Application of corn HSF21 protein in improving plant cold tolerance

Abstract

The application relates to the field of biotechnology, and particularly discloses application of corn HSF21 protein in improving cold resistance of plants. It is found that overexpression of the HSF21 gene in corn can enhance the low-temperature resistance of plants, and thus the application of corn HSF21 protein or a coding gene thereof or biological material containing the coding gene in any of the following aspects is proposed: (1) changing the cold resistance of plants; (2) improving the survival rate of plants in a low-temperature environment; (3) breeding transgenic plants with improved cold resistance; and (4) improving cold-resistant germplasm resources of plants. The application provides a new gene resource for cultivating new low-temperature-resistant plant varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the application of maize HSF21 protein in improving plant cold tolerance. Background Technology

[0002] Maize (Zea mays L.) is an economic crop originating from tropical low-latitude regions. Although it has gradually spread to high-latitude and high-altitude temperate regions during human domestication and cultivation, maize remains highly sensitive to low-temperature damage. Therefore, it is still necessary to cultivate cold-resistant maize varieties when expanding maize cultivation in temperate regions. Maize's sensitivity to low temperatures is mainly due to reduced photosynthetic capacity and metabolic disorders. Short-term exposure of maize seedlings to low temperatures leads to decreased photosynthetic activity, subsequently affecting the transport of assimilates through dissipation mechanisms and the antioxidant system.

[0003] Existing transgenic technology can introduce stress-resistance genes from plants into the genetic material of maize varieties that require improvement, enabling their offspring to exhibit stable, inherited stress resistance and providing superior varietal resources for agricultural production. Researching new genes that regulate cold tolerance in maize is of great significance for maize breeding and subsequent production. Summary of the Invention

[0004] The purpose of this invention is to provide a new application of HSF21 protein in the regulation of cold tolerance in maize.

[0005] This invention provides the application of the maize cold-resistant gene HSF21 and its encoded protein. Maize HSF21 has the highest homology with HSFB1 in Arabidopsis thaliana, but HSFB1 in Arabidopsis thaliana has not yet been found to exhibit a low-temperature phenotype. Through research on the maize cold-related gene HSF21, this invention found that transgenic plants overexpressing this gene exhibit a significantly stronger cold-resistant phenotype than wild-type plants. This invention provides a new gene resource for breeding new cold-resistant plant varieties.

[0006] This invention provides the application of the HSF21 protein and its encoding gene in cold resistance in maize. To discover genes related to cold resistance in maize, this invention screened a maize library of transgenic overexpression lines and observed their phenotypes. It was found that different overexpression gene lines exhibited different phenotypes, and further, several lines overexpressing the HSF21 gene showed obvious cold-resistant phenotypes.

[0007] Specifically, this invention screened overexpressing maize populations, using the relative leaf injury area as an indicator for preliminary screening of low-temperature phenotypes. Overexpressing lines showing the initial phenotype were then re-screened to determine their low-temperature-related phenotypes. By consulting the overexpression information table, the gene number of the overexpressed gene in this line was found to be GRMZM2G139535. Further gene annotation on the MaizeGDB website identified it as the maize transcription factor HSF21. A unified comparison revealed that HSF21 belongs to class B HSF transcription factors, but its function had not been reported. This invention determined that the HSF21 gene may be a key gene for cold tolerance and hardiness in maize. Furthermore, this invention obtained cold-resistant transgenic plants by overexpressing the HSF21 gene in maize.

[0008] The cDNA sequence of the maize HSF21 protein involved in this invention is: i) the nucleotide sequence shown in SEQ ID No. 1; or ii) a nucleotide sequence of the nucleotide sequence shown in SEQ ID No. 1 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; or iii) a nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID NO. 1.

[0009] The maize HSF21 cDNA consists of 1671 bases, and its sequence is shown in SEQ ID No. 1. The gene's reading frame consists of two exons. The amino acid sequence encoded by the maize HSF21 gene is shown in SEQ ID No. 2.

[0010] The corn HSF21 protein of this invention has any one of the following amino acid sequences:

[0011] 1) The amino acid sequence shown in SEQ ID NO.2;

[0012] 2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2.

[0013] It should be understood that those skilled in the art can, based on the amino acid sequence disclosed in this invention, substitute, delete, and / or add one or more amino acids to obtain the mutant sequence of the protein without affecting its activity.

[0014] This invention provides the use of maize HSF21 protein or its encoding gene, or biological material containing its encoding gene, in any of the following aspects:

[0015] (1) Modify the cold tolerance of plants;

[0016] (2) Improve the survival rate of plants under low temperature conditions;

[0017] (3) Select and breed transgenic plants with improved cold tolerance;

[0018] (4) Improve cold-resistant plant germplasm resources.

[0019] Preferably, the goal is to improve the plant's cold tolerance.

[0020] The biological material is an expression cassette, vector, host cell, or recombinant bacteria.

[0021] This invention also provides cloning vectors or various expression vectors containing the HSF21 gene sequence or fragment thereof for plant cold tolerance, host cells containing the vector, transformed plant cells containing the gene sequence or specific fragment thereof, and transgenic plants. The overexpression vector containing the HSF21 gene is a pBCXUN vector containing the Ubi promoter.

[0022] The present invention also provides a method for preparing transgenic plants, which improves the expression level of the HSF21 gene through transgenic methods to obtain plants with enhanced cold resistance.

[0023] The specific method for preparing the transgenic plant includes the following steps:

[0024] (1) Amplify the full-length cDNA sequence of the HSF21 gene (as shown in SEQ ID NO.1);

[0025] (2) Construct an overexpression vector for the HSF21 gene;

[0026] (3) Construct recombinant Agrobacterium containing an overexpression vector of the HSF21 gene;

[0027] (4) Using Agrobacterium infection method, transgenic plants overexpressing HSF21 gene were constructed.

[0028] The plant described in this invention is a monocotyledonous or dicotyledonous plant, preferably rice, wheat, soybean, sorghum, millet, cotton, barley or corn, and more preferably corn.

[0029] The present invention also provides a method for altering the low-temperature resistance of plants, which controls the expression of the maize HSF21 gene in plants through transgenic, hybrid, backcross, self-pollination or asexual reproduction methods.

[0030] The transgenic process involves using DNA homologous recombination technology, Cre / Loxp technology, or Crispr / Gas9 technology to silence or reduce the expression level of the maize HSF21 gene, thereby obtaining transgenic plant lines.

[0031] The transgenic process also includes introducing a recombinant expression vector containing the maize HSF21 gene into maize using methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electrical conductivity, and Agrobacterium-mediated transformation to obtain transgenic maize lines.

[0032] As one embodiment of the present invention, the specific method for constructing a low-temperature resistant transgenic plant is as follows:

[0033] 1) Extract total RNA from maize, reverse transcribe to obtain cDNA, use cDNA as template to amplify the HSF21 gene with primers, construct the amplification product into the expression vector pBCXUN, and name the obtained recombinant expression vector pBCXUN-HSF21.

[0034] 2) Agrobacterium EHA105 was transformed with pBCXUN-HSF21, and then the transformed Agrobacterium was used to infect maize callus tissue to obtain transgenic maize seedlings resistant to low temperature.

[0035] The nucleotide sequences of the primers described in step 1) are shown in SEQ ID No. 3 and 4.

[0036] The infected maize plants are preferably those with the LH244 homozygous genotype. Overexpression of the HSF21 gene of this invention resulted in maize exhibiting a low-temperature resistant phenotype.

[0037] The expression vector is the pBCXUN vector, which is modified from the plasmid pCAMBIA1300 by inserting a hygromycin resistance gene into pCAMBIA1300.

[0038] This invention cloned the HSF21 gene, constructed transgenic plants overexpressing HSF21 and Crispr / Cas9 mutant materials, and verified that HSF21 participates in regulating maize's cold tolerance; overexpression of HSF21 enables maize to acquire stronger low-temperature tolerance. This invention provides new gene resources for breeding new cold-tolerant plant varieties and lays a theoretical foundation for studying the mechanism of maize's response to low-temperature stress. Attached Figure Description

[0039] Figure 1 The figure shows the results of HSF21 gene overexpression test in the WT group and maize overexpression lines in Example 2 of the present invention; in the figure, *** represents P<0.001.

[0040] Figure 2 These are photographs of the plant growth of the WT group and the maize overexpression lines after low-temperature treatment and recovery in Example 3 of this invention;

[0041] Figure 3 This is a statistical chart of ion leakage rates in the WT group and maize overexpression lines in Example 3 of the present invention.

[0042] Figure 4 This is a schematic diagram of two types of HSF21 gene knockout in Example 4 of the present invention;

[0043] Figure 5 These are photos of the plant growth of the WT group and the maize HSF21 gene knockout lines after low-temperature treatment recovery in Example 4 of this invention.

[0044] Figure 6 This is a statistical chart of ion leakage rate in the WT group and the maize HSF21 gene knockout line in Example 4 of the present invention. Detailed Implementation

[0045] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0046] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, 21), or as recommended by the manufacturer's instructions.

[0047] The main reagents used in the following examples were: various restriction endonucleases, Taq DNA polymerase, T4 ligase, Pyrobest Taq enzyme, and KOD were purchased from NEB, Toyobo, and other biotechnology companies; dNTPs were purchased from Genestar; plasmid miniprep kits and agarose gel extraction kits were purchased from Shanghai Jierui Biotechnology Co., Ltd.; agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), rifampin (Rif), and other antibiotics, as well as glucose, BSA, LBMedium, etc., were purchased from Sigma, Bio-Rad, and other companies; reagents used for real-time quantitative PCR were purchased from TaKaRa; and all other chemical reagents used in the examples were imported or domestically produced analytical grade reagents. Primers used in the examples were synthesized by Huada Biotechnology Co., Ltd., and related sequencing was performed.

[0048] Example 1: Construction and detection of HSF21 gene overexpression vector

[0049] Total RNA was extracted from B73 maize (Zea mays L.), and cDNA was obtained by reverse transcription. Using the cDNA as a template and F and R primers, the HSF21 gene was amplified. The primers contained restriction enzyme sites. After restriction enzyme digestion, the amplified DNA was ligated into an overexpression vector. The construction method of the HSF21 gene overexpression vector is as follows:

[0050] (1) Total RNA was extracted from B73 maize using the Magen RNA Extraction Kit. The specific steps were described in the kit instructions.

[0051] (2) Use the Thermo Reverse Transcription Kit to reverse transcribe RNA into cDNA. Refer to the kit instructions for specific steps.

[0052] (3) Using maize cDNA as a template and F and R as primers, amplify the cDNA of HSF21 (as shown in SEQ ID NO.1, whose encoded amino acid sequence is shown in SEQ ID NO.2). The amplified product is then electrophoresed and gel-cleaved for recovery. The recovery method is as described in the kit instructions of Tiangen Company.

[0053] The primers used to amplify the HSF21 gene cDNA are:

[0054] Upstream primer F: 5'-ATGGGAGAAGCGGCCGCGGC-3' (SEQ ID No. 3);

[0055] Downstream primer R: 5'-ATTCTCGCCGCCGCACCGGC-3' (SEQ ID No. 4).

[0056] (4) The recovered HSF21 gene cDNA and pBCXUN vector were double-digested with Xba I and Cla I, and the digestion products were recovered by electrophoresis and gel extraction. The recovered products were ligated using T4 ligase. The HSF21 gene was ligated into the pBCXUN vector to drive the expression of the HSF21 gene using the Ubi promoter.

[0057] The pBCXUN vector is obtained by linking a hygromycin resistance gene into the commercial vector pCAMBIA1300 as the backbone (Guo et al., 2018 Stepwise cis-regulatory changes in ZCN8 contribute to maize flowering-time adaptation. Current Bio. 28, 3005–3015); at the same time, the promoter of the maize ubiquitin gene Ubi is cloned into the vector through enzyme digestion and ligation to drive the transcription of downstream overexpressed genes.

[0058] (5) Take 5 μL of the product from the enzyme digestion-ligation system and transform it into competent E. coli cells. Screen on LB plates containing 50 μg / mL kanamycin. Identify single clones by colony PCR and select positive clones for sequencing. The recombinant expression vector with correct sequencing is named pBCXUN-HSF21. After digesting the plasmid obtained in the previous step, perform electrophoresis detection. The specific method is as follows: digest pBCXUN-HSF21 with XbaI and ClaI, electrophores with 1% agarose gel at 120V and 50mA, and then scan and image using a UVP GelDocumentation gel analysis system.

[0059] Example 2: Construction and detection of plants overexpressing the HSF21 gene

[0060] The pBCXUN vector containing the HSF21 gene described in Example 1 was transformed into Agrobacterium EHA105 strain (Ma et al., 2009, Enhanced tolerance to chilling stress in OsMYB3R-2 transgenic rice is mediated by alteration in cell cycle and ectopic expression of stress genes. Plant Physiol. 150, 244–256), and then infected with maize LH244 callus tissue to obtain transgenic seedlings. The specific method is as follows: Agrobacterium containing the target vector was inoculated into 100 mL of LB triple-antibiotic liquid culture medium (Kan 50 μg / mL, Rif 50 μg / mL, Gen 50 μg / mL), and cultured overnight at 28℃ with shaking. When the OD600 value reached 1.0-2.0, the cells were collected by centrifugation at 50 g for 15 min at room temperature. The cells were then resuspended in 2 mL of transformation solution (1 / 2 MS, 5% sucrose, 40 μL Silwet L-77). Corn callus tissue was immersed in the Agrobacterium transformation solution and sealed. The tissue was then placed back on a light-protected culture rack and allowed to grow normally until plants emerged. The resulting seeds were then screened and subjected to low-temperature stress treatment experiments.

[0061] In this embodiment, overexpression lines OE-1 and OE-2 with high expression levels were isolated. Specifically, real-time quantitative PCR was used to detect the HSF21 gene expression in the obtained overexpression lines OE-1 and OE-2. The specific method is as follows:

[0062] 1) Extract total RNA from plants and reverse transcribe it to obtain cDNA.

[0063] 2) After diluting the cDNA obtained from reverse transcription 5 times, perform real-time quantitative PCR using the Takara kit. The reaction system included: 2×SYBR Premix ExTaq buffer, 0.2 μL DyII, 0.4 μL Primer (F1 / R1), 2 μL cDNA template, and finally add ddH2O to a final volume of 20 μL. After thorough mixing, the mixture was placed in an ABIPRISM 75 real-time quantitative PCR instrument for two-step PCR amplification. The reaction conditions were: 95℃ for 30 s; 95℃ for 5 s; 60℃ for 40 s; 40 cycles.

[0064] The sequences of primers F1 and R1 (primers for qRT-PCR) are as follows:

[0065] F1: 5'-CTGACCAAGACGCACCAGAT-3' (SEQ ID No. 5);

[0066] R1: 5'-GGAGGAGAAGTTGCAGTGCT-3' (SEQ ID No. 6).

[0067] After the PCR reaction is completed, according to 2 -Δ(ΔCt) The principle was used to calculate and plot the relative expression levels between the wild-type (WT group) and overexpression lines (OE). Three biological replicates were performed, and the trends were similar across the three replicates. Simultaneously with the amplification of the identified genes, the UBI gene was amplified as an internal control for each sample. The test results are shown below. Figure 1 ,from Figure 1 The results showed that the expression level of the overexpression strain was significantly higher than that of the WT control group.

[0068] Example 3: Detection of low-temperature resistance in plants overexpressing the HSF21 gene

[0069] First, seeds from the WT group (wild-type maize) and OE-1 and OE-2 obtained in Example 2 were sown in small pots (10cm long, 10cm wide, and 10cm high) containing black soil, imported soil, and vermiculite (mass ratio 1:1:1). Five seeds were placed in each pot, covered with 2cm of soil, and placed on a tray. The pots were watered until the soil was completely moist and placed in a 23°C incubation room with 16 hours of light and 8 hours of darkness. After 14 days of growth, the plants were treated at 4°C for 4 days until the second leaf withered and shriveled. They were then removed and placed in a 23°C incubation room for two days to recover before photographing and collecting samples for ion leakage rate analysis. Three seedlings from each overexpression line (OE) and wild type (WT) were taken for testing, and three biological replicates were performed.

[0070] Representative photos of plant growth after low-temperature treatment recovery in the WT group and maize overexpression lines are shown below. Figure 2As shown (the left image is the control group without low-temperature treatment, and the right image is the experimental group after low-temperature treatment and recovery). The results showed that the wild-type WT leaves were severely wilted, dried out, and even unable to stand upright, while the overexpression lines OE-1 and OE-2 only had slight damage to the leaf tips and remained upright, showing a low-temperature resistant phenotype.

[0071] In this embodiment, the ion leakage rate was statistically analyzed by measuring the relative conductivity of the leaves, L = (S1-S0) / (S2-S0)*100%. After low-temperature treatment, an entire corn plant was placed in a 15ml centrifuge tube containing 10ml of distilled water. The tube was evacuated using a vacuum pump for 30 minutes, then placed on a shaker at room temperature for 1 hour. The initial conductivity value, S1, was then measured using a conductivity meter. The sample was then placed in a boiling water bath for 15 minutes, removed, and placed on a shaker for 2 hours. The conductivity was measured again and recorded as S2. S0 represents the conductivity of the blank control distilled water.

[0072] The results are as follows Figure 3 As shown in Table 1, compared with wild-type WT plants, the ion leakage rates of overexpression lines OE-1 and OE-2 were reduced by 41.6% and 41.9%, respectively (the average difference between the ion leakage rates of wild-type WT plants and overexpression lines in three trials), reaching a significant difference (P<0.05), indicating that overexpression of the HSF21 gene can enhance the cold resistance of maize.

[0073] Table 1. Ion leakage rate values ​​(%) from three independent experiments.

[0074] WT OE-1 OE-2 72.59425 31.93767 33.469953 79.60829 37.30582 37.022639 67.67797 25.72814 23.825957

[0075] Example 4: Detection of low-temperature resistance in plants with the HSF21 gene knocked out

[0076] To further investigate the regulatory role of HSF21 in maize cold tolerance, the HSF21 gene in wild-type maize LH244 was knocked out using CRISPR / Cas9 technology. Two mutant lines were obtained: hsf21-1 and hsf21-2. See the diagram for the knockout illustration. Figure 4 Among them, the hsf21-1 strain has a 43bp deletion from position 79 to position 121 of the first exon of the HSF21 gene, while the hsf21-2 strain has a 22bp deletion from position 2313 to position 2334 of the second exon of the HSF21 gene.

[0077] Low-temperature treatment was applied to the wild-type WT group and the gene knockout lines hsf21-1 and hsf21-2. The specific experimental method was as follows: seeds of the WT group (wild-type maize) and the gene knockout lines hsf21-1 and hsf21-2 were sown in small pots (10cm long, 10cm wide, and 10cm high) containing black soil, imported soil, and vermiculite (mass ratio 1:1:1). Five seeds were placed in each pot, covered with 2cm of soil, and placed on a tray. The pots were watered until the soil was completely moist and placed in a 23℃ incubator with 16 hours of light and 8 hours of darkness. After 14 days of growth, the plants were treated at 4℃ for 3 days, then moved back to a 23℃ incubator for two days to recover. Photos were taken and samples were collected for ion leakage rate analysis. Three seedlings from each knockout line and WT were collected for testing, and three biological replicates were performed.

[0078] Representative photos of plant growth after low-temperature treatment and recovery in the WT group and hsf21 gene knockout lines are shown below. Figure 5 As shown (the left image is the control group without low-temperature treatment, and the right image is the experimental group after low-temperature treatment and recovery). The results showed that the wild-type WT only had slight leaf tip damage and remained upright, while the hsf21 gene knockout strains had severely wilted, dried, curled, and even unable to stand upright, exhibiting a cold-sensitive phenotype.

[0079] In this embodiment, the ion leakage rate was statistically analyzed by measuring the relative conductivity of the leaves, L = (S1-S0) / (S2-S0)*100%. After low-temperature treatment, an entire corn plant was placed in a 15ml centrifuge tube containing 10ml of distilled water. The tube was evacuated using a vacuum pump for 30 minutes, then placed on a shaker at room temperature for 1 hour. The initial conductivity value, S1, was then measured using a conductivity meter. The sample was then placed in a boiling water bath for 15 minutes, removed, and placed on a shaker for 2 hours. The conductivity was measured again and recorded as S2. S0 represents the conductivity of the blank control distilled water.

[0080] Ion leakage rate reflects cell membrane integrity; test results are as follows: Figure 6 As shown in Table 2, compared with the WT group, the ion leakage rates of the knockout lines hsf21-1 and hsf21-2 increased by 46.7% and 47.8%, respectively (the average difference between the ion leakage rates of wild-type WT plants and knockout lines in three trials), reaching a statistically significant difference (P<0.05). This indicates that the degree of cell damage was significantly higher in the HSF21 group than in the WT group, and that knocking out the HSF21 gene weakens the cold tolerance of maize. This further demonstrates that the HSF21 knockout lines (hsf21-1 and hsf21-2) are sensitive to low temperatures.

[0081] Table 2. Ion leakage rate values ​​(%) from three independent experiments.

[0082] WT hsf21-1 hsf21-2 23.81285 66.25987 69.62581 27.60788 83.53274 80.20139 34.2886 76.07954 79.18962

[0083] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of maize HSF21 protein overexpression or its encoding gene, or biological material containing its encoding gene, in any of the following aspects: (1) Improve the cold resistance of plants; (2) Improve the survival rate of plants under low temperature conditions; (3) Select and breed transgenic plants with improved cold tolerance; (4) Improve cold-resistant plant germplasm resources; The plant is corn; the corn HSF21 protein has the amino acid sequence shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The amino acid sequence of the corn HSF21 protein is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The nucleotide sequence of the cDNA of the corn HSF21 protein is shown in SEQ ID NO.1, or in a nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID NO.

1.

4. The application according to claim 1, characterized in that, The biological material is an expression cassette, vector, host cell, or recombinant bacteria.

5. A method for altering the low-temperature resistance of plants, characterized in that, The expression of the maize HSF21 gene in plants is controlled by transgenic, hybrid, backcross, self-pollination, or asexual reproduction methods; the plant's low-temperature resistance is reduced by silencing or lowering the expression level of the maize HSF21 gene, or by overexpressing the maize HSF21 gene to improve the plant's low-temperature resistance; the plant is maize.

6. The method according to claim 5, characterized in that, The transgenic process involves using DNA homologous recombination technology, Cre / Loxp technology, or Crispr / Cas9 technology to silence or reduce the expression level of the maize HSF21 gene, thereby obtaining transgenic plant lines.

7. The method as described in claim 5, characterized in that, The transgenic process involves introducing a recombinant expression vector containing the maize HSF21 gene into maize using methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electrical conductivity, or Agrobacterium-mediated transformation to obtain transgenic maize lines.

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