Application of rice endosperm dominant expression gene IRE1 in rice quality heat resistance at natural high temperature in field

Abstract

The invention relates to application of a rice endosperm dominant expression gene IRE1 in rice quality heat resistance at natural high temperature in a field. The receptor gene IRE1 of the UPR is identified from rice, the function of the receptor gene IRE1 in forward regulation of rice quality heat resistance is successfully identified through a transgenic material, chalkiness is remarkably lower than that of a wild type by overexpressing the IRE1 gene at a high temperature, the protein content is remarkably increased, amylose is remarkably reduced, and the rice quality heat resistance is remarkably improved. The ratio of the storage protein content to the amylose content is obviously increased, and good high-temperature resistance is shown. A new gene resource is provided for rice quality breeding under the global warming background, and the gene has very important significance for cultivating high-temperature-resistant rice varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular breeding, and particularly relates to the application of the rice endosperm-preferential expression gene IRE1 in the heat tolerance of rice quality under natural high temperature in the field. Background Art

[0002] Global food security is not only closely related to yield, but also to quality, which determines human nutrition intake, market value, and the income of farmers. Against the backdrop of global warming, high-temperature records are constantly broken, leading to the deterioration of grain quality, including grain appearance, milling, cooking and eating quality, nutritional characteristics, and market value. Rice (Oryza sativa L.) is one of the top three crops in the world in terms of food production, and nearly half of the world's population regards rice as their staple food. In recent years, people's material and cultural living standards have improved a lot. People's requirements for rice quality have also changed unconsciously. High-quality rice should not only have a good taste, but also be green, healthy, and good-looking. High yield and high quality of rice are important goals of basic research, crop genetic improvement, and application, but there is a certain "contradiction" between the two, and it is often impossible to have both. However, in the actual process of variety breeding, rice yield has always been the focus of breeders' attention, while rice quality has rarely been taken seriously. Among the currently approved rice varieties, the quality is uneven, and the proportion of high-quality rice is relatively low, especially in the early and middle-season rice in the middle and lower reaches of the Yangtze River. In recent years, frequent high-temperature weather has posed a severe challenge to the high yield and high quality of rice. Against this background, the problem of poor rice quality in China has become increasingly exposed. The evaluation of rice quality shows little difference worldwide. Among them, appearance quality is easy to be noticed because it is obvious, and cooking and eating quality has also become a trait that consumers will not ignore because it is relatively easy to distinguish. The other two are rarely noticed because ordinary consumers have no access to them. The first two are the evaluation indicators of rice grades and also the most important rice quality traits. Appearance quality is mainly determined by three factors. The first is grain shape, that is, the shape of rice; the second is transparency, that is, the degree of light transmission of rice; the third is chalkiness, that is, the white and opaque part of rice. The unfolded protein response (UPR) induced by abiotic stresses such as high temperature will inevitably affect crop physiological metabolism and grain development, usually reducing crop yield and quality. Abnormal endosperm development will have a direct impact on the appearance quality and cooking and eating quality of rice. One of the most intuitive manifestations is the formation of chalkiness. Chalkiness is a poor trait that seriously affects rice quality, and its formation reasons are complex and diverse. Biotic and abiotic stresses, unstable filling speed, abnormal starch granule size in the endosperm, and irregular protein body arrangement will all form chalkiness. High temperature during the filling period has a serious impact on japonica rice varieties. High temperature will lead to abnormal endosperm development of rice, and temperatures above 26°C are likely to cause chalkiness and reduced grain weight in rice. Brown rice with severe chalkiness is poor in polishing quality and taste. Rice chalkiness greatly reduces the eating quality and economic value of rice and is one of the most important quality traits of rice. If high-temperature heat damage occurs frequently in the main rice-producing areas, it will lead to serious production reduction and a significant decrease in rice quality.

[0003] The endoplasmic reticulum (ER) is an important organelle involved in protein quality control and cellular homeostasis. The accumulation of unfolded proteins leads to ER stress and subsequently activates the Unfolded Protein Response (UPR), which triggers a set of genes to overcome the accumulation of unfolded proteins in the ER. Of course, ER stress agents are a type that causes ER stress in plants. Multiple abiotic stresses, such as heat and salt stresses, and biotic agents can induce the UPR. However, few studies have revealed the relationship between the UPR and endosperm development, especially the molecular basis of the UPR regulated by the sensor IRE1 of the UPR in a high-temperature environment. However, the mechanism by which this sensor responds to high temperature to ensure normal endosperm development is unclear. Therefore, it is urgent to reveal the regulatory network of how the IRE1 gene responds to high temperature to ensure normal endosperm development in a high-temperature environment, which will contribute to the development and utilization of new high-quality heat-resistant rice varieties. Summary of the Invention

[0004] In the present invention, the sensor gene IRE1 (LOC_Os07g28820) of the UPR was identified from rice, and its function in positively regulating the heat resistance of rice quality was successfully identified through transgenic materials, providing new gene resources for rice quality breeding under the background of global warming, which is of great significance for cultivating heat-resistant rice varieties.

[0005] The purpose of the present invention is to provide the application of the sensor IRE1 gene of the UPR in rice and its encoded protein in improving the heat resistance of rice quality (such as chalky appearance quality and storage substance content, etc.) in a natural high-temperature environment.

[0006] The present invention provides the application of a rice IRE1 protein, a nucleic acid molecule encoding the protein, an expression cassette containing the nucleic acid molecule, a recombinant vector, a transgenic cell line or a recombinant bacterium in any of the following:

[0007] A1) Regulating the heat resistance of rice quality;

[0008] A2) Preparing a product for regulating the heat resistance of rice quality;

[0009] A3) Cultivating heat-resistant rice germplasm with good quality;

[0010] A4) Preparing a product for cultivating heat-resistant rice germplasm with good quality;

[0011] The amino acid sequence of the IRE1 protein is shown as SEQ ID NO.3.

[0012] Furthermore, the nucleotide sequence encoding the IRE1 protein is shown as SEQ ID NO.2, and the genomic sequence of IRE1 is shown as SEQ ID NO.1.

[0013] Furthermore, rice quality includes chalky appearance quality and storage substance content. Among them, chalky appearance quality includes chalky rice rate and chalkiness degree; storage substances include storage proteins and amylose, and storage proteins include glutelin precursors and glutelins.

[0014] Furthermore, the heat tolerance of rice quality is improved by enhancing the expression, function or activity of the IRE1 gene or its encoded protein in rice, that is, IRE1 reduces the chalkiness of rice under high temperature by maintaining a higher proportion of storage protein to amylose content in the endosperm, thereby positively regulating the heat tolerance of rice quality under high temperature.

[0015] The present invention provides a method for enhancing and / or reducing rice quality under high temperature, by enhancing and / or reducing the expression, function or activity of the IRE1 gene or its encoded protein in rice to enhance and / or reduce the heat tolerance of rice quality, and the protein sequence encoded by the IRE1 gene is shown in SEQ ID NO.3.

[0016] Furthermore, the nucleotide sequence encoding the IRE1 protein is shown in SEQ ID NO.2.

[0017] Furthermore, IRE1 reduces the chalky quality of rice under high temperature by maintaining a higher proportion of storage protein to amylose content in the endosperm, thereby positively regulating the heat tolerance of rice quality under high temperature.

[0018] The present invention provides a method for cultivating heat-tolerant rice germplasm with good quality, including enhancing, increasing or upregulating the expression level of the gene encoding the IRE1 protein and / or the expression, function or activity of the IRE1 protein in the recipient rice, and then obtaining rice germplasm with significantly better heat tolerance of quality than the recipient rice, with significantly increased storage protein, significantly reduced amylose, and significantly increased ratio of storage protein to amylose content, showing better high temperature resistance. The protein sequence encoded by the IRE1 gene is shown in SEQ ID NO.3.

[0019] Furthermore, the nucleotide sequence encoding the IRE1 protein is shown in SEQ ID NO.2.

[0020] Furthermore, heat-tolerant rice germplasm with good quality is obtained by transgenic means.

[0021] Beneficial effects: A new gene IRE1 that regulates rice quality (chalkiness appearance quality, storage substance content, etc.) under natural high temperature was cloned in rice. It positively regulates the heat tolerance of rice quality by maintaining the relative steady-state balance of endosperm storage substance content under high temperature in the field. Field trials showed that overexpression of IRE1 under high temperature can significantly reduce chalkiness, significantly increase protein content, and significantly reduce amylose content, resulting in a significant increase in the ratio of storage protein to amylose content, showing good high-temperature resistance. It provides a new gene resource for high-quality rice breeding and also provides a technical reference for cloning related genes in other crops. Brief Description of the Drawings

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use 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 limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 Expression patterns of the IRE1 gene in different tissues of the japonica rice variety Nipponbare (NIP), where Figure A shows the expression levels of IRE1 in different tissues of rice, and Figure B shows the expression levels of IRE1 in the endosperm from 7 to 42 days after fertilization.

[0024] Figure 2 Editing types and genotypes of the IRE1 knockout lines and expression levels of the IRE1 overexpression lines, where Figure A shows the expression level detection of three IRE1 overexpression line materials, and Figure B shows the gene editing types of three IRE1 knockout lines.

[0025] Figure 3 Chalkiness quality phenotypes of the IRE1 gene knockout lines and IRE1 overexpression lines under different field temperature conditions, where Figure A shows the chalkiness quality scanning images of two batches of materials in Wuhan in 2023, and Figure B shows the chalkiness quality statistical charts of two batches of materials in Wuhan in 2023.

[0026] Figure 4 Storage protein, amylose content of the IRE1 gene knockout lines and IRE1 overexpression lines, and IRE1 positively regulates the heat tolerance of rice quality by affecting the expression of genes related to storage substance synthesis, where Figure A shows the storage protein, amylose content, and the ratio of the two storage substance contents of the IRE1 gene knockout lines, ZH11 wild type, and IRE1 overexpression lines, and Figure B shows the protein levels of Wx and glutelin detected in the genetic material of IRE1. Detailed Embodiments

[0027] The following examples are only used to illustrate the technical solutions of the present invention more clearly. Therefore, they are only examples and cannot be used to limit the protection scope of the present invention. It should be noted that unless otherwise specified, the technical terms or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art to which the present invention belongs. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0028] Example 1 Natural high temperature treatment in the field during the rice filling period

[0029] In order to subject each rice genetic material to natural high temperature and normal temperature treatments during the filling period, the sowing periods of each genetic material were planted in batches, and the planting times of each were adjusted according to the length of the heading period of each material. Generally, sowing was carried out once every about half a month, and every effort was made to ensure that heading and the early stage of filling occurred during the high temperature period in the fields of Wuhan. For materials with a relatively long heading period (>95 days), sowing was usually carried out in the middle or late April; for materials with a relatively short heading period (<80 days), sowing was carried out around mid-May. Different sowing dates enabled genetic materials at different growth stages to uniformly start heading and filling in the middle or late July or early August. During this period, extremely high temperature weather exceeding 35°C often occurred in Wuhan, and this record has lasted for more than 60 years. For the normal temperature treatment experiment, all materials were sown in mid-June or at the end of June. In this way, short-heading materials usually started heading and filling in the first or second ten days of September, while long-growth-stage materials started heading and filling in the second or third ten days of September. During this period, the average temperature in Wuhan was relatively low and normal (about 30°C). The dynamic air temperature data automatically recorded every 5 minutes from June to October at Huazhong Agricultural University and from February to May in the field of the Nanshan Base in Lingshui City was measured using a USB temperature and humidity recorder (USB-TH). The early filling stage is the most sensitive period for rice endosperm. Therefore, the daily average temperature during the early filling stage, 6 - 12 days after flowering of each material, was statistically analyzed from 9 am to 7 pm. When the chalkiness rate of the rice of the sensitive material japonica rice ZH11 increased significantly, it indicated that these materials had undergone high temperature treatment during the filling period. On the contrary, when the sensitive wild-type materials still maintained a relatively low grain chalkiness rate, it indicated that they were filling under normal temperature.

[0030] Example 2 Identification of rice quality (chalkiness appearance quality, storage substance content, etc.) under natural field temperature

[0031] Before examining quality traits such as rice chalkiness, the harvested mature seeds are fully dried or sun-dried and stored at room temperature for at least three months. Rice chalkiness, including belly white, heart white, and back white, is generally detected by visual inspection. The mature and dry seeds are threshed and hulled into brown rice. Randomly select 100 intact rice grains, and the percentage of rice grains with chalkiness among them is counted and expressed as the grain chalkiness rate (GCR). Place the rice with chalkiness flat and visually estimate the percentage of the projected area of the chalky part in the whole rice's projected area, which is expressed as the rice chalkiness area (GCA). Chalkiness degree (GCD) is the product of the grain chalkiness rate and the chalkiness area. Chalkiness in rice will reduce the appearance, milling, cooking, and eating quality of rice, as well as the head rice yield. Therefore, the increase in chalkiness is the most direct indicator of rice quality, and high temperature is extremely likely to cause sensitive changes in chalkiness. Therefore, lower rice chalkiness at high temperature indicates stronger high-temperature tolerance of rice quality. Use chemical methods to measure the total protein (the sum of four major storage proteins) content of polished rice flour and the amylose content of polished rice flour.

[0032] The experimental procedure is as follows:

[0033] 1. Measuring the content of four proteins in rice by chemical method

[0034] 1) Take the rice sample to be tested, hull the sample into brown rice and then grind it into polished rice flour;

[0035] 2) Weigh 0.02 g of polished rice flour into a 2 mL centrifuge tube;

[0036] 3) Add 800 μL of albumin extraction solution, mix well with an oscillator for 2 h, centrifuge at 12000 r / min at 4 °C for 15 min, aspirate 700 μL of the supernatant into a new 2 mL centrifuge tube, add another 600 μL of albumin extraction solution, mix well with an oscillator for 2 h, centrifuge at 12000 r / min at 4 °C for 15 min, and aspirate 600 μL of the supernatant into a 2 mL centrifuge tube;

[0037] 4) Add 700 μL of globulin extraction solution, mix well with an oscillator for 2 h, centrifuge at 12000 r / min at 4 °C for 15 min, aspirate 700 μL of the supernatant into a new 2 mL centrifuge tube, add another 600 μL of globulin extraction solution, mix well with an oscillator for 2 h, centrifuge at 12000 r / min at 4 °C for 15 min, and aspirate 600 μL of the supernatant into a 2 mL centrifuge tube;

[0038] 5) Add 1 mL of prolamin extraction solution, mix well with an oscillator for 2 h, centrifuge at 12000 r / min at 4 °C for 15 min, aspirate 1 mL of the supernatant into a new 2 mL centrifuge tube, add another 1 mL of prolamin extraction solution, mix well with an oscillator for 2 h, centrifuge at 12000 r / min at 4 °C for 15 min, and aspirate 1 mL of the supernatant into a 2 mL centrifuge tube;

[0039] 6) Add 500 μL of gluten extract, mix well on an oscillator for 2 h, centrifuge at 12,000 r / min at 4 °C for 15 min, pipette 500 μL of the supernatant into a new 2 mL centrifuge tube, add another 500 μL of gluten extract, mix well on an oscillator for 2 h, centrifuge at 12,000 r / min at 4 °C for 15 min, and pipette 500 μL of the supernatant into a 2 mL centrifuge tube; (When measuring the gluten concentration with an ELISA plate, it needs to be diluted 10 times. Pipette 200 μL into a 2 mL centrifuge tube and add 1.8 mL of distilled water to mix well.)

[0040] 7) Measure the total protein content with an ELISA reader (absorbance value 595); Pipette 40 μL of each sample into an ELISA tube. For gluten, pipette 40 μL after dilution, then add 200 μL of the dye solution in the dark, centrifuge at low speed, store in the dark, and detect with an ELISA instrument.

[0041] Preparation of solutions:

[0042] Albumin extract: 900 mL of dd H2O + 100 mL of 0.1 mol / L Tris-HCl (pH = 7.5)

[0043] Globulin extract: 200 mL of 5 mol / L NaCl + 100 mL of 0.1 mol / L Tris-HCl (pH = 7.5)

[0044] Prolamin extract: 700 mL of absolute ethanol + 100 mL of 0.01 mol / L EDTA-2Na + 200 mL of ddH2O

[0045] Gluten extract: 0.05 mol / L NaOH

[0046] Coomassie Brilliant Blue G-250 dye: Weigh 50 mg of Coomassie Brilliant Blue and dissolve it in 25 mL of 95% ethanol. After complete dissolution, add 50 mL of 85% (W / V) phosphoric acid, and make up the volume to 500 mL with distilled water. Store in a brown reagent bottle in the dark.

[0047] 2. Measurement of amylose content in rice by chemical method

[0048] 1) Weigh 4 g of NaOH and place it in a beaker. Add 50 mL of distilled water to dissolve it, transfer it to a 100 mL volumetric flask, and make up the volume to 100 mL.

[0049] 2) Weigh 6.005 g of glacial acetic acid and place it in a beaker. Add 50 mL of distilled water to dissolve it, transfer it to a 100 mL volumetric flask, and make up the volume to 100 mL.

[0050] 3) Weigh 2 g of KI and dissolve it in 100 mL of water. After complete dissolution, add 0.2 g of iodine flakes and dissolve them thoroughly.

[0051] 4) Weigh 0.01 g of the sample and send it to the bottom of the test tube together with the reference standards with contents of 0.4%, 10.6%, 16.2%, and 26.5%. Label the 10 mL capped test tube (ensure 2 - 3 accurate replicates).

[0052] 5) Add 0.1 mL of absolute ethanol and mix well.

[0053] 6) Add 0.9 mL of 1 mol / L NaOH solution, shake well, and cover with the lid.

[0054] 7) Place in a water bath at 100 °C and boil for 10 min. After taking it out, cool to room temperature, add distilled water to make up to 10 mL, and shake with an oscillator.

[0055] 8) Prepare a mixture of 9.2 mL of distilled water + 0.1 mL of 1 mol / L acetic acid solution + 0.2 mL of iodine solution, mix well by shaking. Pipette 190 μL of the mixture into the enzyme - labeled tube, and then pipette 10 μL of the sample solution into the enzyme - labeled tube and stir evenly.

[0056] 9) Use the blank solution to adjust the zero point of the spectrophotometer at 620 nm, and then measure the absorbance value of the sample solution.

[0057] Draw a standard curve with the reference standards and calculate the amylose content.

[0058] Example 3: The gene IRE1 that is preferentially expressed in rice endosperm is highly expressed under high temperature and can resist high temperature

[0059] Using the RiceXPro rice expression profile database, it was found that IRE1 is preferentially highly expressed in the endosperm ( Figure 1 ). The genomic sequence of IRE1 is shown as SEQ ID NO.1, the amino acid sequence of the IRE1 protein is shown as SEQ ID NO.3, and the nucleotide sequence encoding the IRE1 protein is shown as SEQ ID NO.2.

[0060] Construct the IRE1 gene knockout family and the IRE1 over - expression family ( Figure 2 )

[0061] 1. Construction of the CRISPR vector

[0062] Use the CRISPR - Cas9 system of Yun - De Zhao from Huazhong Agricultural University to construct the U6 - U3 - CRISPR dual - target vector. If the first base of the sgRNA sequence is G, use the OsU6 promoter; if the first base of the sgRNA sequence is A, use the OsU3 promoter.

[0063] 2. Construction of the over - expression vector

[0064] The steps for constructing the IRE1 overexpression vector using the overexpression vector pCAMBIA2300 of our laboratory are as follows:

[0065] First, the target gene IRE1 coding region was amplified, and the forward primer sequence was designed to be IRE1-OE-PAGE-F in Table 1, and the reverse primer was IRE1-OE-PAGE-R. Then, the homologous recombinase was used for connection, and the connection product was transferred into the competent E. coli DH5α for plate plating. Subsequently, single clones were selected for shake sequencing, and plasmids with correct sequencing and no mutation were selected for genetic transformation of japonica rice.

[0066] Agrobacterium-mediated genetic transformation of japonica rice: insert the target gene IRE1 into the T-DNA region of the modified Ti plasmid, use Agrobacterium infection to achieve the transfer and integration of exogenous genes into rice callus, and then regenerate transgenic plants through tissue culture technology to achieve the purpose of overexpressing the target gene. The specific experimental steps (all processes are sterile) are as follows:

[0067] 1. Inducing callus: Prepare sterile induction medium in advance; remove the husks from the seeds, soak them in 75% ethanol for 1 minute, then soak them in 0.15% HgCl2 for 15-25 minutes, and finally wash them with sterile dH2O for 5-10 times; inoculate 8 to 12 seeds into each bottle of medium, and culture them in the dark for 40 to 50 days to induce the formation of callus tissue;

[0068] 2. Subculture: From the induced callus, pick the light yellow, granular, dry, and vigorous callus tissue and transfer it to the subculture medium for dark culture for 20 days;

[0069] 3. Pre-culture: From the subculture callus, pick the light yellow, granular, dry, and vigorous callus tissue and transfer it to the pre-culture medium for dark culture for 3 days;

[0070] 4. Infection and co-cultivation: ① 2 days before the experiment, streak the Agrobacterium strain containing the target gene on a plate containing antibiotics to activate it; ② Scrape the streaked cultured Agrobacterium into 1 / 2N6 suspension medium (add 100 μl AS + 2 ml 50% glucose), shake and culture at 28°C, 200 rpm for 30 minutes until the OD 600 ≈0.4; ③ While shaking the culture, collect the pre-cultured callus into a 250ml sterile conical flask; ④ Pour the Agrobacterium culture solution into the callus and soak it for 30 minutes; ⑤ Pour out the culture solution, dry the callus surface and dry it naturally for 3-4 hours; ⑥ Use a spoon to spread the fully dried callus evenly on the co-culture medium, seal it with sealing glue, and culture it in the dark at 19℃ for 3 days.

[0071] 5. Washing: ① Transfer the co-cultivated callus to a washing cup, pour in sterile distilled water until the callus is completely submerged, cover the lid and shake for 20-30 seconds, then pour out the sterile distilled water. Repeat this 2-3 times until the water in the washing cup is clear; ② Pour out the sterile distilled water, add sterile distilled water containing 500 mg / L Cn, and shake at 200 rpm for 30 minutes; ③ Pour out dH2O, absorb the water on the surface of the callus and dry it naturally.

[0072] 6. Screening: S1: ① When the callus is dried, add 400μl CN+250μl Hn+5ml 50% glucose to each bottle of screening medium and turn it upside down; ② After the callus is dry, spread it evenly on the screening medium, seal it with sealing glue, and culture it in a dark culture room for 20 days. S2: ① Prepare the screening medium, add 300μl CN+250μl Hn+5ml 50% glucose to each 250ml medium, turn it upside down; ② Select dry callus without Agrobacterium contamination from S1 medium and transfer it to a plate, and place it sparsely on S2 screening medium; ③ Culture it in the dark for 20 days, observe whether fresh and tender yellow resistant callus grows, if there is no resistant callus, continue to transfer the plate to do S3.

[0073] 7. Differentiation: Pick a small piece of resistant callus that is light yellow, dense, dry and attached to the culture medium and place it on the differentiation medium. Culture it in the light for 40 days to differentiate into seedlings (about 5-10 cm high).

[0074] 8. Rooting: Pull out the differentiated seedlings from the differentiation medium, cut off the excessively long leaves and roots with scissors, and transplant them into the rooting medium. Culture them in the light culture room for 15 to 20 days, and proceed to the next step after the roots have grown sufficiently.

[0075] 9. Hardening of seedlings: ① After the transformed seedlings grow vigorously, remove the sealing film of the rooting tube, add a certain amount of tap water, and harden the seedlings under light culture for 4-7 days; ② Gently remove the transformed seedlings from the rooting tube, wash the attachment culture medium on the roots, and transplant them to the field to complete the genetic transformation process. Subsequently, the expression level of the generated transgenic seedlings was detected, and three strains with higher expression levels were selected for subsequent phenotypic identification.

[0076] By examining the chalky phenotype of IRE1 knockout and IRE1 overexpression families, it was found that the quality of IRE1 overexpression families under high temperature was significantly better than that of the wild type and its phenotype under normal temperature, verifying that IRE1 overexpression can resist high temperature. Therefore, it is determined that IRE1 is involved in high temperature response and is highly expressed in the endosperm, and it is speculated that IRE1 may be involved in the development of seed endosperm under high temperature.

[0077] Table 1 Primers used for gene function verification of the present invention

[0078]

[0079] Example 4: Application of IRE1 gene in rice in regulating rice quality under field high-temperature environment

[0080] To verify the effect of IRE1 on endosperm development under high temperature, field natural high-temperature treatment was carried out using IRE1 gene knockout lines and IRE1 overexpression lines. Under the normal temperature condition (26.3 °C) in Wuhan in 2023, compared with the wild-type japonica rice ZH11, the chalkiness of the IRE1 knockout lines and IRE1-overexpression significantly increased ( Figure 3 ), and at the same time, the protein content significantly decreased and the amylose increased, resulting in a significant decrease in the ratio of storage protein to amylose content ( Figure 4 A). Under high temperature conditions (33.6 °C), the chalkiness of the wild-type ZH11 and the IRE1 knockout lines both significantly increased ( Figure 3 ), showing a floury phenotype, and the ratio of storage protein to amylose content significantly decreased; while the chalkiness of the IRE1 overexpression lines significantly decreased, the storage protein significantly increased, the amylose significantly decreased, and the ratio of storage protein to amylose content significantly increased ( Figure 4 A), showing better high-temperature resistance.

[0081] To verify whether IRE1 affects the expression of genes related to storage protein / starch synthesis, the protein levels of Wx and glutelin in the genetic material of IRE1 were further analyzed. Immunoblot analysis of Wx and glutelin in mature seeds of IRE1 transgenic lines was performed using anti-Wx and anti-αGlu antibodies.

[0082] The steps of SDS-PAGE analysis and immunoblot analysis of mature grain proteins are as follows: Grind mature brown rice into powder, suspend an equal amount of 0.02 g of rice flour in 500 μL of extraction solution (4% SDS, 4 M urea, 5% β-mercaptoethanol, and 125 mM Tris-HCl (pH = 6.8)), extract for 2 hours, centrifuge for 10 minutes to obtain the supernatant protein solution. The supernatant protein was analyzed by SDS-PAGE, and then Coomassie Brilliant Blue staining and photography were performed. For immunoblotting, an equal amount of supernatant protein from each sample was separated by SDS-PAGE and immunoblotted with specific antibodies (anti-αGlu and anti-Wx). Anti-actin antibody was used as a loading control. The band intensity on each gel was quantified using ImageJ software.

[0083] The results showed that ( Figure 4B), at high temperature, the abundance of Wx protein in the IRE1 overexpression lines was lower than that in the WT, while the expression level of glutelin was higher than that in the WT. However, the opposite trend was observed in the IRE1 knockout lines, indicating that IRE1 negatively regulates the expression of Wx and positively regulates the expression of 57 kDa glutelin precursor and 40 kDa α-glutelin at high temperature. The transgenic evidence of these genetic materials shows that IRE1 positively regulates the heat tolerance of rice quality at high temperature by maintaining a higher ratio of storage protein to amylose content in the endosperm, thereby reducing the chalkiness quality of rice at high temperature. That is, the present invention provides new gene resources for rice quality breeding under the background of global warming and has very important significance for cultivating high-temperature resistant rice varieties.

[0084] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solutions of the present invention, and these simple variations all belong to the protection scope of the present invention.

Claims

1. Use of a rice IRE1 protein, a nucleic acid molecule encoding the protein, an expression cassette containing the nucleic acid molecule, a recombinant vector, a transgenic cell line or a recombinant bacterium in any of the following: A1) Regulating the heat tolerance of rice grain quality; A2) Preparing a product for regulating the heat tolerance of rice grain quality; A3) Cultivating heat-tolerant rice germplasm with good quality; A4) Preparing a product for cultivating heat-tolerant rice germplasm with good quality; The amino acid sequence of the IRE1 protein is shown as SEQ ID NO.

3.

2. The application according to claim 1, wherein The nucleotide sequence encoding the IRE1 protein is shown as SEQ ID NO.

2.

3. The application according to any one of claims 1-2, characterized in that, Rice grain quality includes chalky appearance quality and storage substance content. Among them, chalky appearance quality includes chalky rice rate and chalkiness degree; storage substances include storage proteins and amylose. Storage proteins include proglutelin and glutelin.

4. The application according to claim 3, characterized in that, Improve the heat tolerance of rice quality by enhancing the expression, function or activity of the IRE1 gene or its encoded protein in rice.

5. A method for enhancing rice quality and / or reducing rice quality at high temperatures, characterized in that, Enhance and / or reduce the heat tolerance of rice quality by enhancing and / or reducing the expression, function or activity of the IRE1 gene or its encoded protein in rice. The protein sequence encoded by the IRE1 gene is shown as SEQ ID NO.

3.

6. The method according to claim 5, characterized in that The nucleotide sequence encoding the IRE1 protein is shown as SEQ ID NO.

2.

7. The method according to any one of claims 5-6, characterized in that, IRE1 positively regulates the heat tolerance of rice grain quality at high temperature by maintaining a higher ratio of storage protein to amylose content in the endosperm, thereby reducing the chalky quality of rice grains at high temperature.

8. A method for cultivating heat-resistant rice germplasm with good quality, characterized in that, Including enhancing, increasing or upregulating the expression level of the gene encoding the IRE1 protein and / or the expression, function or activity of the IRE1 protein in the recipient rice, and then obtaining rice germplasm with significantly better heat tolerance of quality than the recipient rice, with significantly increased storage protein, significantly reduced amylose, and a significantly increased ratio of storage protein to amylose content, showing better high-temperature resistance. The protein sequence encoded by the IRE1 gene is shown as SEQ ID NO.

3.

9. The method according to claim 8, wherein The nucleotide sequence encoding the IRE1 protein is shown as SEQ ID NO.

2.

10. The method according to any one of claims 8-9, characterized in that, Obtain heat-tolerant rice germplasm with good quality by transgenic means.

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