Method for improving low temperature stress tolerance of rice, gene and application thereof

Abstract

The application discloses a method for improving the low-temperature stress resistance of rice, a gene and application thereof. Specifically disclosed is a low-temperature stress resistance gene CTB18 of rice, wherein the nucleotide sequence of the gene is shown as SEQ ID NO:1, and the amino acid sequence of the gene is shown as SEQ ID NO:2. The application obtains a low-temperature sensitive mutant of the rice booting stage by performing chemical mutagenesis on the cold-tolerant rice variety Longdao 5 in the booting stage. Under the condition of low-temperature treatment, the anther of the mutant ctb18-1 develops abnormally, the pollen grains are aborted, and the seed setting rate of the plant is reduced. The application separates and clones the key gene CTB18 for regulating the cold tolerance of the rice booting stage from the mutant, provides a new gene resource for cultivating the low-temperature resistant rice variety, has important significance for the field of rice molecular breeding, and provides an efficient breeding mode for creating the transgenic plant variety, germplasm resource and hybridization parent with low-temperature stress resistance.

Description

Technical Field

[0001] This invention belongs to the field of rice molecular breeding, specifically relating to a method, gene, and application for improving the ability of rice to withstand low temperature stress. Background Technology

[0002] Food security is a major strategic issue of overall importance, concerning my country's national economic development, social stability, and national self-reliance. Rice (Oryza sativa L.) is one of the world's most important food crops, a staple food for half the world's population. In my country, the rice planting area is approximately 30.18 million hectares, accounting for 26.9% of the total grain planting area; rice production is 208.56 million tons, accounting for 33.9% of total grain production. Therefore, stable and high-yield rice production plays a vital role in ensuring my country's food security.

[0003] Rice originated in tropical and subtropical regions and is sensitive to low temperatures. Rice-growing areas in my country are widely distributed, with a latitude difference of approximately 34 degrees and an altitude difference of approximately 2700 meters. Rice suffers chilling injury at different growth stages, resulting in yield losses of 3 to 5 million tons annually. Delayed chilling injury occurring during the vegetative growth stage can be effectively prevented through cultivation techniques such as dry-land rearing and sparse planting. However, barrier chilling injury (causing pollen abortion and pollination obstruction) caused by short-term abnormal low temperatures during the booting stage is unavoidable once it occurs, severely impacting grain filling and leading to significant yield reductions. Particularly in the Northeast rice-growing region, a major commodity grain base in my country, barrier chilling injury occurs almost every year, seriously threatening rice production safety. A deep understanding of the molecular mechanisms of rice's response to low temperatures during the booting stage, and subsequently developing cold-resistant rice varieties through molecular design breeding or rapid domestication methods, is a crucial approach to fundamentally solving the problem of barrier chilling injury.

[0004] Because chilling injury in rice is caused by short-term abnormally low temperatures occurring at specific growth and development stages, a stable and accurate assessment system for cold tolerance during the booting stage is lacking, making precise phenotypic identification during genetic analysis extremely difficult, resulting in slow progress in research on the mechanisms of cold tolerance during the booting stage of rice. Currently, genetic resources for cold tolerance during the booting stage of rice are scarce and can be used for genetic breeding. Therefore, it is urgent to discover a low-temperature stress gene that can improve cold tolerance during the booting stage of rice, which is of great significance for breeding cold-resistant and stable-yielding rice varieties. Summary of the Invention

[0005] To address the shortcomings of existing technologies, identifying and applying cold-resistant genes during the rice booting stage to the breeding of low-temperature-tolerant rice varieties is a crucial approach to fundamentally solving the problem of cold damage in rice. Therefore, this invention proposes a method, gene, and its application for improving the low-temperature stress tolerance of rice.

[0006] This invention provides a rice low-temperature stress resistant gene, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0007] This invention uses the cold-tolerant rice variety Longdao 5 (Oryza sativa L. subsp. japonica Longdao 5, LD5) as the research object. Through ethyl mesylate (EMS) mutagenesis and MutMap analysis, a gene controlling cold tolerance during the booting stage of rice was cloned for the first time and named CTB18. Genetic complementation experiments using CRISPR / Cas9 gene editing to create knockout mutants and exogenously introduced CTB18 further demonstrated that the CTB18 gene regulates cold tolerance during the booting stage of rice.

[0008] The present invention also provides a rice low-temperature stress resistant protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0009] The present invention also provides a recombinant vector containing the nucleotide sequence.

[0010] The present invention also provides a recombinant genetically engineered bacterium containing the aforementioned recombinant vector, such as Agrobacterium.

[0011] The present invention also provides primers for amplifying the rice low-temperature stress resistance gene.

[0012] The present invention also provides a method for improving the ability of plants to withstand low temperature stress, by introducing the rice low temperature stress resistance gene, the recombinant vector, or the recombinant genetically engineered bacteria into the plant.

[0013] The present invention also provides a method for preparing transgenic rice with improved resistance to low temperature stress, wherein the recombinant engineered bacteria are used to transform plants, and transgenic plants with improved resistance to low temperature stress are screened.

[0014] The present invention also provides the application of the rice low-temperature stress-resistant gene, the recombinant vector, or the recombinant genetically engineered bacteria in rice breeding.

[0015] In one or more embodiments, the rice breeding variety is a cold-resistant rice variety.

[0016] In one or more embodiments, the rice breeding method includes transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0017] This invention also relates to a method for creating homozygous allelic mutants of the CTB18 gene using gene editing technology, including introducing a gene editing vector containing the CTB18 gene sgRNA sequence into LD5 material. During the creation of the CTB18 mutant material using the gene editing method, a frameshift mutation occurs in the amino acid sequence encoded by the CTB18 gene, leading to premature termination.

[0018] The present invention also relates to a method for creating transgenic plants containing the CTB18 gene. This includes the steps of introducing a recombinant vector containing the CTB18 gene into ctb18-1 and a method for detecting newly created CTB18 gene transgenic positive plants.

[0019] This invention also relates to the use of the gene, the gene recombination vector, the Agrobacterium, or the protein in enhancing the cold tolerance of rice during the booting stage. The rice material used in this invention is preferably the cold-tolerant rice variety Longdao 5 during the booting stage.

[0020] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0021] (1) This invention clones a cold-resistant regulatory gene in rice during the booting stage, providing new gene resources for breeding low-temperature resistant rice varieties, which is of great significance to the field of rice molecular breeding.

[0022] (2) This invention provides an efficient breeding method for creating transgenic plant varieties, germplasm resources, and hybrid parents based on the CTB18 gene that are resistant to low temperature stress. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a flowchart of the mutant screening process in Embodiment 1 of the present invention.

[0025] Figure 2 This is a technical flowchart of Embodiment 2 of the present invention.

[0026] Figure 3 These are photographs of anther morphology, pollen activity, and seed set of wild-type LD5 and mutant ctb18-1 under normal growth conditions and low-temperature treatment in Example 1 of this invention.

[0027] Figure 4The results show the statistical results of pollen grain activity and seed setting rate of wild-type LD5 and mutant ctb18-1 under normal growth conditions and low temperature treatment in Example 1 of this invention.

[0028] Figure 5 This describes the CTB18 gene location and CTB18 gene structure in Examples 2 and 3 of the present invention. Figure 5 Figure a shows a schematic diagram of the CTB18 gene located using the MutMap method. Figure 5 b represents the CTB18 gene structure diagram, and labels the location and mutation type of the mutants ctb18-1, ctb18crp-1, and ctb18crp-2.

[0029] Figure 6 These are photographs of anther morphology, pollen activity, and seed set of wild-type LD5, mutants ctb18crp-1, ctb18crp-2, and transgenic complementary plants ctb18-1 CTB18-Flag and ctb18-1 CTB18-GFP under normal growth conditions and low-temperature treatment, as shown in Example 3 of this invention.

[0030] Figure 7 The results are statistical results of pollen grain activity and seed setting rate of wild-type LD5, mutants ctb18crp-1, ctb18crp-2, and transgenic complementary plants ctb18-1 CTB18-Flag and ctb18-1 CTB18-GFP under normal growth conditions and low temperature treatment in Example 3 of this invention.

[0031] Figure 8 This is a map of the vector XF2807 used for CTB18 transgenic complementation verification in Example 3 of the present invention.

[0032] Figure 9 This is a map of the vector XF2809 used for CTB18 transgenic complementation verification in Example 3 of the present invention. Detailed Implementation

[0033] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0034] This invention involves EMS mutagenesis of LD5 seeds to obtain mutagenic M0 seeds, which are then sown. M1 generation individual plant seeds and M2 line seeds are harvested and subjected to natural low-temperature screening to obtain 22 candidate lines of low-temperature-sensitive mutants at the booting stage. These lines are then subjected to low-temperature treatment (15℃) and detailed identification at the Harbin Rice Pot Farm and artificial climate chamber. Under normal growth conditions (natural temperature and light), the plants are grown to the booting stage, then subjected to low-temperature treatment for 7 days in the artificial climate chamber. After treatment, the plants are moved to normal conditions. At the flowering stage, unopened florets are collected to observe anther morphology and pollen grain activity. Pollen grains from candidate individual plants are stained with 1% I2-KI and observed under an optical microscope to determine pollen activity. At seed maturity, low-temperature-sensitive mutants at the booting stage are screened by statistically analyzing seed set rate. Finally, the booting-stage low-temperature-sensitive mutant ctb18-1 is obtained. The mutant screening process is as follows: Figure 1 As shown.

[0035] F1 seeds were harvested by crossing the selected mutant ctb18-1 with the wild-type LD5, and the F1 population was then self-pollinated to produce the F2 population. The cold tolerance segregation ratio at the booting stage in the F2 population was 109 cold-tolerant plants to 40 cold-intolerant plants (X1). 2 =0.60, p=0.74). Equal amounts of leaves from 27 cold-sensitive individual plants were mixed, and genomic DNA was extracted and resequencing was performed. MutMap analysis revealed a significant SNP-index peak on chromosome 3. Comparison with the rice MSU (http: / / rice.uga.edu / ) database showed that this peak was located on the first nucleotide of an intron in the LOC_Os03g22950 gene. This SNP mutation resulted in abnormal intron retention and frameshift mutation. MSU database prediction indicated that the CTB18 gene encodes an acyl carrier protein (ACP), and CTB18 has two exons and one intron.

[0036] The nucleotide sequence of the rice cold tolerance regulation gene CTB18 during the booting stage is shown in SEQ ID NO:1 (which includes its promoter sequence, 5′-UTR, CDS containing introns, and 3′-UTR). This gene encodes the CTB18 protein, and the amino acid sequence of the CTB18 protein is shown in SEQ ID NO:2.

[0037] To verify the function of the CTB18 gene, a CRISPR / Cas9 gene editing target for CTB18 was designed, and a gene editing vector was constructed. Wild-type rice LD5 was transformed using Agrobacterium-mediated transformation to obtain homozygous knockout mutants ctb18crp-1 and ctb18crp-2. The anther development morphology, pollen activity, and seed setting of the mutants ctb18crp-1 and ctb18crp-2 after low-temperature treatment were observed and analyzed. The results showed that, like the mutant ctb18-1, they exhibited a low-temperature sensitivity phenotype during the booting stage, characterized by abnormal anther development, reduced pollen activity, and decreased seed setting rate, demonstrating that the CTB18 gene is a key gene regulating cold tolerance during the booting stage of rice. Simultaneously, to verify the function of the CTB18 gene through genetic complementation, the recombinant vectors proCTB18:CTB18-Flag and proCTB18:CTB18-GFP were constructed and transformed into ctb18-1 using Agrobacterium-mediated transformation. Positive transgenic plants ctb18-1 CTB18-Flag and ctb18-1 CTB18-GFP were obtained through genotyping. After low-temperature treatment, anther development morphology, pollen grain activity, and seed setting were observed and statistically analyzed. The results showed that the exogenously introduced CTB18 gene could revert to the abnormal anther development, pollen grain abortion, and reduced seed setting rate phenotype of the mutant ctb18-1, further proving that the CTB18 gene is indeed the gene regulating cold tolerance during the booting stage.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1: Obtaining the mutant

[0041] Soak 10,000 rice LD5 seeds in tap water at room temperature for 12 hours, rinse thoroughly with tap water and air dry for 6 hours, then soak again in tap water for 12 hours. After draining the tap water, add 0.75% EMS and treat for 12 hours, rinsing the seeds with tap water 6 times, 10 minutes each time. Finally, rinse with running water for 1.5 hours to remove residual EMS. Figure 1Cleaned M0 mutagenized seeds were sown in May 2015 at an experimental base in Hailun City, Heilongjiang Province. Transplanting occurred 25 days after sowing, and normal field management was implemented. Before heading, the rice was surrounded by plastic film to prevent cross-pollination. In October 2015, approximately 4000 M1 individual seed samples with good seed setting were harvested. In December 2015, approximately 4000 M1 seed samples were sown at a base in Lingshui, Hainan Province, and over 2700 M2 seed samples were harvested in April 2016. The M2 lines were sown in a field in Hailun City, Heilongjiang Province, and subjected to natural low-temperature screening. Twenty-two candidate mutant lines sensitive to low temperature during the booting stage were obtained. Subsequently, low-temperature treatment (15℃) and detailed identification were conducted at a rice pot farm and artificial climate chamber in Harbin. Under normal growth conditions, the plants reached the booting stage and were then subjected to low-temperature treatment (15℃) for 7 days in an artificial climate chamber. After treatment, the plants were moved to normal growing conditions and allowed to grow until the flowering stage. Unopened florets were collected to observe the anther morphology and pollen grain activity. When the seeds matured, the seed setting rate was calculated.

[0042] The screening criteria for mutants are as follows: Under normal growth conditions, the anthers are yellow, and their morphology and length are similar to those of the wild-type LD5. The pollen grain activity is normal, and the seed setting rate is similar to that of the wild-type LD5. However, after low-temperature treatment, the anthers turn white and become curved, the anther length is shorter than that of LD5, the pollen grain activity is significantly reduced, and the seed setting rate is significantly lower than that of LD5. The seed setting rate refers to the percentage of plump seeds on each ear at seed maturity.

[0043] The results are as follows Figure 3 and Figure 4 As shown, under normal growth conditions, the seed setting rate of both LD5 and the mutant did not change significantly. However, after low-temperature treatment, the seed setting rate of the mutant was significantly lower than that of LD5. A low-temperature-sensitive mutant during the booting stage was obtained through screening and named ctb18-1.

[0044] Example 2: Obtaining candidate genes

[0045] The selected low-temperature-sensitive mutant ctb18-1 during the booting stage was crossed with the wild-type LD5, and F1 seeds were harvested. The F1 population was then self-crossed to harvest the F2 population, as follows: Figure 2 As shown. The segregation ratio of cold tolerance during the booting stage in the F2 field population was 109 cold-tolerant plants to 40 cold-intolerant plants (X... 2 =0.60, p=0.74). Equal amounts of leaves from 27 cold-intolerant individual plants were mixed, and genomic DNA was extracted and resequencing was performed.

[0046] The results are as follows Figure 5 As shown in figure a, MutMap analysis revealed a significant SNP-index peak on chromosome 3. Figure 5As shown in b, comparison with the rice MSU (http: / / rice.uga.edu / ) database revealed that the SNP mutation is located on the first nucleotide of the intron in the LOC_Os03g22950 gene. This SNP mutation leads to abnormal intron retention and frameshift mutation. MSU database prediction shows that CTB18 encodes an acyl carrier protein (ACP) with two exons and one intron.

[0047] Example 3: Functional Verification of Candidate Genes

[0048] To verify the function of the LOC_Os03g22950 gene, a CRISPR / Cas9 gene editing target was designed and a recombinant vector was constructed. Wild-type LD5 cells were transformed using Agrobacterium-mediated transformation. CTb18crp-1 and CTb18crp-2, Cas9-free materials with homozygous mutations in the LOC_Os03g22950 gene, were obtained through gene isolation. Figure 5 As shown in b.

[0049] Mutant materials obtained by low-temperature treatment during the booting stage, according to Figure 6 and Figure 7 The results showed that the mutants ctb18crp-1 and ctb18crp-2 exhibited a low-temperature sensitivity phenotype during the booting stage, similar to ctb18-1, with abnormal anther development, reduced pollen activity and seed setting rate. This proved that the gene LOC_Os03g22950 is the candidate gene CTB18 obtained through screening and is a key gene regulating cold tolerance during the booting stage of rice.

[0050] To further validate the function of the candidate gene CTB18, genomic DNA from wild-type rice LD5 was used as a template. The 3'-UTR region of the CTB18 gene was amplified using primers shown in Table 1 (SEQ ID NO:3+SEQ ID NO:5 and SEQ ID NO:4+SEQ ID NO:5), and then ligated into the linearized vector XF675, which was double-digested with PstI and HindIII, to obtain intermediate vectors. The vectors were then double-digested with EcoRI and NcoI, and the promoter and coding regions of the CTB18 gene were amplified using primers shown in SEQ ID NO:6 and SEQ ID NO:7. These amplified regions were then ligated into linearized vectors via Gibson assembly to obtain the final vectors XF2807 and XF2809. The vector maps are shown below. Figure 8 and Figure 9 As shown.

[0051] Table 1 Primer sequences used for constructing the CTB18 gene complementation vector.

[0052]

[0053]

[0054] DH5α was transformed, and positive clones were selected for sequencing. The correctly sequenced recombinant plasmid was transformed into Agrobacterium EHA105 via electroporation, infecting ctb18-1 callus tissue to obtain transgenic positive seedlings ctb18-1 CTB18-Flag and ctb18-1CTB18-GFP. Transgenic T0 generation plants were planted in Hainan, and T1 generation seeds were harvested. T1 generation transgenic positive single-plant seeds, ctb18-1, and wild-type LD5 were planted in a potted plantation in Harbin. Under normal growth conditions, when the plants reached the heading stage, they were transferred to an artificial climate chamber for low-temperature treatment for 7 days, and then returned to the potted plantation for normal cultivation. At the flowering stage, before the florets opened and released pollen, anthers and pollen grains were collected to observe the effect of low-temperature treatment on their development. At seed maturity, the seed set rate was calculated.

[0055] The results are as follows Figure 6 As shown, under normal growth conditions, the anthers of transgenic positive seedlings carrying the CTB18 gene are yellow, and the anther length is similar to that of the wild type LD5. After low-temperature treatment, the exogenously introduced CTB18 gene can restore the phenotype of the mutant ctb18-1, which has white and shortened anthers, making the anther color, shape and length similar to the wild type. At the same time, the exogenously introduced CTB18 gene also restores the phenotype of reduced pollen activity after low-temperature treatment of the mutant ctb18-1.

[0056] The statistical results of the fruit setting rate are as follows: Figure 7 As shown, the exogenously introduced gene CTB18 can revert to the phenotype of reduced seed setting rate in the mutant ctb18-1 after low-temperature treatment. These data demonstrate that the gene CTB18 can complement the low-temperature sensitivity phenotype of the mutant ctb18-1 during the booting stage, proving that the CTB18 gene is a key regulatory gene for low-temperature tolerance during the booting stage of rice.

[0057] In summary, this invention isolated and cloned a gene, CTB18, that regulates cold tolerance during the booting stage of rice. Further in-depth research demonstrated that CTB18 is a key regulatory gene for low-temperature tolerance during the booting stage of rice. Under normal growth conditions, the CTB18 mutant showed no significant morphological differences in growth and development compared to the wild-type LD5. This invention provides new germplasm and gene resources for breeding cold-tolerant rice varieties and is of great significance to the field of rice molecular breeding.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0059]

[0060]

[0061] 。

Claims

1. A method for improving the tolerance of rice to low-temperature stress, characterized in that, Introduce low-temperature stress-resistant genes into rice; The nucleotide sequence of the rice low-temperature stress-resistant gene is shown in SEQ ID NO:

1.

2. A method for preparing transgenic plants with resistance to low-temperature stress, characterized in that, Transformed plants with recombinant engineered bacteria and screened to obtain transgenic plants that are resistant to low temperature stress; The recombinant engineered bacteria contains a rice low-temperature stress tolerance gene, the nucleotide sequence of which is shown in SEQ ID NO:1; The genetically modified plant is genetically modified rice.

3. Application of rice low-temperature stress tolerance gene in rice low-temperature stress tolerance breeding, wherein the nucleotide sequence of the rice low-temperature stress tolerance gene is shown in SEQ ID NO:

1.

4. The application according to claim 3, characterized in that, The rice varieties bred to withstand low-temperature stress are cold-resistant rice varieties.

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