Application of malate dehydrogenase gene in improving rice high temperature tolerance

Abstract

The application discloses application of a malate dehydrogenase gene in improving high-temperature tolerance of rice, and the sequence of the malate dehydrogenase gene is shown as SEQ ID NO. 2. Through obtaining a rice strain with the malate dehydrogenase gene and an insertion mutant strain, the following experimental phenotypes are obtained: overexpression of OsMDH can obviously improve the high-temperature stress tolerance of rice in the heading stage, so that the plant can normally grow when encountering high-temperature stress, the overexpression transgenic strain is weaker in spike degeneration compared with a wild type, and the seed filling is good; the function loss of the OsMDH gene reduces the high-temperature stress tolerance of rice in the heading stage, the insertion mutant transgenic strain is more serious in spike degeneration compared with the wild type, and the seed setting rate and filling are obviously decreased.

Description

Technical Field

[0001] This invention relates to the application of malate dehydrogenase genes in improving the heat resistance of rice, and belongs to the field of biogenetic technology. Background Technology

[0002] Rice is one of my country's most important food crops. With the increasing levels of greenhouse gases such as carbon dioxide, which have strong heat-absorbing properties, global temperatures are rising, and it is projected that by 2100, global temperatures will rise to 3-6°C. Against the backdrop of global warming, frequent heat waves have made the cultivation of heat-resistant, high-quality rice varieties a key focus. Rising temperatures severely threaten crop growth and production, causing premature aging and yellowing of functional leaves, reduced enzyme activity, decreased photosynthetic efficiency, and reduced photosynthetic assimilate transport capacity, resulting in underdeveloped grains and lower thousand-grain weight. It is estimated that high temperatures lead to a 15-35% reduction in crop yields annually.

[0003] In my country, rice cultivation is characterized by "indica in the south and japonica in the north." Japonica rice exhibits better cold and drought resistance, while indica rice is more heat-tolerant, resistant to strong light, and tolerant of humidity, displaying heat tolerance similar to C4 plants such as maize. C3 plants are mainly distributed in temperate and cold regions, while C4 plants originated in the hot and arid tropical and subtropical regions. Compared to C3 plants, C4 plants have weaker photorespiration, which contributes to their stronger adaptability to high temperatures. The leaf structure and key enzyme activities of C4 plants may enhance the efficiency of photosynthetic product transport, water allocation, and stress response. The transition from the C3 to the C4 photosynthetic pathway is relatively simple, evolving independently and polymorphically within each family and genera of angiosperms. Among monocotyledonous grasses, maize and rice are believed to have branched out approximately 50 million years ago, with one branch evolving into C3 crops such as rice and wheat, and the other into C4 crops such as maize and sorghum. Around 10,000 years ago, rice and maize were gradually domesticated into their current cultivated varieties. The genomes of maize and rice exhibit good collinearity between chromosomes and strong conservation of gene sequences. This suggests at the molecular evolutionary level that C3 rice has the potential to evolve into C4 crops with high heat and light tolerance. Therefore, to mitigate the effects of high temperatures on plants, it is necessary to propose methods to improve plant stress resistance and related traits. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of malate dehydrogenase gene, which reduces the tolerance of rice to high temperature stress during the heading stage, in improving the high temperature tolerance of rice.

[0005] Technical solution: To solve the above technical problems, the present invention provides the application of malate dehydrogenase gene in improving the high temperature resistance of rice, wherein the malate dehydrogenase gene sequence is shown in SEQ ID NO.2.

[0006] This invention also provides the application of an overexpression vector containing a malate dehydrogenase gene in improving the heat resistance of rice, the sequence of which is shown in SEQ ID NO.2.

[0007] The overexpression vector includes pCAMBIA1300(GFP)-OsMDH.

[0008] This invention also provides the application of recombinant bacteria containing the malate dehydrogenase gene in improving the heat resistance of rice. The recombinant bacteria containing the malate dehydrogenase gene are obtained by introducing the overexpression vector pCAMBIA1300(GFP)-OsMDH into the host bacterial strain. The pCAMBIA1300(GFP)-OsMDH is obtained by introducing the malate dehydrogenase gene with the sequence shown in SEQ ID NO.2 into pCAMBIA1300(GFP).

[0009] The present invention also provides a method for obtaining heat-resistant rice using a malate dehydrogenase gene, wherein wild-type rice is infected with a recombinant bacterium containing a malate dehydrogenase gene to obtain transgenic rice containing a malate dehydrogenase gene, the sequence of which is shown in SEQ ID NO.2.

[0010] The method for preparing the recombinant bacteria containing the malate dehydrogenase gene includes the following steps:

[0011] (1) Using the cDNA of japonica rice variety dongjin as a template, PCR amplification was performed, double enzyme digestion was performed, and the vector was ligated with the backbone vector to obtain the recombinant expression vector.

[0012] (2) Introduce the recombinant expression vector described in step (1) into the host bacteria to obtain recombinant bacteria containing malate dehydrogenase.

[0013] The sequences of the primer pairs used for PCR amplification are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0014] The present invention also provides a method for verifying the high-temperature resistance of rice obtained by the method, comprising the following steps:

[0015] (1) Using wild-type rice growing at the same time as the control group, the rice seeds obtained by the above method were soaked in water and cultured to germinate, and then resistance was screened with hygromycin. After germination, the seedlings were transferred to soil for soil cultivation.

[0016] (2) High temperature treatment was applied to soil-grown seedlings to measure the photosynthetic rate, canopy temperature and stomatal opening of the leaves, and the activity of peroxidase-related enzymes in the leaves was analyzed.

[0017] (3) When the photosynthetic rate of the rice obtained by the method is slower than that of the control group, the stomatal opening of the leaves is smaller than that of the control group, and the activity of peroxidase is higher than that of the control group, it indicates that the rice has the ability to withstand high temperatures.

[0018] The peroxide-related enzymes include one or more of malondialdehyde-MDA, hydrogen peroxide-H2O2, ascorbate peroxidase-APX, superoxide dismutase-SOD, peroxidase-POD, or catalase-CAT.

[0019] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention obtains experimental phenotypes by acquiring rice lines transgenic with the malate dehydrogenase (OsMDH) gene and insertion mutant lines: Overexpression of OsMDH can significantly improve the tolerance of rice to high temperature stress during the heading stage, enabling the plant to grow normally when encountering high temperature stress. The overexpressing transgenic lines have weaker panicle degeneration and better grain filling compared to wild-type lines. The loss of function of the OsMDH gene reduces the tolerance of rice to high temperature stress during the heading stage. The insertion mutant transgenic lines have more severe panicle degeneration compared to wild-type lines, and the seed setting rate and filling rate are significantly reduced. 2. The protein and its encoding gene of the present invention have important theoretical and practical significance for the study of plant stress resistance mechanisms and the improvement of plant stress resistance and related traits. They will play an important role in the genetic engineering improvement of plant stress resistance and have broad application prospects. Attached Figure Description

[0020] Figure 1 The results of OsMDH gene overexpression detection;

[0021] Figure 2 Identification results of OsMDH insertion mutant plants;

[0022] Figure 3 Analysis of canopy temperature of OsMDH transgenic plants and control plants after 3 days of high temperature: a) plant morphology; b) temperature of different plant tissues; c) flag leaf surface temperature.

[0023] Figure 4 Analysis of stomatal aperture and photosynthetic rate of OsMDH transgenic plants in the high-temperature group (3 days and 5 days of high temperature) and the control group: a) stomatal open area; b) leaf surface temperature; c) transpiration rate; d) stomatal conductance; e) intracellular CO2 concentration.

[0024] Figure 5Analysis of peroxide-related enzyme activities in OsMDH transgenic plants under high-temperature conditions (3 days and 5 days) and the control group: a) Malondialdehyde (MDA) content; b) Hydrogen peroxide (H2O2) content; c) Ascorbate peroxidase (APX) content; d) Peroxidase (POD) content; e) Catalase (CAT) content; f) Superoxide dismutase (SOD) content.

[0025] Figure 6 Analysis of seed setting rate and thousand-grain weight of OsMDH transgenic plants in high-temperature group (high temperature for 3 days and 5 days) and control group: a) temperature of the control group and high-temperature treatment group for one day; b) seed setting rate of OsMDH transgenic plants in the control group; c) thousand-grain weight of OsMDH transgenic plants in the control group; d) seed setting rate of OsMDH transgenic plants in the high-temperature treatment group; e) thousand-grain weight of OsMDH transgenic plants in the high-temperature treatment group. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0027] The following uses the japonica rice variety “(Oryza sativa cv.Dongjin)” (seeds stored in this experiment) as an example to illustrate the specific implementation method of this method.

[0028] (I) OsMDH Overexpression Vector

[0029] Construction of OsMDH overexpression vector pCAMBIA1300(GFP)-OsMDH

[0030] The amino acid sequence of malate dehydrogenase OsMDH is shown in SEQ ID NO.1:

[0031] MAAIDLSSPARSSPAPLSPRRGSLHLLLRRPRRPTLRCSLDAAPKQAQAQGPPAAVAAE

[0032] EAPTARKECYGVFCTTYDLRADEKTKSWKSLVNVAVSGAAGMISNHLLFKLASGEVF

[0033] GPDQPIALKLLGSERSIQALEGVAMELEDSLYPLLREVSIGIDPYVVFEDAEWALLIGA

[0034] KPRGPGMERSALLDINGQIFAEQGKALNSVASRNVKVIVVGNPCNTNALICLKNAPNI

[0035] PAKNFHALTRLDENRAKCQLALKAGVFYDKVSNMTIWGNHSTTQVPDFLNAKINGR

[0036] PVKEVIKDTKWLEDEFTKTVQKRGGVLIQKWGRSSAASTAVSIVDAIRSLVNPTPEGD

[0037] WFSTGVYTTGNPYGIAEDIVFSMPCRSKGDGDYELVKDVAMDDFLWERIKKSEAELLAEKRCVAHLTGEGNAFCDLPGDTMLPGEM*

[0038] The CDS sequence of the malate dehydrogenase gene is shown in SEQ ID NO.2:

[0039]

[0040] The 5' primer was designed starting from the ATG coding start site of the OsMDH gene (chromosome 8, CDS coordinates: 28141042-28146270, LOC_Os08g44810), and the 3' primer was designed before the stop codon.

[0041] Primer 1: SEQ ID NO.3: 5'-aaaCCATGG CGATGGCGGCAATCGATCTCTC -3';

[0042] Primer 2: SEQ ID NO.4: 5'-aaaCAGATCTG CATTTCTCCTGGAAGCATGG -3';

[0043] In primer 1, the CCATGG sequence is the restriction site for restriction endonuclease Nco I, and the underlined sequence CGATGGCGGCAATCGATCTCTC is the coding sequence for the OsMDH gene. In primer 2, the CAGATCTG sequence is the restriction site for restriction endonuclease Bg II, and the underlined sequence CATTTCTCCTGGAAGCATGG is the coding sequence for the OsMDH gene. These primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0044] Using cDNA from the japonica rice variety (Oryza sativa cv. Dongjin) as a template, PCR amplification was performed using the specific primers described above (1 cycle at 95℃ for 5 min; 45 cycles at 95℃ for 45 s, 58℃ for 1 min, and 72℃ for 1.5 min; 1 cycle at 72℃ for 10 min). The PCR amplification products were recovered and double-digested with restriction endonucleases Nco I and Bgl II (using the standard Takara restriction endonuclease system). The digested products were recovered. pCAMBIA1300(GFP) was double-digested with restriction endonucleases Nco I and Bgl II, and the backbone vector was recovered. The digested products and the backbone vector were ligated, and then sequenced. The sequencing results showed that the pCAMBIA1300(GFP)-OsMDH vector was obtained.

[0045] (II) Obtaining OsMDH transgenic plants

[0046] The recombinant expression vector pCAMBIA1300(GFP)-OsMDH was introduced into Agrobacterium EHA105 by electroporation to obtain recombinant Agrobacterium.

[0047] Recombinant Agrobacterium containing pCAMBIA1300(GFP)-OsMDH was used to infect callus tissue of japonica rice (Oryza sativac v. Dongjin). The infected callus tissue was blotted dry on sterile filter paper and then transferred to a standard co-culture medium for incubation at 24°C in the dark for 4 days. The washed callus tissue was then transferred to a selective medium containing 60 mg / L hygromycin for resistance selection. The selected resistant callus tissue was transferred to a pre-differentiation medium for 7 days and then to a standard differentiation medium for light culture. When the seedlings reached 4 cm in height, they were transferred to test tubes containing standard rooting medium and grown for 2-3 weeks. After 2-3 days of hardening off, the well-grown seedlings were transplanted into a greenhouse (T0 generation). The seedlings were then subcultured to obtain the T1 generation.

[0048] (III) Identification of OsMDH transgenic plants

[0049] Two-week-old wild-type rice (Oryza sativa cv. Dongjin) and T1 generation rice seedlings were collected, and mRNA was extracted and reverse transcribed to obtain cDNA. The expression level of OsMDH in rice was analyzed using quantitative real-time PCR (forward primer: SEQ ID NO.5: 5'-GGGTGATGGTGACTACGAAC-3'; reverse primer: SEQ ID NO.6: 5'-CTCCTGGAAGCATGGTGTCT-3'). The expression level of OsMDH gene in wild-type rice (WT) was taken as 1, and the expression levels of OsMDH gene in overexpression lines OX-1 and OX-2 were as follows: Figure 1 As shown in the figure. The results showed that the expression level of the OsMDH gene in both overexpression lines was more than 10 times that of wild-type rice.

[0050] (iv) Obtaining the OsMDH mutant

[0051] Transcript sequences of the OsMDH gene were analyzed and purchased from the Korean rice t-DNA mutant database (PFG_1E-05243 and PFG_2A-20731), with a genetic background of the japonica rice variety dongjin (WT).

[0052] Using three pairs of primers—primer F1, primer F2, and primer F3—and leaf DNA from different strains as templates, the mutants KO-1 (PFG_1E-05243) and KO-2 (PFG_2A-20731) were identified.

[0053] Primer F1: SEQ ID NO.7: 5'-ATGGACACTACACTACACCT-3';

[0054] Primer R1: SEQ ID NO.8: 5'-CATACTTAGTGTTACAGGGG-3';

[0055] Primer F2: SEQ ID NO.9: 5'-CTAGAGTCGAGAATTCAGTACA-3'.

[0056] The results of the identification are as follows Figure 2 As shown, the results indicate that both lines are OsMDH gene T-DNA insertion mutants.

[0057] (iv) Analysis of the heat tolerance of OsMDH transgenic plants

[0058] T1 generation transgenic rice seeds of OsMDH were harvested. Two overexpression lines (OX-1, OX-2) and two insertion mutant lines (KO-1, KO-2) were selected for simulated high-temperature stress treatment at the heading stage. Seeds were soaked in water for 3 days to induce germination, and then screened for resistance using 50 mg / L hygromycin. Three days after germination, seedlings were transplanted into soil for soil cultivation, with wild-type dongjin rice grown at the same time serving as a control. The experiment was repeated three times.

[0059] High-temperature treatment (e.g., for soil-grown seedlings that have grown to 14 weeks) Figure 6 a. Control group: 0-4: 27°C, 5-8: 30°C, 9-12: 32°C, 13-16: 34°C, 17-20: 31°C, 21-24: 30°C; High-temperature treatment group: 0-4: 33°C, 5-8: 36°C, 9-12: 38°C, 13-16: 40°C, 17-20: 37°C, 21-24: 36°C. After 7 days of treatment, the photosynthetic rate, canopy temperature, and stomatal aperture of the leaves were measured every other day, and the activities of peroxidase-related enzymes in the leaves (malondialdehyde-MDA, hydrogen peroxide-H2O2, ascorbate peroxidase-APX, superoxide dismutase-SOD, peroxidase-POD, catalase-CAT) were further analyzed.

[0060] OsMDH can inhibit stomatal opening in rice leaves and reduce photosynthetic rate. Figure 4 The plant maintains a low canopy temperature. Figure 3 ), and increase the activity of peroxidase-associated enzymes ( Figure 5 This treatment alleviated high-temperature stress damage. After 7 days of high temperature treatment, and one week after being removed from the greenhouse, the overexpression lines treated with high temperature showed stronger heat tolerance than the wild type, while the mutant lines showed greater sensitivity than the wild type, thus increasing the number of grains per ear and the thousand-grain weight. Figure 6 This indicates that OsMDH can improve the tolerance of rice to high temperature stress. The results are as follows: Figure 3-6 As shown. Among them, Figure 4 CT represents the control group, and HT represents the high-temperature treatment group.

Claims

1. The application of overexpression of malate dehydrogenase gene in improving heat tolerance of rice during the heading stage, characterized in that, The malate dehydrogenase gene sequence is shown in SEQ ID NO.

2.

2. The application of an overexpression vector containing the malate dehydrogenase gene in improving the high-temperature tolerance of rice during the heading stage, characterized in that... The malate dehydrogenase gene sequence is shown in SEQ ID NO.

2.

3. The application according to claim 2, characterized in that, The overexpression vector includes pCAMBIA1300-GFP-OsMDH.

4. The application of recombinant bacteria containing the malate dehydrogenase gene in improving the high-temperature tolerance of rice during the heading stage, characterized in that... The recombinant bacteria containing the malate dehydrogenase gene are obtained by introducing the overexpression vector pCAMBIA1300-GFP-OsMDH into the host bacterial strain. The pCAMBIA1300-GFP-OsMDH is obtained by introducing the malate dehydrogenase gene with the sequence shown in SEQ ID NO.2 into pCAMBIA1300-GFP.

5. A method for obtaining heat-resistant rice at the heading stage using a malate dehydrogenase gene, characterized in that, Wild-type rice was infected with a recombinant bacterium containing the malate dehydrogenase gene to obtain transgenic rice containing the malate dehydrogenase gene, the sequence of which is shown in SEQ ID NO.

2.

6. The method according to claim 5, characterized in that, The method for preparing the recombinant bacteria containing the malate dehydrogenase gene includes the following steps: (1) Using the cDNA of the japonica rice variety dongjin as a template, PCR amplification was performed, followed by double enzyme digestion and ligation with the backbone vector to obtain the recombinant expression vector; (2) Introduce the recombinant expression vector described in step (1) into the host bacteria to obtain recombinant bacteria containing malate dehydrogenase.

7. The method according to claim 6, characterized in that, The sequences of the primer pairs used for PCR amplification are shown in SEQ ID NO.3 and SEQ ID NO.

4.

8. A method for verifying the high-temperature resistance of rice obtained by the method according to any one of claims 5 to 7, characterized in that, Includes the following steps: (1) Using wild-type rice growing at the same time as the control group, the rice seeds obtained by the method described in any one of claims 5 to 7 were soaked in water and cultured to germinate, and then resistance was screened with hygromycin. After germination, the seedlings were transferred to soil for soil cultivation. (2) High temperature treatment was applied to soil-grown seedlings to measure the photosynthetic rate, canopy temperature and stomatal opening of the leaves, and the activity of peroxidase-related enzymes in the leaves was analyzed. (3) When the photosynthetic rate of the rice obtained by the method of any one of claims 5 to 7 is slower than that of the control group, the stomatal opening of the leaves is smaller than that of the control group, and the activity of peroxidase is higher than that of the control group, it indicates that the rice has the ability to withstand high temperatures.

9. The method according to claim 8, characterized in that, The peroxide-related enzymes include one or more of malondialdehyde-MDA, hydrogen peroxide-H2O2, ascorbate peroxidase-APX, superoxide dismutase-SOD, peroxidase-POD, or catalase-CAT.

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