Use of a high temperature tolerant gene zmc tu2 of maize
By overexpressing the ZmCTU2 gene in the maize line B104, the problem of insufficient resistance to high temperature stress in maize was solved, and the high temperature tolerance and growth performance of maize plants were significantly improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HENAN AGRICULTURAL UNIVERSITY
- Filing Date
- 2022-08-31
- Publication Date
- 2026-06-19
AI Technical Summary
The current technology has limited identification and transgenic verification of heat-resistant genes in maize, resulting in insufficient resistance of maize to high-temperature stress, which affects maize yield and food security.
The heat-resistant gene ZmCTU2 was identified and overexpressed in the maize line B104. A recombinant vector was constructed and introduced into maize embryos to infect them with Agrobacterium tumefaciens, resulting in transgenic plants overexpressing ZmCTU2. The expression was then carried out using the maize polyubiquitin gene promoter.
It significantly improved the resistance of maize plants to high temperature stress, enhanced the survival rate and growth status of plants, and the expression value of ZmCTU2 gene increased by 46-18 times in transgenic plants.
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Figure CN116376967B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology technology, specifically relating to the application of a maize heat-resistant gene ZmCTU2. Background Technology
[0002] With population growth and industrialization, global warming has become an issue that cannot be ignored. Maize (Zea mays L.) is one of my country's most important food crops, as well as a crucial feed and industrial raw material. In 2020, my country's maize planting area exceeded 40 million hectares, with a yield exceeding 260 million tons, ranking first among all food crops. Maize production plays a pivotal role in my country's food security. Although maize is a warm-loving crop, excessively high temperatures can have numerous adverse effects on its growth and development. In recent years, the frequency, duration, and affected area of extreme heat events in my country have been increasing, leading to severe poor grain filling in maize in many regions. Moreover, these high-temperature events typically occur from June to August, precisely during the critical period of growth, development, and yield formation for summer maize in the Huang-Huai-Hai Plain. High-temperature heat damage has become one of the major hazards limiting maize yield. Therefore, identifying heat-resistant genes in maize, elucidating the molecular mechanisms of maize's response to high-temperature stress, and breeding varieties based on these genes are effective measures to ensure my country's food security in the context of global warming, and are an urgent task for Chinese maize researchers.
[0003] Plant resistance to heat stress is a highly complex process involving intricate transcriptional regulatory networks, precise protein homeostasis control, translational regulation, post-translational protein modification, and signal cascade amplification, among other responses. In recent years, with the development of molecular biology techniques and the application of genome-wide association studies (GWAS), some heat-resistant genes in maize have been gradually discovered. However, due to the complexity of heat tolerance evaluation systems and limitations of maize transgenic technology, the number of identified maize heat-resistant genes is currently very limited, and those with transgenic functional verification are even fewer.
[0004] Therefore, a maize heat-resistant gene ZmCTU2 was identified in this invention. Overexpression of ZmCTU2 in the maize line B104 can significantly improve the resistance of B104 to high-temperature stress. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing an application of the maize heat-resistant gene ZmCTU2. In this invention, a maize heat-resistant gene ZmCTU2 was identified. Overexpression of ZmCTU2 in the maize line B104 can significantly improve the resistance of B104 to high-temperature stress.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: an application of the maize heat-resistant gene ZmCTU2, characterized in that the maize heat-resistant gene ZmCTU2 is used to obtain heat-resistant maize plants, the nucleotide sequence of the full-length coding region of the maize heat-resistant gene ZmCTU2 is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2.
[0007] Preferably, the method for obtaining heat-resistant maize plants using the maize heat-resistant gene ZmCTU2 is as follows:
[0008] S1. The pTF101.1-3×Flag vector was digested with BamHI / SpeI double enzymes and reacted at 7℃ for 6 hours. The digestion product was separated by 1% agarose gel, and an 11.54Kb band was cut off. The product was recovered using a DNA gel recovery kit to obtain the recovered product of pTF101.1-3×Flag vector digested with BamHI / SpeI double enzymes.
[0009] S2. Using the cDNA library of maize inbred line B73 as a template, PCR reaction was performed with specific primers F and R, and 2×KOD Mix as buffer to clone the full-length coding region of the maize heat-resistant gene ZmCTU2. After PCR amplification, the region was separated with 1% agarose gel, and a band of about 1380 bp was excised. The PCR product was recovered using a DNA gel recovery kit to obtain the PCR recovery product of the full-length coding region of the maize heat-resistant gene ZmCTU2.
[0010] The PCR reaction system consisted of 10 μL of 2×KOD Mix, 1 μL of specific primer F, 1 μL of specific primer R, 1 μL of cDNA library from maize inbred line B73, and sterile ultrapure water to a final volume of 20 μL. The PCR amplification conditions were as follows: 98℃ pre-denaturation for 10 min; 98℃ denaturation for 10 s, 55℃ annealing for 10 s, 68℃ extension for 40 s, for 40 cycles; and 68℃ extension for 10 min. The nucleotide sequence of specific primer F is shown in SEQ ID NO:3; the nucleotide sequence of specific primer R is shown in SEQ ID NO:4.
[0011] S3. Using homologous recombination, the full-length coding region of the maize heat-resistant gene ZmCTU2 obtained in S2 was ligated from the PCR recovery product of the pTF101.1-3×Flag vector obtained in S1 to the BamHI / SpeI site of the pTF101.1-3×Flag vector digested with BamHI / SpeI. The promoter used was the maize polyubiquitin gene promoter. The ligation reaction was carried out at 50℃ for 25 min to obtain the ligation product. The ligation product was transformed into E. coli, amplified and multiplied, and extracted using a plasmid extraction kit to obtain a successfully ligated vector with the correct sequence. This vector was named pTF101.1-ZmCTU2.
[0012] S4. After introducing the pTF101.1-ZmCTU2 vector obtained in S3 into Agrobacterium EHA105, recombinant Agrobacterium was used to infect the immature embryos of maize inbred line B104. The infected immature embryos were induced to form callus tissue, and then differentiated into plants on a culture medium containing glyphosate herbicide. The resulting plants were ZmCTU2 overexpressing transgenic plants, which are heat-resistant maize plants.
[0013] Preferably, the enzyme digestion system in S1 is: BamHI 1 μL, SpeI 1 μL, Cutsmart buffer 5 μL, pTF101.1-3×Flag vector 10 μL, and sterile ultrapure water to make up to 50 μL.
[0014] Preferably, the homologous recombination system in S3 is: 5 μL of 2×Hieff Clone Enzyme Premix, 2 μL of the recovered product of the pTF101.1-3×Flag vector double digested with BamHI / SpeI, 1 μL of the recovered product of the full-length coding region of the maize heat-resistant gene ZmCTU2 PCR, and sterile ultrapure water to a final volume of 10 μL.
[0015] Compared with the prior art, the present invention has the following advantages:
[0016] 1. This invention identifies a maize heat stress-related gene, ZmCTU2. Overexpression of ZmCTU2 in maize strain B104, which does not have heat stress resistance, can significantly improve the plant's resistance to heat stress.
[0017] 2. In the ZmCTU2 OE#3 and ZmCTU2 OE#5 transgenic plants obtained in this invention, the expression value of the ZmCTU2 gene increased by 46 and 18 times, respectively, compared with the wild-type maize inbred line B104. High-temperature treatment of B104 seedlings with two leaves and one bud and the overexpression line revealed no significant difference in growth status between ZmCTU2 OE#3, ZmCTU2 OE#5, and B104 before treatment. After high-temperature treatment, the growth status of ZmCTU2 OE#3 and ZmCTU2 OE#5 plants was significantly better than that of B104; their survival rate and fresh weight were significantly higher than those of B104 plants. This indicates that overexpression of ZmCTU2 in the maize line B104, which lacks heat tolerance, can significantly improve the plant's resistance to high-temperature stress.
[0018] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description
[0019] Figure 1 This is a framework diagram of the pTF101.1-ZmCTU2 recombinant vector expression in Example 1.
[0020] Figure 2 This is the PCR identification result of the ZmCTU2 overexpressing transgenic plant in Example 1.
[0021] Figure 3 This represents the expression level of ZmCTU2 in the transgenic positive plants identified in Example 1.
[0022] Figure 4 This is a comparison diagram of the growth of ZmCTU2 OE#3, ZmCTU2 OE#5 and B104 before and after high-temperature treatment in Example 1.
[0023] Figure 5 This is a comparison chart of plant survival rate and fresh weight after high-temperature treatment of ZmCTU2 OE#3, ZmCTU2 OE#5 and B104 in Example 1. Detailed Implementation
[0024] Example 1
[0025] In this embodiment, the maize heat-resistant gene ZmCTU2 is used to obtain heat-resistant maize plants. The nucleotide sequence of the full-length coding region of the maize heat-resistant gene ZmCTU2 is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2.
[0026] The method for obtaining heat-resistant maize plants using the maize heat-resistant gene ZmCTU2 is as follows:
[0027] S1. The pTF101.1-3×Flag vector was digested with BamHI / SpeI double enzymes and reacted at 7℃ for 6 hours. The digestion product was separated by 1% agarose gel, and an 11.54Kb band was cut off. The product was recovered using a DNA gel recovery kit to obtain the recovered product of pTF101.1-3×Flag vector digested with BamHI / SpeI double enzymes.
[0028] The enzyme digestion system was as follows: BamHI 1 μL, SpeI 1 μL, Cutsmart buffer 5 μL, pTF101.1-3×Flag vector 10 μL, and sterile ultrapure water to a final volume of 50 μL.
[0029] S2. Using the cDNA library of maize inbred line B73 as a template, PCR reaction was performed with specific primers F and R, and 2×KOD Mix as buffer to clone the full-length coding region of the maize heat-resistant gene ZmCTU2. After PCR amplification, the region was separated with 1% agarose gel, and a band of about 1380 bp was excised. The PCR product was recovered using a DNA gel recovery kit to obtain the PCR recovery product of the full-length coding region of the maize heat-resistant gene ZmCTU2.
[0030] The PCR reaction system consisted of 10 μL of 2×KOD Mix, 1 μL of specific primer F, 1 μL of specific primer R, 1 μL of B73 cDNA library, and sterile ultrapure water to a final volume of 20 μL. The PCR amplification conditions were as follows: 98℃ pre-denaturation for 10 min; 98℃ denaturation for 10 s, 55℃ annealing for 10 s, 68℃ extension for 40 s, for 40 cycles; and 68℃ extension for 10 min. The nucleotide sequence of specific primer F is shown in SEQ ID NO:3; the nucleotide sequence of specific primer R is shown in SEQ ID NO:4.
[0031] S3. Using homologous recombination, the full-length coding region of the maize heat-resistant gene ZmCTU2 obtained in S2 was ligated from the PCR-recovered product of the pTF101.1-3×Flag vector obtained in S1 to the BamHI / SpeI site of the pTF101.1-3×Flag vector digested with BamHI / SpeI. The promoter used was the maize polyubiquitin promoter. The ligation reaction was carried out at 50℃ for 25 min to obtain the ligation product. The ligation product was transformed into E. coli, amplified, and extracted using a plasmid extraction kit to obtain a successfully ligated and correctly sequenced recombinant vector, named pTF101.1-ZmCTU2. The expression framework of this recombinant vector is as follows: Figure 1As shown; the pTF101.1-ZmCTU2 vector can express the ZmCTU2 protein shown in SEQ ID NO:2; in addition, the recombinant vector can also express the bar gene, using the CaMV35S promoter, which enables the following positive overexpression transgenic plants to grow on a medium containing glyphosate herbicide;
[0032] The homologous recombination system consisted of: 5 μL of 2×HieffClone Enzyme Premix, 2 μL of the recovered product of the pTF101.1-3×Flag vector digested with BamHI / SpeI, 1 μL of the recovered product of the full-length coding region of the maize heat-resistant gene ZmCTU2, and sterile ultrapure water to a final volume of 10 μL.
[0033] S4. After introducing the pTF101.1-ZmCTU2 vector plasmid obtained in S3 into Agrobacterium EHA105, recombinant Agrobacterium was used to infect the immature embryos of maize inbred line B104. The infected immature embryos were induced to form callus tissue, and then differentiated into plants on a medium containing glyphosate herbicide. The resulting plants were ZmCTU2 overexpressing transgenic plants, which are heat-resistant maize plants, denoted as ZmCTU2-OE.
[0034] The preparation method of the culture medium (1L) containing glyphosate herbicide is as follows: 100mL 10×LS macroelements (30mM CaCl2·2H2O, 188mM KNO3, 206mM NH4NO3, 15mM MgSO4·7H2O, 12.5mM KH2PO4); 10mL 100×LS microelements (9.3mM MnSO4·H2O, 3.7mM ZnSO4·7H2O, 0.01mM CoCl2·6H2O, 0.01mM CuSO4·5H2O, 0.1mM NaMoO4·2H2O, 0.5mM KI, 10mM H3BO3); 10mL 100×LS Fe salts (10mM FeSO4·7H2O, 10mM Na2EDTA); 10mL 100×LS vitamins (0.41mM niacin, 0.3mM VB1, 0.24mM VB6, 55.5mM inositol); 15mL 100mg / L 2,4-D; 0.7g proline; 0.5g MES; 20g sucrose; 8g agar; sterilize at 121℃ for 15min, cool to 50℃, add 1.0ml 250g / L Cb (final concentration 250mg / L) or 1.0ml 250g / L Cef (final concentration 250mg / L), 0.1ml 100mM AgNO3, 0.85ml 175g / L glyphosate (final concentration 87.5mg / L), pour into 30ml plates, and store in the dark at room temperature (20℃~25℃).
[0035] Identification of transgenic positive plants of ZmCTU2-OE obtained in this embodiment: Genomic DNA of the overexpressing transgenic plant ZmCTU2-OE obtained in this embodiment was amplified by PCR using primer Ubi-F on the vector framework and primer ZmCTU2-R on the gene. The PCR reaction system was: 10 μL of 2×KOD Mix, 1 μL of specific primer Ubi-F, 1 μL of specific primer ZmCTU2-R, and template DNA. 1 μL of sterile ultrapure water was added to bring the volume to 20 μL; the PCR amplification reaction conditions were: 98℃ pre-denaturation for 10 min; 98℃ denaturation for 10 s, 55℃ annealing for 10 s, 68℃ extension for 20 s, for 40 cycles; the amplification product was obtained by extending the product at 68℃ for 10 min; the amplification product was separated using a 1% agarose gel and identified by PCR. The presence or absence of a 560 bp band determined whether the transgenic plant was a positive transgenic plant, using genomic DNA from the non-transgenic maize inbred line B104 as a control; the PCR identification results of the ZmCTU2-OE transgenic positive plants obtained in this example are as follows. Figure 2 The results showed that non-transgenic plants (maize inbred line B104) could not show a 750bp electrophoretic band, while ZmCTU2-OE transgenic positive plants showed a clear 560bp electrophoretic band; the nucleotide sequence of primer Ubi-F is shown in SEQ ID NO:5; the nucleotide sequence of primer ZmCTU2-R is shown in SEQ ID NO:6.
[0036] To identify the expression level of ZmCTU2 in the above-mentioned transgenic positive plants: The identified transgenic positive plants were selected, with the non-transgenic maize inbred line B104 as a control. RNA was extracted from both plants. Total RNA was extracted using the trizol method. Before sampling, the tweezers, scissors, and blades used for sampling were flammed with an alcohol lamp. The material used for RNA extraction was removed, wrapped in aluminum foil, and quickly frozen in liquid nitrogen. Samples not immediately used for RNA extraction could be transferred to an ultra-low temperature freezer at -80°C for later use. An appropriate amount of tissue was thoroughly ground into powder in a mortar and mortar, and 1 mL of the powder was added... The trizol extract was rapidly rotated in a mortar and pestle to ensure the trizol fully covered the plant tissue. After melting, the mixture was thoroughly ground until clear and transparent. The mixture was then transferred to a 1.5 mL RNase-free centrifuge tube and incubated at room temperature for 5 min. It was then centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was transferred to a new centrifuge tube, and 1 / 5 volume of chloroform was added. The mixture was vigorously shaken for 15 s and incubated at room temperature for 5 min. It was then centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was transferred to a new centrifuge tube, and an equal volume of isopropanol was added. The mixture was gently mixed and incubated at room temperature for 10 min. It was then centrifuged at 12,000 rpm for 10 min at 4 °C and the supernatant was discarded. The precipitate was washed with 75% ethanol and centrifuged at 12,000 rpm for 5 min at 4 °C and the ethanol was discarded. The residual ethanol was carefully removed with a pipette tip and the mixture was dried at room temperature for 3-5 min until the RNA edges became transparent. 30 μL of DEPC water was added for reverse transcription.
[0037] Synthesis of first-strand cDNA: The concentration of RNA was measured using a Nanodrop instrument. 1 μg of total RNA was digested with 1 μL of DNase I (Thermo Scientific) at 37°C for 30 min, and then denatured at 70°C for 10 min. After that, the mixture was placed on ice. The reagents required for reverse transcription were added according to the dosage of the Promega reverse transcription kit. The mixture was incubated at 42°C for 1 h, and then denatured at 95°C for 5 min. The reaction system was immediately placed on ice to separate the first strand of cDNA from the RNA template. Four volumes of sterile ultrapure water were added, and the mixture was stored at -20°C for later use.
[0038] qRT-PCR detection and analysis: Using first-strand cDNA as a template, Roche's SYBR Green Master I enzyme premix and qRT primers for the ZmCTU2 gene (ZmCTU2 qF and ZmCTU2 qR) were added, and PCR amplification was performed using the ThermoFisher STEP ONEPLUS system. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 5 s; annealing temperature of the corresponding primers (55℃) for 15 s; extension at 72℃ for 10 s; 45 cycles. Simultaneously, using first-strand cDNA as a template, Roche's SYBR Green Master I enzyme premix and qRT primers for the internal control gene EF1A (EF1A qF and EF1A qR) were added, and PCR amplification was performed using the ThermoFisher STEP ONEPLUS system. PCR amplification was performed using the PLUS system. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 5 s; annealing temperature of the corresponding primers (55℃) for 15 s; extension at 72℃ for 10 s; 45 cycles. The number of cycles required for the ZmCTU2 gene and the internal control gene EF1A to reach maximum fluorescence value in each sample was determined using the above PCR reaction. Finally, the number of cycles for the maize gene EF1A was used as an internal control for each sample, and 2... EF1A基因的循环数 / 2 ZmCTU2基因的循环数 The formula calculates the relative expression value of the ZmCTU2 gene in each sample. Then, the relative expression value of the ZmCTU2 gene in the maize inbred line B104 sample is set to 1, and the expression value of the ZmCTU2 gene in the overexpression line sample relative to that in the maize inbred line B104 sample is calculated. The results are as follows: Figure 3 As shown, the expression levels of ZmCTU2 were high in positive plants No. 3 and No. 5, and were denoted as ZmCTU2 OE#3 and ZmCTU2OE#5, respectively.
[0039] The nucleotide sequence of ZmCTU2 qF is shown in SEQ ID NO:7; the nucleotide sequence of ZmCTU2 qR is shown in SEQ ID NO:8; the nucleotide sequence of EF1A qF is shown in SEQ ID NO:9; and the nucleotide sequence of EF1A qR is shown in SEQ ID NO:10.
[0040] Depend on Figure 3 It can be seen that the expression value of the ZmCTU2 gene in the ZmCTU2 OE#3 and ZmCTU2 OE#5 transgenic plants increased by 46 and 18 times respectively compared with the wild-type maize inbred line B104; the obtained ZmCTU2 OE#3 and ZmCTU2 OE#5 are plants with overexpression of ZmCTU2, which will be used for subsequent comparison of high temperature resistance.
[0041] High-temperature treatment was applied to seedlings of the wild-type maize inbred line B104 (two leaves and one heart) and the ZmCTU2 OE#3 and ZmCTU2 OE#5 overexpression lines obtained in this example. The specific method of high-temperature treatment was as follows: maize seeds were sown in sterilized nutrient soil and cultured at 26℃ for 15 hours of light followed by 9 hours of darkness for 10 days until the two-leaf-one-heart stage was reached; then, they were transferred to a high-temperature incubator for 5 days (45℃ for 15 hours of light, 37℃ for 9 hours of darkness); after the treatment, the maize was transferred to a normal incubator and recovered at 26℃ for 15 hours of light followed by 9 hours of darkness for 10 days; then, the survival rate (number of surviving plants / total number of plants × 100%) and fresh weight (fresh weight of the aboveground parts of the plants) of ZmCTU2 OE#3, ZmCTU2 OE#5, and B104 were counted.
[0042] The results showed that the growth of ZmCTU2 OE#3, ZmCTU2 OE#5, and B104 obtained in this embodiment was not significantly different before treatment. After high-temperature treatment, the growth of ZmCTU2 OE#3 and ZmCTU2 OE#5 plants was significantly better than that of B104. Figure 4 (As shown); the survival rate and fresh weight of ZmCTU2 OE#3 and ZmCTU2 OE#5 plants were significantly higher than those of B104 plants (as shown); Figure 5 As shown in the figure, this indicates that overexpression of ZmCTU2 in the maize line B104 can significantly improve the plant's resistance to high temperature stress.
[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.
Claims
1. A heat-resistant gene for maize ZmCTU2 The application is characterized by, The corn heat-resistant gene ZmCTU2 The heat-resistant gene in maize is used to obtain heat-resistant maize plants. ZmCTU2 The nucleotide sequence of the full-length coding region is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2.