Application of zmbzip61 protein or its coding gene in regulating low temperature stress tolerance of maize

Abstract

The present application relates to the technical field of plant breeding, and particularly relates to application of ZmbZIP61 protein or its coding gene in regulating low-temperature stress tolerance of corn. The present application finds that the ZmbZIP61 protein is related to the low-temperature stress tolerance of plants, and the low-temperature stress tolerance of plants can be effectively improved by increasing the expression level of the ZmbZIP61 protein in the plants. The ZmbZIP61 protein and its application provided by the present application can be used for cold-resistant plant breeding, and a new gene target and resource are provided. The present application not only has important significance for the study of the cold tolerance molecular mechanism of corn, but also has important 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 ZmbZIP61 protein or its encoding gene in regulating maize's tolerance to low-temperature stress. 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 quite sensitive to cold during its growth, especially in the early autotrophic growth stages. Under low temperatures, the activity of C4 and Calvin cycle enzymes in maize photosynthesis is inhibited, promoting dissipation mechanisms and affecting the antioxidant defense of maize leaves. Furthermore, cold stress can also affect the development of chloroplasts and meristems, causing irreparable damage to later production.

[0003] Currently, existing technologies for understanding the physiological mechanisms related to cold tolerance in maize are not yet fully mature, but some cold-tolerant QTLs in maize seedlings have been discovered. Transgenic technology allows the introduction of endogenous or exogenous stress-resistance genes into the genetic material of maize varieties requiring improvement, enabling their offspring to exhibit stably inherited stress resistance. This may help to ultimately elucidate the mechanisms of cold tolerance. While some studies on maize cold-tolerant genes have been reported, developing more genes with superior cold-resistance functions is crucial for obtaining maize varieties with excellent cold-tolerant phenotypes.

[0004] In plants, basic region / leucine zipper motif (bZIP) transcription factors play important roles in multiple biological processes and have been reported to regulate processes such as pathogen defense, light and stress signal transduction, seed maturation, and flower development. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides the application of ZmbZIP61 protein or its encoding gene in regulating the ability of maize to tolerate low temperature stress.

[0006] In this invention, a preliminary screening of low-temperature phenotypes is conducted in a multi-gene overexpression maize population using the relative leaf injury area as an indicator. Overexpression lines with low-temperature tolerance obtained from the initial screening are then re-screened to determine their low-temperature-related phenotypes. For the lines with significant low-temperature tolerance obtained from the screening, their overexpressed genes are identified.

[0007] Through the above screening process, the target gene GRMZM2G137046 (ZmbZIP61) identified in this invention may be related to maize's tolerance to low-temperature stress. Further experimental results demonstrate that ZmbZIP61 can positively regulate plant cold resistance; increasing the expression level of ZmbZIP61 protein in plants can significantly improve plant cold resistance.

[0008] In a first aspect, the present invention provides the ZmbZIP61 protein, or its encoding gene, or the use of the gene containing the encoding gene in regulating the ability of maize to tolerate low-temperature stress.

[0009] The ability to withstand low-temperature stress described in this invention can also be understood as cold resistance, that is, the ability to withstand temperatures above or below zero, specifically including reducing ion leakage rate or reducing leaf damage rate.

[0010] The present invention further provides the application of ZmbZIP61 protein, or its encoding gene, or biological materials containing its encoding gene in the cultivation of plants with high cold resistance and / or high growth performance.

[0011] The present invention further provides the application of ZmbZIP61 protein, or its encoding gene, or biological materials containing its encoding gene in the improvement of plant cold-resistant germplasm resources.

[0012] Furthermore, by increasing the expression level of the ZmbZIP61 protein in the plant, the plant's tolerance to low-temperature stress is improved.

[0013] Furthermore, cold-resistant strains were bred by hybridizing ZmbZIP61 protein overexpression lines with other lines.

[0014] Furthermore, the ZmbZIP61 protein comprises any one of the following amino acid sequences:

[0015] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0016] (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.

[0017] The amino acid sequence shown in SEQ ID NO.1 is the amino acid sequence of the ZmbZIP61 protein from maize. Those skilled in the art can obtain mutants of the ZmbZIP61 protein with the same function as the amino acid sequence shown in SEQ ID NO.1 by substituting, deleting and / or adding one or more amino acids, without affecting its activity, based on the amino acid sequence shown in SEQ ID NO.1 and conventional techniques in the art such as the conservation of amino acids.

[0018] Furthermore, the gene encoding the ZmbZIP61 protein includes any one of the following nucleotide sequences:

[0019] (1) The nucleotide sequence shown in SEQ ID NO.2;

[0020] (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;

[0021] (3) A nucleotide sequence that can hybridize with a nucleotide sequence such as SEQ ID NO.2 under strict conditions.

[0022] The nucleotide sequence shown in SEQ ID NO.2 is the cDNA sequence of the ZmbZIP61 protein in maize, consisting of 988 bases. The reading frame of this gene is from position 105 to position 803 at the 5' end. This reading frame consists of 4 exons and 3 introns. Considering codon degeneracy, all nucleotide sequences encoding the ZmbZIP61 protein are within the scope of protection of this invention.

[0023] Furthermore, the plant is a monocotyledonous or dicotyledonous plant, such as corn, rice, wheat, cotton or soybean, preferably corn.

[0024] Furthermore, the biological material is an expression cassette, a vector, or a transgenic cell.

[0025] This invention also protects various vectors (including plasmids) and transgenic cells that include the gene encoding the ZmbZIP61 protein. The transgenic cells do not include plant cells capable of independently developing into complete plants, and are therefore not considered new plant varieties.

[0026] Secondly, the present invention provides a method for cultivating cold-resistant transgenic plants, comprising:

[0027] Regulate the expression level of ZmbZIP61 protein in the aforementioned plants;

[0028] The ZmbZIP61 protein comprises any one of the following amino acid sequences:

[0029] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0030] (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.

[0031] Furthermore, the expression level of ZmbZIP61 protein in the plant can be regulated using any of the following methods:

[0032] Genetically modified organisms, hybridization, backcrossing, self-pollination, or asexual reproduction;

[0033] The preferred methods for transgenic technology include: Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated transformation.

[0034] Furthermore, by increasing the expression level of ZmbZIP61 protein in the plant, the cold resistance of the plant is improved.

[0035] Furthermore, by introducing an overexpression vector containing the encoding gene of the ZmbZIP61 protein into the plant through the transgenic method, the expression level of the ZmbZIP61 protein in the plant is increased.

[0036] As a preferred embodiment, the present invention provides a method for breeding cold-resistant transgenic maize, comprising:

[0037] (1) Extract total RNA from maize, reverse transcribe to obtain cDNA, use cDNA as template and the sequence shown in SEQ ID NO.3-4 as primer to amplify the CDS sequence of the ZmbZIP61 gene, and ligate the amplification product to the plant expression vector pCUN to obtain the recombinant expression vector.

[0038] (2) Agrobacterium was transformed using the recombinant expression vector obtained in step (1) to obtain recombinant Agrobacterium;

[0039] (3) The recombinant Agrobacterium obtained in step (2) was used to infect maize callus tissue, and positive transgenic plants were screened to obtain cold-resistant transgenic maize.

[0040] The present invention has the following beneficial effects:

[0041] This invention has discovered that the ZmbZIP61 protein can positively regulate plant cold resistance. By increasing the expression level of ZmbZIP61 protein, the ability of plants to tolerate low-temperature stress can be effectively improved. Based on this, this invention constructed a transgenic maize line overexpressing the ZmbZIP61 gene. Compared with wild-type maize, this plant showed significantly improved cold resistance.

[0042] The application of the ZmbZIP61 protein provided by this invention in regulating the ability of plants to tolerate low temperature stress is of great significance in the field of plant breeding. It can be used to cultivate cold-resistant plant varieties and effectively increase plant yield. Attached Figure Description

[0043] 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.

[0044] Figure 1 The results show the expression level detection of the ZmbZIP61 gene in the overexpression lines OE-11 and OE-12 provided in Example 2 of this invention, where CK represents wild-type maize plants, and OE-11 and OE-12 represent the overexpression lines OE-11 and OE-12, respectively.

[0045] Figure 2 This is a diagram showing the growth of wild-type maize plants, overexpression lines OE-11 and OE-12 after low-temperature treatment, as provided in Example 3 of this invention; where CK represents wild-type maize plants, and OE-11 and OE-12 represent overexpression lines OE-11 and OE-12, respectively.

[0046] Figure 3 This is a statistical chart showing the ion leakage rate results of wild-type plants, overexpression lines OE-11 and OE-12 in Example 3 of the present invention; where CK represents wild-type maize plants, and OE-11 and OE-12 represent overexpression lines OE-11 and OE-12, respectively. Detailed Implementation

[0047] 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.

[0048] Unless otherwise specified, the experimental methods involved in the following examples are conventional methods in the art. For example, you can refer to Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 21) or follow the conditions recommended in the manufacturer's instructions.

[0049] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available, for example:

[0050] In the following examples, the pBSK vector is a commonly used cloning vector that can be obtained through commercial purchase.

[0051] 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).

[0052] 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).

[0053] 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.

[0054] All other chemical reagents used in the following examples were imported or domestically produced analytical grade reagents. The primers used in the following examples were synthesized by Huada Pharmaceutical Co., Ltd. and subjected to relevant sequencing.

[0055] Example 1

[0056] This embodiment constructs and identifies the ZmbZIP61 gene overexpression vector, specifically including the following steps:

[0057] This invention identified a ZmbZIP61 overexpression line with significant cold tolerance by screening for cold-tolerant maize lines in a maize library of transgenic overexpression lines. Four transformation events of ZmbZIP61 were observed in the transgenic overexpression maize library. Real-time quantitative PCR identification revealed that two of these lines showed significant upregulation of ZmbZIP61 gene expression. These two lines did not exhibit obvious growth and developmental phenotypes, but both showed significant cold tolerance.

[0058] The present invention further analyzes the coding region sequence of the maize ZmbZIP61 gene, designs primers F and R based on the coding region sequence, amplifies the coding region of the gene, and ligates it into the overexpression vector pCUN with a 35S promoter (this vector is obtained by ligating a hygromycin resistance gene between the SalI and Kpn restriction sites of pCAMBIA1300).

[0059] The primer sequences used are as follows:

[0060] Upstream primer F: 5'-ATGCAGGAGCAGGCGGCGAG-3' (SEQ ID NO.3);

[0061] Downstream primer R: 5'-TTGGCCGTGGCCCTCTCCAC-3' (SEQ ID NO.4).

[0062] The specific method for ligating the ZmbZIP61 gene into the pCUN vector with a 35S promoter is as follows: First, using cDNA as a template, ZmbZIP61 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 ZmbZIP61-pBSK. ZmbZIP61 is then digested with SalI and KpnI and recovered, and ligated into the pCUN vector. The ligation product is named 35S:ZmbZIP61.

[0063] After digestion with enzymes, the obtained plasmid was analyzed by electrophoresis. Specifically, 35S:ZmbZIP61 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 ZmbZIP61 was successfully constructed.

[0064] Example 2

[0065] This embodiment constructs and identifies maize overexpressing the ZmbZIP61 gene, specifically including the following steps:

[0066] The pCUN vector (35S:ZmbZIP61) containing the ZmbZIP61 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... 600 When the value is 1.0-2.0, the bacterial cells are collected by centrifugation at 50×g for 15 min at room temperature; the bacterial cells are resuspended in 2 mL of transformation solution (1 / 2 MS, 5% sucrose, 40 μL Silwet L-77); the corn callus tissue is immersed in the Agrobacterium transformation solution and sealed. It is then placed back on a light-cured culture rack for normal growth until plants emerge. The resulting seeds are then screened and subjected to low-temperature stress treatment experiments.

[0067] In this embodiment, overexpression lines OE-11 and OE-12 were isolated. The gene expression level of ZmbZIP61 in the obtained overexpression lines OE-11 and OE-12 was detected by real-time quantitative PCR. The specific method is as follows:

[0068] (1) Extract total RNA from maize and reverse transcribe it to obtain cDNA.

[0069] (2) After diluting the cDNA obtained by reverse transcription by 5 times, real-time quantitative PCR was performed using the Takara kit. The reaction system used was: 2×SYBR Premix ExTaq buffer, 0.2μL LdyII, 0.4μL Primer (F / R), 2μL cDNA template, and finally ddH2O to make up to 20μL.

[0070] After thorough mixing, the samples were placed in an ABIPRISM 75 real-time quantitative PCR instrument for two-step PCR amplification. The reaction conditions were: 95℃ for 30 s; 5℃ for 5 s, 60℃ for 40 s, for 40 cycles. Simultaneously with the amplification of the identified gene, each sample was amplified using UBI as an internal control. After the PCR reaction was completed, the results were analyzed according to step 2. -Δ(ΔCt) The relative expression levels between wild-type and overexpression lines were calculated based on the principle of [previous method] and plotted for analysis. The results are as follows: Figure 1 As shown, the results indicate that the ZmbZIP61 gene was upregulated by approximately 3-fold and 7-fold in OE-11 and OE-12, respectively.

[0071] Example 3

[0072] This embodiment verifies the low-temperature resistance of maize lines overexpressing the ZmbZIP61 gene, including the following procedures:

[0073] 1. In this invention, seeds of wild-type maize (control group CK) and overexpression lines OE-11 and OE-12 were sown in small pots (10cm long, 10cm wide, and 10cm high) containing black soil, imported soil, and vermiculite (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 then 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℃ until the second leaf withered and shriveled. They were then removed and placed in a 23℃ incubator for two days to recover before being photographed and samples were collected for ion leakage rate analysis. In each experiment, 3-5 seedlings were used, and each independent experiment was repeated three times.

[0074] Phenotypic characteristics of wild-type plants and overexpression lines OE-11 and OE-12 after recovery from low-temperature treatment are as follows: Figure 2 As shown, compared with the control, the wilting of leaves in the overexpression lines OE-11 and OE-12 was significantly reduced, indicating that both overexpression lines OE-11 and OE-12 exhibited a low-temperature resistant phenotype.

[0075] 2. 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.

[0076] The results are as follows Figure 3 As shown, compared with wild-type plants (70%), the ion leakage rates of overexpression lines OE-11 and OE-12 were 35% and 25%, respectively, which were significantly lower than those of wild-type plants, indicating that overexpression of the ZmbZIP61 gene can enhance the frost resistance of maize.

[0077] 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 ZmbZIP61 protein, or its encoding gene, or biological material containing its encoding gene in regulating the ability of maize to tolerate low-temperature stress; wherein the application is: to improve the ability of maize to tolerate low-temperature stress by increasing the expression level of the ZmbZIP61 protein in the maize. The ZmbZIP61 protein has the amino acid sequence shown in SEQ ID NO.

1.

2. Application of ZmbZIP61 protein, or its encoding gene, or biological materials containing its encoding gene in the breeding of highly cold-resistant maize; The ZmbZIP61 protein has the amino acid sequence shown in SEQ ID NO.

1.

3. Application of ZmbZIP61 protein, or its encoding gene, or biological materials containing its encoding gene in the improvement of cold-resistant maize germplasm resources; The ZmbZIP61 protein has the amino acid sequence shown in SEQ ID NO.

1.

4. The application according to any one of claims 1-3, characterized in that, The gene encoding the ZmbZIP61 protein has the nucleotide sequence shown in SEQ ID NO.

2.

5. The application according to any one of claims 1-3, characterized in that, The biological material is an expression cassette, vector, or transgenic cell.

6. A method for cultivating cold-resistant transgenic plants, characterized in that, include: Increase the expression level of ZmbZIP61 protein in the plant; The ZmbZIP61 protein has the amino acid sequence shown in SEQ ID NO.1; The plant in question is corn.

7. The method according to claim 6, characterized in that, Increase the expression level of ZmbZIP61 protein in the plant 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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