Application of PtoHSFC1 gene in regulating and controlling cold resistance and heat resistance of poplar

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a PtoHSFC1 gene in regulating and controlling cold resistance and heat resistance of a poplar, and the nucleotide sequence of the PtoHSFC1 gene is shown as SEQ ID NO.1. Through bioinformatics analysis, inducible expression analysis, target gene overexpression and temperature treatment of a knockout plant, the result shows that the heat resistance and cold resistance of the populus tomentosa can be improved by improving the expression level of the PtoHSFC1 gene.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving PtoHSFC1 Application of genes in regulating the cold and heat resistance of poplar trees. Background Technology

[0002] Poplar, belonging to the genus *Populus* in the family Salicaceae, is an important forestry resource. It plays a vital role in timber production and processing, as well as in shelterbelt construction, with China boasting the world's largest scale of poplar plantation cultivation. Beyond its timber use, poplar exhibits significant advantages in industrial processing, bringing considerable economic, ecological, and social benefits. In recent years, with the continuous expansion of research into the molecular mechanisms of plant stress responses and breakthroughs in molecular biology techniques, research on plant stress resistance has leaped from the traditional physiological and biochemical levels to the molecular level, providing theoretical support and technical pathways for the innovative development of plant stress resistance genetic engineering. Due to its rapid growth rate, high biomass accumulation efficiency, wide geographical distribution, mature asexual reproduction system, and relatively simple genome structure, poplar has become a representative model plant system in forest physiological research and genetic engineering-mediated molecular breeding. Because of its multifaceted advantages, poplar has been established as a model system for functional genomics research in woody plants, applied not only in traditional forest cultivation but also in molecular design breeding and forest development research.

[0003] In nature, both biotic and abiotic stresses affect plant growth. Against the backdrop of global warming, continuously rising environmental temperatures pose significant stress to plant growth and development. To cope with high-temperature stress, plants have evolved multi-level defense strategies, such as adjusting leaf angle to reduce solar radiation absorption, regulating stomatal opening to decrease transpiration rates, or activating heat shock protein expression through hormone signaling pathways to enhance cellular thermal stability and repair capabilities. In plants, HSF transcription factors play a crucial role in high-temperature stress. Plant heat shock transcription factors (HSFs) are widely present in various plant species and are important components in plant signaling pathways for resisting abiotic stress. They not only participate in the response and adaptation processes to various abiotic stresses by directly regulating the expression of heat shock proteins and related genes through transcription, but also mediate many plant life processes. However, the role of HSFs in poplar varies, and the role of HSFC1 in poplar is uncertain. Summary of the Invention

[0004] Reference 1, “Zhang Aidong, Tian Zimeng, Xiao Kai, et al. Genome-wide identification of Amaranth Hsf family genes and their expression analysis under high temperature stress [J]. Anhui Agricultural Sciences, 2025, 53(21):91-99,” reported that AtrHsf02, AtrHsf06, and AtrHsf19 showed extremely low expression levels under short-term heat shock (6, 12 h) and long-term high temperature stress (72 h), almost undetectable, indicating no heat resistance. Reference 2, “Wang Xueyi, Gu Yongmei, Zhang Xuemei, et al. Bioinformatics and stress response expression analysis of Populus HSF family genes [J]. Journal of Beijing Forestry University, 2021, 43(02):34-45,” reported that in Populus simonii, after 12, 24, and 48 hours of heat shock, the expression levels of AtrHsf02, AtrHsf06, and AtrHsf19 were extremely low, almost undetectable, indicating no heat resistance. After high-temperature treatment, no relative expression levels of PsnHSF1 and PsnHSF29 were detected. PsnHSF29 in Reference 2 is the same gene as HSFC1 in this invention. Reference 1 indicates that although Hsf is an important element in the signaling pathway for plant resistance to abiotic stress, the regulatory effects of genes in the Hsf family on heat or cold tolerance are not universal. Reference 2 shows that HSFC1 has no regulatory effect on heat tolerance in Populus tomentosa, indicating that whether HSFC1 has a regulatory effect on heat or cold tolerance in poplar is unknown, making the role of HSF in Populus tomentosa also unknown. To solve the above technical problems, this invention provides... PtoHSFC1 Application of genes or their expression promoters in regulating cold and heat resistance in poplar trees.

[0005] PtoHSFC1 The application of genes or their expression promoters in regulating the cold and heat resistance of poplar trees, wherein the regulation is to enhance the cold and heat resistance of poplar trees. PtoHSFC1 Overexpression of the gene can effectively enhance its heat resistance. PtoHSFC1 Overexpression of the gene can effectively enhance its antifreeze properties, therefore it is proposed that... PtoHSFC1 Application of genes in regulating the cold and heat resistance of poplar trees.

[0006] PtoHSFC1

[0007] Preferably, the expression promoter comprises: containing the PtoHSFC1 Recombinant plasmids containing the gene or containing the gene PtoHSFC1 Recombinant bacteria.

[0008] Preferably, the recombinant plasmid is the product of the... PtoHSFC1 The gene is obtained by linking it to a plant expression vector.

[0009] Preferably, the plant expression vector is Flag-HA.

[0010] Preferably, the recombinant bacteria are obtained by introducing the recombinant plasmid into Agrobacterium.

[0011] Preferably, a method for enhancing the cold and heat resistance of poplar trees includes... PtoHSFC1 The steps for introducing genes into poplar cells or plants.

[0012] Preferably, enhancing the heat resistance or cold resistance of poplar trees is reflected in reducing the degree of leaf damage after the poplar trees are subjected to temperature stress.

[0013] Preferably, enhancing the heat resistance or cold tolerance of poplar trees is reflected in the improved light energy conversion efficiency of poplar trees after being subjected to temperature stress.

[0014] Preferably, the poplar is white poplar.

[0015] Using the PtoHSFC1 Methods for genetically enhancing the heat or cold resistance of poplar trees include the following steps: Will include PtoHSFC1 Gene overexpression vectors are introduced into poplar recipient cells or tissues, where they are regenerated into transgenic plants; PtoHSFC1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses Populus tomentosa as research material to clone Populus tomentosa. PtoHSFC1 Genes. Their function was investigated through bioinformatics analysis, induced expression analysis, target gene overexpression, and temperature treatment of knockout plants. The main results are as follows:

[0017] (1) The phylogenetic tree construction results show that Populus tomentosa PtoHSFC1 With hairy poplar PtrHSFC1 Silver Poplar PalHSFC1 ,Tangerine CsHSFC1 and soybeans GmHSFC1 6. They have a high degree of homology.

[0018] (2) Subcellular localization experiments showed PtoHSFC1 The protein is located in the cell nucleus and cytoplasm.

[0019] (3) The results of promoter response element analysis show that PtoHSFC1 Gene promoters have ABA-responsive elements, photoresponsive elements, GA-responsive elements, IAA-responsive elements, and many other responsive elements.

[0020] (4) PtoHSFC1 Morphological observations of overexpression in Populus tomentosa indicate that... PtoHSFC1 After the gene was overexpressed in Populus tomentosa, the overexpressing plants under normal conditions were taller and had thicker stems than the wild type, while the knockout lines were the opposite of the overexpressing plants. Figure 6 ).

[0021] (5) After treatment at 45℃, the leaves of the plants showed varying degrees of scorching. DAB staining revealed that... PtoHSFC1 -OE plants have the smallest damaged leaf area, while PtoHSFC1 -KO plants exhibited extensive leaf scorching and dehydration. Simultaneously, this invention also measured relevant physiological indicators, finding that under high-temperature stress, PtoHSFC1 The REC and MDA of -OE plants were significantly lower than those of WT plants, while PtoHSFC1 -KO plants showed significantly higher efficiency than WT plants, with relative conductivity (REC) and malondialdehyde (MDA) content being important indicators of membrane damage. The Fv / Fm ratio directly reflects the maximum light energy conversion efficiency of PSII. ΦPSII reflects the actual light energy conversion efficiency of the plant in PSII. Under environmental stress, the PSII efficiency of plants decreases; low temperature and freezing stress damage the membrane system and alter PSII electron transport. However, PtoHSFC1 -OE plants had higher Fv / Fm and ΦPSⅡ values ​​than WT plants. PtoHSFC1 -KO plants were significantly lower than WT ( Figure 7 ),and PtoHSFC1 Overexpressing plants showed only slight damage at 45°C. In this invention, PtoHSFC1 -OE plants consume more excess light energy as heat to prevent light damage than WT plants, while PtoHSFC1 The photosynthetic system of -KO plants is more severely damaged under high-temperature stress than that of WT plants. Furthermore, the activity of photosystem II (PSII) is significantly inhibited when plants are subjected to high-temperature stress. This inhibitory effect is likely caused by the excessive accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS). Therefore, it can be concluded that the photosynthetic system of -KO plants is more severely damaged under high-temperature stress than that of WT plants. PtoHSFC1 Overexpression of the gene can effectively enhance its heat resistance.

[0022] (6) After low-temperature and freezing treatment, the leaves of all plants showed varying degrees of frost damage. The frost damage was less severe in the overexpressing plants than in the wild type, while the opposite was true for the knockout plants. DAB staining revealed... PtoHSFC1 -OE plants have the smallest damaged leaf area, while PtoHSFC1 -The leaves of the KO plant showed extensive frost damage. This invention also measured relevant physiological indicators and found that under low temperature and freezing stress, PtoHSFC1 The REC and MDA of -OE plants were significantly lower than those of WT plants, while [[ID=3 -KO plants were significantly higher than WT plants. This suggests that the population of Populus tomentosa was significantly higher. ​ This may have improved cell membrane integrity under low-temperature stress. Under environmental stress, the efficiency of PSII in plants decreases; low temperature and freezing stress disrupt the membrane system and alter PSII electron transport. However, ​ -OE plants had higher Fv / Fm and ΦPSⅡ values ​​than WT plants. ​ -KO plants were significantly lower than WT plants, and ​ Overexpressing plants showed only slight damage under low temperature conditions. ​ In this invention, ​ -OE plants consume more excess light energy as heat to prevent light damage than WT plants, while ​ The photosynthetic system of -KO plants is more severely damaged under low-temperature stress than that of WT plants. Furthermore, the activity of photosystem II (PSII) is significantly inhibited when plants are subjected to freezing stress. This inhibitory effect is likely caused by the excessive accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS). Therefore, it can be concluded that the photosynthetic system of -KO plants is more severely damaged under low-temperature stress than that of WT plants. ​ Overexpression of the gene can effectively enhance its antifreeze properties. Attached Figure Description

[0023] ​ Why the white poplar ​ Amino acid sequence pairs.

[0024] ​ Induced by exogenous factors ​ The expression, where (a) represents the expression under PEG induction. ​ Changes in expression levels, (b) for Nacl-induced changes. ​ Changes in GA expression levels, (c) under the induction of GA hormone ​ Changes in expression levels, (d) for Cold-induced changes. ​ Changes in expression levels; (e) Heat-induced changes ​ Changes in expression levels; (f) JA-induced changes ​ Changes in expression levels.

[0025] ​ Why the white poplar​ Subcellular localization.

[0026] ​ Why the white poplar ​ -OE strain identification, where (a) is the DNA positive identification of the obtained overexpression positive plants; (b) is the expression level of OE-3, OE-9, and OE-12. Note: The numbers above are the numbers of different strains.

[0027] ​ for ​ -KO strain identification.

[0028] ​ Wild-type white poplar, ​ -OE and ​ - The growth phenotype of KO plants, where A represents the growing plant, B represents the plant height, and C represents the plant stem diameter.

[0029] ​ To determine the phenotypic and physiological indicators and enzyme activity of plants treated at 45℃, where A represents plant phenotypic, B represents relative conductivity, C represents MAD content, D represents leaves after DAB staining, E represents ΦPSII, F represents SOD, G represents POD, and H represents Pro physiological indicators and enzyme activity.

[0030] ​ Phenotypic and physiological parameters of plants treated at 0℃ are given, where A represents plant phenotype, B represents leaves after DAB staining, C represents relative conductivity, D represents MAD content, E represents Fv / Fm, and F represents ΦPSII. ​ For the enzyme activity assay performed at 0℃, A represents SOD, B represents POD, C represents Pro, and D represents the expression level of PtoCOR47.

[0031] ​ The images show the phenotypic and physiological parameters of leaves treated at -7℃. A represents the leaf phenotype, B represents the leaf after DAB staining, C represents the expression level of PtoCOR47 in leaves of live plants after cold treatment, and D represents the Fv / Fm ratio. ​ For the determination of enzyme activity after treatment at -7℃, A represents MAD content, B represents SOD, C represents the expression level of PtoCOR47 in detached leaves after freezing, D represents POD, and E represents Pro.

[0032] ​ A map of the plant expression vector Flag-HA.

[0033] Note: WT represents wild-type white poplar. Detailed Implementation

[0034] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0035] Example 1 1. Bioinformatics analysis of the HSF gene family (1) HSF protein phylogenetic tree and homology analysis A search for *Populus tomentosa* (hairy poplar) was conducted in the NCBI database at https: / / www.ncbi.nlm.nih.gov / . ​ HSF s Arabidopsis thaliana ( ) ​ HSF s ), rice ( ​ HSF s ),tomato( Sl HSF s ) and corn ( ​ HSF s ), soybean (GmHSF s The amino acid sequences of the above species, including citrus (CsHSFs), were analyzed using DNAMAN software. ​ As shown, all species of HSF proteins possess four relatively conserved functional sequences at their intracellular N-terminus. These conserved sequences indicate that they play an important biological role in the function of HSF.

[0036] (2) Promoter analysis Retrieved from the NCBI database ​ The promoter sequence, approximately 2000 bp upstream of the gene coding sequence, was analyzed using the online analysis software PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). ​ Online predictive analysis was performed on a 2000bp promoter sequence.

[0037] The results obtained from statistical analysis of various response elements are as follows: ​ As shown, ​ The promoter sequence includes 90 cis-acting elements, of which 50 are CAAT-BOX related to the enhancer, 2 are ABRE (ABA-responsive elements), and 12 are photoresponsive elements. This invention focuses on the function of these two elements and conducts experiments related to biological stress.

[0038] Table 1: ​Promoter cis-acting element 2. Induction by exogenous hormones ​ Analysis of expression results ​ Induced by exogenous factors ​ The expression, where (a) represents the expression under PEG induction. ​ Changes in expression levels, (b) for Nacl-induced changes. ​ Changes in GA expression levels, (c) under the induction of GA hormone ​ Changes in expression levels, (d) for Cold-induced changes. ​ Changes in expression levels; (e) Heat-induced changes ​ Changes in expression levels; (f) JA-induced changes ​ Changes in expression levels.

[0039] In (a) and (b), 20% PEF and 100 mM NaCl were added to the nutrient solutions, respectively, while the control group consisted of plants under normal nutrient solution conditions. In (d) and (e), wild-type plants were treated at 4℃ and 37℃, respectively, while the control group was treated at room temperature (22℃). Total RNA was extracted from the samples, reverse transcribed, and then measured by RT-PCR. ​ Gene expression. (c) and (f) show two-month-old hydroponically grown wild-type Populus tomentosa plants, one group sprayed with 0.3 mM GA and 0.1 mM JA, and the other group sprayed with tap water as a control. Results are as follows. ​ As shown.

[0040] After adding 20% ​​PEF and 100mM NaCl to the nutrient solution ​ Gene expression levels showed a slight increase from 0 to 4 hours, then gradually increased from 4 to 12 hours, reaching a maximum of 15-fold at 12 hours, and then gradually decreased. ​ a, b). During low-temperature treatment ​ Gene expression levels decreased slightly from 0 to 2 hours, gradually increased from 2 to 16 hours, reached a maximum of 9-fold at 16 hours, and then decreased. ​ d). When treating at 37°C ​ Gene expression levels gradually increased from 0 to 9 hours, reaching a maximum of 15-fold at 9 hours, then decreased from 9 to 12 hours, and stabilized at 9.8-fold from 12 to 24 hours. ​ e). After applying GA and JA externally. ​Gene expression levels decreased slightly from 0 to 2 hours. GA treatment showed a slow increase from 2 to 6 hours, a slight decrease from 6 to 8 hours, followed by a rapid increase, reaching a 12-fold increase at 12 hours. JA treatment showed an increase from 2 to 10 hours, reaching a maximum of 35-fold at 10 hours, followed by a sharp decrease from 10 to 12 hours. ​ c, f). These results indicate that under various induction conditions, ​ It plays a dominant role in responding to these hormones.

[0041] 3. Subcellular localization analysis To clarify ​ Where are the proteins encoded by genes located, by... ​ The full-length CDS sequence of the gene was ligated into the plant transient expression vector pTEX-GFP to construct a 35S:pTEX- ​ -GFP fusion protein expression vector. This plasmid and the empty 35S:GFP vector were co-transformed with the membrane-localizing marker plasmid (35S:pSAT4A-AtRCI2A-mcherry) via PEG4000-mediated transformation into protoplasts of cultivated Populus tomentosa, and incubated at 25℃ for 16-20 h. Fluorescence was then observed using a laser confocal fluorescence microscope. GFP green fluorescence signal was observed at 488 nm excitation light, Nucleus red fluorescence signal at 555 nm, and Chloroplast blue fluorescence signal at 405 nm. ​ As shown, from left to right, the fields are dark field (GFP, Nucleus, Chloroplast), bright field, and merge field (Merge). The results of transient transformation of Populus tomentosa protoplasts using subcellular localization vectors demonstrate that... ​ Proteins are located in the cell nucleus and cytoplasm. ​ ).

[0042] Example 2 Acquisition and identification of genetically modified Populus tomentosa 1. ​ Gene cloning Materials preparation: Leaves of wild-type Populus tomentosa that have grown for 3 months were selected. Total RNA was extracted using the Trizol method, and cDNA was synthesized using a reverse transcription kit. Primer design: based on Populus tomentosa ​ Gene sequence (retrieved from NCBI database), design ​ Full-length CDS cloning primers PCR amplification: Using cDNA as a template, PCR amplification was performed using high-fidelity DNA polymerase. The reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; and 72℃ final extension for 10 min. Sequence verification: The PCR product was ligated into the pMD19-T vector, transformed into E. coli DH5α, and positive clones were selected for sequencing to obtain... ​ The full-length CDS sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.

[0043] 2. Obtaining and identifying transgenic Populus tomentosa overexpressed with this gene. For detailed identification ​ Based on the functions of Populus tomentosa, primers OE- were designed to clone the overexpressed gene. ​ -F / R, was synthesized at BGI Genomics, and the amplified DNA was ligated using T4 DNA ligase. ​ The full-length CDS sequence of the gene was ligated into the plant expression vector Flag-HA to construct an overexpression vector. The map of Flag-HA can be found here. ​ The ligation product was transformed into E. coli DH5α, and positive clones were screened by kanamycin resistance. The correctness of the vector construction was verified by PCR and sequencing.

[0044] PtoHSFC1-F: 5'-ATGATGGAGGCTAATAACAACAA-3', denoted as SEQ ID NO.2; PtoHSFC1-R: 5'-CTAAAAGCCACCTCCCAAAAGTG-3', denoted as SEQ ID NO.3.

[0045] Wild-type Populus tomentosa was transformed using the Agrobacterium leaf disc infection method. After several stages of tissue culture, DNA was extracted from the seedlings in the tissue culture bottles. The marker gene neomycin phosphotransferase II was then screened using the Flag-HA vector. ​ The gene fragment (GenBank accession number: AF485783) was amplified, and the expected fragment length was 1020bp for positive identification.

[0046] like ​ As shown, several positive Populus tomentosa plants were selected, total RNA was extracted, and after reverse transcription, the expression level was identified by qRT-PCR. Three independent transgenic overexpression lines with relatively high expression levels, namely OE-3, OE-9 and OE-12, were obtained, with expression levels of 21-fold, 45-fold and 30-fold, respectively. These three lines were then rapidly propagated to obtain a sufficient number of plants for subsequent experimental treatments.

[0047] 3. Obtaining and identifying genetically modified Populus tomentosa Target design: based on ​ Gene CDS sequences (exon1 and exon2 regions) were used to design three CRISPR / Cas9 knockout target sites. The three target site sequences were inserted into the pCRISPR vector backbone to construct the multi-target knockout vector pCRISPR-PtoHSFC1. The knockout vector was transformed into Agrobacterium EHA105 competent cells. Positive Agrobacterium strains were screened for resistance to Rifampicin and Kanamycin, and the correctness of the target insertion was verified by PCR.

[0048] like ​ As shown, the CRISPR / Cas9 site-specific knockout method was used to construct... ​ Gene knockout vectors were introduced into wild-type Populus tomentosa via Agrobacterium infection. Sequencing of the regrowth plants identified two knockout lines, lines 5 and 17. Sequencing results showed that two bacteria in line 5 had a 322bp deletion between the first and third target sites; while three bacteria in line 17 had a 321bp deletion between the first and third target sites.

[0049] Functional verification 1. Analysis of the heat resistance of Populus tomentosa ​ Belonging to the HSF family, it has been confirmed in many studies to have stress resistance capabilities. (This is for research purposes.) ​ Does it have a similar function and is overexpressed in Populus tomentosa? ​ After tissue culture, transgenic and wild-type Populus tomentosa plants with consistent growth conditions were treated at 45°C for 3 months. ​ As shown, after 12 hours of treatment, the leaves of the overexpressing plants showed the least degree of leaf scorching, while the leaves of the knockout plants exhibited extensive scorching and curling. DAB staining further revealed that the relative damaged area of ​​the leaves of the overexpressing plants was significantly lower than that of the wild-type Populus tomentosa, while the damaged area of ​​the leaves of the knockout plants was significantly higher than that of the wild-type. Phenotypically, as... ​ Genetically modified white poplar has enhanced heat resistance to poplar trees.

[0050] Simultaneously, relevant physiological indicators were measured. Fv / Fm and ΦPSⅡ were measured using a Li-6800 photosynthesis system before and after treatment. The Fv / Fm ratio directly reflects the maximum light energy conversion efficiency of PSII. ΦPSⅡ reflects the actual light energy conversion efficiency of the plant in PSII. Before treatment, there were no significant differences in Fv / Fm and ΦPSⅡ between WT and transgenic plants. After treatment at 45℃, ​-OE's Fv / Fm and ΦPSⅡ are significantly higher than WT's, while ​ -KO plants were significantly lower than WT plants. Simultaneously, leaf samples from the same stem nodes were used to measure the activities of SOD, POD, and Pro. No significant differences were found before treatment, but after treatment... ​ -OE has higher SOD, POD, and Pro activity values ​​than WT, while ​ -KO showed the opposite results. Using a conductivity meter to measure REC (relative conductivity), OE plants showed significantly lower REC than WT plants (reduced by 20%-30%), while KO plants showed significantly higher REC than WT plants (increased by 40%-50%). Using the thiobarbituric acid method to determine MDA content, OE plants showed lower MDA content than WT plants (reduced by 15%-25%), while KO plants showed higher MDA content than WT plants (increased by 30%-40%).

[0051] In summary, ​ It can improve the heat resistance of Populus tomentosa.

[0052] 2. Analysis of the cold resistance of Populus tomentosa like ​ As shown, transgenic and wild-type Populus tomentosa plants with similar growth conditions were subjected to 0℃ low-temperature treatment for 24 hours. After treatment, it was found that… ​ -OE showed less wilting compared to WT, while ​ -KO showed a significant decline compared to WT. For example... ​ Leaf positions of WT and transgenic plants were taken and frozen at -7°C for 3 hours, followed by 3 hours of recovery at room temperature. Results were observed... ​ -OE frostbite severity is lower compared to WT, while ​ -KO showed severe frost damage compared to WT. DAB staining of leaves that had undergone low-temperature and freezing treatment made the damage even more pronounced. ​ -OE blades have a lower relative damaged area than WT blades, while ​ -KO blades are damaged to a much greater extent than WT blades, such as ​ As shown in the figure. Phenotypic, the transgenic white poplar showed enhanced resistance to cold in poplar trees.

[0053] Simultaneously, relevant physiological indicators were measured, revealing no significant difference in Fv / Fm between WT and transgenic plants before treatment. After low-temperature and freezing treatment, ​ -OE's Fv / Fm is significantly higher than WT's, while ​ -KO plants were significantly lower than WT plants. Simultaneously, leaf samples from the same stem nodes were used to measure the activities of SOD, POD, and Pro. No significant differences were found before treatment, but after treatment... ​ -OE has higher SOD, POD, and Pro activity values ​​than WT, while ​-KO showed the opposite results. OE plants had lower REC and MDA levels than WT plants, while Fv / Fm, ΦPSⅡ, and the activities of SOD, POD, and Pro were higher than WT plants, consistent with the trend of high-temperature treatment.

[0054] 3. Overexpression ​ Analysis of the influence of Populus tomentosa on extreme temperature resistance overexpression ​ The genetically modified organism (GMO) enhanced the cold tolerance of poplar trees. To further clarify... ​ To investigate the molecular mechanism, wild-type and transgenic Populus tomentosa with consistent growth were cultured in nutrient solution for two months. Leaves from the same node were then selected, and total RNA was extracted. After reverse transcription, qPCR was performed to detect the expression of the poplar cold-resistance-related gene COR47. It was found that PtoCOR47 expression increased after low-temperature and freezing treatment. ​ The expression level of PtoCOR47 in OE leaves was significantly higher than that in WT plants, and the expression level of PtoCOR47 in OE plants was 5-8 times that in WT plants. ​ -KO is lower than WT, and KO is 0.2-0.3 times WT.

[0055] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0056] Although preferred embodiments of the invention 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 both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0057] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. PtoHSFC1 The application of the gene or its expression promoter in regulating the cold and heat tolerance of poplar, characterized in that, PtoHSFC1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the regulation is to enhance the cold tolerance and heat tolerance of poplar.

2. Use according to claim 1, characterized in that, The expression promoter includes a recombinant plasmid containing the PtoHSFC1 gene or a recombinant bacteria containing the PtoHSFC1 gene.

3. Use according to claim 2, characterized in that, The recombinant plasmid is obtained by ligating the PtoHSFC1 gene into a plant expression vector.

4. Use according to claim 3, characterized in that, The plant expression vector is Flag-HA.

5. Use according to claim 2, characterized in that, The recombinant bacteria are obtained after the recombinant plasmid is introduced into Agrobacterium.

6. Use according to claim 1, characterized in that, Methods of enhancing cold tolerance and heat tolerance in poplar trees include introducing into a poplar cell or plant PtoHSFC1 the step of introducing the gene into the poplar cell or plant.

7. Use according to claim 1, characterized in that, Enhancing the heat resistance or cold tolerance of the poplar is manifested in that the poplar has reduced leaf damage after temperature stress.

8. The use according to claim 1, characterized in that, Enhancing the heat resistance or cold tolerance of the poplar is manifested in that the poplar has improved light energy conversion efficiency after temperature stress.

9. The use according to claim 1, characterized in that, The poplar is Populus tomentosa.

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