Application of ZmBX11 protein or coding gene thereof in regulating and controlling low-temperature stress tolerance of corn

Abstract

The invention relates to an application of a ZmBX11 protein or a coding gene thereof in regulating and controlling the low-temperature stress tolerance of corn. By improving the expression level of the ZmBX11 protein in the plant, the low-temperature stress tolerance of the plant can be effectively improved. The ZmBX11 protein and the application thereof provided by the invention can be used for breeding cold-resistant plants, and new gene targets and resources are provided. The method not only has important significance on research on a cold-resistant 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, specifically to the application of ZmBX11 protein or its encoding gene in regulating maize's ability to tolerate 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 enzymes and enzymes in the Calvin cycle during 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. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an application of the ZmBX11 protein or its encoding gene in regulating the tolerance of maize to low-temperature stress. This invention has found a correlation between the ZmBX11 protein and the plant's tolerance to low-temperature stress; increasing the expression level of ZmBX11 protein in plants can effectively improve their tolerance to low-temperature stress. The ZmBX11 protein and its application provided by this invention can be used for breeding cold-resistant plants, offering a new gene target and resource. This is not only significant for the study of the molecular mechanisms of cold tolerance in maize but also of great value in the field of plant breeding.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The use of ZmBX11 protein, or its encoding gene, or biological material containing its encoding gene in any of the following: P1) Application in improving the ability of plants to tolerate low temperature stress; P2) Application in cultivating plants with high cold resistance and / or high growth performance; P3) Application in the improvement of plant cold-resistant germplasm resources; The application is achieved by increasing the expression level of the ZmBX11 protein in plants; The ZmBX11 protein includes any one of the following amino acid sequences. (1) The amino acid sequence as shown in SEQ ID NO.1; the ZmBX11 protein consists of the amino acid sequence as shown in SEQ ID NO.2. ZmBX11 Gene encoding.

[0006] (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids as shown in SEQ ID NO.1.

[0007] The gene encoding the ZmBX11 protein includes any one of the following nucleotide sequences: (1) The nucleotide sequence as shown in SEQ ID NO.2; (2) A nucleotide sequence that encodes a protein with the same function, obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.2; (3) A nucleotide sequence that can hybridize with a nucleotide sequence as shown in SEQ ID NO.2 under strict conditions.

[0008] The plant is a monocotyledonous or dicotyledonous plant, preferably corn.

[0009] Based on the above scheme, the biological material is an expression cassette, vector, or transgenic cell containing the nucleic acid molecule shown in SEQ ID NO.2.

[0010] A method for cultivating cold-resistant transgenic plants, characterized in that the method is achieved by increasing the expression level of ZmBX11 protein in the plant; The amino acid sequence of the ZmBX11 protein includes any one of the following amino acid sequences: (1) The amino acid sequence as shown in SEQ ID NO.1; (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids as shown in SEQ ID NO.1.

[0011] Preferably, the expression level of ZmBX11 protein in the plant is regulated by any of the following methods: Genetically modified organisms, hybridization, backcrossing, self-pollination, or asexual reproduction; The preferred methods for transgenic technology include: Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated transformation.

[0012] The beneficial effects of the application of the ZmBX11 protein or its encoding gene in regulating the ability of maize to tolerate low-temperature stress are as follows: This invention has discovered that the ZmBX11 protein can positively regulate plant cold resistance. By increasing the expression level of ZmBX11 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 ZmBX11 gene. Compared with wild-type maize, this plant showed significantly improved cold resistance.

[0013] The application of the ZmBX11 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

[0014] The present invention includes the following figures: Figure 1 For the overexpression lines #1 and #2 provided in Example 2 of this invention ZmBX11 Gene expression level detection results, where WT represents wild-type maize plants, and #1 and #2 represent... ZmBX11 Overexpression lines #1 and #2.

[0015] Figure 2 This is a diagram showing the growth of wild-type maize plants, overexpression lines #1 and #2 after low-temperature treatment, as provided in Example 3 of this invention; where WT represents wild-type maize plants, and #1 and #2 represent overexpression lines #1 and #2, respectively.

[0016] Figure 3 This is a statistical chart showing the ion leakage rate results of wild-type plants and overexpression lines #1 and #2 in Example 3 of the present invention; where WT represents wild-type maize plants, and #1 and #2 represent overexpression lines #1 and #2, respectively. Detailed Implementation

[0017] 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.

[0018] 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: A Laboratory Manual (Sambrook J & Russell DW, 21) or follow the conditions recommended in the manufacturer's instructions.

[0019] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available, for example: In the following examples, the pBSK vector is a commonly used cloning vector that can be obtained through commercial purchase.

[0020] 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 maizefowering-time adaptation. Current Bio. 28, 3005–3015).

[0021] Agrobacterium strain EHA105 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 ectopic expression of stress genes. Plant Physiol. 150, 244–256).

[0022] The primers used in the following examples were synthesized by a third-party company and subjected to relevant sequencing. Example 1 This embodiment constructs and identifies the ZmBX11 gene overexpression vector, specifically including the following steps: This invention, through screening a maize line with a cold-resistant phenotype in a maize library of transgenic overexpression lines, discovered a maize line with a significantly cold-resistant phenotype. ZmBX11 Overexpression lines. In the maize library of transgenic overexpression lines, ZmBX11 There were a total of 4 transformation events. Real-time quantitative PCR identification revealed that 2 of the lines underwent transformation. ZmBX11 The gene expression levels were significantly upregulated. These two lines did not show obvious growth and development phenotypes, but both showed obvious cold tolerance phenotypes.

[0023] This invention further relates to corn ZmBX11 The gene coding region sequence was analyzed, and primers F and R were designed based on the coding region sequence to amplify the coding region of the gene and ligate it into the overexpression vector pCUN with a 35S promoter (this vector was obtained by ligating the hygromycin resistance gene between the SalI and KpnI restriction sites of pCAMBIA1300).

[0024] The primer sequences used are as follows: Upstream primer F: 5'-ATGGCACTCATCATGCAGGA-3' (SEQ ID NO.3); Downstream primer R: 5'-AGGATAGACCTCAGTAGTAG-3' (SEQ ID NO.4).

[0025] The specific method for ligating the ZmBX11 gene into the pCUN vector with a 35S promoter is as follows: First, using cDNA as a template, ZmBX11 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 ZmBX11-pBSK. ZmBX11 is then digested with SalI and KpnI and recovered, and ligated into the pCUN vector. The ligation product is named 35S: ZmBX11.

[0026] The obtained plasmid was digested with enzymes and then subjected to electrophoresis. Specifically, 35S:ZmBX11 was digested with SalI and KpnI, followed by electrophoresis on a 1% agarose gel at 120 V and 50 mA, and then imaged using a UVP Gel Documentation system. The results showed that the pCUN overexpression vector of ZmBX11 was successfully constructed.

[0027] Example 2 This embodiment constructs and identifies maize overexpressing the ZmBX11 gene, specifically including the following steps: The pCUN vector (35S: ZmBX11) containing the ZmBX11 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. 600When 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 and allowed to grow normally until plants emerge. The resulting seeds are then screened for low-temperature stress treatment experiments.

[0028] In this embodiment, overexpression lines #1 and #2 were isolated. The gene expression level of ZmBX11 in the obtained overexpression lines #1 and #2 was detected by real-time quantitative PCR. The specific method is as follows: (1) Extract total RNA from maize and reverse transcribe it to obtain cDNA.

[0029] (2) After diluting the cDNA obtained by reverse transcription by 2 times, perform real-time quantitative PCR using the Takara kit. The reaction system used is: 2×SYBR Premix ExTaq buffer, 0.2μL DyII, 0.4μL Primer (F / R), 2μL cDNA template, and finally add ddH2O to make up to 20μL.

[0030] After thorough mixing, the samples were placed in an ABI PRISM 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 ZmBX11 gene was upregulated by approximately 9-fold and 14-fold in #1 and #2, respectively.

[0031] Example 3 This embodiment verifies the low-temperature resistance of maize lines overexpressing the ZmBX11 gene, including the following procedures: 1. In this invention, seeds of wild-type maize (control group WT) and overexpression lines #1 and #2 were sown in pots measuring 30 cm long, 20 cm wide, and 15 cm high, filled with black soil, imported soil, and vermiculite (1:1:1). Twelve seeds of each genotype were placed in each pot, covered with 2 cm of soil, and placed on a tray. The pots were watered until the soil was completely moist and then placed in a 25°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 until the second leaf wrinkled and wilted. They were then removed and placed in a 25°C incubation room for two days to recover before photographing and collecting samples to statistically analyze the degree of leaf damage. Each independent experiment was repeated three times.

[0032] Phenotypes of wild-type plants and overexpression lines #1 and #2 after recovery from low-temperature treatment are as follows: Figure 2 As shown, compared with the control, the leaf damage of overexpression lines #1 and #2 was significantly reduced, indicating that both overexpression lines #1 and #2 exhibited a low-temperature tolerant phenotype.

[0033] 2. In this embodiment, the formula for calculating the area of ​​the relatively undamaged leaf is: (1 - B / A) × 100%, where A represents the total area of ​​the second leaf and B is the area of ​​obvious damage.

[0034] The results are as follows Figure 3 As shown, compared with the wild-type plant WT (40%), the relative undamaged leaf area of ​​overexpression lines #1 and #2 were 60% and 65%, respectively, both significantly higher than that of the wild-type plant, indicating that overexpression... ZmBX11 Genes can enhance the cold resistance of corn.

[0035] 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.

[0036] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. The use of ZmBX11 protein, or its encoding gene, or biological material containing its encoding gene, in any of the following: P1) Application in improving the ability of plants to tolerate low temperature stress; P2) Application in cultivating plants with high cold resistance and / or high growth performance; P3) Application in the improvement of plant cold-resistant germplasm resources; The application is achieved by increasing the expression level of the ZmBX11 protein in plants; The amino acid sequence of the ZmBX11 protein is shown in SEQ ID NO.1, and the ZmBX11 protein is composed of the amino acid sequence shown in SEQ ID NO.

2. ZmBX11 Gene encoding.

2. The application as described in claim 1, characterized in that, The biomaterial is an expression cassette containing the nucleic acid molecule shown in SEQ ID NO.

2.

3. A method for cultivating cold-resistant transgenic plants, characterized in that, The method is achieved by increasing the expression level of ZmBX11 protein in plants; The amino acid sequence of the ZmBX11 protein is shown in SEQ ID NO.1.

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