Application of maize ACP2 protein and its encoding gene in improving plant cold resistance

Abstract

This invention relates to the field of plant genetic engineering technology, specifically disclosing the application of maize ACP2 protein and its encoding gene in improving plant cold resistance. This invention discovers that overexpressing the ACP2 gene in maize can enhance the plant's low-temperature tolerance, thus proposing the application of maize ACP2 protein or its encoding gene, or biological materials containing its encoding gene, in improving plant cold resistance. This invention provides new genetic resources for breeding new cold-resistant plant varieties and lays a theoretical foundation for studying the mechanisms of maize's response to low-temperature stress.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to the application of maize ACP2 protein and its encoding gene in improving plant cold resistance. Background Technology

[0002] Maize (Zea mays L.) is an economic crop originating in tropical low-latitude regions. Although it has gradually entered temperate regions at higher latitudes and altitudes during human domestication and cultivation, maize remains highly sensitive to low-temperature damage. Therefore, expanding maize cultivation in temperate regions still requires the breeding of cold-resistant maize varieties. 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 assimilate transport through dissipation mechanisms and the antioxidant system. Transgenic technology can introduce stress-resistance genes into the genetic material of maize varieties requiring improvement, enabling their offspring to exhibit stable heritable stress resistance and providing superior varietal resources for agricultural production.

[0003] Therefore, it is necessary to research and provide new maize genes for the application of regulating maize cold tolerance in order to solve the problems of existing technologies. Summary of the Invention

[0004] The purpose of this invention is to provide a new genetic resource for breeding new cold-resistant plant varieties and its application.

[0005] This invention provides the application of the maize cold-resistance gene ACP2 and its encoded protein. Maize ACP2 shows the highest homology with ACP2 in Arabidopsis thaliana, but no low-temperature phenotype has been found in Arabidopsis ACP2. While the peanut ACP2 gene has been found to be significantly induced by low temperature, no direct genetic phenotypic evidence has been provided. Furthermore, it is generally known in the art that there is no necessary relationship between changes in gene expression levels and phenotype. For example, this invention found that the expression level of the maize MYB41 gene is induced by low temperature, but transgenic materials overexpressing MYB41 do not show a significant low-temperature phenotype. In addition, the homology between maize ACP2 protein and peanut APC2 protein is low, with only about 40% sequence homology, making it unlikely that the proteins will have similar functions.

[0006] This invention aims to discover genes related to cold tolerance and cold resistance in maize. A maize library of transgenic overexpression lines was specifically screened, and their phenotypes were observed. Different overexpression lines exhibited different phenotypes. Further investigation revealed that several lines overexpressing the ACP2 gene all displayed a significant cold-tolerant phenotype. Further research on the maize ACP2 gene showed that transgenic plants overexpressing this gene had a significantly higher cold-tolerant phenotype than wild-type plants, thus providing insights into the application of the ACP2 protein and its encoding gene in maize cold tolerance and cold resistance. The ACP2 gene provided by this invention offers a genetic resource for breeding new cold-tolerant plant varieties.

[0007] Specifically, this invention screens 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 are 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 is found to be GRMZM2G136262. Further analysis using Arabidopsis homologous gene annotations on the MaizeGDB website identifies it as the maize protein ACP2. A unified comparison reveals that ACP2 belongs to the acyl carrier protein family, but its function has not been reported. This invention confirms that the ACP2 gene may be a key gene for cold tolerance in maize. This invention provides the maize cold-resistant gene ACP2, and by overexpressing the ACP2 gene in maize, cold-resistant transgenic plants can be obtained.

[0008] The cDNA sequence of the maize ACP2 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 ACP2 cDNA consists of 948 bases, and its sequence is shown in SEQ ID No. 1. The gene's reading frame consists of four exons. The amino acid sequence encoded by the maize ACP2 gene is shown in SEQ ID No. 2.

[0010] The corn ACP2 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; or

[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 application of maize ACP2 protein or its encoding gene, or biological materials containing its encoding gene, in improving plant cold resistance, breeding transgenic plants with improved cold resistance, improving cold-resistant plant germplasm resources, and increasing plant survival rate under low-temperature conditions.

[0015] The biological material is an expression cassette, vector, host cell, or recombinant bacteria.

[0016] This invention also provides cloning vectors or various expression vectors containing the ACP2 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 ACP2 gene is a pBCXUN vector containing the Ubi promoter.

[0017] The present invention also provides a method for preparing transgenic plants, which improves the expression level of the ACP2 gene through transgenic methods to obtain plants with enhanced cold resistance.

[0018] The specific method for preparing the transgenic plant includes the following steps:

[0019] 1) Amplify the full-length cDNA sequence of the ACP2 gene (as shown in SEQ ID NO.1);

[0020] 2) Construct an overexpression vector for the ACP2 gene;

[0021] 3) Construct recombinant Agrobacterium containing an overexpression vector of the ACP2 gene;

[0022] 4) Transgenic plants overexpressing the ACP2 gene were constructed using the Agrobacterium infection method.

[0023] The present invention relates to the application of the ACP2 protein and its encoding gene in plants, wherein the plants are monocotyledonous or dicotyledonous plants, preferably rice, wheat, soybean, sorghum, millet, cotton, barley or corn, and more preferably corn.

[0024] This invention also provides a method for constructing cold-resistant transgenic maize, which uses transgenic, hybrid, backcross, self-pollination or asexual reproduction methods to enable maize to express or overexpress the maize ACP2 gene.

[0025] The transgenic process involves introducing a recombinant expression vector containing the ACP2 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.

[0026] In an embodiment of the present invention, the specific method for constructing a low-temperature resistant transgenic plant is as follows:

[0027] 1) Extract total RNA from maize, reverse transcribe to obtain cDNA, use cDNA as template, F and R as primers to amplify the ACP2 gene, construct the amplification product into the expression vector pBCXUN, and name the obtained recombinant expression vector pBCXUN-ACP2.

[0028] 2) Agrobacterium EHA105 was transformed with pBCXUN-ACP2, and then the transformed Agrobacterium was used to infect maize callus tissue to obtain transgenic maize seedlings resistant to low temperature.

[0029] The nucleotide sequences of primers F and R in step 1) are shown in SEQ ID No. 3 and 4. The infected maize is preferably a maize plant with the LH244 homozygous genotype. Overexpression of the ACP2 gene of this invention resulted in maize exhibiting a low-temperature resistant phenotype.

[0030] The expression vector is the pBCXUN vector, which is modified from the plasmid pCAMBIA1300 by inserting a hygromycin resistance gene into pCAMBIA1300.

[0031] This invention cloned the ACP2 gene, constructed transgenic plants overexpressing ACP2, and verified that ACP2 participates in regulating maize's cold tolerance; overexpression of ACP2 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

[0032] Figure 1 The figure shows the results of ACP2 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.

[0033] 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.

[0034] 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; in the chart, *** represents P<0.001.

[0035] Figure 4This is a statistical chart showing the relative leaf damage area of ​​the WT group and the maize overexpression line in Example 3 of this invention. In the figure, ** represents P<0.01. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Example 1: Construction and detection of ACP2 gene overexpression vector

[0040] In this embodiment, 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 ACP2 gene was amplified. The primers contained restriction enzyme sites, and after enzyme digestion, the aCP2 gene was ligated into an overexpression vector. The specific construction method of the ACP2 gene overexpression vector is as follows:

[0041] (1) Total RNA was extracted from B73 maize using the Magen RNA Extraction Kit. The specific steps were described in the kit instructions.

[0042] (2) Use the Thermo Reverse Transcription Kit to reverse transcribe RNA into cDNA. Refer to the kit instructions for specific steps.

[0043] (3) Using maize cDNA as a template and F and R as primers, amplify the cDNA of ACP2 (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-cleaned for recovery. The recovery method is as described in the kit instructions of Tiangen Company.

[0044] The primers used to amplify the ACP2 gene cDNA are:

[0045] Upstream primer F: 5'-ATGGCGCACTCCCTCGCCG-3' (SEQ ID No. 3);

[0046] Downstream primer R: 5'-CTAGACAACCTTTGGAGGCG-3' (SEQ ID No. 4).

[0047] (4) The recovered ACP2 gene cDNA and pBCXUN vector (the pBCXUN vector is obtained by ligating 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); simultaneously, the promoter of the maize ubiquitin gene Ubi was cloned into the vector via enzyme digestion and ligation to drive the transcription of downstream overexpressed genes) were double-digested with Xba I and Cla I. The digestion products were then recovered by electrophoresis and gel extraction. The recovered products were ligated using T4 ligase. The ACP2 gene was ligated into the pBCXUN vector, and the expression of the ACP2 gene was driven by the Ubi promoter.

[0048] (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-ACP2. After digesting the plasmid obtained in the previous step, perform electrophoresis detection. The specific method is as follows: digest pBCXUN-ACP2 with XbaI and ClaI, electrophores with 1% agarose gel at 120V and 50mA, and then scan and image using a UVP Gel Documentation gel analysis system.

[0049] Example 2: Construction and detection of plants overexpressing the ACP2 gene

[0050] The pBCXUN vector containing the ACP2 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 was 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 callus 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.

[0051] In this embodiment, overexpression lines OE#1 and OE#2 with high expression levels were isolated, and the gene expression of ACP2 in the obtained overexpression lines OE#1 and OE#2 was detected by real-time quantitative PCR. The specific method is as follows:

[0052] 1) Extract total RNA from plants and reverse transcribe it to obtain cDNA.

[0053] 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 ABI PRISM 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.

[0054] The sequences of primers F1 and R1 (primers for qRT-PCR) are as follows:

[0055] F1: 5'-GGACTGCTATCACTGGCGAA-3' (SEQ ID No. 5);

[0056] R1: 5'-GATGTCCTGTGCGCTTGTTT-3' (SEQ ID No. 6).

[0057] 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.

[0058] Example 3: Detection of low-temperature resistance in plants overexpressing the ACP2 gene

[0059] First, seeds from the WT group (wild-type maize) and OE#1 and OE#2 obtained in Example 2 were sown separately in small pots (10cm long, 10cm wide, and 10cm high) filled with 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 experimental groups were subjected to a 4°C low-temperature treatment 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 and relative leaf damage area statistics.

[0060] The plant growth of the WT group and maize overexpression lines after low-temperature treatment recovery is as follows: Figure 2 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 leaves of the wild-type WT were severely wilted, dried up, 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.

[0061] 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.

[0062] The results show that... Figure 3As 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 42.6% and 42.1%, respectively (the average difference in ion leakage rates between wild-type WT plants and overexpression lines in three trials), reaching a statistically significant difference (P < 0.001), indicating that overexpression of the ACP2 gene can enhance the cold resistance of maize. Three seedlings from each overexpression line (OE) and wild-type (WT) were used for measurement, and three biological replicates were performed.

[0063] Table 1. Ion leakage rate values ​​from three independent experiments.

[0064] WT OE#1 OE#2 79.33％ 41.79％ 45.95％ 81.25％ 30.24％ 32.54％ 82.39％ 43.15％ 38.07％

[0065] In this embodiment, the relative leaf damage area is calculated by attaching cold-treated corn leaves to A4 paper with glue sticks, taking a photo, processing the photo in ImageJ software, setting a scale, circling the damaged area of ​​the leaf, clicking to measure, and recording it as A1; circling the entire circumference of the leaf, clicking to measure, and recording it as A2. The relative leaf damage area can be calculated by dividing A1 by A2 and multiplying by 100%.

[0066] The results show that... Figure 4 As shown in Table 2, compared with wild-type WT plants, the relative leaf damage area of ​​overexpression lines OE#1 and OE#2 decreased by 38.9% and 39.2%, respectively (the average difference in relative leaf damage area between wild-type WT plants and overexpression lines in three trials), reaching a statistically significant difference (P < 0.01), indicating that overexpression of the ACP2 gene can enhance the cold resistance of maize. Three seedlings from each overexpression line (OE) and wild-type (WT) were used for measurement, and three biological replicates were performed.

[0067] Table 2. Relative leaf damage area in three independent experiments.

[0068] WT OE#1 OE#2 76.37％ 37.42％ 34.02％ 69.02％ 45.98％ 45.19％ 87.39％ 32.60％ 35.85％

[0069] 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. The application of maize ACP2 protein or its encoding gene, or biological materials containing its encoding gene, in improving plant cold resistance; the amino acid sequence of the maize ACP2 protein is shown in SEQ ID NO.2; the plant is maize.

2. The application of maize ACP2 protein or its encoding gene, or biological materials containing its encoding gene, in improving plant survival rate under low temperature conditions; the amino acid sequence of the maize ACP2 protein is shown in SEQ ID NO.2; the plant is maize.

3. The application of maize ACP2 protein or its encoding gene, or biological materials containing its encoding gene, in the improvement of cold-resistant plant germplasm resources; the amino acid sequence of the maize ACP2 protein is shown in SEQ ID NO.2; the plant is maize.

4. The application of maize ACP2 protein or its encoding gene, or biological materials containing its encoding gene, in the breeding of transgenic plants with improved cold resistance; the amino acid sequence of the maize ACP2 protein is shown in SEQ ID NO.2; the plant is maize.

5. The application according to any one of claims 1-4, characterized in that, The nucleotide sequence of the cDNA of the maize ACP2 protein is shown in SEQ ID NO.

1.

6. The application according to any one of claims 1-4, characterized in that, The biological material is an expression cassette, vector, host cell, or recombinant bacteria.

7. A method for constructing cold-resistant transgenic maize, characterized in that, The maize ACP2 gene is expressed or overexpressed in maize through transgenic, hybrid, backcross, self-pollination or asexual reproduction methods. The amino acid sequence of the maize ACP2 protein encoded by the maize ACP2 gene is shown in SEQ ID NO.

2.

8. The method as described in claim 7, characterized in that, The transgenic process involves introducing a recombinant expression vector containing the maize ACP2 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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