Rice cold-tolerant gene CT4.1 and its application

Abstract

The invention discloses a rice cold-resistance gene CT4.1 and an application thereof, and belongs to the technical field of plant genetic engineering. The invention provides an application of the rice cold-resistance gene CT4.1 in improving the cold resistance of plants, and the nucleotide sequence of the rice cold-resistance gene CT4.1 is shown in SEQ ID NO.1. The invention also provides an application of a protein encoded by the rice cold-resistance gene CT4.1 in improving the cold resistance of plants, and the amino acid sequence of the protein encoded by the rice cold-resistance gene CT4.1 is shown in SEQ ID NO.3. The invention obtains an overexpressed transgenic plant by overexpressing the gene CT4.1, and the survival rate of the transgenic plant after low temperature stress is significantly increased compared with that of wild rice plants, thereby providing new gene resources and breeding resources for rice breeding, and having important application value for effectively improving the cold resistance of rice by using the gene resources through genetic breeding and genetic engineering methods.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a rice cold-resistant gene CT4.1 and an application thereof. Background Art

[0002] Rice ( Oryza sativa L.) is one of the most important crops, originating from tropical and subtropical regions and is sensitive to cold stress. In high-latitude and high-altitude areas, cold stress seriously restricts the growth and production of rice. The direct seeding technology of early rice in the double-season rice area in the south has significant cost savings and efficiency gains, and is very popular among farmers. However, the cold tolerance of early rice varieties is not strong. In order to avoid problems such as rotten seeds and rotten seedlings caused by "late spring cold", the direct seeding time of early rice needs to be about one week later than the seedling transplanting. As a result, the direct seeding technology of early rice can only use early-maturing or medium-early-maturing varieties, which limits the improvement of early rice yield and quality. Even so, in extreme weather, there are still serious losses. Improving the cold tolerance of rice can not only ensure the stable yield of the double-season rice area in the south, but also expand the ecological area of ​​rice cultivation.

[0003] Rice cold tolerance is a complex quantitative trait regulated by micro-effect polygenes. Different scholars have located more than 250 cold tolerance QTLs, such as qLTG3-1, bZIP73, Ctb1, COLD1, qCTS-9, Ghd8, CTB4a, OsGSTZ2, qPSR10 and HAN1, using different mapping populations and cold treatment conditions. Among them, the natural variation of COLD1, CTB4a, qPSR10 and HAN1 genes has been proven to enhance the adaptability of rice to low temperature environments. However, natural alleles with cold tolerance are still rarely found. Most of them are cold tolerance genes identified from genetic populations constructed from japonica-indica rice or natural populations. Their cold tolerance is difficult to break through the existing biological low temperature tolerance of japonica rice, and there are certain limitations in production applications. Jiangxi Dongxiang wild rice (abbreviated as "Dongye", DY) has extremely strong cold resistance. Discovering its new cold-resistant genes has important application value for the breeding of modern rice cold-resistant varieties and the study of the molecular basis of cold resistance. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a rice cold-tolerant gene CT4.1 and its application, which are used to improve the survival rate of rice plants under low temperature stress.

[0005] To achieve the above object, the present invention provides the use of a rice cold-tolerant gene CT4.1 in improving plant cold tolerance. The nucleotide sequence of the rice cold-tolerant gene CT4.1 is shown in SEQ ID NO.1.

[0006] Preferably, the plant is one of rice, wheat, soybean, sorghum, millet, barley or corn.

[0007] The present invention also provides the use of the protein encoded by the rice cold-tolerant gene CT4.1 in improving the cold tolerance of plants. The amino acid sequence of the protein encoded by the rice cold-tolerant gene CT4.1 is shown in SEQ ID NO.3.

[0008] Preferably, the plant is one of rice, wheat, soybean, sorghum, millet, barley or corn.

[0009] The present invention also provides the use of an expression box, a vector or a recombinant bacterium containing the rice cold-tolerant gene CT4.1 in improving the cold-tolerant property of plants.

[0010] The present invention also provides application of the rice cold-resistant gene CT4.1 in cultivating cold-resistant transgenic plants.

[0011] The present invention also provides application of the rice cold-resistant gene CT4.1 in improving plant cold-resistant germplasm resources.

[0012] The present invention also provides the use of the rice cold-resistant gene CT4.1 in improving the low-temperature survival rate of plants, wherein the plant is one of rice, wheat, soybean, sorghum, millet, barley or corn.

[0013] The present invention provides a method for improving the cold tolerance of rice by utilizing the rice cold tolerance gene CT4.1. The rice is made to express or over-express the rice cold tolerance gene CT4.1 by genetic modification, hybridization, backcrossing, selfing or asexual reproduction. The nucleotide sequence of the rice cold tolerance gene CT4.1 is shown in SEQ ID NO.1.

[0014] Preferably, the transgenic comprises introducing a recombinant expression vector containing the rice cold-tolerant gene CT4.1 into rice using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated methods to obtain a transgenic rice line.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects:

[0016] The present invention provides a rice cold-tolerant gene CT4.1 and an application thereof in improving the cold tolerance of rice. Overexpression transgenic plants are obtained by overexpressing the gene CT4.1. Compared with wild rice plants, the survival rate of the transgenic plants after low-temperature stress is significantly increased, thereby providing new gene resources and breeding resources for rice breeding, and having important application value for effectively improving the cold tolerance of rice by utilizing the gene resources through genetic breeding and genetic engineering methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a diagram showing the expression level identification results of the rice cold-tolerant gene CT4.1 overexpression material and the gene CT4.1 in wild rice materials;

[0019] Figure 2 This is a comparison of the phenotypes of rice cold-tolerant gene CT4.1 overexpression materials and wild rice materials after low temperature stress treatment;

[0020] Figure 3 This is a statistical chart of the survival rate of rice cold-tolerant gene CT4.1 overexpression materials and wild rice materials after low temperature stress treatment;

[0021] Figure 4 This is a statistical chart of the survival rate of rice cold-tolerant gene CT4.1 knockout materials and wild rice materials after low temperature stress treatment. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Sources of materials used in the present invention: Indica rice variety Gan Zao Xian 49 (GZX49) was obtained from the Rice Research Institute of Jiangxi Academy of Agricultural Sciences; FastStart Universal SYBR Green Master was purchased from Roche, USA; and japonica rice Zhonghua 11 (ZH11) was obtained from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences.

[0028] Example 1

[0029] 1. Cloning of gene CT4.1

[0030] 1. Extract genomic DNA from rice variety Dongye, use primers 5'-ATGGTGCTCCAACACAGTGATC-3' (SEQ ID NO.4) and 5'-TTAGAAGATGACGGAGATGCAC-3' (SEQ ID NO.5) for polymerase chain reaction, sequence the obtained PCR product, and obtain the nucleotide sequence of rice cold tolerance gene CT4.1, which consists of 1355 bases, as shown in SEQ ID NO.1. The PCR program is as follows: 94℃ pre-denaturation for 5min; 35 cycles (94℃ denaturation for 30s; 55℃ annealing for 30s; 72℃ extension for 6min), 72℃ extension for 10min.

[0031]

[0032] 2. RNA was extracted from the leaves of rice variety Dongye, reverse transcribed into cDNA, and polymerase chain reaction was performed using primers 5'-ATGGTGCTCCAACACAGTGATC-3' (SEQ ID NO.6) and 5'-TTAGAAGATGACGGAGATGCAC-3' (SEQ ID NO.7). The amplified product size was 1260 bp. The obtained PCR product was sequenced and analyzed to obtain the coding sequence (CDS) of gene CT4.1, which consists of 1260 bases, and the nucleotide sequence is shown in SEQ ID NO.2. The PCR program is as follows: 94℃ pre-denaturation for 5min; 30 cycles (94℃ denaturation for 30s; 55℃ annealing for 30s; 72℃ extension for 1min), 72℃ extension for 7min.

[0033]

[0034] 3. The CDS sequence (SEQ ID NO.2) was translated using Primer 3 software (http: / / frodo.wi.mit.edu / ) to obtain the amino acid sequence of the protein, encoding 419 amino acids. The amino acid sequence is shown in SEQ ID NO.3.

[0035] SEQ ID NO.3: MVLQHSDRMDSLFSPQTSWVSGPIIVGAGPSGLAVAASLREQGVPFTMLERADCIASLWQKRTYDRLKLHLPKQFCELPRMAFPAHYPEYPTRRQFIDYLEDYAAAFDINPLFGHTVLSARYDETSGLWRVRASSSAGAEMEYIGSWLVVATGENAESVVPDIPGIDGFGGEVVHVADYKSGEAYRGKRVLVVGCGNSGMEVSLDLC DHGARPAMVVRDAVHVLPREVLGKSTFELAVLLMSWLPLWLVDKILVLLAWLVLGNLAKLGIRRPATGPLELKNTTGRTPVLDYGALARIRSGEITVVPGVARFGK GFAELADGRVIALDAVVLATGYRSNVPQWLQGNDFFNKDGYPKTAFPNGWKGESGLYAVGFTRRGLSGASADAMRAAKDLARVWKEATKPTKKSTACHRRCISVIF.

[0036] The above primers were synthesized by Shanghai Bioengineering, and the sequences were determined by Shanghai Bioengineering. DNA and RNA extraction, PCR and reagent formulation are all conventional techniques in the art.

[0037] 2. Overexpression of the cold-tolerant gene CT4.1 improves the cold tolerance of rice seedlings

[0038] 1. Construction of overexpression vector

[0039] According to the cDNA sequence of the rice cold-tolerant gene CT4.1, see the full-length coding sequence of SEQ ID NO.2, the homologous recombination linkers on both sides of the KpnI and BamHI double restriction sites of the pC1300S vector were introduced into the upstream and downstream primers respectively, and the primers were designed as follows:

[0040] OX-F:5'-gagctttcgcgagctcggtaccATGGTGCTCCAACACAGTGATC-3' (SEQ ID NO.8);

[0041] OX-R:5'-caggtcgactctagaggatccTTAGAAGATGACGGAGATGCAC-3' (SEQ ID NO. 9).

[0042] The cold-resistant gene CT4.1 was used as a template (SEQ ID NO.1) and the primers OX-F and OX-R were used for polymerase chain reaction. The product size was 1398 bp. The PCR program was as follows: 94℃ pre-denaturation for 5 min; 30 cycles (94℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 1 min), and 72℃ extension for 7 min. The CT4.1 gene fragment amplified and separated by PCR was connected to the pC1300S vector after double digestion with KpnI and BamHI by homologous recombination. After sequencing and comparison, it was transformed into Agrobacterium EHA105 to obtain the cold-resistant gene CT4.1 overexpression vector.

[0043] 2. Obtaining overexpressing transgenic plants

[0044] The cold-tolerant gene CT4.1 overexpression vector was transferred into the indica rice variety Ganzaoxian 49 (GZX49) through Agrobacterium-mediated genetic transformation. After selective culture, differentiation, rooting, and seedling hardening, T 0 All transgenic materials were propagated and stable genetic T 1 Generation material. For a certain T 1 For the transgenic lines of the first generation, if the T 2 All the plants of the T 1 The first generation of transgenic lines were homozygous transgenic lines. Three homozygous transgenic lines OX1, OX2, and OX3 were selected for subsequent analysis.

[0045] 3. Identification of overexpressing transgenic plants

[0046] (1) Expression level identification

[0047] The 2-leaf seedlings were taken, total RNA was extracted, reverse transcribed to obtain cDNA, and fluorescent real-time quantitative PCR was performed. The primers used to detect the expression of gene CT4.1 were:

[0048] RT-F:5'-CCCCTTGGTTAAATTCGTTGTT-3' (SEQ ID NO.10);

[0049] RT-R: 5'-AGCAACGTAGTATGATCACACT-3' (SEQ ID NO. 11).

[0050] The reagent used for quantitative analysis was FastStart Universal SYBR Green Master (ROX). The instrument used was the real-time fluorescence quantitative PCR instrument ViiA7 from Applied Biosystems, USA. Ubqtin gene was used as an internal reference.

[0051] like Figure 1 As shown, WT represents the wild-type rice GZX49, and OX1, OX2, and OX3 represent transgenic lines overexpressing the cold-tolerant gene CT4.1. In the transgenic lines overexpressing the cold-tolerant gene CT4.1, the relative expression level of the gene CT4.1 was significantly higher than that of the wild-type rice GZX49.

[0052] (2) Identification of cold-tolerant phenotypes

[0053] Seedling cultivation: Select 100 healthy and plump seeds and seal them in a parchment bag, soak them at 30℃ for 2 days, and germinate them for 1 day. Mix nutrient soil, perlite and vermiculite with water in a mass ratio of 8:1:1. Put the mixed soil into a black small square box of 6cm×6cm×7cm. Select 30 healthy seeds with white spots, sow them in the small square box, and then cover them with soil (set 3 replicates). Put the small square box in a bread tray and place it in a 30℃ precision artificial climate box (KBWF240, parameters 0℃~70℃), set the light for 16h and the dark for 8h every day, and cultivate the seedlings to the one-leaf and one-core stage (8~10d).

[0054] The seedlings at the one-leaf-one-core stage were placed in a 4℃ low-temperature incubator for 84 hours, and then placed in a 30℃ precision artificial climate box with 16 hours of light and 8 hours of darkness per day, and resumed growth for 7 days. The number of surviving individual plants and the total number of seedlings of the test materials were counted, and the average survival rate of the seedlings of 3 replicates was used as the identification index of cold tolerance in the seedling stage. Seedling survival rate (%) = (number of surviving seedlings / total number of seedlings) × 100%.

[0055] like Figure 2 As shown, WT represents wild rice GZX49, OX1, OX2 and OX3 represent transgenic plants overexpressing the cold-tolerant gene CT4.1, and the scale bar is 5 cm. After low temperature stress, the growth of transgenic plants overexpressing the cold-tolerant gene CT4.1 was significantly better than that of wild rice GZX49.

[0056] like Figure 3 As shown, WT represents the wild-type rice GZX49 plant, OX1, OX2 and OX3 represent transgenic plants overexpressing the cold-tolerant gene CT4.1. After low temperature stress, the survival rate of transgenic plants overexpressing the cold-tolerant gene CT4.1 was significantly higher than that of the wild-type rice GZX49 plant.

[0057] 3. Crispr knockout of cold-tolerant gene CT4.1 reduces cold tolerance of rice seedlings

[0058] 1. Construction of CRISPR knockout vector

[0059] According to the nucleotide sequence of the rice cold-tolerant gene CT4.1 (SEQ ID NO.1), the target site for knocking out the cold-tolerant gene CT4.1 was designed through the website http: / / skl.scau.edu.cn / targetdesign / . The designed target site is: 5'-GTGCGGCAACTCCGGTATGG-3' (SEQ ID NO.12). Homologous recombination linkers on both sides of the KpnI restriction site of the pCXUN_CAS9 vector were introduced into the upstream and downstream primers respectively. The designed primers are:

[0060] U3-F:5'-CCCCTTTCGCCAGGGGTACCgtaattcatccaggtctccaag-3' (SEQ ID NO. 13);

[0061] U3-R:5'-TACGAATTCGAGCTCGGTACCgctgtgccgtacgacggtacg-3' (SEQ ID NO. 14);

[0062] Crispr-F:5'-GTGCGGCAACTCCGGTATGGgttttagagctagaaatagcaagtta-3' (SEQ IDNO.15);

[0063] Crispr-R:5'-CCATACCGGAGTTGCCGCACgccacggatcatctgcacaactc-3' (SEQ ID NO. 16).

[0064] Using OsU3 as a template, primers U3-F and Crispr-R were used for polymerase chain reaction to obtain PCR product 1, with a product size of 476 bp. The PCR program was as follows: 94°C pre-denaturation for 5 min; 30 cycles (94°C denaturation for 30 s; 55°C annealing for 30 s; 72°C extension for 30 s), and 72°C extension for 7 min.

[0065] Then, using OsU3 as a template, primers U3-R and Crispr-F were used for polymerase chain reaction to obtain PCR product 2, with a product size of 368 bp. The PCR program was as follows: 94°C pre-denaturation for 5 min; 30 cycles (94°C denaturation for 30 s; 55°C annealing for 30 s; 72°C extension for 30 s), and 72°C extension for 7 min.

[0066] Finally, the mixture of PCR product 1 and PCR product 2 was used as a template, and primers U3-F and U3-R were used for polymerase chain reaction to obtain the final PCR product with a size of 824 bp. The PCR program was as follows: 94℃ pre-denaturation for 5 min; 30 cycles (94℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 1 min), 72℃ extension for 7 min. The final product separated by PCR amplification was connected to the pCXUN_CAS9 vector after KpnI digestion by homologous recombination. After sequencing and alignment, it was transformed into Agrobacterium EHA105 to obtain the gene CT4.1 Crispr knockout vector.

[0067] 2. Obtaining Crispr knockout plants

[0068] The gene CT4.1 Crispr knockout vector was transferred into japonica rice Zhonghua 11 (ZH11) through Agrobacterium-mediated genetic transformation. After selective culture, differentiation, rooting, and seedling hardening, T 0 All transgenic materials were propagated to obtain homozygous mutations and stable inheritance of T cells without Cas9 protein residues. 1 Three homozygous transgenic lines, CR1, CR2, and CR3, were selected for subsequent analysis.

[0069] 3. Identification of Crispr knockout plants

[0070] (1) Genotype identification

[0071] Take the seedlings at the 2-leaf stage and extract DNA. Use primers sq-F and sq-R for polymerase chain reaction, and the amplified product size is 429bp. The obtained PCR product was sequenced to analyze the editing of the gene CT4.1 target site. The PCR program is as follows: 94℃ pre-denaturation for 5min; 32 cycles (94℃ denaturation for 30s; 55℃ annealing for 30s; 72℃ extension for 30s), 72℃ extension for 7min. The primer sequences used are as follows:

[0072] sq-F: 5'-ATGGAGTACATTGGCAGCTGG-3' (SEQ ID NO. 17);

[0073] sq-R: 5'-GGTGGTGTTCTTGAGCTCGAG-3' (SEQ ID NO. 18).

[0074] ZH11 represents the wild plant of japonica rice Zhonghua 11, and CR1, CR2, and CR3 represent the cold-tolerant gene CT4.1 Crispr knockout lines. In the cold-tolerant gene CT4.1 Crispr knockout lines, the gene CT4.1 has homozygous mutations at the target site.

[0075] The sequencing results of the target sites of the Crispr knockout materials were as follows: the reference gene of the wild plant Zhonghua 11 of japonica rice represented by ZH11 was: 5'-CGTCGTCGGGTGCGGCAACTCCGGTATGGAGGTGTCGCTGG (SEQ ID NO.19); the CR1 knockout strain replaced the 27th base of the reference gene with a single base, "T" replaced with "A"; the CR2 knockout strain deleted the double bases "A" and "G" at the 30th and 31st bases of the reference gene; the CR3 knockout strain deleted the single base "A" at the 18th base of the reference gene.

[0076] (2) Identification of cold-tolerant phenotypes

[0077] Take the seeds, soak them at 30℃ for 2 days, germinate them for 1 day, then sow 25 germinated seeds in a small black square box filled with nutrient soil, and culture them in a light incubator (lighting time is 16h / d, temperature is 30℃) until the 2-leaf stage, and take pictures before treatment. The 2-leaf seedlings were placed in a 4℃ low-temperature incubator for 96h, then returned to the light incubator for recovery and cultivation for 1 week, and the photos were taken and the survival rate was calculated. Three replicates were performed for each material.

[0078] like Figure 4 As shown in the figure, ZH11 represents the wild-type japonica rice Zhonghua 11, and CR1, CR2, and CR3 represent the cold-tolerant gene CT4.1 Crispr knockout plants. After low temperature stress, the survival rate of the cold-tolerant gene CT4.1 Crispr knockout plants was significantly lower than that of the wild-type japonica rice Zhonghua 11.

[0079] In summary, the present invention discovered that the cold-tolerant gene CT4.1 can be used to enhance the cold-tolerant performance of rice and improve the survival rate of rice under low temperature stress.

[0080] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of rice cold-tolerant gene CT4.1 in improving cold tolerance of rice, characterized in that: The nucleotide sequence of the rice cold-tolerant gene CT4.1 is shown in SEQ ID NO.

1.

2. The use of the protein encoded by the rice cold-tolerant gene CT4.1 as claimed in claim 1 in improving the cold tolerance of rice, characterized in that: The amino acid sequence of the protein encoded by the rice cold-tolerant gene CT4.1 is shown in SEQ ID NO.

3.

3. Use of an expression cassette, vector or recombinant bacterium containing the rice cold-tolerant gene CT4.1 as claimed in claim 1 in improving the cold-tolerant property of rice.

4. Use of the rice cold-tolerant gene CT4.1 as claimed in claim 1 in breeding cold-tolerant transgenic rice.

5. Use of the rice cold-tolerant gene CT4.1 as claimed in claim 1 in improving rice cold-tolerant germplasm resources.

6. Use of the rice cold-tolerant gene CT4.1 as claimed in claim 1 in improving the survival rate of rice in low temperatures.

7. A method for improving the cold tolerance of rice using the cold tolerance gene CT4.1 of rice as claimed in claim 1, characterized in that: The rice is made to overexpress the rice cold-tolerant gene CT4.1 by a transgenic method. The nucleotide sequence of the rice cold-tolerant gene CT4.1 is shown in SEQ ID NO.

1.

8. The method for improving cold tolerance of rice according to claim 7, characterized in that: The transgenic method comprises introducing a recombinant expression vector containing the rice cold-tolerant gene CT4.1 into rice by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated method to obtain a transgenic rice strain.

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