Application of genes NAT2 and NAT3 in improving heat tolerance of rice

Abstract

The application relates to the technical field of genetic engineering, and discloses application of genes NAT2 and NAT3 in improving heat resistance of rice. The genomic DNA sequence of the gene NAT2 is shown as SEQ ID NO. 1, and the genomic DNA sequence of the gene NAT3 is shown as SEQ ID NO. 2. The survival rate of the obtained gene editing plants at the seedling stage and the seed setting rate of the plants at the reproductive stage are improved under high-temperature stress conditions by gene editing mutation of the genes NAT2 and NAT3, and the yield of the rice under extreme high-temperature conditions is protected.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of genes NAT2 and NAT3 in improving the heat resistance of rice. Background Technology

[0002] Rice, as one of the world's most important food crops, is facing increasingly severe challenges from high temperatures. Particularly in major rice-growing regions like China, rising temperatures have become a significant factor affecting rice yields. Rice exhibits high sensitivity to heat stress at all stages of its growth cycle, especially during the reproductive stage, where temperature fluctuations have a particularly significant impact on pollen activity, directly leading to a substantial decrease in seed setting rate. Through long-term natural evolution, rice has developed a complex set of mechanisms to balance its growth and development with the need for heat resistance. These mechanisms encompass multiple levels, from sensing heat signals, signal transduction, and downstream gene expression regulation to related metabolic and physiological responses. Nevertheless, many unknowns remain regarding the genetic basis of rice heat resistance, requiring further scientific research to elucidate its significance. This research is crucial for breeding more heat-resistant rice varieties and developing effective cultivation and management strategies to ensure the stability and sustainability of rice yields against the backdrop of global climate change.

[0003] This invention identified two negative regulators of rice heat tolerance: genes NAT2 (LOC_Os03g05290) and NAT3 (LOC_Os06g22960). Double mutants of these genes exhibited significantly enhanced high-temperature resistance. In the molecular mechanism by which NAT2 and NAT3 regulate rice heat tolerance, the double mutants of NAT2 and NAT3 rapidly accumulate ROS in the plant under high-temperature conditions, strengthening the early high-temperature response. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide the use of genes NAT2 and NAT3 in production practice, namely, their application in improving the heat resistance of rice.

[0005] The genes NAT2 and NAT3 provided by this invention encode two potential aquaporins, with MSU gene numbers LOC_Os03g05290 and LOC_Os06g22960, respectively. The full-length genomic DNA of gene NAT2 contains 1917 bases, as shown in SEQ ID NO.1, and the full-length genomic DNA of gene NAT3 contains 1541 bases, as shown in SEQ ID NO.2. The coding region of gene NAT2 is 753 bases long, as shown in SEQ ID NO.3, and the coding region of gene NAT3 is 747 bases long, as shown in SEQ ID NO.4. Protein NAT2 contains 250 amino acids, as shown in SEQ ID NO.5, and protein NAT3 contains 248 amino acids, as shown in SEQ ID NO.6. Proteins NAT2 and NAT3 contain multiple transmembrane domains (TMDs).

[0006] This invention provides an application of genes NAT2 and NAT3 in altering the heat resistance of rice. Specifically, sgRNAs targeting genes NAT2 and NAT3 are designed, and CRISPR-CAS9 genome editing technology is used to induce base mutations, deletions, or insertions at specific locations in the rice genome, thereby obtaining rice plants with altered genome sequences.

[0007] As an application of genes NAT2 and NAT3 in improving the heat resistance of rice, knocking out genes NAT2 and NAT3 in rice (ZH11) resulted in transgenic plants with significantly higher seedling survival rates under heat stress compared to wild-type plants. By using gene editing technology to substitute, delete, or insert bases in the coding regions of rice genes NAT2 and NAT3, frameshift mutations or premature translation termination occur in the encoded proteins, thereby inactivating NAT2 and NAT3 proteins. This promotes rapid accumulation of ROS in rice plants, enhances the early response to high temperatures, and strengthens the plant's resistance to high temperatures, resulting in rice materials with enhanced high-temperature resistance.

[0008] In the double mutant material dm-1 with NAT2 and NAT3 knockout genes, the nucleotide sequence of the NAT2 coding region is shown in SEQ ID NO. 7, and the amino acid sequence encoding the protein is shown in SEQ ID NO. 8; the nucleotide sequence of the NAT3 coding region is shown in SEQ ID NO. 9, and the amino acid sequence encoding the protein is shown in SEQ ID NO. 10. In the double mutant material dm-2 with NAT2 and NAT3 knockout genes, the nucleotide sequence of the NAT2 coding region is shown in SEQ ID NO. 11, and the amino acid sequence encoding the protein is shown in SEQ ID NO. 12; the nucleotide sequence of the NAT3 coding region is shown in SEQ ID NO. 13, and the amino acid sequence encoding the protein is shown in SEQ ID NO. 14.

[0009] In the heat stress experiment involved in this invention, the heat stress temperature used during the rice seedling stage was 45°C. The field experiment was conducted in Changxing County, Huzhou City, Zhejiang Province, and local temperature changes were recorded during the planting period.

[0010] The specific solution of the present invention is as follows:

[0011] The application of genes NAT2 and NAT3 in heat stress tolerance of rice provided by this invention, the genomic DNA sequences of genes NAT2 and NAT3 are as follows:

[0012] i) The nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2; or

[0013] ii) Nucleotide sequences of SEQ ID NO.1 and SEQ ID NO.2 that have been substituted, deleted, and / or have one or more nucleotides added and express the same functional protein; or

[0014] iii) Nucleotide sequences that have more than 85% homology with the nucleotide sequences of i) or ii) and express the same functional protein.

[0015] The present invention provides NAT2 and NAT3 genes for controlling rice heat stress resistance. The gene sequences are derived from the rice variety ZH11. The full-length coding region of gene NAT2 is 753 bases, as shown in SEQ ID NO.3, and the full-length coding region of gene NAT3 is 747 bases, as shown in SEQ ID NO.4. Protein NAT2 contains 250 amino acids, as shown in SEQ ID NO.5, and protein NAT3 contains 248 amino acids, as shown in SEQ ID NO.6. Further research revealed that genes NAT2 and NAT3 play important roles in rice's heat tolerance under heat stress.

[0016] The application of genes NAT2 and NAT3 in improving rice high-temperature resistance: Simultaneous mutation of NAT2 and NAT3 genes in rice resulted in gene-edited plants with significantly improved seedling survival rate under heat stress, enhancing rice's resistance to natural high-temperature climate in the field and increasing rice yield.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] This invention reveals for the first time the application of genes NAT2 and NAT3 in improving the high-temperature resistance of rice. Using gene editing technology, NAT2 and NAT3 were mutated in the rice variety ZH11. The resulting mutant material exhibited significantly higher high-temperature resistance compared to the wild type, and the survival rate of the double-mutant rice seedlings under high-temperature stress during the seedling stage was significantly higher than that of the wild-type ZH11. In field trials, under the influence of high summer temperatures, the seed setting rate and yield of the wild-type ZH11 decreased significantly, while the number of tillers, seed setting rate, and yield of the mutant rice were all significantly higher than those of the wild-type ZH11. Further physiological experiments showed that after exposure to high-temperature stress, the ROS content in the leaves of the double-mutant rice seedlings increased rapidly, prompting the closure of stomata on the leaves, thereby achieving water conservation within the rice seedlings under high-temperature conditions and improving the rice's tolerance to high temperatures. The gene and manipulation techniques provided by this invention have great application potential in improving the heat resistance of rice. They can produce cultivated rice materials that do not contain transgenic vectors and whose traits can be stably inherited, and have high application value. Attached Figure Description

[0019] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0020] Figure 1 This describes the expression of genes NAT2 and NAT3 induced by high temperature.

[0021] Figure 2 The heat tolerance phenotypes of ZH11 seedlings and gene knockout mutants dm-1 and dm-2 were compared.

[0022] Figure 3 To compare the survival rates of seedlings from control ZH11 with those from gene knockout mutants dm-1 and dm-2;

[0023] Figure 4 This refers to the temperature changes in Changxing during the field experiment.

[0024] Figure 5 To photograph and record the field phenotypes of rice after maturity, comparing ZH11 with gene knockout mutants dm-1 and dm-2.

[0025] Figure 6After the field experiment, photographs were taken to record the phenotypes of single rice plants of control ZH11 and gene knockout mutants dm-1 and dm-2.

[0026] Figure 7 To collect agronomic traits of control ZH11 and gene knockout mutants dm-1 and dm-2 rice after the field experiment;

[0027] Figure 8 NBT staining experiments confirmed that under high temperature stress, the superoxide anion content in the leaves of rice seedlings of gene knockout mutants dm-1 and dm-2 increased rapidly compared with the control ZH11.

[0028] Figure 9 The results of H2O2 content detection in rice seedling leaves of ZH11 and gene knockout mutants dm-1 and dm-2 confirmed that under high temperature stress, the superoxide anion content in the leaves of rice seedlings of gene knockout mutants dm-1 and dm-2 increased rapidly compared with the control ZH11.

[0029] Figure 10 This demonstrated that proteins NAT2 and NAT3 have the ability to transport H2O2 in a yeast system.

[0030] Figure 11 To observe under cryo-electron microscopy that, compared with the control ZH11, the gene knockout mutants dm-1 and dm-2 rice seedlings had a higher degree of stomatal closure on their leaves under high temperature stress;

[0031] Figure 12 To statistically analyze the ratio of open and closed stomata on rice leaves of control ZH11 and gene knockout mutants dm-1 and dm-2 under cryo-electron microscopy.

[0032] Figure 13 The gene knockout mutants dm-1 and dm-2 showed significant high-temperature resistance in both the seedling and reproductive stages. Detailed Implementation

[0033] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions or according to the conditions recommended in the reagent manufacturer's instructions.

[0034] Example 1: Knocking out genes NAT2 and NAT3 enhances heat resistance in rice.

[0035] To investigate the roles of genes NAT2 and NAT3 in heat stress response, this invention created NAT2 / NAT3-CRISPR transgenic plants. This example uses the japonica rice variety ZH11.

[0036] The creation of NAT2 / NAT3-CRISPR plants can follow conventional methods for cultivating transgenic plants, including the following steps: A. Constructing a CRISPR-Cas9 expression vector targeting the above genes and introducing the expression vector into Agrobacterium; B. Transforming the callus tissue of the target plant using the Agrobacterium infection method to obtain transgenic plants. The specific operational steps are as follows:

[0037] (1) Construction of CRISPR-CAS9 vector

[0038] A method for knocking out rice genes using CRISPR-Cas9 technology was employed. The nucleotide sequence of the sgRNA targeting the NAT2 gene was designed as 5'-ACGCCGCCTTGAGTGCACCCGGG-3' (SEQ ID NO.15), and the nucleotide sequence of the sgRNA targeting the NAT3 gene was designed as 5'-ACCAGCCCGGCCGGGTCAAGCGG-3' (SEQ ID NO.16). A dual sgRNA vector targeting both NAT2 and NAT3 genes was constructed by ligating the Cas9 expression plasmid using enzyme digestion and ligation.

[0039] (2) Transformation of Agrobacterium tumefaciens with CRISPR-CAS9 vector

[0040] The constructed CRISPR-CAS9 vector was transformed into Agrobacterium strain EHA105 using the freeze-thaw method. Genetically engineered bacteria containing this CRISPR-CAS9 vector were obtained by screening on LB plates using kanamycin and rifampin.

[0041] (3) Screening transgenic plants by infecting rice protoplasts with Agrobacterium tumefaciens

[0042] Agrobacterium infection of rice callus tissue, followed by co-culture, screening, differentiation, rooting, and hardening-off of the transformed material into transgenic seedlings. After transplanting and selection with hygromycin, T0 generation NAT2 / NAT3-CRISPR transgenic positive seedlings were obtained. PCR amplification and gene sequencing identified two homozygous double mutant plants, dm-1 and dm-2. The mutant lines selected from the T0 generation were propagated to obtain several seeds of the two homozygous mutants.

[0043] The primer sequences used to identify mutations at the target location are:

[0044] sq-NAT2-F 5'-GGCAGTGACAACACACAAGG-3'(SEQ ID NO.17)sq-NAT2-R 5'-ACAGCCTCACACGATCAATT-3'(SEQ ID NO.18)sq-NAT3-F 5'-TCCTCTGCTCACTCAGCTCT-3'(SEQ ID NO.19)sq-NAT3-R 5'-GGGCGATCCAGTAGAAGACG-3'(SEQ ID NO.20)

[0045] In the dm-1 material, a double mutant material with the NAT2 and NAT3 genes knocked out:

[0046] The nucleotide sequence of the NAT2 coding region is shown in SEQ ID NO.7;

[0047] The amino acid sequence of the protein encoded by the mutant form NAT2 is shown in SEQ ID NO. 8;

[0048] The nucleotide sequence of the NAT3 coding region is shown in SEQ ID NO.9.

[0049] The amino acid sequence of the protein encoded by the mutant form NAT3 is shown in SEQ ID NO.10.

[0050] In the dm-2 material, a double mutant material with the NAT2 and NAT3 genes knocked out:

[0051] The nucleotide sequence of the NAT2 coding region is shown in SEQ ID NO.11;

[0052] The amino acid sequence of the protein encoded by the mutant form NAT2 is shown in SEQ ID NO.12;

[0053] The nucleotide sequence of the NAT3 coding region is shown in SEQ ID NO.13;

[0054] The amino acid sequence of the protein encoded by the mutant form NAT3 is shown in SEQ ID NO.14.

[0055] This invention investigated the heat tolerance of ZH11 and NAT2 NAT3 double mutants (dm-1, dm-2) in seedlings. Wild-type ZH11 and the double mutants dm-1 and dm-2 were cultured in a plant incubator for 7 days, then treated at 45℃ for 3 days. The seedlings were then transferred back to normal conditions for recovery for 7 days. Photos were taken and recorded at three time points: before treatment, at the end of treatment, and 7 days after recovery. The final survival rate after recovery was also calculated. The results showed that, compared to ZH11, the double mutant plants exhibited significant resistance to high temperatures, and their survival rate was significantly higher than that of the control wild-type ZH11. Phenotypic results are shown below. Figure 2 Survival rate comparison can be found in Figure 3 In summary, these results indicate that genes NAT2 and NAT3 play an important role as regulatory genes for heat tolerance in rice, and the genes and manipulation techniques provided in this invention have great application potential in improving the heat tolerance of rice.

[0056] This invention conducted a field experiment on the ZH11 and NAT2NAT3 double mutants (dm-1, dm-2) under high-temperature summer conditions: wild-type ZH11 and the double mutants dm-1 and dm-2 were sown in the Changxing Agricultural Experimental Field, with 25 rice seedlings planted in each experimental plot, and each plot covering an area of ​​2.25 square meters. During the field experiment, local temperature changes in Changxing were recorded, and the field phenotypes of the ZH11 and NAT2NAT3 double mutants (dm-1, dm-2) were photographed. The double mutants dm-1 and dm-2 were taller than the wild-type ZH11. Figure 5 After the field experiment, agronomic traits such as tiller number, total number of grains per plant, number of grains per plant with seed, seed setting rate, thousand-grain weight, and yield per experimental plot were statistically analyzed. The results showed that the double mutants dm-1 and dm-2 had significantly higher tiller number, seed setting rate, and yield per experimental plot than the wild type ZH11. Figure 7 These results confirm that knocking out the genes NAT2 and NAT3 can significantly improve the rice's tolerance to high temperatures.

[0057] This invention further explores the molecular mechanisms by which genes NAT2 and NAT3 regulate rice resistance to high-temperature stress. NBT staining experiments revealed that, after high-temperature treatment, compared to the wild-type control ZH11, the superoxide anion content in the leaves of rice seedlings from the gene knockout mutants dm-1 and dm-2 increased rapidly. Figure 8 Furthermore, the H2O2 content in the leaves of wild-type ZH11 and gene knockout mutants dm-1 and dm-2 rice seedlings after short-term high-temperature stress treatment was detected using an H2O2 probe. The results showed that under high-temperature stress, compared with the control ZH11, the H2O2 content in the leaves of gene knockout mutants dm-1 and dm-2 rice seedlings increased rapidly. Figure 9 Subsequently, the ability of proteins NAT2 and NAT3 to transport H2O2 was confirmed in a yeast system, see [link to relevant documentation]. Figure 10 The proportions of open and closed stomata on leaves of wild-type ZH11 and gene knockout mutants dm-1 and dm-2 rice seedlings under high-temperature stress were observed and statistically analyzed under cryo-electron microscopy. The results showed that, compared with the control ZH11, the gene knockout mutants dm-1 and dm-2 rice seedlings exhibited a higher degree of stomatal closure. Figure 12 The above results indicate that proteins NAT2 and NAT3 can regulate rice heat tolerance by transporting H2O2 as a second messenger molecule.

[0058] Finally, it should be emphasized that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations and extensions possible. Anyone with ordinary technical knowledge in the art can directly deduce or reasonably conceive of all other possible variations based on the disclosure of the present invention, and these variations should also be considered part of the scope of protection of the present invention.

Claims

1. The application of genes NAT2 and NAT3 in improving heat resistance in rice, characterized in that, In rice, gene editing technology is used to knock out the NAT2 and NAT3 genes, thereby inactivating their functions and improving the rice's heat resistance. NAT2 The genomic DNA sequence is shown in SEQ ID NO.1, gene NAT3 The genomic DNA sequence is shown in SEQ ID NO.

2.

2. The application of genes NAT2 and NAT3 according to claim 1 in improving the heat resistance of rice, characterized in that, Specifically, it includes: Using gene editing technology to modify rice genes NAT2 and NAT3 The coding region of the gene undergoes base substitution, deletion, or insertion. NAT2 The base sequence of the coding region is shown in SEQ ID NO.3, gene. NAT3 The coding region base sequence is shown in SEQ ID NO.4, which makes the rice gene... NAT2 and NAT3 The coding regions of these proteins can cause frameshift mutations or premature translation termination, leading to the inactivation of NAT2 and NAT3 proteins.

3. The application of genes NAT2 and NAT3 according to claim 2 in improving the heat resistance of rice, characterized in that, The gene editing technology described uses CRISPR-Cas9 gene editing technology.

Images