omics database. Based on homologous sequence alignment, lily HSF family members were obtained, and a phylogenetic tree was constructed using IQTREE software.

[0052] The subcellular localization process of LdHSFC1 in step (2) is as follows: First, the LdHSFC1 open reading frame with the terminator removed is recombined with the pCAMBIA1300-GFP vector and transformed into Agrobacterium competent cells GV3101 and cultured by shaking. Then, Agrobacterium solution containing pCAMBIA1300-GFP and pCAMBIA1300-LdHSFC1-GFP is mixed with bacterial solution containing nuclear localization signal RFP-NLS, respectively. After collecting and resuspending the bacteria, they are injected into tobacco leaves. After 48 h, the expression of GFP signal in the injected leaves is observed using a laser confocal microscope to determine the localization of LdHSFC1 protein in the cell.

[0053] The transcriptional activity analysis process in step (2) is as follows: The open reading frame of the LdHSFC1 gene was constructed into the pGBKT7 vector and pBD-VP16 vector to obtain the recombinant expression vectors pGBKT7-LdHSFC1 and pBD-LdHSFC1-VP16, respectively. Subsequently, the recombinant plasmids pGBKT7-GAL4 (positive control), pGBKT7 (negative control), pGBKT7-LdHSFC1 and pBD-LdHSFC1-VP16 were transformed into yeast AH109 competent cells, respectively, and evenly spread on SD / -Trp medium. Then, positive clones were picked and spotted on solid medium of SD / -Trp, SD / -Trp-His, SD / -Trp-His+3AT (10 mM), SD / -Trp-His+3AT (20 mM) and SD / -Trp-His + X-α-gal for plate detection to observe the growth of yeast cells and detect β-galactosidase activity.

[0054] The process of analyzing the expression pattern of the LdHSFC1 gene in step (3) is as follows: select 'White Paradise' tissue culture seedlings with similar growth status and perform heat shock treatment at 37℃ for different durations. Set up 3 biological replicates for each sample. Use the Trizol method to extract RNA from 'White Paradise' leaves, reverse transcribe it into cDNA, and then perform RT-qPCR experiment of LdHSFC1. Select lily 18S rRNA as internal reference gene.

[0055] The process of silencing LdHSFC1 in step (4) to improve the heat resistance of lilies is as follows: construct the pTRV2-LdHSFC1 recombinant plasmid and transfer it into Agrobacterium GV3101. After overnight culture by shaking, collect the bacteria, resuspend them, and adjust the OD600 of Agrobacterium to 1.0. Then, mix pTRV1 with pTRV2 and pTRV2-LdHSFC1 at a ratio of 1:1 respectively. Infect the lily petals by vacuuming. After infection, pour out the bacterial solution and wash it with deionized water. Then place it in a petri dish (0.4% water agar) and culture it in the dark for 1 day. Equilibrate under normal light for 2 days. After heat treatment at 42℃, identify the heat resistance phenotype and determine the physiological indicators.

[0056] The beneficial effects of this invention are:

[0057] This invention addresses the heat sensitivity of lilies by identifying a superior gene that enhances their heat tolerance, thereby enabling the development of more heat-resistant lily varieties for better application in the cut flower market and landscaping. Simultaneously, it provides important theoretical basis and a superior gene reserve for lilies and other plants to mitigate the adverse effects of high-temperature stress through gene regulation technology. Attached Figure Description

[0058] Figure 1Evolutionary analysis of LdHSFC1.

[0059] Figure 2 Subcellular localization analysis of LdHSFC1;

[0060] In this study, the empty GFP vector served as a negative control, and RFP-NLS served as a nuclear localization target signal with a scale bar of 50 μm.

[0061] Figure 3 This study analyzed the transcriptional activation activity of LdHSFC1.

[0062] Figure 4 The expression level of LdHSFC1 in lily leaves under heat shock at 37℃ for different time periods.

[0063] Figure 5 Silencing the LdHSFC1 gene in lily petals;

[0064] Among them, A: expression level of silenced LdHSFC1 in lily petals; B: observation of the effect of silenced LdHSFC1 on the heat resistance phenotype and DAB staining of lily petals under RT and HS (42°C) conditions; C: determination of the ion permeability of lily petal extract under RT and HS conditions by silencing LdHSFC1. Detailed Implementation

[0065] The present invention will be further described in detail below with reference to specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0066] Example 1 Cloning and Sequence Analysis of the LdHSFC1 Gene

[0067] RNA extraction and gene cloning: First, RNA was extracted from lily leaves using the TRizol (Invitrogen, USA) method. Then, the mRNA was reverse transcribed into cDNA using a reverse transcription kit (R323-01, Vazyme). High-fidelity PCR amplification was then used to clone the open reading frame of LdHSFC1 found in the lily genome using the cDNA as a template (primer sequences F: ATGGCCTTCTCTGGATCTTCAAAAGA (SEQ ID NO:3), R: CTAGAAGAATCCCTCGCCAAGAATACA (SEQ ID NO:4)). Conserved domain analysis was then performed on the correctly cloned sequences. Phylogenetic tree construction: Arabidopsis HSF family members were obtained from the TAIR website (https: / / www.arabidopsis.org / ), and lily genome sequences were obtained from the LGD (https: / / lgd.njau.edu.cn / lily / ) Liliales multi-omics database. Based on homology alignment, lily HSF family members were identified, and a phylogenetic tree was constructed using IQTREE software.

[0068] Example 2: Subcellular localization and transcriptional activity analysis of LdHSFC1

[0069] Subcellular localization vector construction and expression: Using pGBKT7-LdHSFC1 as a template, specific primers pCAMBIA1300-LdHSFC1-GFP-F and pCAMBIA1300-LdHSFC1-GFP-R (primer sequences are shown in Table 2) with homologous sequences and SalI and KpnI restriction sites were designed. The LdHSFC1 open reading frame with the stop codon removed was amplified by PCR. After purification and recovery of the amplification product, the pCAMBIA1300-GFP vector (pCAMBIA1300-35S-EGFP, BioWind) was double-digested with SalI and KpnI, and the digestion products were recovered. The PCR product and the digested vector were ligated by homologous recombination, transformed into E. coli, plated on LB solid selection medium containing kanamycin, and positive single clones were picked for sequencing verification to obtain the recombinant vector pCAMBIA1300-LdHSFC1-GFP.

[0070] The recombinant vector pCAMBIA1300-LdHSFC1-GFP was transformed into Agrobacterium competent cells GV3101. After inverted culture at 28°C for 2-3 days, positive single clones were picked and cultured by shaking. Then, Agrobacterium culture containing pCAMBIA1300-GFP and pCAMBIA1300-LdHSFC1-GFP was mixed with bacterial culture containing nuclear localization signal RFP-NLS at a volume ratio of 3:1. After resuspending the bacteria, they were injected into tobacco leaves. GFP signal expression in the injected leaves was observed using a laser confocal microscope (LSM800, Zeiss, Germany) after 48 h to determine the localization of LdHSFC1 protein in cells.

[0071] Example 3: Transcriptional activity analysis of LdHSFC1

[0072] Transcriptional activity analysis: The open reading frame of the LdHSFC1 gene was constructed into the pGBKT7 vector (Beijing Cooler Master Technology Co., Ltd.) and pBD-VP16 (Baosai Biotechnology) to obtain the recombinant expression vectors pGBKT7-LdHSFC1 and pBD-LdHSFC1-VP16, respectively.

[0073] Detailed process of constructing the recombinant expression vector pGBKT7-LdHSFC1:

[0074] Using cDNA from Lanzhou lily leaves as a template, primers pGBKT7-LdHSFC1-F and pGBKT7-LdHSFC1-R (primer sequences are shown in Table 2) containing homologous sequences and restriction enzyme sites EcoRI and BamHI were designed for PCR amplification, and the products were purified and recovered. The pGBKT7 vector was digested with restriction endonucleases EcoRI and BamHI, and the digested products were purified and recovered. Subsequently, the digested products were ligated and transformed into *E. coli*, plated on LB solid selection medium containing kanamycin, and positive single clones were selected and sequenced to obtain the recombinant vector pGBKT7-LdHSFC1.

[0075] Detailed process of constructing the recombinant expression vector pBD-LdHSFC1-VP16:

[0076] Using the recombinant vector pGBKT7-LdHSFC1 as an amplification template, primers pBD-LdHSFC1-F and pBD-LdHSFC1-R (primer sequences are shown in Table 2) containing homologous sequences and the restriction enzyme site Stu I were designed for PCR amplification, and the products were purified and recovered. The pBD-VP16 vector was digested with the restriction endonuclease Stu I, and the digested product was purified and recovered, then ligated with the PCR amplification product and transformed into E. coli. The transformed bacteria were plated on LB solid selection medium containing kanamycin, and positive single clones were selected and sequenced to obtain the recombinant vector pBD-LdHSFC1-VP16.

[0077] Subsequently, the recombinant plasmids pGBKT7-GAL4 (positive control, Beijing Huayueyang), pGBKT7 (negative control), pGBKT7-LdHSFC1, and pBD-LdHSFC1-VP16 were transformed into competent yeast AH109 cells, and evenly spread on SD / -Trp solid medium. They were incubated upside down at 30°C, and yeast colonies grew after 2-3 days. Then, positive clones were picked and spotted onto solid medium containing SD / -Trp, SD / -Trp-His, SD / -Trp-His+3AT (10 mM), SD / -Trp-His+3AT (20 mM), and SD / -Trp-His +X-α-gal for plate testing to observe the growth of yeast cells and detect β-galactosidase activity.

[0078] Example 4: Analysis of the expression pattern of the LdHSFC1 gene

[0079] The expression pattern analysis of the LdHSFC1 gene was performed using tissue culture seedlings of 'White Paradise' with similar growth stages and ages. The seedlings were treated with heat shock at 37℃ for different durations, and each sample was replicated three times. RNA was extracted from 'White Paradise' leaves using the Trizol method. After removing the genomic DNA, the quality and concentration of RNA were measured. The RNA was reverse transcribed into cDNA and then used for RT-qPCR experiments of LdHSFC1. The 18S rRNA of lily was selected as the internal reference gene (primer sequences are shown in Table 1).

[0080] Table 1 Primers for real-time quantitative PCR

[0081] Primer Primer sequence (5′-3′) LdHSFC1-F TTTCTCCCACGTTCTCCTCC (SEQ ID NO:13) LdHSFC1-R AATGTGTGGTAGGAGGTGGG (SEQ ID NO:14) 18S rRNA-F AGTTGGTGGAGCGATTTGTCT (SEQ ID NO:15) 18S rRNA-R CCTGTTATTGCCTCAAACTTCC (SEQ ID NO:16)

[0082] Example 5: VIGS (Virus Induced Gene Silencing) to silence the LdHSFC1 gene

[0083] Transient silencing of the LdHSFC1 gene in lily petals:

[0084] A specific fragment from the LdHSFC1 sequence was selected, and primers pTRV2-LdHSFC1-F and pTRV2-LdHSFC1-R (primer sequences are shown in Table 2) were designed to amplify this specific fragment. Using pGBKT7-LdHSFC1 as a template, PCR amplification was performed, and the product was purified and recovered. The pTRV2 vector was digested with restriction endonucleases at Xba I and Kpn I sites. The digested product was purified and recovered, then ligated and transformed into *E. coli*. The transformed plasmid was plated on LB solid selection medium containing kanamycin, and positive single clones were selected and sequenced to obtain the pTRV2-LdHSFC1 recombinant plasmid.

[0085] The pTRV2-LdHSFC1 recombinant plasmid was transformed into Agrobacterium GV3101. After overnight culture by shaking, the bacteria were collected, resuspended, and the Agrobacterium OD600 was adjusted to 1.0. Then, pTRV1 was mixed with pTRV2 and pTRV2-LdHSFC1 at a 1:1 ratio, and the lily petals were infected by vacuum method. After infection, the bacterial solution was poured out and washed with deionized water. Then, it was placed in a petri dish (0.4% water agar) and cultured in the dark for 1 day. It was then equilibrated under normal light for 2 days. After heat treatment at 42℃, the heat resistance phenotype was identified and the physiological indicators were measured.

[0086] Table 2 Primers used for plasmid construction

[0087] Primer Primer sequence (5′-3′) pCAMBIA1300-LdHSFC1-GFP-F AGAAAGCTTCTGCAGGGGCCCGGGGTCGACATGGCCTTCTCTGGATCTTCAAAAGA (SEQ ID NO:5) pCAMBIA1300-LdHSFC1-GFP-R CAGCTCCTCGCCCTTGCTCACCATGGTACCTTACTAGAAGAATCCCTCGCCAAGAATACA (SEQ ID NO:6) pGBKT7-LdHSFC1-F GACCTGCATATGGCCATGGAGGCCGAATTCATGGCCTTCTCTGGATCTTCAAAAGA (SEQ ID NO:7) pGBKT7-LdHSFC1-R GTTATGCGGCCGCTGCAGGTCGACGGATCCCTAGAAGAATCCCTCGCCAAGAATACA (SEQ ID NO:8) pBD-LdHSFC1-F AGACAGTTGACTGTATCGCCGACCGGTATGGCCTTCTCTGGATCTTCAAAAGA (SEQ ID NO:9) pBD-LdHSFC1-R ATTTAATGAAACCAGAGTTAAAGGCCTCTAGAAGAATCCCTCGCCAAGAATA (SEQ ID NO:10) pTRV2-LdHSFC1-F GATTCTGTGAGTAAGGTTACCGAATTCTCTAGAATGGCCTTCTCTGGATCTTCAAAAGA (SEQ ID NO:11) pTRV2-LdHSFC1-R GCCCGGGCCTCGAGACGCGTGAGCTCGGTACCCTAGAAGAATCCCTCGCCAAGAATACA (SEQ ID NO:12)

[0088] Example 6: Determination of ion permeability and DAB staining of lily petal extract

[0089] Fresh lily petals (0.5 g each) from the high-temperature treated and control samples were collected and placed in centrifuge tubes containing 10 ml of ultrapure water. Vacuum treatment was applied until the petals completely sank to the bottom. The centrifuge tubes were then placed in a 28°C constant-temperature shaking incubator and shaken at 200 rpm for 1 h. The conductivity of the extract was measured using a conductivity meter and recorded as the initial value S1. After measurement, the centrifuge tubes were placed in a water bath at 100°C for 20 min, allowed to cool naturally, and then shaken on a shaker for approximately 2 h. The conductivity of the extract was measured again and recorded as S2. The ultrapure water conductivity value S0 served as a blank control. Three replicates were performed. Relative ion permeability = (S1-S0) / (S2-S0). To visualize the H2O2 content in lily cells, DAB staining was performed on lily petals treated with different methods. First, DAB staining solution was infiltrated into the lily petals using a vacuum method. The petals were then stained in the dark for 24 h, and finally, the pigment was removed using 75% ethanol.

[0090] Results Analysis

[0091] 1. Cloning and sequence analysis of the LdHSFC1 gene

[0092] To investigate the biological characteristics and functions of the LdHSFC1 gene, it was cloned from lily leaves. Its open reading frame (ORF) is 864 bp, encoding 287 amino acids. Comparison with NCBI and TAIR websites revealed that the LdHSFC1 gene belongs to the HSF gene family and shows high identity with HSFC1 proteins from other species, preliminarily identifying it as the HSFC1 gene from lilies. To further investigate the phylogenetic relationship between LdHSFC1 and members of the Arabidopsis HSF family, a phylogenetic tree was constructed using IQTREE software. The phylogenetic tree results showed that LdHSFC1 is most closely related to At3G24520 (AtHSFC1). Figure 1 ).

[0093] 2. Subcellular localization and transcriptional activity analysis of LdHSFC1

[0094] To further investigate the potential role of the LdHSFC1 gene in plant cells, subcellular localization experiments were conducted to clarify its location within plant cells. The results showed that the GFP-LdHSFC1 fluorescent signal was mainly concentrated in the nucleus of tobacco cells, while the positive control showed green fluorescence throughout the entire cell. Figure 2 These phenomena provide strong evidence for the conclusion that LdHSFC1 is a nuclear localization protein. Yeast one-hybrid experiments showed that yeast cells expressing BD-LdHSFC1 or the empty BD vector could not grow on SD / -Trp-His medium, but the positive control expressing GAL4 and yeast cells expressing BD-VP16 grew well, indicating that LdHSFC1 lacks transcriptional activation activity in yeast cells. Figure 3 Furthermore, yeast cells expressing BD-LdHSFC1-VP16 showed significantly inhibited growth compared to BD-VP16, indicating that LdHSFC1 functions as a transcriptional repressor in yeast cells.

[0095] 3. Analysis of LdHSFC1 expression pattern under heat treatment conditions

[0096] To investigate the expression pattern of LdHSFC1 under heat treatment conditions, real-time quantitative PCR analysis was performed on heat-treated lily leaves. RT-qPCR results showed that the expression level of LdHSFC1 did not change significantly in the early stage of heat treatment (0–0.5 h), but began to increase after 1 h. The relative expression level of the LdHSFC1 gene was highest at 12 h of heat shock treatment. Figure 4 Therefore, it is speculated that high temperature can activate the expression of LdHSFC1.

[0097] 4. VIGS silencing LdHSFC1 improves the heat resistance of lilies.

[0098] To investigate the function of the LdHSFC1 gene in lily heat stress, the LdHSFC1 gene was silenced in lily petals using VIGS. The results showed that under normal conditions, there was no significant difference in the condition of petals between the control TRV2 and LdHSFC1-silenced lilies. However, after heat treatment, the petals of the control TRV2 lily suffered significantly more severe heat damage. Simultaneously, under room temperature conditions, there was no significant difference in the relative ion permeability of the petal extracts from the control TRV2 and LdHSFC1-silenced lilies. Figure 5 However, under heat shock conditions, the relative ion permeability of the lily petal extract transformed with pTRV1 / pTRV2-LdHSFC1 was significantly lower than that of the control group, indicating that the control group petals had more cell effluent and more severe damage compared to petals with silenced LdHSFC1. DAB staining revealed that after high-temperature treatment, the control group petals showed a deeper reddish-brown color compared to the experimental group, indicating that the petals with VIGS-silenced LdHSFC1 contained less H2O2 than the control group petals. In conclusion, VIGS-silenced LdHSFC1 can improve the heat resistance of lilies.

[0099] This invention addresses the heat sensitivity of lilies by identifying a gene, LdHSFC1, that can enhance the heat tolerance of lilies. Silencing this gene improves the heat tolerance of lilies. Therefore, this invention provides significant theoretical and practical value for cultivating new heat-resistant lily germplasm and for molecular breeding of heat-resistant lilies.

[0100] sequence list

[0101] SEQ ID NO:1

[0102] ATGGCCTTCTCTGGATCTTCAAAAGAGAAGGTACTGGGGAGACAGAAGAAGCCTGTGTCCAGCGAAATGGAAGGGAACAACAGTCGAAGCAGCAGTAGACATCAGAATCAGCTGGTGGCGCCTTTTGTGGCCAAGACTTACGAGATGGTGAGCGATACTACAAATGATGTGCTGATCCGTTGGGGGAGCGCAAACAACAGCTTTATCGTACTCAACCACGCCCTCTTCTCCCAAGTTCTCCTCCCCTCTTACTTCAAGCACAGCAACTTCTCCAGTTTCATCCGTCAGCTTAACACTTACGGATTTAGAAAGGTGGATCCTGACAGGTGGGAATTCGCCCATGAGTCATTCGTCCGTGGGCAGACTCACCTCCTACCACACATAGTCCGACGGACATCTAAAAAAGTAGACTGTGATGATGCAGAGGACACGAAGTTACTGCAAGAGGTTGGACGGCTGAGGAAAGCGCGGCAAGCTCTTGAAGAGGAGCTGCAGGACATAAGCAAGCGAGTGCAGGTTGCCCAGAGGAAGTCACAACAGATAATGTCCTTCCTCTCGAAGGTGGCCGAGGACCCCGACCACTTGACCCGCGTCATATCCTCCAAGAAGAAGCAACTAACAGAAAACAAGAAGAGGCGCCTCTTGGTCTCGCCTCGGCATCAAACCTGGCAAAATCAGTACCCAACTAATAGTCTGCCTACTATAACCAGCAATGATGATGATGTAGCTGTTGTGCCGACAAGCCCATTTGATCAAGCCGGAAGCAAGCTGATAATGTCACCAGAGCTCGATATTAATGAACACAGTTCACGGGTGATGACTCCAGGGTTCCCCTTCTGTATTCTTGGCGAGGGATTCTTCTAG

[0103] SEQ ID NO:2

[0104] MAFSGSSKEKVLGRQKKPVSSEMEGNNSRSSSRHQNQLVAPFVAKTYEMVSDTTNDVLIRWGSANNSFIVLNHALFSQVLLPSYFKHSNFSSFIRQLNTYGFRKVDPDRWEFAHESFVRGQTHLLPHIVRRTSKKVDCDDAEDTKLLQEVGRLRKARQALEEELQDISKRVQVAQRKSQQIMSFLSKVAEDPDHLTRVISSKKKQLTENKKRRLLVSPRHQTWQNQYPTNSLPTITSNDDDVAVVPTSPFDQAGSKLIMSPELDINEHSSRVMTPGFPFCILGEGFF。

Claims

1. The LdHSFC1 gene with the nucleotide sequence shown in SEQ ID NO:1 is used in at least one of the following a1) and a2): a1) Its application in improving the heat resistance of lilies; a2) Application in cultivating new lily germplasm with improved heat resistance.

2. The LdHSFC1 protein encoded by the LdHSFC1 gene is used in at least one of the following a1) and a2): a1) Its application in improving the heat resistance of lilies; a2) Application in cultivating new lily germplasm with improved heat resistance; The LdHSFC1 protein is as shown in c1) or c2) below: c1) A protein having the amino acid sequence shown in SEQ ID NO:2; c2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in c1).

3. The biological material that inhibits the expression of the LdHSFC1 gene with the nucleotide sequence shown in SEQ ID NO:1, is used in at least one of the following a1) and a2): a1) Its application in improving the heat resistance of lilies; a2) Application in cultivating new lily germplasm with improved heat resistance; The biomaterial is at least one of the following: d1) - d10): d1) DNA molecules used to suppress LdHSFC1 gene expression; d2) An expression cassette containing the DNA molecule described in d1); d3) A recombinant vector containing the DNA molecule described in d1) or the expression cassette described in d2); d4) Recombinant microorganisms containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3); d5) A transgenic plant cell line containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3); d6) Transgenic plant tissue containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3); d7) A transgenic plant organ containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3); d8) A transgenic plant containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3); d9) Regenerative cells, tissue cultures, or protoplasts produced from cells or tissue cultures of the transgenic plants described in d8); d10) Propagation material of the transgenic plants as described in d8).

4. The application according to any one of claims 1-3, characterized in that, By inhibiting the expression of the LdHSFC1 gene in lilies or reducing the activity and / or content of the LdHSFC1 protein, the heat resistance of lilies can be improved or new lily germplasm with improved heat resistance can be cultivated.

5. A method for improving the heat resistance of lilies, characterized in that, Inhibiting the expression of the LdHSFC1 gene in lilies with nucleotide sequences as shown in SEQ ID NO:1 or reducing the activity and / or content of LdHSFC1 protein can improve the heat resistance of lilies. The LdHSFC1 protein is as shown in c1) or c2) below: c1) A protein having the amino acid sequence shown in SEQ ID NO:2; c2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in c1).

6. A method for cultivating new lily germplasm with improved heat resistance, characterized in that, Inhibit the expression of the LdHSFC1 gene in lilies with nucleotide sequences as shown in SEQ ID NO:1 or reduce the activity or content of LdHSFC1 protein to screen for plants with improved heat resistance. The LdHSFC1 protein is as shown in c1) or c2) below: c1) A protein having the amino acid sequence shown in SEQ ID NO:2; c2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in c1).

7. The method according to claim 5 or 6, characterized in that, The expression of the LdHSFC1 gene can be inhibited by RNA interference, VIGS virus-induced gene silencing, gene editing, or antisense RNA technology.

8. The LdHSFC1 gene with a nucleotide sequence as shown in SEQ ID NO:

1.

9. The LdHSFC1 protein encoded by the LdHSFC1 gene according to claim 8, characterized in that, The LdHSFC1 protein is the protein shown in c1) or c2) below: c1) A protein having the amino acid sequence shown in SEQ ID NO:2; c2) A fusion protein with the same function is obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in c1).

10. A biomaterial for silencing the LdHSFC1 gene of claim 8, characterized in that, The biomaterial is at least one of the following: d1)-d5): d1) DNA molecules used to suppress LdHSFC1 gene expression; d2) An expression cassette containing the DNA molecule described in d1); d3) A recombinant vector containing the DNA molecule described in d1) or the expression cassette described in d2); d4) Recombinant microorganisms containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3); d5) A transgenic plant cell line containing the DNA molecule described in d1), or the expression cassette described in d2), or the recombinant vector described in d3).

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