Use of zmbhlh76 protein or its coding gene in regulating plant stress resistance

Abstract

The present application relates to the technical field of plant breeding, and particularly relates to application of ZmbHLH76 protein or a coding gene thereof in regulation of plant stress resistance. The ZmbHLH76 protein can also be applied to cultivation of high-stress-resistance or high-yield plants and improvement of plant stress resistance germplasm resources. The ZmbHLH76 protein comprises an amino acid sequence as shown in SEQ ID NO. 1. The present application screens a ZmbHLH76 protein closely related to plant stress resistance, and the stress resistance of plants can be effectively regulated by regulating the expression level of the protein in plants, thereby providing a new gene target and resource for cultivation of new high-stress-resistance plants, which has important application value in the field of plant breeding.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to the application of the ZmbHLH76 protein or its encoding gene in regulating plant stress resistance. Background Technology

[0002] Maize (Zea mays L.) belongs to the genus Zea in the family Poaceae and is an important food and forage crop with high yields. Maize is highly sensitive to cold, especially during its early autotrophic growth stages. Low temperatures inhibit the activity of C4 and Calvin cycle enzymes in maize photosynthesis, promoting dissipation mechanisms and affecting the antioxidant defense of maize leaves. Cold stress affects the development of chloroplasts and meristems, causing irreparable damage to later production. The physiological mechanisms of cold tolerance in maize remain largely unknown, but current research has identified cold tolerance QTLs in maize seedlings. Transgenic technology allows the introduction of endogenous or exogenous stress-resistance genes into the genetic material of maize varieties requiring improvement, resulting in offspring exhibiting stably inherited stress resistance. This may help clarify the mechanisms of cold tolerance. Although some studies on maize cold-resistance genes have been reported, developing genes with superior cold-resistance functions is of great significance for obtaining maize varieties with excellent cold-resistance phenotypes.

[0003] bHLH transcription factors are a class of proteins with broad biological functions. Their name comes from their unique basic helix-loop-helix (bHLH) domain, a structure that facilitates DNA binding and protein dimerization. bHLH transcription factors are one of the largest gene families shared by all three eukaryotic kingdoms and are widely distributed in plants, animals, and fungi. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides the application of the ZmbHLH76 protein or its encoding gene in regulating plant stress resistance.

[0005] This invention employs a preliminary screening of low-temperature phenotypes in a multi-gene overexpression maize population, using the relative leaf injury area as an indicator. Overexpression lines exhibiting low-temperature tolerance are then re-screened to determine their low-temperature-related phenotypes. The overexpressed genes of lines showing significant low-temperature tolerance are identified. Through this screening, this invention determines that the gene GRMZM2G112629 (ZmbHLH76) may be associated with maize's tolerance to low-temperature stress, and ZmbHLH76 belongs to the bHLH transcription factor family. Further experimental demonstrations show that the ZmbHLH76 protein negatively regulates plant cold resistance; reducing the expression level of the ZmbHLH76 protein in plants can improve their cold resistance.

[0006] In a first aspect, the present invention provides the application of the ZmbHLH76 protein, or its encoding gene, or biological materials containing its encoding gene in regulating plant stress resistance.

[0007] The present invention further provides the application of the ZmbHLH76 protein, or its encoding gene, or biological material containing its encoding gene in the cultivation of plants with high stress resistance and / or high yield.

[0008] The present invention further provides the application of ZmbHLH76 protein, or its encoding gene, or biological materials containing its encoding gene in the improvement of plant stress-resistant germplasm resources.

[0009] Furthermore, the stress resistance of the plant is improved by reducing the expression level of the ZmbHLH76 protein in the plant.

[0010] Furthermore, the ZmbHLH76 protein comprises any one of the following amino acid sequences:

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

[0012] (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.1.

[0013] Furthermore, the gene encoding the ZmbHLH76 protein includes any one of the following nucleotide sequences:

[0014] (1) The nucleotide sequence shown in SEQ ID NO.2;

[0015] (2) A nucleotide sequence that encodes a protein with the same function, obtained by substituting, deleting or inserting one or more nucleotides, as shown in SEQ ID NO.2;

[0016] (3) A nucleotide sequence that can hybridize with a nucleotide sequence such as SEQ ID NO.2 under strict conditions.

[0017] The nucleotide sequence shown in SEQ ID NO.2 is the cDNA sequence of the ZmbHLH76 protein in maize, consisting of 1288 bases. The gene reading frame consists of two exons. Considering codon degeneracy, all nucleotide sequences encoding the ZmbHLH76 protein are within the scope of protection of this invention.

[0018] Furthermore, the stress resistance refers to cold resistance; the plant is corn.

[0019] Furthermore, improving the plant's stress resistance includes its ability to resist or tolerate temperatures above or below freezing. Specifically, this can manifest as reducing the relative area of ​​leaf injury or decreasing the ion leakage rate of the leaves.

[0020] Furthermore, the biological material is an expression cassette, a vector, or a transgenic cell.

[0021] This invention also protects various vectors (including plasmids) and transgenic cells that include the gene encoding the ZmbHLH76 protein. The transgenic cells do not include plant cells capable of independently developing into complete plants, and are therefore not considered new plant varieties.

[0022] Furthermore, the carrier is a pCUN carrier.

[0023] Secondly, the present invention provides a method for regulating plant stress resistance, comprising:

[0024] Regulate the expression level of ZmbHLH76 protein in the plant;

[0025] The ZmbHLH76 protein comprises any one of the following amino acid sequences:

[0026] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0027] (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.1.

[0028] Furthermore, the gene encoding the ZmbHLH76 protein includes any one of the following nucleotide sequences:

[0029] (1) The nucleotide sequence shown in SEQ ID NO.2;

[0030] (2) A nucleotide sequence that encodes a protein with the same function, obtained by substituting, deleting or inserting one or more nucleotides, as shown in SEQ ID NO.2;

[0031] (3) A nucleotide sequence that can hybridize with a nucleotide sequence such as SEQ ID NO.2 under strict conditions.

[0032] Furthermore, the expression level of the ZmbHLH76 protein in the plant can be regulated using any of the following methods:

[0033] Genetically modified organisms, hybridization, backcrossing, self-pollination, or asexual reproduction;

[0034] The preferred methods for transgenic technology include one or more of the following: Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated transformation.

[0035] Furthermore, the expression level of the ZmbHLH76 protein in the plant can be increased by constructing the gene encoding the ZmbHLH76 protein into an expression vector and introducing it into the plant (based on any of the transgenic methods described above).

[0036] Furthermore, the aforementioned resistance is cold resistance.

[0037] Furthermore, the plant in question is corn.

[0038] The present invention has the following beneficial effects:

[0039] This invention identified a protein related to plant stress resistance—ZmbHLH76—through phenotypic screening in a multi-gene overexpression maize population. Overexpression of this protein in plants significantly reduced their low-temperature resistance. The discovery of this protein's function provides a new gene target and resource for breeding cold-resistant plant varieties, and is of great significance for studying the molecular mechanisms of cold tolerance in maize. It also lays a theoretical foundation for researching the mechanisms by which plants respond to low-temperature stress and the molecular mechanisms by which they resist adverse environments. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 The results show the expression level detection of the ZmbHLH76 gene in the overexpression lines OE#5 and OE#6 provided in Example 2 of this invention, where CK represents wild-type maize plants, and OE#5 and OE#6 represent overexpression lines, respectively.

[0042] Figure 2 This is the plant growth of the overexpression lines OE#5 and OE#6 provided in Example 3 of the present invention after recovery from low temperature treatment; wherein, CK represents wild-type maize plants, and OE#5 and OE#6 represent two overexpression lines respectively.

[0043] Figure 3 This is a statistical chart showing the relative leaf injury area of ​​the overexpression lines OE#5 and OE#6 provided in Example 3 of the present invention; where CK represents wild-type maize plants, and OE#5 and OE#6 represent two overexpression lines respectively.

[0044] Figure 4 This is a statistical chart showing the ion leakage rate results of the overexpression lines OE#5 and OE#6 provided in Example 3 of the present invention; where CK represents wild-type maize plants, and OE#5 and OE#6 represent two overexpression lines respectively. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0046] Unless otherwise specified, the experimental methods involved in the following examples are conventional methods in the art, such as referring to Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 21), or following the conditions recommended in the manufacturer's instructions.

[0047] Unless otherwise specified, all experimental reagents used in the following examples are commercially available, such as:

[0048] pBSK vectors are commonly used cloning vectors and can be purchased commercially.

[0049] The pCUN vector was obtained by inserting a hygromycin resistance gene between the restriction sites of pCAMBIA1300 (Guo et al., 2018 Stepwise cis-regulatory changes in ZCN8 contribute to maize fowering-time adaptation. Current Bio. 28, 3005–3015);

[0050] Agrobacterium strain EHA105 was kindly provided by the Crop Functional Genomics Platform of the College of Biological Sciences, China Agricultural University, and is commercially available (Ma et al., 2009, Enhanced tolerance to chilling stress in OsMYB3R-2 transgenic rice is mediated by alteration in cell cycle and ectopicexpression of stress genes. Plant Physiol. 150, 244–256).

[0051] In the following examples, 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, LB medium, 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 following examples were imported or domestically produced analytical grade reagents.

[0052] The primers used in the following examples were synthesized by BGI Genomics and sequenced.

[0053] Example 1

[0054] This embodiment constructs and identifies the ZmbHLH76 gene overexpression vector, specifically including the following steps:

[0055] This invention identified a ZmbHLH76 overexpression line with significant cold tolerance by screening for cold-tolerant maize lines in a maize library of transgenic overexpression lines. Six transformation events of ZmbHLH76 were observed in the transgenic overexpression maize library. Real-time quantitative PCR identification revealed that two of these lines showed significant upregulation of the ZmbHLH76 gene expression. These two lines did not exhibit obvious growth and developmental phenotypes, but both showed significant cold sensitivity.

[0056] This invention analyzes the coding region sequence of the maize ZmbHLH76 gene, designs primers F and R based on the coding region sequence, amplifies the coding region of this gene, and ligates it into the overexpression vector pCUN with a 35S promoter. The specific primer sequences are as follows:

[0057] Upstream primer F: 5'-ATGAAGAGCCGGAGGCAGAGC-3' (Seq ID NO.3);

[0058] Downstream primer R: 5'-GACGAGGATCGTCGGAGCAAGC-3' (Seq ID NO.4).

[0059] The specific method for ligating the ZmbHLH76 gene into the pCUN vector with a 35S promoter is as follows: First, using cDNA as a template, ZmbHLH76 is amplified using upstream primer F and downstream primer R. The PCR product is then ligated into the pBSK vector, and the ligation product is named ZmbHLH76-pBSK. ZmbHLH76 is then digested with SalI and KpnI and recovered, and ligated into the pCUN vector. The ligation product is named 35S:ZmbHLH76.

[0060] After digestion with enzymes, the obtained plasmid was analyzed by electrophoresis. Specifically, 35S ZmbHLH76 was digested with SalI and KpnI, followed by electrophoresis on a 1% agarose gel at 120V and 50mA, and then imaged using a UVP Gel Documentation system. The results showed that the pCUN overexpression vector of ZmbHLH76 was successfully constructed.

[0061] Example 2

[0062] This embodiment describes the construction and identification of maize overexpressing the ZmbHLH76 gene, including the following procedures:

[0063] The pCUN vector (35S: ZmbHLH76) containing the ZmbHLH76 gene, constructed in Example 1, was transformed into Agrobacterium EHA105 strain, and then infected maize callus tissue to obtain transgenic seedlings. Specifically, the 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°C with shaking until OD was reached. 600 The bacterial cells were collected by centrifugation at 50×g for 15 min at room temperature, with a value of 1.0-2.0. The cells were then resuspended in 2 mL of transformation buffer (1 / 2 MS, 5% sucrose, 40 μL Silwet L-77). Corn callus tissue was immersed in the Agrobacterium transformation buffer and sealed. The tissue was then returned to a light-treated culture rack and allowed to grow normally until plants emerged. The resulting seeds were then screened and subjected to low-temperature stress treatment experiments.

[0064] In this embodiment, overexpression lines OE#5 and OE#6 were isolated. The gene expression of ZmbHLH76 in the obtained overexpression lines OE#5 and OE#6 was detected by real-time quantitative PCR. The specific method is as follows:

[0065] (1) Extract total RNA from maize and reverse transcribe it to obtain cDNA.

[0066] (2) After diluting the cDNA obtained from reverse transcription 5-fold, real-time quantitative PCR was performed using a Takara kit. The reaction system was: 2×SYBR Premix ExTaq buffer, 0.2 μL DyII, 0.4 μL Primer (F / R), 2 μL cDNA template, and finally, ddH2O was added 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. While amplifying the identified gene, UBI was used as an internal control for each sample for simultaneous amplification. After the PCR reaction was completed, the results were processed according to step 2. -Δ(ΔCt) The relative expression levels between wild-type and overexpression lines were calculated and plotted based on the principle.

[0067] The results are as follows Figure 1 As shown, the results indicate that the ZmbHLH76 gene was upregulated by more than 200-fold in OE#5 and OE#6, respectively.

[0068] Example 3

[0069] This embodiment tests the low-temperature resistance of maize overexpressing the ZmbHLH76 gene, and includes the following procedure:

[0070] This invention first involves sowing seeds of wild-type maize (control group CK) and overexpression lines OE#5 and OE#6 in small pots (10cm long, 10cm wide, and 10cm high) filled with black soil, imported soil, and vermiculite (1:1:1). Five seeds are placed in each pot, covered with 2cm of soil, and placed on a tray. The pots are watered until the soil is completely moist and then placed in a 23℃ incubator with 16 hours of light and 8 hours of darkness. After 14 days of growth, the seeds are treated at 4℃ until the second leaf wrinkles and wilts. They are then removed and placed in a 23℃ incubator for two days to recover before photographing and collecting samples for statistical analysis of the relative damaged area and ion leakage rate of the leaves. In each experiment, 3-5 seedlings are used, and each independent experiment is repeated three times.

[0071] Phenotypic characteristics of wild-type plants and overexpression lines OE#5 and OE#6 after recovery from low-temperature treatment are as follows: Figure 2 As shown, compared with wild-type maize, the overexpression lines OE#5 and OE#6 exhibited significantly enhanced leaf wilting, indicating that both overexpression lines OE#5 and OE#6 showed a cold-sensitive phenotype.

[0072] In this embodiment, the statistical method for the relative damaged area of ​​the leaf is as follows: After cold treatment, the corn leaf is glued to an A4 sheet of paper, then photographed. The photograph is processed in ImageJ software. A ruler is set, and the damaged area of ​​the leaf is circled and measured, recorded as A1. The entire circumference of the leaf is circled and measured, recorded as A2. The ratio of the relative damaged area is then calculated as A1 / A2. The results are as follows: Figure 3 As shown, compared with wild-type plants (50%), the relative leaf injury area of ​​overexpression lines OE#5 and OE#6 exceeded 80%, which was significantly higher than that of wild-type plants. This indicates that overexpression of the ZmbHLH76 gene can weaken the frost resistance of maize.

[0073] In this embodiment, the ion leakage rate was statistically analyzed by detecting the relative conductivity of the leaves, L = (S1 - S0) / (S2 - S0). 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. The results are as follows: Figure 4 As shown, compared with wild-type plants (40%), the ion leakage rates of overexpression lines OE#5 and OE#6 were 80% and 79%, respectively, which were significantly higher than those of wild-type plants. This indicates that overexpression of the ZmbHLH76 gene can weaken the frost resistance of maize.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the ZmbHLH76 protein, or its encoding gene, or biological material containing its encoding gene, in regulating plant cold resistance, characterized in that... By increasing the expression level of the ZmbHLH76 protein in the plant, the cold resistance of the plant was reduced; The amino acid sequence of the ZmbHLH76 protein is shown in SEQ ID NO.1; The plant in question is corn.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the ZmbHLH76 protein is shown in SEQ ID NO.

2.

3. The application according to claim 1 or 2, characterized in that, The biological material is an expression cassette, vector, or transgenic cell.

4. A method for reducing the cold resistance of corn, characterized in that, include: Increase the expression level of ZmbHLH76 protein in the maize; The amino acid sequence of the ZmbHLH76 protein is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that, Increase the expression level of ZmbHLH76 protein in maize using any of the following methods: Genetically modified, hybridized, backcrossed, or self-crossed; The transgenic material includes one or more of the following methods: Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated transformation.

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