Safflower heat shock transcription factor CtHsf-06 as well as coding gene and application thereof

Abstract

The invention discloses a safflower heat shock transcription factor CtHsf-06 as well as a coding gene and application thereof, and aims to solve the technical problem that the current safflower high-temperature-resistant gene resources and germplasm resources are deficient. According to the application, on the basis of a heat shock transcription factor CtHsf-06 which is screened from a safflower Hsf gene family and is obviously subjected to high-temperature stress up-regulation, the transcriptional level of the heat shock transcription factor CtHsf-06 in response to high-temperature stress, a promoter cis-acting element and a subcellular site of the heat shock transcription factor CtHsf-06 are analyzed. The CtHsf-06 gene is located in a cell nucleus, transcription of the CtHsf-06 gene is influenced by hormone signals and adversity stress, and the CtHsf-06 gene plays an important role in high-temperature response of safflower carthamus; the heterologous expression can improve the high-temperature resistance of the yeast; the overexpression of the corn protoplast can inhibit the increase of the content of H2O2 and MDA at high temperature and promote the improvement of the activity of CAT and APX enzymes, thereby improving the high-temperature tolerance of the corn protoplast. According to the application, the function and action mechanism of the CtHsf-06 and the coding gene thereof for resisting high-temperature stress are verified for the first time, and a solid theoretical basis is provided for the production of safflower carthamus to cope with high-temperature stress damage and breeding of new high-temperature-resistant varieties.

Description

Technical Field

[0001] This invention application relates to the field of molecular breeding technology, specifically to a safflower heat shock transcription factor CtHsf-06, its encoding gene, and its applications. Background Technology

[0002] With the intensification of global climate change and the increasing frequency of extreme heat events, heat stress has become a significant factor affecting crop growth, yield, and quality. Safflower ( Carthamus tinctorius Safflower (L.), also known as prickly safflower or wild safflower, is an annual herbaceous plant belonging to the genus Safflower in the family Asteraceae. As an important oilseed crop and medicinal plant in my country, its seeds are rich in unsaturated fatty acids, and its petals contain active ingredients such as hydroxysafflower yellow pigment A, making it widely used in food, medicine, and other fields. However, safflower is sensitive to high temperatures; sustained high temperatures can lead to reduced photosynthetic efficiency, decreased pollen fertility, inhibited oil synthesis, and even premature aging, severely impacting yield and quality. Therefore, elucidating the molecular mechanism of safflower's heat resistance and utilizing genetic engineering technology to improve its high-temperature resistance is of great significance for ensuring the sustainable development of the safflower industry.

[0003] Plants have developed a complex set of heat stress response mechanisms over a long period of evolution, among which heat shock transcription factors (HSFs) are the core hubs regulating heat tolerance. HSFs sense high-temperature signals and activate the expression of downstream heat shock proteins (HSPs) and other stress-related genes, helping cells maintain protein homeostasis and repair damaged proteins, thereby improving plant heat tolerance. Currently, the HSF family has been extensively studied in crops such as Arabidopsis thaliana, rice, and maize, but functional studies in safflower remain relatively scarce. For example, Arabidopsis thaliana AtHSFA1s has been shown to be a major regulator of heat stress response, and its overexpression lines exhibit significantly enhanced heat tolerance; rice OsHSFA2d improves heat tolerance by activating the expression of antioxidant enzyme genes (such as APX and CAT) and reducing the accumulation of reactive oxygen species (ROS); maize ZmHSFA6a is significantly upregulated under high temperature stress, and its overexpression can improve the survival rate and biomass of transgenic plants; in addition, some studies have used CRISPR-Cas9 gene editing or overexpression technology to modify HSFs, which has significantly improved the high temperature adaptability of crops.

[0004] Although the function of HSF in various crops has been validated, current research still faces the following limitations: species-specific differences, with HSF functions potentially diverging in different plants, and the heat tolerance regulatory mechanism of safflower HSF remaining unclear, lacking systematic functional studies; gene redundancy and functional overlap, as the HSF family has numerous members (e.g., 21 in Arabidopsis and 31 in maize), with some genes exhibiting redundant functions, resulting in limited regulatory effects of single genes; and insufficient application and translation, with most studies focusing only on functional validation in model plants (e.g., Arabidopsis), lacking practical application cases in economic crops such as safflower. Therefore, locating and cloning the safflower-specific HSF gene, elucidating its heat tolerance molecular mechanism, and developing corresponding molecular breeding strategies are of great significance for breeding heat-resistant safflower varieties and addressing the challenges of climate change.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] This invention addresses the current technical problem of scarce heat-resistant gene and germplasm resources in safflower. Through systematic screening of the safflower Hsf gene family, a heat shock transcription factor CtHsf-06 that is significantly upregulated under high-temperature stress was identified. The transcriptional level, promoter region cis-acting elements, and subcellular sites of CtHsf-06 in response to high-temperature stress were analyzed, and finally, the regulatory role of CtHsf-06 in the heat tolerance of safflower was determined. CtHsf-06 The gene is located in the cell nucleus, and its transcriptional activity is affected by hormonal signals and abiotic stress, playing an important role in the high-temperature response of safflower. Heterologous expression of the gene can improve the tolerance of yeast to high-temperature stress; overexpression of the gene can inhibit the increase of H2O2 and MDA content at high temperatures, promote the increase of CAT and APX enzyme activities, and thus improve the high-temperature tolerance of maize protoplasts. This invention provides important genetic resources and technical support for stress-resistance breeding in safflower, maize, wheat, tobacco, or Arabidopsis thaliana.

[0007] The first aspect disclosed in this application relates to a heat shock transcription factor gene. CtHsf-06 Its DNA sequence is shown in SEQ ID NO. 1.

[0008] The second aspect disclosed in this application relates to a heat shock transcription factor CtHsf-06, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The third aspect disclosed in this application discloses the heat shock transcription factor gene. CtHsf-06 Or the use of the heat shock transcription factor CtHsf-06 in any of the following or in the preparation of reagents having any of the following functions: (1) Improve the high-temperature tolerance of plants; (2) High temperature stress inhibits the increase of H2O2 and / or MDA in plant protoplasts; (3) High temperature stress increases the activity of CAT and / or APX enzymes in plants; (4) Improve the high temperature tolerance of yeast.

[0010] The fourth aspect disclosed in this application discloses the heat shock transcription factor gene. CtHsf-06 Or the application of its expression vectors in the selection and breeding of plant varieties / lines resistant to high temperature stress.

[0011] In some embodiments of this disclosure, the heat shock transcription factor gene is... CtHsf-06 Overexpression.

[0012] In some embodiments of this disclosure, the plant is safflower, corn, wheat, tobacco, or Arabidopsis thaliana.

[0013] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: 1. Filling the gap in heat-resistance gene resources in safflower: For the first time, CtHsf-06, a member of the Hsf family responding to high-temperature stress, was cloned from safflower. Its gene sequence contains stress and hormone response elements such as TC-rich repeats, ABRE, and CGTCA-motif, indicating that its transcriptional activity is regulated by high temperature and ABA signaling. This discovery provides a key gene resource for the study of the molecular mechanism of heat resistance in safflower, overcoming the limitation of the lack of heat-resistance genes in safflower in existing technologies.

[0014] 2. Clarifying gene function and mechanism of action: Through subcellular localization (nuclear localization) and heterologous functional verification (enhanced yeast thermoresistance), it was confirmed that CtHsf-06 directly participates in the high-temperature stress response. Overexpression experiments showed that it can significantly improve the thermoresistance of maize protoplasts, indicating that this gene maintains protein homeostasis and cellular oxidative balance under high temperatures by regulating the activity of oxidative damage markers (such as H2O2 and MDA) and ROS scavenging enzymes (such as APX and CAT), providing direct evidence for elucidating the molecular mechanism of safflower thermoresistance.

[0015] 3. Advancing the process of molecular design breeding: Based on the heat-resistant function of CtHsf-06, molecular marker-assisted selection technology can be developed to accelerate the breeding of new heat-resistant safflower varieties. Compared with traditional breeding, the application of this gene can shorten the breeding cycle and precisely improve the yield stability of safflower under high-temperature conditions, meeting the supply demand of medicinal resources and oil crops under climate change.

[0016] 4. Cross-species application potential: CtHsf-06 is located in the cell nucleus in both monocot and dicotyledonous plants, and heterologous expression enhances the heat tolerance of yeast, indicating its functional conservation. This gene can serve as a broad-spectrum stress-resistance gene resource for improving the heat tolerance of crops such as maize and wheat, expanding its economic value in the field of agricultural biotechnology. Attached Figure Description

[0017] Figure 1 As shown in one embodiment of this application CtHsf Expression of gene family members under high temperature stress, where: A represents... CtHsf Expression of gene family members under high temperature stress; BE values ​​are respectively CtHsf-06 , CtHsf-16 , CtHsf-18 , CtHsf-22 Expression under high temperature stress.

[0018] Figure 2 This paper analyzes the cis-acting elements of the CtHsf-06 promoter in one embodiment of this application, where: the gray arrow represents the sequence of CtHsf-06 2000 bp upstream of the start codon ATG; the sequences in parentheses correspond to various cis-acting elements; and the positive and negative signs represent the positive and negative chains, respectively.

[0019] Figure 3 High temperature stress and ABA in one embodiment of this application CtHsf-06 The effects of high temperature stress on expression, including: A, the effect of high temperature stress on... CtHsf-06 The effects of B high temperature stress and ABA on expression; CtHsf-06 Effects on expression; CK, untreated normal safflower sample; H, high temperature; ABA, abscisic acid.

[0020] Figure 4 This is a subcellular localization analysis of CtHsf-06-GPF in one embodiment of this application, wherein: A and B are transient transformation systems of maize protoplasts and tobacco leaves, respectively; Bright, bright field; GFP, green fluorescence; mCherry, fluorescent red fluorescence; Merged, superimposed image; GFP is excited by a 488 nm laser and monitored at 525 nm; mCherry is excited by a 561 nm laser and monitored with a 580 nm long-pass filter.

[0021] Figure 5 As shown in one embodiment of this application CtHsf-06 Heterologous expression of the gene in *Saccharomyces cerevisiae* strain BY4741, including: A, comparison of growth of empty vector yeast and recombinant vector yeast under different temperature conditions (30℃, 37℃, 39℃, 42℃); B, OD values ​​of empty vector yeast and recombinant vector yeast at 0, 1, 3, 6, 12 and 24 h under different temperature conditions.600 value.

[0022] Figure 6 In one embodiment of this application, overexpression under high temperature stress CtHsf-06 Effects on maize protoplasts and determination of physiological indicators, including: A, CtHsf-06 A, Effect of overexpression on protoplast heat resistance; B, Statistical analysis of protoplast damage rate after high-temperature treatment; C, Overexpression under high-temperature stress. CtHsf-06 Effects on the content of H2O2 and MDA in maize protoplasts and the activities of CAT and APX enzymes. Detailed Implementation

[0023] The following examples illustrate specific implementations of the present invention. However, these examples are merely for illustrative purposes and do not limit the scope of the invention in any way.

[0024] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and materials involved are all commercially available conventional products; unless otherwise specified, the test and detection methods involved are all conventional methods.

[0025] Example 1: Safflower Hsf Identification of gene families and expression patterns under high temperature stress Using 22 species from Arabidopsis thaliana AtHsfs 31 in corn ZmHsfs 25 in rice OsHsfs Protein sequences of gene family members were used as query sequences and preliminarily screened by BLASTP alignment with the whole genome data of safflower using TBtools software (E-value ≤ 10). -5 After removing incomplete domains and redundant sequences, further screening was conducted using InterPro (https: / / www.ebi.ac.uk / interpro) and the CDD conserved domain database (http: / / www.ncbi.nlm.nih.gov / cdd) to determine whether the samples contained the core conserved domains of plant Hsf—DNA-binding domain (DBD) and oligomerization domain (OD) / HR-A / B region. A total of 22 domains were identified. CtHsf Gene family members are systematically numbered and named according to their location and order on the chromosome. CtHsf01 - CtHsf22 Their physicochemical properties were predicted and analyzed (Table 1).

[0026] Table 1CtHsf Physicochemical properties of gene family members .

[0027] In order to analyze CtHsf To investigate the expression of gene family members under high-temperature stress, safflower seedlings with 4–6 true leaves were subjected to 42°C high-temperature stress treatment for 0, 0.5, 1, 2, 4, 6, and 8 hours. Samples from at least five seedlings were pooled, then flash-frozen in liquid nitrogen and used for transcriptome analysis. The FPKM of genes identified in safflower was used to analyze the expression of these genes. CtHsfs The expression patterns under high temperature stress were analyzed.

[0028] The results are as follows Figure 1 As shown in A, in 22 CtHsfs middle, CtHsf-06 , CtHsf-16 , CtHsf-18 and CtHsf-22 The transcriptional level of these molecules was significantly upregulated under high-temperature stress. Then, RT-qPCR (primer sequences are shown in Table 2) was used to verify their expression under high-temperature stress, and the results were as follows: Figure 1 As shown in BE, except CtHsf-22 outside, CtHsf-06 , CtHsf-16 , CtHsf-18 The expression patterns under high-temperature stress were similar to those in the transcriptome, both being significantly induced by high temperature. CtHsf-06 The effect was most pronounced under high temperature stress.

[0029] Table 2 Primer Sequences .

[0030] Example 2: Safflower CtHsf-06 Analysis of cis-acting elements in gene promoters In order to analyze CtHsf-06 To determine whether the transcriptional level of safflower responds to high-temperature stress, cis-regulatory elements in its promoter sequence were analyzed. First, whole-genome data of safflower were used to identify… CtHsf-06 The gene sequence and its transcription start site were obtained, and the 2000 bp sequence upstream of the transcription start site was extracted as the query sequence. The cis-acting elements were analyzed using the PlantCARE database (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html).

[0031] The results are as follows Figure 2 As shown, CtHsf-06There was one active element associated with defense and stress (TC-richrepeats, TAAGAGAGGAA) and 14 active elements associated with ABA response (ABRE, ACGTG); in addition, there were 10 active elements responsive to methyl jasmonate (CGTCA-motif, CGTCA and TGACG-motif, TGACG) and one active element responsive to low-temperature stress (LTR, CCGAAA). This indicates that... CtHsf-06 Its transcriptional activity may be affected by hormonal signals and abiotic stress, thus playing an important role.

[0032] Example 3: Safflower CtHsf-06 Gene expression in response to high temperature stress and ABA To further determine CtHsf-06 To investigate whether gene expression was affected by high temperature and ABA, simulated field high-temperature treatment was applied to 4-6 leaf safflower seedlings (starting at 28℃, increasing by 2℃ every 1 hour until reaching 42℃, maintained for 2 hours, for a total of 8 hours). Total RNA was then extracted from the 5th leaf, and its expression under high-temperature stress was analyzed using RT-qPCR. Simultaneously, seedlings were pretreated with ABA for 4 hours, then treated at 42℃ for 8 hours, after which total RNA was extracted from the 5th leaf, and its expression was analyzed using RT-qPCR. The results are as follows: Figure 3 As shown CtHsf-06 It was clearly induced by high temperature stress and ABA, indicating that it is related to abiotic stress of safflower, especially high temperature stress.

[0033] Example 4: Safflower CtHsf-06 Subcellular site analysis of genes All biochemical reactions occur in different cellular compartments; therefore, subcellular localization plays a crucial role in gene function. Based on predictions from the Plant Protein Subcellular Localization Prediction Website (http: / / www.csbio.sjtu.edu.cn / bioinf / plant-multi),... CtHsf-06 The subcellular sites are located in the cell nucleus. Therefore, in this example, a fusion vector 35S:CtHsf-06-GFP labeled with green fluorescent protein and a fusion vector 35S:AtIMP4-mCherry labeled with cherry red protein, whose nuclear localization is known, were constructed. Using a maize protoplast transformation system and a tobacco transient transformation system, the subcellular sites were identified by observing fluorescence through laser confocal microscopy.

[0034] The results are as follows Figure 4 As shown, the fluorescence signal of CtHsf-06-GPF overlaps with the AtIMP4-mCherry signal, which is known to be located in the nucleus in Arabidopsis, in both maize protoplasts and tobacco leaves, indicating that... CtHsf-06The subcellular sites are located in the nucleus in both monocots and dicots, and this result is consistent with previous predictions.

[0035] Example 5: CtHsf-06 Heterologous overexpression improves the heat resistance of yeast. Yeast possesses a eukaryotic expression system, allowing for the in vitro validation and screening of the phenotypic function of stress-resistance genes in plants and animals, including those subjected to salt stress, drought stress, temperature stress, and heavy metal stress. In this example, the temperature-sensitive yeast strain BY4741 was used. The recombinant vector pYES2-NTB-CtHsf-06 was transformed into the BY4741 yeast strain. Then, single clones of the experimental group strain pYES2-NTB-CtHsf-06 and the control strain pYES2-NTB were resuspended in 2 mL of sterile water, and the OD was adjusted. 600 The value was 0.5, so a gradient dilution (10) was performed. 0 10 -1 10 -2 The culture medium was inoculated into SG-Ura solid and liquid media and incubated in incubators at temperatures of 30℃, 37℃, 39℃, 42℃, and 45℃, respectively. OD values ​​were recorded during the incubation process. 600 The value and growth status.

[0036] The results are as follows Figure 5 As shown, the control yeast strain grew at 30℃ but not at 37℃, 39℃, 42℃, and 45℃. The yeast strain containing the recombinant plasmid grew at 30℃, 37℃, and 39℃ but not at 42℃ and 45℃. The yeast strain containing the recombinant plasmid performed better than the control yeast strain under high temperature stress, and its OD... 600 The value also reflects the foreign gene CtHsf-06 Expression in yeast also enhances tolerance to high temperatures. Therefore, CtHsf-06 Heterogeneous sources improve the yeast’s tolerance to high temperature stress.

[0037] Example 6: CtHsf-06 Overexpression of [a substance] improves the heat resistance of maize protoplasts. Malondialdehyde (MDA) content reflects the degree of lipid peroxidation in plant cell membranes; an elevated MDA level indicates increased damage to the membrane system. Excessive accumulation of H2O2 can directly or indirectly oxidize biomolecules such as nucleic acids and proteins, and damage cell membranes, accelerating cell senescence and disintegration. Catalase (CAT) and ascorbate peroxidase (APX) are important enzymatic antioxidant systems in plants, responsible for scavenging H2O2. Therefore, to further determine... CtHsf-06To investigate whether this method could improve plant heat tolerance, this study utilized a transient protoplast transformation system. The overexpression vector OE-CtHsf-06 was transformed into maize leaf protoplasts, incubated at room temperature for 16 hours, and then incubated under high-temperature stress (38℃) for 30 minutes. The damage caused by high-temperature stress to the protoplasts was then observed, and the contents of MDA and H2O2, as well as the activities of CAT and APX, were measured.

[0038] The results are as follows Figure 6 As shown, in the control group under high-temperature stress, the protoplasts shrank and deformed, indicating that they were significantly damaged by high-temperature stress, resulting in the loss of protoplast activity. Overexpression of [the substance / product]... CtHsf-06 It significantly reduced the damage to protoplasts caused by high temperature; furthermore, compared with the control, overexpression significantly reduced the damage. CtHsf-06 It also significantly inhibited the increase in H2O2 and MDA content induced by high temperature in protoplasts, and increased the activity of CAT and APX enzymes induced by high temperature, which further illustrates... CtHsf-06 It can improve the heat resistance of plants.

[0039] Although some preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0040] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of its inventive concept. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A heat shock transcription factor gene CtHsf-06 whose DNA sequence is shown as SEQ ID NO.

1.

2. A heat shock transcription factor CtHsf-06, the amino acid sequence of which is shown as SEQ ID NO.

2.

3. The heat shock transcription factor gene of claim 1 CtHsf-06 or the use of the heat shock transcription factor CtHsf-06 of claim 2 in any one of the following or in the preparation of an agent having the function of any one of the following: (1) improving the high temperature tolerance of plants; (2) inhibiting the increase of H2O2 or / and MDA in plant protoplasts under high temperature stress; (3) improving the activity of CAT or / and APX enzymes in plants under high temperature stress; (4) improving the high temperature tolerance of yeast.

4. The heat shock transcription factor gene of claim 1 CtHsf-06 Or the application of its expression vectors in the selection and breeding of plant varieties / lines resistant to high temperature stress.

5. Use according to claim 3 or 4, characterized in that, transcription factor gene CtHsf-06 overexpression.

6. Use according to claim 3 or 4, characterized in that, The plant is safflower, corn, wheat, tobacco or Arabidopsis.

Images