Mulberry heat shock transcription factor MaHSF1 as well as coding gene and application thereof

Abstract

The invention discloses a mulberry heat shock transcription factor MaHSF1 as well as a coding gene and application thereof, and aims to solve the technical problem that high-temperature-resistant gene resources and germplasm resources of mulberry are deficient at present. On the basis of the heat shock transcription factor MaHSF1 which is screened from a mulberry HSF gene family and is obviously subjected to high-temperature stress up-regulation and heterologous expression of which can improve the high-temperature resistance of the yeast, the over-expression of the heat shock transcription factor MaHSF1 can inhibit the increase of the content of malondialdehyde MDA at high temperature; the activity of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and the content of osmotic regulation substance free proline are improved, so that the high-temperature resistance of arabidopsis thaliana is improved; the expression of the promoter has developmental stage specificity and heat stress induction characteristics, which indicates the accuracy of the regulation mechanism of the promoter. According to the application, the function and the action mechanism of the MaHSF1 and the coding gene thereof for resisting the high-temperature stress damage are verified for the first time, and a solid theoretical basis is provided for the mulberry production to cope with the high-temperature stress damage and the breeding of new high-temperature-resistant varieties.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and genetic engineering technology, specifically relating to a mulberry heat shock transcription factor MaHSF1, its encoding gene, and its applications. Background Technology

[0002] With the intensification of global warming, high-temperature stress has become one of the major abiotic stress factors restricting plant growth and development, and reducing crop yield and quality. As sessile organisms, plants cannot escape high-temperature environments by moving; therefore, they have evolved complex stress regulatory networks to cope with high-temperature stress. Among these, heat shock factors (HSFs) are the core family of transcription factors mediating plant heat tolerance. HSFs regulate the expression of heat shock proteins (HSPs) and other stress-related genes by recognizing and binding to cis-acting elements in the promoter regions of downstream heat-responsive genes, thereby enhancing the plant's tolerance to high temperatures. Based on structural characteristics, plant HSFs can be divided into three categories: A, B, and C. Category A HSFs mostly act as transcription activators, playing a key role in heat tolerance regulation, with relatively little research on the HSFA6b subclass. Currently, HSF genes have been cloned from various plants such as Arabidopsis thaliana, rice, and tea, but the functions of HSF genes in different species exhibit species specificity, and the regulatory mechanisms of most HSF genes are not yet fully elucidated.

[0003] Mulberry is an important perennial woody economic plant, serving not only as a primary feed source for silkworms but also in a wide range of applications, including medicinal use, animal feed, functional foods, and ecological protection. While mulberry trees have a wide distribution and have developed certain environmental adaptability through long-term evolution, high-temperature stress can still lead to leaf wilting, decreased photosynthetic efficiency, and hindered growth, severely impacting their yield and ecological value.

[0004] Currently, research on the identification and function of HSF genes in mulberry trees is relatively limited. Therefore, identifying heat-resistant genes in mulberry trees to provide new genetic resources and technical approaches for the genetic improvement of plant heat resistance is of significant theoretical and practical value. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a mulberry heat shock transcription factor. MaHSF1 Another technical problem that this invention aims to solve is to provide mulberry heat shock transcription factors. MaHSF1 Applications of genes.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A mulberry heat shock transcription factor MaHSF1 The gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] This invention also provides a mulberry heat shock transcription factor. MaHSF1 The gene expresses a protein whose amino acid sequence is shown in SEQ ID NO.2.

[0008] This invention also provides a mulberry heat shock transcription factor. MaHSF1 The promoter of the gene, the sequence of which is shown in SEQ ID NO.3.

[0009] This invention also provides a mulberry heat shock transcription factor. MaHSF1 Application of genes in enhancing plant tolerance to high temperature stress.

[0010] A further improvement lies in the fact that enhancing the plant's tolerance to high-temperature stress refers to: inhibiting the increase of plant MDA under high-temperature stress, increasing the activities of SOD, POD, and CAT in the plant, and increasing the accumulation of free proline in the plant. Specifically, this includes the following steps: (1) Constructing a transcription factor containing the mulberry heat shock described above MaHSF1 Gene overexpression vectors: The overexpression vector contains MaHSF1 A stable overexpression vector for Arabidopsis thaliana with a specific sequence, named pCAMBIA1300-MaHSF1, was used to amplify the [specific sequence]. MaHSF1 The primer sequences for the specific sequence are SEQ ID NO.4 and SEQ ID NO.5: 1300- MaHSF1 -F:aggacaggtacccggggatcATGAAGTCTCAGTTTGTGCG; 1300- MaHSF1 -R: atcgtctttgtagtcgtcgatCTTGGGGCTAGAGGCCAAA; Among them, the lowercase letters are the carrier homologous arms; (2) Transform the overexpression vector constructed in step (1) into plant cells; (3) The plant cells obtained in step (2) were treated at 42℃ for 24 hours to improve the plant’s high temperature tolerance.

[0011] This invention also provides a mulberry heat shock transcription factor. MaHSF1 Application of gene promoters in plant tissue expression or high-temperature response expression.

[0012] A further improvement is that the expression in plant tissues refers to: extremely low basal activity in the early stages of vegetative growth, with expression regulated by developmental stage; and the expression in high-temperature response refers to: heat stress-specific induction of mulberry heat shock transcription factors. MaHSF1The gene promoter is significantly expressed in vascular tissues and reproductive organs, exhibiting heat-responsive characteristics. Specifically, it includes the following steps: (1) Constructing a transcription factor containing the mulberry heat shock described above MaHSF1 overexpression vectors for gene promoters: The overexpression vector contains MaHSF1 A stable overexpression vector of Arabidopsis thaliana containing a gene promoter-specific sequence was named pBI121-ProMaHSF1 amplification of the MaHSF1 The primer sequences for the gene promoter-specific sequence are SEQ ID NO. 6 and SEQ ID NO. 7: pBI121-ProMaHSF1-F:tatgaccatgattacgccaagctAAGTGTCTTTAAGAGGAGGC; pBI121-ProMaHSF1-R:ggactgaccacccggggatcTTTCACTACAACTCAATCTTGATTT; Among them, the lowercase letters are the carrier homologous arms; (2) Transform the overexpression vector constructed in step (1) into plant cells; (3) The plant cells obtained in step (2) under normal developmental conditions have highly specific and dynamic expression patterns that depend on the developmental stage; or, (4) The plant cells obtained in step (2) were treated at 42℃ for 3h and 6h respectively, and their expression patterns showed obvious high temperature stress induction characteristics.

[0013] Furthermore, the plant cells involved in the above applications are all Arabidopsis thaliana cells.

[0014] The beneficial effects of this invention are: This invention analyzes the mulberry HSF family sequences using bioinformatics tools, and for the first time isolates and clones all 20 HSF members. Furthermore, it identifies gene expression patterns in mulberry under high-temperature stress and in yeast heterologous transformation, identifying gene groups in plants that regulate high-temperature responses, a field with relatively limited research. HSFA6b transcription factor MaHSF1 For cloned MaHSF1 Gene overexpression vectors were constructed and transformed into Arabidopsis thaliana; MaHSF1Transgenic Arabidopsis thaliana and wild-type plants were simultaneously treated in a 42℃ light incubator for 24 hours. Phenotypic observation revealed that the growth of transgenic plants was less affected by high-temperature stress, while the growth of wild-type plants was inhibited, with significant leaf wilting. Physiological indicators showed that this gene maintains protein homeostasis and cellular oxidation balance under high temperatures by regulating oxidative damage markers (such as MDA), ROS-scavenging enzymes (such as SOD, POD, and CAT), and osmotic regulators (such as free proline). MaHSF1 The gene promoter was used to construct an expression vector, which was then transformed into Arabidopsis thaliana. GUS staining revealed that the vector exhibited developmental stage specificity and high-temperature stress-induced characteristics. The results indicated that mulberry trees... MaHSF1 Genes can enhance a plant's tolerance to high temperatures, providing direct evidence for elucidating the molecular mechanism of heat tolerance in mulberry trees, and have important application value in molecular breeding to improve the high-temperature stress of plants. Attached Figure Description

[0015] Figure 1 The relative expression of 20 cloned mulberry HSF members under high temperature stress.

[0016] Figure 2 Phenotypic observation of 20 mulberry HSF members heterologously transformed yeast under high temperature stress; A: growth at normal temperature of 30℃; B: growth under high temperature stress of 53℃.

[0017] Figure 3 For transformation MaHSF1 Phenotypic observation and analysis of Arabidopsis thaliana under high temperature stress; A: Phenotypic observation of transgenic Arabidopsis thaliana and wild-type before and after stress; B: Relative expression level determination; C: MDA content determination; D: SOD activity determination; E: POD activity determination; F: CAT activity determination; G: Free proline content determination.

[0018] Figure 4 For transformation MaHSF1 Analysis of promoter expression in Arabidopsis thaliana tissues and its response to high temperature stress.

[0019] Figure 5 For transformation CaMV35S Analysis of promoter expression in Arabidopsis thaliana tissues and its response to high temperature stress. Detailed Implementation

[0020] The genome sequences of *Sang* 32, *Populus tomentosa*, and rice were downloaded from the NCBI Genome Database; the genome sequence of *Morus alba* was downloaded from MorusDB; the genome database of *Ficus hirta* was downloaded from the China National Center for Biotechnology Information; and the genome data of *Arabidopsis thaliana* was downloaded from the TAIR database. Hidden Markov Model (HMM) maps of the characteristic HSF domain (PF00047) retrieved from the Pfam database (http: / / pfam.xfam.org / ) were used to perform HMM searches across the entire annotated protein database using TBtools software with default parameters. The obtained candidate HSF proteins were further validated using the online program CD-search (https: / / www.ncbi.nlm.nih.gov / cdd) in the Conserved Domains Database (CDD). Protein sequences with intact HSF domains were aligned using ClustalW and MEGAX. The physicochemical properties of the HSF gene family were analyzed using Tbtools (Table 1).

[0021] Table 1 MaHSF Physicochemical properties of gene family members

[0022] To perform functional analysis on the 20 HSF members, primers were designed to clone the actual sequences of the 20 members.

[0023] To analyze the expression of the mulberry HSF gene under high temperature stress, mulberry seedlings with 6 to 8 true leaves were subjected to high temperature stress at 45℃ for 0, 3, and 6 hours. Leaf tissues were collected, flash-frozen in liquid nitrogen, and then used for real-time quantitative PCR analysis.

[0024] The results are as follows Figure 1 As shown, MaHSF3 and MaHSF7 The effect was downregulated in leaves treated with high temperature for 3 hours and 6 hours, while MaHSF5 The expression level showed a downward trend after 3 hours of high-temperature treatment, and an upward trend after 6 hours. MaHSF15 Expression showed no significant changes before and after high-temperature stress. Furthermore, with prolonged high-temperature treatment, the expression of 16 other HSF genes in mulberry trees showed an upregulation trend. Among them, MaHSF1 , MaHSF2 , MaHSF5 , MaHSF9 , MaHSF12 and MaHSF16 The expression level increased more significantly from 3h to 6h under heat stress.

[0025] To quickly obtain the heat resistance results of 20 MaHSFs members, the full-length coding regions of the 20 MaHSFs members were inserted into the pYES2 vector to obtain the recombinant plasmid MaHSFs-pYES2. After sequencing verification, the above fusion vector and the empty vector pYES2 were transformed into yeast using the LiAc / PEG method. Yeast transformants were screened using solid synthetic uracil-deficient medium (SD / –Ura) supplemented with 2% glucose. Single clones of the empty vector pYES2 and the recombinant plasmid MaHSFs-pYES2 were inoculated into liquid SD / –Ura medium containing 2% glucose and cultured overnight at 30°C until OD500. 600 Reaching 0.5. For in GAL1 Promoter-induced MaHSF Gene expression was performed by diluting yeast suspension cells at a volume ratio of 1:100 in liquid SD / –Ura medium containing 2% galactose and culturing overnight. OD was then expressed in fresh SG / –Ura medium. 600 Adjusted to 0.7. In the heat stress treatment, yeast cells were treated at 53°C for 4 hours, followed by the preparation of 10... 0 Up to 10⁻ 4 Gradual dilutions were prepared, with 10 μL of each sample spotted onto solid SD / –Ura plates. The plates were incubated at 30°C for 2–5 days, and photographs were taken to record the growth status of the yeast spots.

[0026] The results showed that all transformed yeast cells could grow normally at 30℃. Figure 2 (A). However, after heat stress treatment at 53℃ for 4 hours, 19 of the 20 MaHSF expression strains showed enhanced heat resistance to varying degrees, with only MaHSF7 failing to provide any protection. Figure 2 (B). Notably, MaHSF1, MaHSF12, MaHSF13, MaHSF15, MaHSF16, and MaHSF17 exhibit particularly outstanding heat resistance. (Combined with these...) MaHSF We prioritize the expression level of genes under heat stress. MaHSF1 , MaHSF12 and MaHSF16 These are key candidate genes for subsequent functional identification. Among them, MaHSF1 , MaHSF12 and MaHSF1 6 were respectively compared with Arabidopsis thaliana HSFA6b , HSFA1a and HSFB2a They share the same origin. Because... HSFA6b Its function under heat stress requires further analysis; therefore, this study selected... MaHSF1 Conduct in-depth analysis.

[0027] The specific embodiments of the present invention will be described below with reference to specific examples. However, the following embodiments are only used to illustrate the present invention in detail and do not limit the scope of the present invention in any way.

[0028] Example 1: MaHSF1 Analysis of how overexpression improved the heat resistance of Arabidopsis thaliana. For evaluation MaHSF1 To investigate their role in heat stress tolerance, we obtained 11 independent Arabidopsis transgenic overexpression lines (named OE-1 to OE-11) through Agrobacterium-mediated transformation combined with hygromycin screening. Three representative lines (OE-3, OE-6, and OE-9) were randomly selected, with wild-type (WT) plants as controls, to observe morphological differences. Figure 3 As shown in Figure A, under normal growth conditions, no significant phenotypic differences were observed between WT and transgenic lines; however, after 24 hours of continuous heat stress at 42℃, WT plants exhibited severe wilting and visible damage, while... MaHSF1 The overexpression lines showed only mild symptoms of damage, indicating a significantly enhanced heat tolerance. qRT-PCR analysis confirmed that under heat stress, the three transgenic lines exhibited... MaHSF1 Expression was significantly upregulated ( Figure 3 B).

[0029] To further assess cell damage and antioxidant capacity, we measured several physiological indicators. Under normal conditions, there were no significant differences in MDA content and SOD, POD, and CAT activities between WT and transgenic plants. Figure 3 (C–F); however, after heat stress, the MDA content of the transgenic lines decreased by 41%–59% compared to WT (C–F). Figure 3 The C values ​​indicate a reduction in oxidative membrane damage. Simultaneously, the activities of SOD, POD, and CAT in the overexpression lines were increased by 23%–35%, 11%–13%, and 23%–33%, respectively, compared to WT. Figure 3 (D–F). Furthermore, the accumulation of free proline in transgenic lines after heat stress increased by 64%–91% compared to WT (D–F). Figure 3 (G).

[0030] The above results collectively demonstrate that MaHSF1 It can enhance the heat stress tolerance of Arabidopsis thaliana, and its mechanism of action may be related to strengthening the antioxidant defense system and promoting the synthesis of osmotic protective substances.

[0031] Example 2: MaHSF1 Tissue expression and high-temperature response of gene promoter transformed Arabidopsis thaliana To explore MaHSF1 The transcriptional regulation and expression dynamics were studied, and a model was constructed based on the transcriptional regulation and expression dynamics of the gene. MaHSF1 Promoter-driven GUS reporter gene transgenic Arabidopsis line (named ProMaHSF1::GUSA total of 11 transgenic lines (L1-L11) were obtained, and one independent T3 generation line L8 was randomly selected for further analysis.

[0032] Histochemical GUS staining results showed that its expression pattern was highly specific and dynamic, and dependent on developmental stage and environmental signals. No GUS activity was detected in 7-day-old seedlings under either normal growth conditions or after 6 hours of heat stress. Figure 4 Similarly, no expression was observed in the leaves and roots of 2-week-old seedlings under non-stress conditions; however, after 6 hours of heat treatment at this stage, clear GUS signals appeared in the root tissues, indicating an early heat-induced response. Figure 4 (B). In plants at the 4-week rosette stage, this induction pattern was more refined: no basal activity was observed at room temperature, while after 6 hours of heat stress treatment, strong GUS staining was precisely confined to the veins (B). Figure 4 (C). After entering the reproductive growth stage (5 weeks of age), the expression pattern changes significantly: constitutive C was detected in multiple tissues, including roots, stems, leaves, and flowers. ProMaHSF1 active( Figure 4 Furthermore, heat stress (3h and 6h) significantly enhanced GUS intensity in these tissues, indicating that developmental processes and stress signals synergistically enhanced promoter activity. As a control, transgenic lines with constitutive CaMV35S promoter-driven GUS exhibited stable and unchanged GUS activity across all developmental stages and under stress conditions. Figure 5 This confirms that the observed time-space control characteristics originate from MaHSF1 The promoter itself.

[0033] The above results indicate that MaHSF1 Its expression is regulated by developmental stage and exhibits heat-responsive characteristics: its basal activity is extremely low in the early stages of vegetative growth, while heat stress can specifically induce its significant expression in vascular tissues and reproductive organs.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mulberry heat shock transcription factor MaHSF1 Genes, characterized by, The mulberry heat shock transcription factor MaHSF1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A mulberry heat shock transcription factor as described in claim 1 MaHSF1 The gene expression protein, characterized by, Its amino acid sequence is shown in SEQ ID NO.

2.

3. A mulberry heat shock transcription factor as described in claim 1 MaHSF1 Gene promoters are characterized by, Its sequence is shown in SEQ ID NO.

3.

4. A mulberry heat shock transcription factor as described in claim 1 MaHSF1 Application of genes in enhancing plant tolerance to high temperature stress.

5. The application as described in claim 4, characterized in that, The enhancement of plant tolerance to high temperature stress refers to: inhibiting the increase of plant MDA under high temperature stress, increasing the activity of SOD, POD, and CAT in plants, and increasing the accumulation of free proline in plants.

6. The application as described in claim 5, characterized in that, Includes the following steps: (1) Constructing a transcription factor containing the mulberry heat shock described above MaHSF1 Gene overexpression vector: The overexpression vector contains MaHSF1 A stable overexpression vector for Arabidopsis thaliana with a specific sequence, named pCAMBIA1300-MaHSF1, was used to amplify the [specific sequence]. MaHSF1 The primer sequences for the specific sequence are SEQ ID NO.4 and SEQ ID NO.5: 1300- MaHSF1 -F:aggacaggtacccggggatcATGAAGTCTCAGTTTGTGCG; 1300- MaHSF1 -R:atcgtctttgtagtcgtcgatCTTGGGGCTAGAGGCCAAA; Among them, the lowercase letters are the carrier homologous arms; (2) Transform the overexpression vector constructed in step (1) into plant cells; (3) The plant cells obtained in step (2) were treated at 42℃ for 24 hours to improve the plant’s high temperature tolerance.

7. A mulberry heat shock transcription factor as described in claim 3 MaHSF1 Application of gene promoters in plant tissue expression or high-temperature response expression.

8. The application as described in claim 7, characterized in that, The expression in plant tissues refers to the fact that the basic activity is extremely low in the early stages of vegetative growth, and the expression is regulated by the developmental stage. The expression in high-temperature response refers to: heat stress-specific induced mulberry heat shock transcription factor. MaHSF1 The gene promoter is significantly expressed in vascular tissues and reproductive organs and exhibits heat-responsive properties.

9. The application as described in claim 8, characterized in that, Includes the following steps: (1) Constructing a transcription factor containing the mulberry heat shock described above MaHSF1 Overexpression vectors containing gene promoters: The overexpression vectors are those containing... MaHSF1 A stable overexpression vector of Arabidopsis thaliana containing a gene promoter-specific sequence was named pBI121 - Pro MaHSF1, amplified MaHSF1 The primer sequences for the gene promoter-specific sequence are SEQ ID NO. 6 and SEQ ID NO. 7: pBI121-ProMaHSF1-F:tatgaccatgattacgccaagctAAGTGTCTTTAAGAGGAGGC; pBI121-ProMaHSF1-R:ggactgaccacccggggatcTTTCACTACAACTCAATCTTGATTT; Among them, the lowercase letters are the carrier homologous arms; (2) Transform the overexpression vector constructed in step (1) into plant cells; (3) The plant cells obtained in step (2) under normal developmental conditions have highly specific and dynamic expression patterns that depend on the developmental stage; or, (4) The plant cells obtained in step (2) were treated at 42℃ for 3h and 6h respectively, and their expression patterns showed obvious high temperature stress induction characteristics.

10. In the application as described in claim 6 or 9, the characteristic is that, The plant cells used were all Arabidopsis thaliana cells.

Images