CN117402910B - Application of PP2C01 gene in regulation and control of salt tolerance of rice - Google Patents
Application of PP2C01 gene in regulation and control of salt tolerance of rice Download PDFInfo
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 39
- 235000009566 rice Nutrition 0.000 title claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 19
- 230000015784 hyperosmotic salinity response Effects 0.000 title claims abstract description 17
- 240000007594 Oryza sativa Species 0.000 title description 3
- 241000209094 Oryza Species 0.000 claims abstract description 38
- 238000010362 genome editing Methods 0.000 claims abstract description 6
- 230000001105 regulatory effect Effects 0.000 claims abstract description 6
- 230000001276 controlling effect Effects 0.000 claims abstract description 5
- 108091033409 CRISPR Proteins 0.000 claims description 24
- 238000010354 CRISPR gene editing Methods 0.000 claims description 20
- 239000013604 expression vector Substances 0.000 claims description 8
- 241000589158 Agrobacterium Species 0.000 claims description 7
- 230000001404 mediated effect Effects 0.000 claims description 5
- 239000013612 plasmid Substances 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000011426 transformation method Methods 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 238000003209 gene knockout Methods 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 2
- 238000011160 research Methods 0.000 abstract description 2
- 230000035882 stress Effects 0.000 description 10
- 150000003839 salts Chemical class 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- 239000013598 vector Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000036579 abiotic stress Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000008641 drought stress Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
Abstract
The present invention relates toOsPP2C01The application of the gene in regulating and controlling the salt tolerance of rice. Our research has found thatOsPP2C01Gene regulation of salt tolerance of rice byOsPP2C01The salt tolerance of rice can be obviously improved by gene editing. The invention provides a thought for cultivating a new salt-tolerant rice strain.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a method for preparing a plant geneOsPP2C01The application of the gene in regulating and controlling the salt tolerance of rice.
Background
Rice [ (Oryza sativa L.)Oryza sativaL.) is one of the most important food crops in the world, the planting area is about 1/3 of the total area of the food crops, and nearly half of the population in the world uses rice as staple food. However, in the course of rice production, abiotic stresses (e.g., drought stress, salt stress, heavy metal stress, low temperature stress) seriously affect the growth and development of rice, and thus the yield of rice. Salt stress is one of the main abiotic stresses in the rice planting process, and high salt not only causes ionic toxicity and hypertonic stress, but also accompanies secondary stress (such as oxidative damage). The high salt condition can inhibit the normal growth and development of the rice, and even cause death of the rice when serious. Therefore, more key genes involved in regulating and controlling the salt tolerance of the rice are mined, and the rice breeding for resisting high salt stress is bredThe material has important significance for the development of rice breeding and planting industries.
Researchers have now studied to find some genes related to rice salt tolerance, but at presentOsPP2C01The application of the gene in the aspect of regulating the salt tolerance of rice has not been reported yet.
Disclosure of Invention
The present invention aims to provideOsPP2C01The application of the gene in regulating and controlling the salt tolerance of rice.
The technical scheme of the invention is as follows:
OsPP2C01application of gene in regulation and control of salt tolerance of rice, said geneOsPP2C01The genome sequence of the gene is shown as SEQ ID NO.1, the CDS sequence is shown as SEQ ID NO.2, and the protein sequence is shown as SEQ ID NO. 3.
Preferably, in practical use, the gene editing techniques such as CRISPR/Cas9 pairs can be usedOsPP2C01The gene is knocked out to obtain the high salt tolerance rice.
Further preferred, the application is: designing a primer sequence for amplifying a target sequence by taking a sequence shown as SEQ ID NO.4 as a target sequence 1 and a sequence shown as SEQ ID NO.5 as a target sequence 2; PCR amplification is carried out by taking pCBC-MT1T2 plasmid as a template to obtain a PCR product; purifying the PCR product, and recombining to obtain a CRISPR/Cas9-OsPP2C01 expression vector by using an enzyme digestion-connection mode; and transforming the CRISPR/Cas9-OsPP2C01 expression vector with correct sequence into rice by an agrobacterium-mediated transformation method to obtain transformed seedlings.
Preferably, the primer sequences are shown as SEQ ID NO.6 and SEQ ID NO. 7.
Preferably, the variety of rice includes Zhonghua 11.
Compared with the prior art, the invention has the beneficial effects that:
our research has found thatOsPP2C01Gene regulation of salt tolerance of rice byOsPP2C01The salt tolerance of the rice can be obviously improved by gene editing, and the high salt tolerance rice can be obtained. The invention provides a simple and effective technical means for quickly creating a new strain of the brine-tolerant rice.
Drawings
Fig. 1:OsPP2C01gene structure and knockout target information.
Fig. 2:OsPP2C01salt tolerance test results.
Detailed Description
The present invention will be further described with reference to specific examples and drawings for a better understanding of the technical contents of the present invention to those skilled in the art.
EXAMPLE 1 construction of CRISPR/Cas9-OsPP2C01 expression vectorOsPP2C01Obtaining of mutant I, construction of CRISPR/Cas9-OsPP2C01 expression vector
From the slaveOsPP2C01Two 20bp targets are selected in CDS sequence regions of the genes to perform double-target knockout of CRISPR/Cas 9. The target sequence is connected to a CRISPR/Cas9 carrier by a PCR method, and the carrier of CRISPR/Cas9-OsPP2C01 is transformed into flower 11 in a rice variety by an agrobacterium-mediated transformation method to obtainOsPP2C01Deletion mutant ricepp2c01-ko-1Andpp2c01-ko-2) (FIG. 1).
1. The CRISPR/Cas9-OsPP2C01 expression vector is constructed as follows:
for editingOsPP2C01CRISPR vectors of genes express 2 sgrnas: and the coding sequence of the sgRNA1 recognition region and the sgRNA2 recognition region are 148 th to 166 th positions of a sequence 2 (SEQ ID NO. 2), and the coding sequence of the sgRNA2 recognition region is 667 th to 689 th positions of the sequence 2 (SEQ ID NO. 2). Two target sequences were designed as follows:
target 1: CTAATACCACACCCAAGAA (SEQ ID NO. 4)
Target 2: TCTGTCATTTCCGAGTCTCCAGG (SEQ ID NO. 5)
Designing a primer sequence according to the target point, wherein the primer sequence is as follows:
BsF:AATAATGGTCTCAGGCGCTAATACCACACCCAAGAAGTTTTAGAGCTAGAAATAGC（SEQ ID NO.6）
BsR:ATTATTGGTCTCTAAACCCTGGAGACTCGGAAATGACAGACGCTTCTTGGTGCC（SEQ ID NO.7）
PCR amplification was performed using the 100-fold diluted pCBC-MT1T2 plasmid as a template and BsF and BsR as primers to obtain a PCR product. And (3) purifying the PCR product, and obtaining the CRISPR/Cas9-OsPP2C01 expression vector by using the following enzyme digestion-connection mode. Vector construction methods refer to Xing HL, dong L, wang ZP, zhang HY, han CY, liu B, wang XC, chen QJ. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant biol 2014 Nov 29;14:327.
Cleavage-ligation system:
2. authentication
5ul of ligation product was used to transform E.coli competence. Screening was performed on LB plates containing 50. Mu.g/mL kanamycin. Colony PCR identifies monoclonal, selects positive clone, extracts plasmid and sends it to sequence. Sequencing results show that the plasmid of the positive clone is a recombinant CRISPR vector CRISPR/Cas9-OsPP2C01.
2. CRISPR/Cas9-OsPP2C01 transformed rice callus and positive seedling identification
1. Agrobacterium transformation
The recombinant vector CRISPR/Cas9-OsPP2C01 with correct sequencing is transformed into the agrobacterium competent GV3101 strain by an electric excitation method, and bacterial colony PCR identification is carried out for later use after verification.
The single colony of the agrobacteria GV3101/CRISPR/Cas9-OsPP2C01 with correct identification is inoculated into 2-3mL of liquid culture medium containing 100 mug/mL kanamycin and 50 mug/mL rifampicin, shake-cultured overnight at 28 ℃, transferred into a large amount of liquid culture medium containing antibiotics for shake-cultured the next day, and after transferred for several times, bacterial bodies are collected and resuspended to an OD600 of between 0.8 and 1.0. Transferring the fungus GV3101/CRISPR/Cas9-OsPP2C01 into flower 11 by adopting an agrobacterium-mediated method, infecting the young embryo with the agrobacterium tumefaciens GV3101, placing the young embryo invaded by the agrobacterium tumefaciens GV3101 on a selection culture medium for multiple screening to obtain a resistant callus, and regenerating the resistant callus into seedlings to obtain T0 generation transformed seedlings. The Agrobacterium tumefaciens transformation method is described in Zhao, W., zheng, S. & Ling, HQ. An efficient regeneration system and Agrobacterium-mediated transformation of Chinese upland rice cultivar Handao297. Plant Cell Tiss Organ Cult 106.106, 475-483 (2011).
2.OsPP2C01Identification of mutant T0 generation plants
Extracting DNA from T0 generation transgenic rice plant leaves, performing PCR amplification by taking genomic DNA as a template, and sequencing by taking Zhonghua 11 as a control. The amplified products were sequenced and compared with the Zhonghua 11 sequence, and the identified effective mutant lines, respectively designated aspp2c01-ko-1Andpp2c01-ko-2. Wherein:
pp2c01-ko-1deletion of one base "C" at the second target (i.e., deletion of base "C" at position 17 of SEQ ID NO.5, as shown in FIG. 1) results in a frame shift mutation, therebyOsPP2C01Mutant lines obtained by gene knockout are obtained correspondinglyOsPP2C01Mutants of the gene.
pp2c01-ko-2Is a second target point inserted with a T (namely, a T is inserted between 18 th and 19 th positions of SEQ ID NO.5 as shown in figure 1), so that translation is terminated in advance, therebyOsPP2C01Mutant lines obtained by gene knockout are obtained correspondinglyOsPP2C01Mutants of the gene.
Example 2 different strainsOsPP2C01Salt tolerance function verification of gene editing plant
Selecting wild medium flower 11 (ZH 11),pp2c01-ko-1Andpp2c01-ko-2adopts a greenhouse water planting method. Seed 10% H 2 O 2 Sterilizing for 10min, and adding distilled water to remove H on seed surface 2 O 2 Washing, uniformly placing the seeds in a culture dish with sterilizing filter paper, germinating in the dark for 2d in a constant temperature incubator at 37 ℃, and accelerating germination for one day at 28 ℃. The germinated seeds are placed in a water culture box with a filter screen for 7d, and the nutrient solution in the water culture box is replaced every 2 d. Culture growth conditions 28/25 ℃ (day/night, approximately 70% relative humidity), photoperiod 14/10h (day/night), illumination intensity 400 μmol.m -2 ·s -1 . Selecting good-growth rice seedlings with the leaf age of 3-leaf stage, and performing salt stress treatment by using 0.6% NaCl. After 7d of salt stress, compared with ZH11,pp2c01-kothe survival of the lines was significantly improved (fig. 2), indicating knockoutOsPP2C01The gene can obviously improve the salt stress resistance of rice。
Nutrient solution formula table:
when in use, the working solution is prepared at present, each liter of working solution needs 1.25ml of mother solution, and the pH value is 5.5.
The above embodiments are only examples of the present invention, and the present invention is not limited thereto, but any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (3)
1. OsPP2C01The application of the gene in regulating and controlling the salt tolerance of rice is characterized by comprising the following steps: pairing by CRISPR/Cas9 gene editing technologyOsPP2C01Gene knockout is carried out to improve the salt tolerance of the rice;
designing a primer sequence for amplifying a target sequence by taking a sequence shown as SEQ ID NO.4 as a target sequence 1 and a sequence shown as SEQ ID NO.5 as a target sequence 2; PCR amplification is carried out by taking pCBC-MT1T2 plasmid as a template to obtain a PCR product; purifying the PCR product, and recombining to obtain a CRISPR/Cas9-OsPP2C01 expression vector by using an enzyme digestion-connection mode; transforming the CRISPR/Cas9-OsPP2C01 expression vector with correct sequence into rice by an agrobacterium-mediated transformation method to obtain transformed seedlings;
the saidOsPP2C01The CDS sequence of the gene is shown as SEQ ID NO. 2.
2. The use according to claim 1, wherein the primer sequences are shown in SEQ ID No.6 and SEQ ID No. 7.
3. The use according to any one of claims 1-2, wherein the rice variety comprises Zhonghua 11.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102943084A (en) * | 2012-11-28 | 2013-02-27 | 上海市农业生物基因中心 | Rice stress resistance related gene OsPP2C44 and coded protein and application thereof |
| CN116855519A (en) * | 2023-08-05 | 2023-10-10 | 吉林农业大学 | Cyperus esculentus CePP2C19 gene and application thereof |
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| JP2005185101A (en) * | 2002-05-30 | 2005-07-14 | National Institute Of Agrobiological Sciences | VEGETABLE FULL-LENGTH cDNA AND UTILIZATION THEREOF |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102943084A (en) * | 2012-11-28 | 2013-02-27 | 上海市农业生物基因中心 | Rice stress resistance related gene OsPP2C44 and coded protein and application thereof |
| CN116855519A (en) * | 2023-08-05 | 2023-10-10 | 吉林农业大学 | Cyperus esculentus CePP2C19 gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| The Protein Phosphatase Calcineurin Is Essential for NaCl Tolerance of Saccharomyces cerevisiae;Imelda Mendoza et al;THE JOURNAL OF BIOLOGICAL CHEMISTRY;19940325;第269卷(第12期);第8792-8796页 * |
| 基于全基因组水稻蛋白磷酸酶的鉴定、进化和表达分析;杨猛;中国优秀硕士论文电子期刊网;20080601;全文 * |
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