CN117904150A - Application of malate dehydrogenase gene in improving high temperature resistance of rice - Google Patents
Application of malate dehydrogenase gene in improving high temperature resistance of rice Download PDFInfo
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Abstract
The invention discloses application of a malate dehydrogenase gene in improving rice high temperature resistance, wherein the malate dehydrogenase gene sequence is shown as SEQ ID NO. 2. The experimental phenotype is obtained by obtaining the rice strain transformed with the malate dehydrogenase gene and the insertion mutant strain: the over-expression OsMDH can obviously improve the tolerance of the rice heading period to high temperature stress, so that the plant can normally grow when encountering high temperature stress, the degradation of the over-expression transgenic strain is weaker than that of a wild-type spike, and the grains are well enriched; the OsMDH gene function deletion reduces the tolerance of rice heading period to high temperature stress, the degeneration of the insertion mutant transgenic strain is more serious relative to the wild-type spike, and the fruiting rate and the fullness are obviously reduced.
Description
Technical Field
The invention relates to application of a malate dehydrogenase gene in improving high temperature resistance of rice, and belongs to the technical field of biological genes.
Background
Rice is one of the most important grain crops in China. As greenhouse gases such as carbon dioxide, which are highly endothermic, increase year by year, resulting in an increase in surface air temperature, it is expected that the global air temperature will rise to 3-6 ℃ in 2100 years. Under the background of global warming, high-temperature heat waves frequently occur, and the cultivation of high-quality rice varieties with high temperature resistance has become a focus. The increase of air temperature seriously threatens the growth and production of crops, causes the effective functions She Zaocui of plants to yellow, the activity of enzymes to be reduced, the photosynthetic efficiency to be reduced and the photosynthetic assimilation conveying capacity to be reduced, so that the seeds are not full and thousand seed weight to be reduced, and the annual high temperature is estimated to cause 15-35% of crop yield reduction.
The rice in China is distributed in the south, indica and north, the polished round-grained nonglutinous rice has better cold resistance and drought resistance, while the polished long-grained nonglutinous rice is more heat-resistant, strong light-resistant and moisture-resistant, and shows heat resistance similar to C4 plants such as corn. C3 plants are mainly distributed in temperate and cold regions, whereas C4 plants originate in tropical and subtropical regions of high temperature and drought. Compared to C3 plants, C4 plants breathe less light, which promotes C4 plants to adapt to higher temperatures than C3 plants. Leaf structure and key enzyme activity of C4 plants may increase the efficiency of photosynthetic product transport, water partitioning, and stress response. The transition of the photosynthetic pathway from the C3 pathway to the C4 pathway is relatively simple, evolving independently from multiple sources in each family and genus of angiosperms. In monocotyledono She He plants, maize and rice are considered to have evolved in branches about 5 thousands of years ago, one forming C3 plants, such as rice, wheat, and the other forming C4 plants, such as maize, sorghum. Approximately 1 million years ago, rice and corn were gradually acclimatized to the current cultivar, and showed better collinearity between chromosomes and strong conservation between gene sequences in the corn and rice genomes. This indicates from the molecular evolution level that C3 rice has the potential to evolve into high heat resistance and high light resistance of C4 crops. Therefore, in order to facilitate the alleviation of the influence of plant high temperature resistance, it is necessary to propose an improvement method for improving stress tolerance and related traits of plants.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing application of a malate dehydrogenase gene for reducing tolerance to high temperature stress in the heading period of rice in improving the high temperature resistance of the rice.
The technical scheme is as follows: in order to solve the technical problems, the invention provides application of a malate dehydrogenase gene in improving high temperature resistance of rice, and the malate dehydrogenase gene sequence is shown as SEQ ID No. 2.
The invention also provides application of the overexpression vector containing the malate dehydrogenase gene in improving the high temperature resistance of rice, and the malate dehydrogenase gene sequence is shown as SEQ ID No. 2.
Wherein the overexpression vector comprises pCAMBIA1300 (GFP) -OsMDH.
The invention also provides application of the recombinant bacterium containing the malate dehydrogenase gene in improving the high temperature resistance of rice, wherein the recombinant bacterium containing the malate dehydrogenase gene is obtained by introducing an over-expression vector pCAMBIA1300 (GFP) -OsMDH into a host strain, and the pCAMBIA1300 (GFP) -OsMDH is obtained by introducing the malate dehydrogenase gene with a sequence shown as SEQ ID NO.2 into the pCAMBIA1300 (GFP).
The invention also provides a method for obtaining high-temperature resistant rice by utilizing the malate dehydrogenase gene, which comprises the step of infecting wild rice by using recombinant bacteria containing the malate dehydrogenase gene to obtain transgenic rice containing the malate dehydrogenase gene, wherein the malate dehydrogenase gene sequence is shown as SEQ ID NO. 2.
Wherein, the preparation method of the recombinant bacteria containing the malate dehydrogenase gene comprises the following steps:
(1) Carrying out PCR amplification by taking cDNA of japonica rice dongjin as a template, and connecting the cDNA with a skeleton vector by double enzyme digestion to obtain a recombinant expression vector;
(2) And (3) introducing the recombinant expression vector in the step (1) into host bacteria to obtain recombinant bacteria containing malate dehydrogenase.
Wherein, the sequences of the primer pairs for PCR amplification are shown as SEQ ID NO.3 and SEQ ID NO. 4.
The invention also provides a method for verifying the high temperature resistance of the rice obtained by the method, which comprises the following steps:
(1) Taking wild rice growing in the same period as a control group, soaking seeds of the rice obtained by the method in water, culturing to germinate, then carrying out resistance screening by hygromycin, and after germination, transferring seedlings to soil for soil culture;
(2) Performing high-temperature treatment on the soil culture Miao Jin, measuring the photosynthetic rate, canopy temperature and pore opening of the leaf, and analyzing the activity of leaf peroxide-related enzyme;
(3) When the photosynthetic rate of the rice obtained by the method is slower than that of the control group, the leaf pore opening is smaller than that of the control group, and the activity of the peroxide-related enzyme is higher than that of the control group, the rice has high temperature resistance.
Wherein the peroxide-related enzyme comprises one or more of malondialdehyde-MDA, hydrogen peroxide-H 2O2, ascorbate peroxidase-APX, superoxide dismutase-SOD, peroxidase-POD or catalase-CAT.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: 1. the invention obtains the experimental phenotype by obtaining the transgenic malate dehydrogenase (OsMDH) gene rice strain and the insertion mutant strain: the over-expression OsMDH can obviously improve the tolerance of the rice heading period to high temperature stress, so that the plant can normally grow when encountering high temperature stress, the degradation of the over-expression transgenic strain is weaker than that of a wild-type spike, and the grains are well enriched; the OsMDH gene function deletion reduces the tolerance of the rice to high temperature stress in the heading period, the degeneration of the insertion mutant transgenic strain is more serious relative to the wild-type spike, and the fruiting rate and the fullness are obviously reduced; 2. the protein and the coding gene thereof have important theoretical and practical significance for researching the stress tolerance mechanism of plants and improving the stress tolerance and related properties of the plants, and play an important role in improving the stress tolerance genetic engineering of the plants, so that the protein and the coding gene have wide application prospect.
Drawings
FIG. 1 shows the result of OsMDH gene over-expression gene expression test;
FIG. 2 shows the identification of OsMDH insert mutant plants;
FIG. 3 is a plant canopy temperature analysis of OsMDH transgenic plants and control plants after 3 days at high temperature: a is plant morphology; b is the different tissue temperatures of the plants; c is the surface temperature of flag leaves;
FIG. 4 shows the stomatal opening and photosynthetic rate analysis of OsMDH transgenic plants at high Wen Zu (3 days, 5 days at high temperature) and control plants: a is the open area of the air holes; b is the surface temperature of the blade; c is the transpiration rate; d is the air hole conductivity; e is intracellular CO 2 concentration;
FIG. 5 shows the peroxide-related enzyme activity assays for OsMDH transgenic plants at high Wen Zu (3 days, 5 days at high temperature) and control plants: a is malondialdehyde-MDA content; b is hydrogen peroxide-H 2O2 content; c is the ascorbate peroxidase-APX content; d is peroxidase-POD content; e is catalase-CAT content; f is superoxide dismutase-SOD;
FIG. 6 shows high Wen Zu (3 days, 5 days at high temperature) and control set seed setting rate and thousand kernel weight analysis for OsMDH transgenic plants: a is the temperature of one day of the control group and the high temperature treatment group; b is the seed setting rate of the transgenic plants of the control group OsMDH; c is thousand kernel weight of transgenic plants of the control group OsMDH; d is the fruiting rate of the transgenic plants in the high-temperature treatment group OsMDH; e is thousand kernel weight of transgenic plants of high temperature treatment group OsMDH.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
Specific embodiments of the present method will be described below by taking the rice variety (Oryza sativa cv. Dongjin) as an example (seeds are stored in the present experiment).
OsMDH super-expression vector
Construction of OsMDH super-expression vector pCAMBIA1300 (GFP) -OsMDH
The amino acid sequence of malate dehydrogenase OsMDH is shown in SEQ ID NO. 1:
MAAIDLSSPARSSPAPLSPRRGSLHLLLRRPRRPTLRCSLDAAPKQAQAQGPPAAVAAE
EAPTARKECYGVFCTTYDLRADEKTKSWKSLVNVAVSGAAGMISNHLLFKLASGEVF
GPDQPIALKLLGSERSIQALEGVAMELEDSLYPLLREVSIGIDPYVVFEDAEWALLIGA
KPRGPGMERSALLDINGQIFAEQGKALNSVASRNVKVIVVGNPCNTNALICLKNAPNI
PAKNFHALTRLDENRAKCQLALKAGVFYDKVSNMTIWGNHSTTQVPDFLNAKINGR
PVKEVIKDTKWLEDEFTKTVQKRGGVLIQKWGRSSAASTAVSIVDAIRSLVNPTPEGD
WFSTGVYTTGNPYGIAEDIVFSMPCRSKGDGDYELVKDVAMDDFLWERIKKSEAELLAEKRCVAHLTGEGNAFCDLPGDTMLPGEM*
the CDS sequence of the malate dehydrogenase gene is shown in SEQ ID NO. 2:
ATGGCGGCAATCGATCTCTCCTCCCCCGCAAGATCATCGCCGGCGCCTCTCTCCCCGCGCCGCGGCTCCCTGCACCTCCTCCTCCGACGCCCGCGCCGACCCACCCTCCGCTGCTCCCTCGACGCCGCGCCAAAGCAGGCTCAGGCTCAGGGCCCGCCCGCGGCGGTCGCGGCGGAGGAGGCGCCCACCGCGCGCAAGGAGTGCTATGGCGTCTTCTGCACCACCTACGACCTCAGGGCGGATGAGAAGACAAAGTCCTGGAAGAGCTTAGTGAATGTTGCTGTGTCGGGTGCAGCAGGGATGATATCAAATCATCTGCTTTTCAAACTTGCCTCTGGTGAGGTTTTTGGACCGGACCAACCAATAGCACTTAAGTTGTTGGGCTCTGAAAGATCAATACAAGCACTTGAAGGTGTGGCTATGGAATTGGAGGATTCACTGTATCCATTGCTAAGGGAAGTCAGCATTGGTATAGACCCTTATGTAGTCTTCGAAGATGCAGAATGGGCTCTTCTTATTGGGGCTAAGCCCAGAGGTCCTGGAATGGAGCGATCTGCCTTACTAGATATCAACGGTCAAATCTTTGCTGAACAGGGGAAAGCACTTAACTCCGTGGCATCTCGGAATGTGAAGGTCATAGTTGTCGGGAACCCCTGTAACACTAATGCATTGATTTGCTTAAAGAATGCTCCCAACATACCAGCAAAAAACTTTCATGCATTGACGAGGTTGGATGAAAATAGAGCCAAGTGCCAGCTGGCACTAAAAGCGGGTGTATTTTATGACAAAGTATCAAATATGACTATTTGGGGGAACCACTCAACAACTCAGGTTCCTGATTTTTTGAATGCTAAAATTAACGGGAGACCAGTGAAAGAAGTCATTAAAGACACAAAGTGGTTAGAAGATGAATTCACCAAAACAGTTCAAAAGCGTGGAGGAGTACTCATACAAAAATGGGGCAGATCTTCAGCTGCATCAACCGCCGTTTCCATCGTGGATGCTATTAGGTCCCTTGTAAATCCTACCCCAGAAGGCGATTGGTTCTCTACTGGGGTTTATACGACTGGAAATCCTTATGGCATAGCAGAGGACATCGTGTTCAGTATGCCATGCAGGTCAAAGGGTGATGGTGACTACGAACTAGTTAAAGATGTGGCAATGGACGATTTCCTCTGGGAACGGATAAAAAAGAGTGAAGCTGAATTGCTCGCCGAGAAAAGATGCGTTGCCCATCTTACTGGAGAGGGCAATGCATTTTGTGATCTTCCCGGAGACACCATGCTTCCAGGAGAAATGTAG
Starting from the coding start site ATG of the OsMDH gene (chromosome 8, CDS coordinates: 28141042-28146270, LOC_Os08g44810), a 5 'primer was designed, and a 3' primer was designed before the stop codon:
primer 1: SEQ ID NO.3:5'-aaaCCATGGCGATGGCGGCAATCGATCTCTC-3';
primer 2: SEQ ID NO.4:5'-aaaCAGATCTGCATTTCTCCTGGAAGCATGG-3';
The CCATGG sequence in the primer 1 is the restriction enzyme cleavage site of restriction enzyme Nco I, and the underlined sequence CGATGGCGGCAATCGATCTCTC is the coding sequence of OsMDH gene; the CAGATCTG sequence in primer 2 is the restriction enzyme Bg II cleavage site, and the underlined sequence CATTTCTCCTGGAAGCATGG is the OsMDH gene coding sequence. The above primers were synthesized by Beijing Optimuno Co., ltd.
PCR amplification (5 minutes at 95 ℃,1 cycle at 95 ℃,45 seconds at 58 ℃,1 minute at 72 ℃, 1.5 minutes at 45 cycles at 72 ℃ 10 minutes at 72 ℃) was performed using cDNA of japonica rice variety (Oryza sativa cv. Dongjin) as a template, and the PCR amplified product was recovered and double-digested with restriction enzymes Nco I and Bgl II (conventional use system for restriction enzymes by Takara Co.), and the digested product was recovered; the pCAMBIA1300 (GFP) was digested with restriction enzymes Nco I and Bgl II to recover the backbone vector; and connecting the enzyme digestion product with the framework vector, and then sequencing, wherein a sequencing result shows that the pCAMBIA1300 (GFP) -OsMDH vector is obtained.
(II) obtaining OsMDH transgenic plants
The recombinant expression vector pCAMBIA1300 (GFP) -OsMDH is introduced into the agrobacterium EHA105 by using an electric shock method to obtain recombinant agrobacterium.
The recombinant agrobacterium containing pCAMBIA1300 (GFP) -OsMDH is used for transfecting the calli of japonica rice (Oryza sativa cv. Dongjin); the impregnated calli are dried by suction on sterile filter paper and transferred to a conventional co-culture medium for dark culture at 24 ℃ for 4 days; the washed calli were transferred to selection medium containing 60 mg hygromycin per liter for resistance selection; transferring the selected resistant callus to a pre-differentiation culture medium for 7 days, and transferring to a conventional differentiation culture medium for illumination culture; transferring the seedlings to a test tube filled with a conventional rooting culture medium for 2-3 weeks when the seedlings grow to 4cm, and transplanting the seedlings which grow well to a greenhouse (T0 generation) after hardening the seedlings for 2-3 days. And respectively passaging the seedlings to obtain the generation T1.
(III) OsMDH identification of transgenic plants
Wild rice seedlings (Oryza sativa cv. Dongjin) and T1 generation rice seedlings were taken for 2 weeks, mRNA was extracted and reverse transcribed, and the expression level of OsMDH in rice was analyzed by fluorescent quantitative PCR technique after cDNA was obtained (upstream primer: SEQ ID NO.5:5'-GGGTGATGGTGACTACGAAC-3'; downstream primer: SEQ ID NO.6: 5'-CTCCTGGAAGCATGGTGTCT-3'). The expression level of OsMDH gene in the overexpressing lines OX-1 and OX-2 was shown in FIG. 1, with the expression level of OsMDH gene in wild-type rice (WT) as 1. The results show that: the expression quantity of OsMDH genes in the 2 over-expression lines is more than 10 times of that of wild rice.
(IV) OsMDH mutant acquisition
Transcript sequences of OsMDH genes were analyzed and purchased from Korean rice T-DNA mutant library (rice T-DNA insertion seqence database) with the numbers PFG_1E-05243 and PFG_2A-20731, and the genetic background was japonica rice variety dongjin (WT).
By three pairs of primers: the primers F1, F2 and F3 were identified with different strain leaf DNAs as templates to obtain KO-1 (PFG_1E-05243) and KO-2 (PFG_2A-20731) mutants.
Primer F1: SEQ ID NO.7:5'-ATGGACACTACACTACACCT-3';
Primer R1: SEQ ID NO.8:5'-CATACTTAGTGTTACAGGGG-3';
Primer F2: SEQ ID NO.9:5'-CTAGAGTCGAGAATTCAGTACA-3'.
The identification results are shown in fig. 2, and the results show that: each of the 2 lines was OsMDH gene T-DNA insertion mutants.
(IV) high temperature resistance analysis of OsMDH transgenic plants
And harvesting OsMDH T1 generation transgenic rice seeds, and respectively selecting two over-expression strains (OX-1 and OX-2) and two insertion mutant strains (KO-1 and KO-2) to perform heading stage simulated high temperature treatment stress treatment. Seed soaking is carried out for 3 days to enable the seed to germinate, then 50mg/L hygromycin is used for resistance screening, seedlings are moved to soil for soil cultivation after 3 days of germination, and the test is repeated for 3 times by taking a wild type dongjin which grows at the same time as a control.
The soil culture Miao Jin grown to 14 weeks was subjected to high temperature treatment (as shown in FIG. 6a, control: 0-4 at 27 degrees, 5-8 at 30 degrees, 9-12 at 32 degrees, 13-16 at 34 degrees, 17-20 at 31 degrees, 21-24 at 30 degrees; high temperature treatment: 0-4 at 33 degrees, 5-8 at 36 degrees, 9-12 at 38 degrees, 13-16 at 40 degrees, 17-20 at 37 degrees, 21-24 at 36 degrees) for 7 days. Every other day, leaf photosynthetic rate, canopy temperature and stomatal opening were measured and leaf peroxide-related enzyme activities (malondialdehyde-MDA, hydrogen peroxide-H 2O2, ascorbate peroxidase-APX, superoxide dismutase-SOD, peroxidase-POD, catalase-CAT) were further analyzed.
OsMDH can inhibit the stomata opening of rice leaves, reduce photosynthetic rate (figure 4), maintain lower canopy temperature (figure 3), improve activity of peroxide-related enzyme (figure 5), and relieve high temperature stress injury. After 7 days at high temperature, after one week of removal from the greenhouse, the overexpressed strain showed a stronger high temperature resistance than the wild type, whereas the mutant strain showed a more sensitive than the wild type, thereby increasing the number of grains per ear and thousand kernel weight (fig. 6), indicating that OsMDH can increase the tolerance of rice to high temperature stress. The results are shown in FIGS. 3-6. In fig. 4, CT is a control group, and HT is a high temperature treatment group.
Claims (9)
1. The application of the malate dehydrogenase gene in improving the high temperature resistance of rice is characterized in that the malate dehydrogenase gene sequence is shown as SEQ ID NO. 2.
2. The application of the overexpression vector containing the malate dehydrogenase gene in improving the high temperature resistance of rice is characterized in that the malate dehydrogenase gene sequence is shown as SEQ ID No. 2.
3. The use of claim 2, wherein the overexpression vector comprises pCAMBIA1300 (GFP) -OsMDH.
4. The application of the recombinant bacteria containing the malate dehydrogenase gene in improving the high temperature resistance of rice is characterized in that the recombinant bacteria containing the malate dehydrogenase gene is obtained by introducing an over-expression vector pCAMBIA1300 (GFP) -OsMDH into a host strain, and the pCAMBIA1300 (GFP) -OsMDH is obtained by introducing the malate dehydrogenase gene with the sequence shown as SEQ ID NO.2 into the pCAMBIA1300 (GFP).
5. A method for obtaining high-temperature resistant rice by utilizing a malate dehydrogenase gene is characterized in that recombinant bacteria containing the malate dehydrogenase gene are used for infecting wild rice to obtain transgenic rice containing the malate dehydrogenase gene, and the malate dehydrogenase gene sequence is shown as SEQ ID NO. 2.
6. The method according to claim 5, wherein the method for producing the recombinant bacterium containing a malate dehydrogenase gene comprises the steps of:
(1) Carrying out PCR amplification by taking cDNA of japonica rice dongjin as a template, and connecting the cDNA with a skeleton vector by double enzyme digestion to obtain a recombinant expression vector;
(2) And (3) introducing the recombinant expression vector in the step (1) into host bacteria to obtain recombinant bacteria containing malate dehydrogenase.
7. The method of claim 6, wherein the sequences of the PCR amplified primer pairs are set forth in SEQ ID NO.3 and SEQ ID NO. 4.
8. A method of verifying the high temperature resistance of rice obtained by the method of any one of claims 5 to 7, comprising the steps of:
(1) Taking wild rice growing in the same period as a control group, soaking seeds of the rice obtained by the method of any one of claims 5-7 in water for cultivating to germinate, then carrying out resistance screening by hygromycin, and after germination, transferring seedlings to soil for soil culture;
(2) Performing high-temperature treatment on the soil culture Miao Jin, measuring the photosynthetic rate, canopy temperature and pore opening of the leaf, and analyzing the activity of leaf peroxide-related enzyme;
(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 leaf pore opening is smaller than that of the control group, and the activity of the peroxide-related enzyme is higher than that of the control group, it is indicated that the rice has high temperature resistance.
9. The method of claim 8, wherein the peroxide-related enzyme comprises one or more of malondialdehyde-MDA, hydrogen peroxide-H 2O2, ascorbate peroxidase-APX, superoxide dismutase-SOD, peroxidase-POD, or catalase-CAT.
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