Application of maize ms33 protein in regulating cold tolerance of plants

Abstract

The application relates to the technical field of genetic engineering, and particularly discloses application of corn Ms33 protein in regulating cold resistance of plants. It is found that overexpression of the Ms33 gene in corn can enhance the low-temperature resistance of plants, and thus the application of corn Ms33 protein or a coding gene thereof or a biological material containing the coding gene in regulating the cold resistance of corn is proposed. The application lays a certain theoretical foundation for studying the mechanism of corn response to low-temperature stress, and provides a new gene resource for cultivating new varieties of low-temperature resistant plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to the application of maize Ms33 protein in regulating plant cold tolerance. 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] Providing new genetic resources to enhance the cold resistance of maize is of positive significance for the improvement of maize planting resources and actual production. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the maize cold-resistant gene Ms33 and its encoded protein.

[0005] Maize Ms33 shares the highest homology with GPAT3 in Arabidopsis thaliana, belonging to the mitochondrial GPAT gene. There is currently no genetic evidence to support that this gene or its homologs can directly affect plant cold tolerance. Changes in gene expression levels and phenotypes are not necessarily correlated. Even if the expression levels of GPAT genes in certain plants are known to be associated with low-temperature stress, this does not necessarily mean that GPAT genes have the ability to improve plant cold tolerance. For example, studies have found that the expression level of the maize MYB41 gene is induced by low temperature, but transgenic MYB41 materials do not exhibit a significant low-temperature phenotype. Furthermore, the GPAT gene family contains numerous homologous genes. For example, Arabidopsis thaliana has 10 GPAT gene family members. Based on the different subcellular localizations of the enzymes, Arabidopsis thaliana GPAT gene family members can be divided into three categories: plastid GPAT (ATS1), mitochondrial GPAT (AtGPAT1 / 2 / 3), and endoplasmic reticulum GPAT (AtGPAT4 / 5 / 6 / 7 / 8 / 9). Different types of GPAT proteins have highly diverse functions. Although they are all members of the GPAT family, they cannot be simply understood as having similar functions or mutually known functions. This invention, through the study of the maize cold-related gene Ms33, found that transgenic plants overexpressing this gene have a significantly more cold-resistant phenotype than wild-type plants. The Ms33 gene can provide a new gene resource for breeding new cold-resistant plant varieties.

[0006] This invention provides the application of the maize Ms33 protein and its encoding gene in cold resistance in plants. To discover maize genes related to cold resistance, this invention screened a library of transgenic overexpression lines and observed their phenotypes. It was found that different overexpression lines exhibited different phenotypes, and further investigation revealed that several lines overexpressing the Ms33 gene all displayed a clear cold-resistant phenotype.

[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 GRMZM2G070304. Further analysis using gene annotations on the MaizeGDB website identifies it as Ms33 (male sterile33). A unified comparison reveals that Ms33 belongs to the glycerol-3-phosphoacyltransferase family. This invention confirms that the Ms33 gene may be a key gene for cold tolerance and hardiness in maize. This invention provides the maize cold-tolerant gene Ms33, and by overexpressing the Ms33 gene in maize, cold-tolerant transgenic plants were obtained.

[0008] This invention provides the application of maize Ms33 protein or its encoding gene, or biological materials containing its encoding gene, in regulating maize cold tolerance.

[0009] This invention also provides the use of maize Ms33 protein or its encoding gene, or biological materials containing its encoding gene, in any of the following aspects:

[0010] (1) Improve cold-resistant plant germplasm resources;

[0011] (2) Selecting and breeding transgenic plants with improved cold tolerance;

[0012] (3) Improve the survival rate of plants under low temperature conditions.

[0013] The cDNA sequence of the maize Ms33 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.

[0014] The maize Ms33 cDNA consists of 2147 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 Ms33 gene is shown in SEQ ID No. 2.

[0015] The corn Ms33 protein of this invention has any one of the following amino acid sequences:

[0016] 1) The amino acid sequence shown in SEQ ID NO.2; or

[0017] 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.

[0018] 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.

[0019] The biological material is an expression cassette, vector, host cell, or recombinant bacteria.

[0020] This invention also provides cloning vectors or various expression vectors containing the Ms33 gene sequence or fragments thereof for plant cold tolerance, host cells containing the vectors, transformed plant cells containing the gene sequence or specific fragments thereof, and transgenic plants. The overexpression vector containing the MS33 gene is a pBCXUN vector containing the Ubi promoter.

[0021] The present invention also provides a method for preparing transgenic plants, which improves the expression level of the MS33 gene through transgenic methods to obtain plants with enhanced cold resistance.

[0022] The specific method for preparing the transgenic plant includes the following steps:

[0023] (1) Amplify the full-length cDNA sequence of the Ms33 gene (as shown in SEQ ID NO.1);

[0024] (2) Construct an overexpression vector for the Ms33 gene;

[0025] (3) Construct recombinant Agrobacterium containing an overexpression vector of the Ms33 gene;

[0026] (4) Using Agrobacterium infection method, transgenic plants overexpressing Ms33 gene were constructed.

[0027] The present invention relates to the application of the Ms33 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.

[0028] The present invention also provides a method for improving the low-temperature resistance of plants, which improves the expression of the maize Ms33 gene in plants through transgenic, hybrid, backcross, self-pollination or asexual reproduction.

[0029] The transgenic process involves introducing a recombinant expression vector containing the Ms33 gene into maize using methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electrical conductivity, and Agrobacterium-mediated transformation.

[0030] In an embodiment of the present invention, the specific method for constructing a low-temperature resistant transgenic plant is as follows:

[0031] 1) Extract total RNA from maize, reverse transcribe to obtain cDNA, use cDNA as template, F and R as primers to amplify Ms33 gene, construct the amplification product into expression vector pBCXUN, and name the obtained recombinant expression vector pBCXUN-Ms33.

[0032] 2) Agrobacterium EHA105 was transformed with pBCXUN-Ms33, and then the transformed Agrobacterium was used to infect maize callus tissue to obtain transgenic maize seedlings resistant to low temperature.

[0033] The nucleotide sequences of primers F and R in step 1) are shown in SEQ ID No. 3 and 4. The maize callus tissue is preferably derived from maize plants with the LH244 homozygous genotype. Overexpression of the MS33 gene of this invention resulted in maize exhibiting a low-temperature resistant phenotype.

[0034] The expression vector is the pBCXUN vector, which is modified from the plasmid pCAMBIA1300 by inserting a hygromycin resistance gene into pCAMBIA1300.

[0035] This invention cloned the Ms33 gene and constructed transgenic plants overexpressing the Ms33 gene. Cold resistance analysis of the obtained transgenic maize revealed that overexpression of Ms33 improved maize's cold tolerance, giving it stronger low-temperature resilience. This invention provides new gene 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. Attached Figure Description

[0036] Figure 1 The figure shows the results of the Ms33 gene overexpression test in the WT group and the maize overexpression line in Example 2 of the present invention; in the figure, *** represents P<0.001.

[0037] 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.

[0038] Figure 3 This is a statistical chart showing the ion leakage rate of the WT group and the maize overexpression line in Example 3 of the present invention; in the chart, ** represents P<0.01.

[0039] Figure 4 This is a statistical chart showing the relative leaf damage area of ​​the WT group and the maize overexpression line in Example 3 of the present invention; in the figure, ** represents P<0.01, and *** represents P<0.001. Detailed Implementation

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Example 1: Construction and detection of Ms33 gene overexpression vector

[0044] 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 Ms33 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 Ms33 gene overexpression vector is as follows:

[0045] (1) Total RNA was extracted from B73 maize using the Magen RNA Extraction Kit. The specific steps were described in the kit instructions.

[0046] (2) Use the Thermo Reverse Transcription Kit to reverse transcribe RNA into cDNA. Refer to the kit instructions for specific steps.

[0047] (3) Using maize cDNA as a template and F and R as primers, amplify Ms33 cDNA (as shown in SEQ ID NO.1, whose encoded amino acid sequence is shown in SEQ ID NO.2), and recover the amplified product by electrophoresis and gel cutting. The recovery method is in accordance with the instructions of the Tiangen Company kit.

[0048] The primers used to amplify the Ms33 gene cDNA are:

[0049] Upstream primer F: 5'-ATGGCCAAGAAGAGGCTCCTC-3' (SEQ ID No. 3);

[0050] Downstream primer R: 5'-TTATCGGACGATGCCGTCG-3' (SEQ ID No. 4).

[0051] (4) The recovered Ms33 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 Ms33 gene was ligated into the pBCXUN vector, and the expression of the Ms33 gene was driven by the Ubi promoter.

[0052] (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-Ms33. After digesting the plasmid obtained in the previous step, perform electrophoresis detection. The specific method is as follows: digest pBCXUN-MS33 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.

[0053] Example 2: Construction and detection of plants overexpressing the Ms33 gene

[0054] The pBCXUN vector containing the Ms33 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 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.

[0055] In this embodiment, the overexpression lines OE#1 and OE#2, which were isolated with high expression levels, were used to detect the gene expression of Ms33 in the obtained overexpression lines OE#1 and OE#2 using real-time quantitative PCR. The specific method is as follows:

[0056] 1) Extract total RNA from plants and reverse transcribe it to obtain cDNA.

[0057] 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.

[0058] The sequences of primers F1 and R1 (primers for qRT-PCR) are as follows:

[0059] F1: 5'-GCTTCGACCCGCTCTACTAC-3' (SEQ ID No. 5);

[0060] R1: 5'-GGTACTTGTCCTTCCGGGTG-3' (SEQ ID No. 6).

[0061] 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.

[0062] Example 3: Detection of low-temperature resistance in plants overexpressing the Ms33 gene

[0063] 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 subjected to a 4°C low-temperature treatment for 4 days until the second leaf wrinkled and wilted. After that, the plants were removed and placed in a 23°C incubation room for two days to recover. Photos were taken and samples were collected to statistically analyze the ion leakage rate and the relative leaf damage area.

[0064] The plant growth of the WT group and maize overexpression lines (OE#1 and OE#2) 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.

[0065] 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 for 30 minutes using a vacuum pump, then placed on a shaker at room temperature for 1 hour. The initial conductivity was then measured as S1. 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. Three seedlings from each overexpression line (OE#1 and OE#2) and wild-type (WT) were used for the measurement, and three biological replicates were performed.

[0066] 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 decreased by 26.1% and 29.4%, 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<0.01), indicating that overexpression of the Ms33 gene can enhance the cold resistance of maize.

[0067] Table 1. Ion leakage rate values ​​from three independent experiments.

[0068] 77.82％ 47.36％ 40.60％ 75.98％ 53.52％ 46.34％ 67.19％ 41.79％ 45.95％

[0069] In this embodiment, the relative leaf damage area was statistically analyzed by attaching cold-treated corn leaves to A4 paper with glue sticks, taking photos, processing the photos in ImageJ software, setting a scale, circling the damaged area of ​​the leaf, measuring and recording it as A1, circling the entire circumference of the leaf, measuring and recording it as A2. The relative leaf damage area can be calculated by A1 / A2*100%. Three seedlings from each overexpression line (OE#1 and OE#2) and wild type (WT) were taken for measurement, and three biological replicates were performed.

[0070] 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 28.8% and 42.2% respectively (the average difference of relative leaf damage area between wild-type WT plants and overexpression lines in three studies), reaching a significant difference (P<0.01), indicating that overexpression of the Ms33 gene can enhance the cold resistance of maize.

[0071] Table 2. Relative leaf injury values ​​from three independent experiments.

[0072] 74.03％ 47.40％ 30.85％ 73.88％ 31.86％ 28.49％ 65.90％ 48.11％ 27.88％

[0073] 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 overexpression of maize Ms33 protein or its encoding gene, or biological materials containing its encoding gene, in improving the cold resistance of maize; the amino acid sequence of said maize Ms33 protein is shown in SEQ ID NO.

2.

2. Application of maize Ms33 protein overexpression or its encoding gene, or biological materials containing its encoding gene, in any of the following aspects: (1) Improve cold-resistant maize germplasm resources; (2) Breeding transgenic maize with improved cold tolerance; (3) Improve the survival rate of corn under low temperature conditions; The amino acid sequence of the corn Ms33 protein is shown in SEQ ID NO.

2.

3. The application according to claim 1 or 2, characterized in that, The cDNA sequence of the corn Ms33 protein is shown in SEQ ID NO.

1.

4. The application according to claim 1 or 2, characterized in that, The biological material is an expression cassette, vector, host cell, or recombinant bacteria.

5. A method for improving the low-temperature resistance of corn, characterized in that, The expression of the maize Ms33 gene in maize is enhanced by transgenic, hybrid, backcross, or self-crossing methods; the amino acid sequence of the protein encoded by the maize Ms33 gene is shown in SEQ ID NO.

2.

6. The method as described in claim 5, characterized in that, The transgenic process involves introducing a recombinant expression vector containing the maize Ms33 gene into maize using methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electrical conductivity, or Agrobacterium-mediated transformation.

Images