Method for improving seed-setting rate of rice under high-temperature stress environment

Abstract

A method for improving the seed-setting rate of rice under a high-temperature stress environment. Provided is the use of a target protein or a coding gene of the target protein in improving the seed-setting rate of rice grown in a high-temperature stress environment. The target protein is as set forth in SEQ ID NO: 1. High-temperature stress severely reduces the seed-setting rate of rice. The method has a great application and promotion value for cultivating high-yield rice in a high-temperature stress environment.

Description

A method for improving the seed setting rate of rice under high temperature stress environment Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for improving the seed setting rate of rice under a high temperature stress environment. Background Art

[0002] Rice is one of humanity's most important staple crops, with a long history of cultivation and consumption. Half the world's population consumes rice, primarily in Asia, southern Europe, tropical America, and parts of Africa. Rice production ranks third among global staple crops, behind corn and wheat, but its ability to sustain a significant population is why the United Nations designated 2004 as the International Year of Rice.

[0003] China is one of the original origins of rice. Farmland in southern China is primarily paddy fields, with rice being the primary crop. Six thousand years ago, the Hemudu people domesticated rice; 2,000 years ago, rice spread eastward to Japan. Later, rice spread westward to Iran, Greece, and Africa, then to Spain, Italy, France, Germany, and the United Kingdom. After the discovery of the New World, rice made its way to the Americas.

[0004] The optimum temperature for rice growth is around 25°C. When temperatures exceed 30°C, rice begins to lose control, severely impacting growth. When temperatures exceed 32°C, rice enters a state of heat stress, severely impacting growth and yield. Research shows that rice's heat tolerance range is between 30°C and 32°C; temperatures exceeding this range negatively impact rice yield and quality. When temperatures exceed 35°C, rice grows poorly, leaves begin to shrink, new leaves fail to grow, and even scorch and die. SUMMARY OF THE INVENTION

[0005] As a thermophilic crop, rice is extremely sensitive to temperature during the booting and heading stages. The most suitable temperature is 25°C to 30°C, and adverse effects will occur if the daily average temperature is above 30°C. If the booting stage is subjected to continuous high temperatures of above 35°C, the rice floral organs will be underdeveloped, the pollen will be poorly developed, and the vitality will decrease. If the high temperature is above 35°C during the heading and flowering and pollination stages, heat damage will occur, affecting the shedding of pollen and the elongation of pollen tubes, resulting in the inability to fertilize and the formation of empty shells, resulting in a decrease in the fruit set rate, a low thousand-grain weight, or even a total loss of harvest. The technical problem solved by the present invention is to provide a rice breeding method, which is used to cultivate rice germplasm with an increased fruit set rate in a high temperature stress environment. Technical Solutions

[0006] The purpose of the present invention is to provide a method for improving the seed setting rate of rice under high temperature stress environment.

[0007] The present invention provides application of a target protein in improving the seed setting rate of rice grown under a high temperature stress environment.

[0008] The present invention also provides the use of target protein-related biological materials in improving the fruit setting rate of rice grown under high temperature stress environment.

[0009] The present invention also provides the use of target protein-related biological materials in the preparation of transgenic rice; compared with recipient rice, the transgenic rice has an increased fruit setting rate under high temperature stress environment.

[0010] The present invention also provides a method for preparing transgenic rice with improved fruit setting rate under high temperature stress environment, comprising the following steps: introducing and expressing the coding gene of the target protein into the starting rice to obtain transgenic rice with improved fruit setting rate under high temperature stress environment.

[0011] The present invention also provides a method for producing heat-stress-resistant transgenic rice, comprising the steps of introducing and expressing a gene encoding a target protein into a starting rice plant to obtain the heat-stress-resistant transgenic rice. The heat-stress resistance is manifested by an increase in the seed setting rate of the transgenic rice under heat stress relative to the starting rice plant.

[0012] The present invention also provides the use of the target protein in improving the stress tolerance of rice to high temperature stress. The improvement of the stress tolerance to high temperature stress is reflected in inhibiting the reduction of the fruit set rate caused by high temperature stress.

[0013] The present invention also provides the use of target protein-related biological materials in improving the stress tolerance of rice to high temperature stress. The improvement of stress tolerance to high temperature stress is reflected in inhibiting the reduction of seed setting rate caused by high temperature stress.

[0014] Any of the above target proteins is shown in SEQ ID NO: 1.

[0015] The target protein-related biological material is any one of the following (a1) to (a8):

[0016] (a1) a nucleic acid molecule encoding the target protein shown in SEQ ID NO: 1;

[0017] (a2) an expression cassette containing the nucleic acid molecule described in (a1);

[0018] (a3) a recombinant vector containing the nucleic acid molecule described in (a1);

[0019] (a4) a recombinant vector containing the expression cassette described in (a2);

[0020] (a5) a recombinant microorganism containing the nucleic acid molecule described in (a1);

[0021] (a6) a recombinant microorganism containing the expression cassette described in (a2);

[0022] (a7) a recombinant microorganism containing the recombinant vector described in (a3);

[0023] (a8) A recombinant microorganism containing the recombinant vector described in (a4).

[0024] Illustratively, the nucleic acid molecule encoding the target protein shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2.

[0025] Specifically, the nucleic acid molecule is a DNA molecule.

[0026] Illustratively, the gene encoding any of the above target proteins is shown in SEQ ID NO: 2.

[0027] The recombinant vector can specifically be the following recombinant plasmid: a recombinant plasmid obtained by inserting the double-stranded DNA molecule shown in SEQ ID NO: 2 into the multiple cloning site of a plant expression vector.

[0028] The recombinant vector can specifically be the following recombinant plasmid: a recombinant plasmid obtained by inserting the double-stranded DNA molecule shown in SEQ ID NO: 2 into the multiple cloning site of the pCAMBIA1305.1 plasmid.

[0029] The recombinant vector can be specifically the following recombinant plasmid: the small fragment between the KpnI and XbaI enzyme recognition sequences of the pCAMBIA1305.1 plasmid is replaced with the double-stranded DNA molecule shown in SEQ ID NO: 2, while other sequences remain unchanged, to obtain the recombinant plasmid.

[0030] The recombinant microorganism may specifically be a recombinant Agrobacterium obtained by introducing the recombinant vector into Agrobacterium.

[0031] Exemplarily, the Agrobacterium is Agrobacterium EHA105.

[0032] Any of the above-mentioned high temperature stress refers to temperature stress above 35°C.

[0033] Any of the above-mentioned high temperature stress refers to the monthly average maximum temperature during the booting period being above 35°C.

[0034] Any of the above-mentioned high temperature stress refers to a monthly average maximum temperature of 35° C. or above from the tillering stage to the end of the flowering and pollination stage.

[0035] Any of the above-mentioned high temperature stress refers to the monthly average maximum temperature from the tillering stage to the end of the grain filling stage being above 35°C.

[0036] Any of the above-mentioned high temperature stress refers to temperature stress above 37°C.

[0037] Any of the above-mentioned high temperature stress refers to the monthly average maximum temperature during the booting period being above 37°C.

[0038] Any of the above-mentioned high temperature stress refers to a monthly average maximum temperature of 37° C. or above from the tillering stage to the end of the flowering and pollination stage.

[0039] Any of the above-mentioned high temperature stress refers to the monthly average maximum temperature from the tillering stage to the end of the grain filling stage being above 37°C.

[0040] Any of the above-mentioned high temperature stress refers to temperature stress above 40°C.

[0041] Any of the above-mentioned high temperature stress refers to the monthly average maximum temperature during the booting period being above 40°C.

[0042] Any of the above-mentioned high temperature stress refers to a monthly average maximum temperature of 40° C. or above from the tillering stage to the end of the flowering and pollination stage.

[0043] Any of the above-mentioned high temperature stress refers to the monthly average maximum temperature from the tillering stage to the end of the grain filling stage being above 40°C.

[0044] The monthly average maximum temperature refers to the average of the maximum temperatures of each day in that month.

[0045] Exemplarily, any of the above-mentioned rice may be Nipponbare rice. Beneficial effects

[0046] The disclosure of the present invention can be used to cultivate rice germplasm with increased seed setting rate in high temperature stress environment, thereby increasing rice yield in high temperature rice cultivation areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 shows the statistical results of the relative expression levels of target genes.

[0048] FIG2 is a photograph of plants in the field at full maturity.

[0049] Figure 3 is a photo of the development status of grains at the milky stage.

[0050] Figure 4 is a photograph showing the number of grains per plant at the fully mature stage.

[0051] Figure 5 shows the results of the statistical test group's fruit setting rate at the mature stage. Modes for Carrying Out the Invention

[0052] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0053] Unless otherwise noted, the experimental methods in the following examples are conventional methods and were performed according to techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples are commercially available unless otherwise noted. Unless otherwise noted, quantitative assays in the following examples were performed in triplicate, and the results were averaged. The pCAMBIA1305.1 plasmid is a circular plasmid, and its complete sequence is shown in SEQ ID NO: 3. Example

[0054] 1. Construction of recombinant plasmid

[0055] A recombinant plasmid was generated by replacing the small fragment between the KpnI and XbaI restriction enzyme recognition sequences of the pCAMBIA1305.1 plasmid with the double-stranded DNA molecule shown in SEQ ID NO: 2, while maintaining all other sequences unchanged. The recombinant plasmid was verified by sequencing. The DNA molecule shown in SEQ ID NO: 2 encodes the protein shown in SEQ ID NO: 1.

[0056] 2. Preparation of transgenic plants

[0057] 1. Genetic transformation to obtain regenerated plants

[0058] The recombinant plasmid constructed in step 1 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium. Embryogenic calli of rice Nipponbare were genetically transformed with the recombinant Agrobacterium using the Agrobacterium infection method. Resistant calli were then screened (using 100 mg / L hygromycin) for resistance, followed by differentiation and regeneration culture, and finally rooting culture to obtain T0 generation regenerated plants.

[0059] 2. Screening transgenic plants from T0 generation regenerated plants

[0060] The leaves of the T0 generation regenerated plants were taken, genomic DNA was extracted, and PCR identification was performed. The plants that were positive in PCR identification were T0 generation transgenic plants.

[0061] PCR identification method: Using genomic DNA as a template, PCR amplification is performed using a primer pair consisting of primer F1 and primer R1 (the upstream primer and downstream primer correspond to the upstream and downstream positions of the exogenous DNA insertion in the plasmid, respectively). If PCR amplification shows a specific band of 2100bp, the PCR identification is positive.

[0062] Primer F1: 5′-gacgcacaatcccactatcct-3′;

[0063] Primer R1: 5′-caaattgaggatgagaccaagcg-3′.

[0064] 3. Subculture of transgenic plants

[0065] Self-pollinate the T0 transgenic plants and harvest the seeds. Grow these seeds into plants, which are the T1 plants. Extract genomic DNA from leaves of the T1 plants and perform PCR analysis (using the same method as in step 2). Plants that test positive by PCR are considered T1 transgenic plants.

[0066] Self-pollinate the T1 transgenic plants and harvest the seeds. Grow these seeds into plants, which are the T2 generation. Extract genomic DNA from leaves of the T2 plants and perform PCR analysis (using the same method as in step 2). Plants that test positive by PCR are considered T2 transgenic plants.

[0067] For a certain T1 generation transgenic plant, if all T2 generation plants obtained by self-pollination are transgenic plants, the self-pollinated offspring of the T1 generation transgenic plant are a homozygous transgenic line.

[0068] Two homozygous transgenic lines were obtained and named OE-1 and OE-2.

[0069] The T2 generation transgenic plants of the homozygous transgenic line were self-pollinated and the seeds were harvested to obtain the T3 generation seeds.

[0070] 3. Preparation of Transgenic Plants

[0071] The recombinant plasmid was replaced with the pCAMBIA1305.1 plasmid and the operation was carried out according to step 2 to obtain a homozygous empty vector strain.

[0072] 4. Detecting the relative expression of target genes

[0073] The test plants were: Nipponbare rice plants, T2 generation plants of the OE-1 line, T2 generation plants of the OE-2 line, or T2 generation plants of the empty vector line. Nipponbare rice is represented by NIP.

[0074] Total RNA was extracted from leaves of test plants cultured under parallel conditions and reverse transcribed to generate cDNA. Real-time quantitative PCR was used to measure the relative expression of the target gene using the cDNA as a template and the actin gene as an internal reference gene.

[0075] The primers used to amplify the target gene are as follows:

[0076] F2: 5'-gcccaatagctttgatcgtgt-3';

[0077] R2: 5'-gcatatctgaccaaagcttcat-3'.

[0078] The primers used to amplify the internal reference gene are as follows:

[0079] F3: 5'-agcagcatgaagatcaaggtggtc;

[0080] R3: 5'-ccttggcaatccacatctgctg-3'.

[0081] The results are shown in Figure 1 (the vertical axis represents the relative expression of the target gene, sample size N = 3). There was no significant difference in the relative expression of the target gene in the vector-transfected plants compared to the Nipponbare rice plants. Compared to the Nipponbare rice plants, the relative expression of the target gene in the OE-1 strain was significantly increased, and the relative expression of the target gene in the OE-2 strain was even more significantly increased.

[0082] 5. Heat stress test

[0083] The vegetative growth phase of rice lasts approximately 90 days and can be divided into four stages: seedling stage (approximately 35 days), transplanting stage (7-10 days), tillering stage (approximately 30 days), and jointing stage (approximately 15 days). The reproductive growth phase of rice lasts approximately 70 days and can be divided into four stages: booting stage (approximately 15 days), heading stage (approximately 15 days), flowering and pollination stage (15-20 days), and grain filling stage (approximately 20 days). The grain filling stage is divided into three stages: milky stage, sallow stage, and fully ripe stage.

[0084] Experimental location: experimental field in Langfang City, Hebei Province.

[0085] The test seeds were: Nipponbare rice seeds, T3 generation seeds of the OE-1 strain, T3 generation seeds of the OE-2 strain, or T3 generation seeds of the empty vector strain. Nipponbare rice is represented by NIP.

[0086] 1. Take the test seeds and soak them in a 2000-fold diluted solution of 25% cyanobacter-methyl suspension for one day, then soak them in water until they turn white.

[0087] 2. Around May 1, sow the white seeds in the nursery field (the field for growing rice seedlings) and cultivate and manage them normally for about one month.

[0088] 3. Around June 1, transplant the seedlings from step 2 into the rice fields (the fields where rice is grown) and cultivate and manage them normally until the rice plants enter the tillering stage (around June 10).

[0089] 4. Divide the rice fields that have completed step 3 into 6 rectangular plots (each plot is 2m×3m, and each plot has approximately 100 rice plants). Three plots are used as experimental groups (denoted by HS) and three plots are used as control groups (denoted by Control). Greenhouses are built for the rice plants in the three plots of the experimental group, while the three plots of the control group remain as they are and are cultivated and managed normally until they are fully ripe. The greenhouses are then dismantled and the grains are harvested (October 1).

[0090] In step 4, the ambient temperature of each rice group (for greenhouses, the temperature inside the greenhouse) is continuously monitored, as shown in Table 1. The average high temperature in June refers to the average of the maximum daily temperature from the start of grouping in step 4 to the last day of the month, and the average low temperature in June refers to the average of the minimum daily temperature from the start of grouping in step 4 to the last day of the month; the average high temperature in July / August / September refers to the average of the maximum daily temperature in that month, and the average low temperature in July / August / September refers to the average of the minimum daily temperature in that month.

[0091]

[0092] Photographs of fully mature field plants are shown in Figure 2. In the control group, there were no significant differences in panicle phenotypes between the empty vector-transfected plants, the OE-1 strain, and the OE-2 strain compared to the Nipponbare rice. In the experimental group, many panicles in the Nipponbare rice group failed to set normally, had low weight, and showed no panicle drooping. There were no significant differences in panicle phenotypes between the empty vector-transfected plants and the Nipponbare rice group. However, the panicles of the OE-1 strain and the OE-2 strain showed significant panicle drooping compared to the Nipponbare rice group.

[0093] Photographs of grain development at the milky stage are shown in Figure 3. In the control group, the phenotypes of the grains from the empty vector transgenic plants, the OE-1 strain, and the OE-2 strain were not significantly different from those of the Nipponbare rice. In the experimental group, the grains of the Nipponbare rice developed abnormally and were shriveled. The phenotypes of the grains from the empty vector transgenic plants were not significantly different from those of the Nipponbare rice. The grains of the OE-1 strain and the OE-2 strain developed normally.

[0094] Photographs showing the number of grains per plant at full maturity are shown in Figure 4. In the control group, there was no significant difference in the number of grains per plant between the empty vector-transformed plants, the OE-1 line, and the OE-2 line compared to the rice variety Nipponbare. In the experimental group, there was no significant difference in the number of grains per plant between the empty vector-transformed plants and the rice variety Nipponbare. However, the number of grains per plant in the OE-1 and OE-2 line plants increased significantly compared to the rice variety Nipponbare.

[0095] At full maturity, the seed set rate of the experimental groups was calculated (seed set rate = number of spikelets per plant that normally set seeds / total number of spikelets per plant). The results are shown in Figure 5 (the vertical axis represents seed set rate, sample size N = 15). There was no significant difference in seed set rate between the empty vector-transfected plants and the Nipponbare rice variety. However, the seed set rate of both the OE-1 and OE-2 lines was significantly increased compared to the Nipponbare rice variety.

[0096] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0097] CROSS-REFERENCE TO RELATED APPLICATIONS

[0098] This application claims priority to a Chinese patent application filed on March 26, 2024 (application number: 202410349139.0), the entire contents of which are incorporated herein by reference. Industrial Applicability

[0099] The invention discloses a method for cultivating rice germplasm with increased seed setting rate in a high-temperature stress environment, thereby increasing rice yield in high-temperature rice cultivation areas. The invention has significant application and promotion value for cultivating high-yield rice in a high-temperature stress environment.

Claims

1. Use of a target protein in improving the seed setting rate of rice grown under high temperature stress; the target protein is shown in SEQ ID NO:

1.

2. Application of target protein-related biomaterials in improving the seed setting rate of rice grown under high temperature stress environment; The target protein-related biological material is any one of the following (a1) to (a8): (a1) a nucleic acid molecule encoding the target protein shown in SEQ ID NO: 1; (a2) an expression cassette containing the nucleic acid molecule described in (a1); (a3) a recombinant vector containing the nucleic acid molecule described in (a1); (a4) a recombinant vector containing the expression cassette described in (a2); (a5) a recombinant microorganism containing the nucleic acid molecule described in (a1); (a6) a recombinant microorganism containing the expression cassette described in (a2); (a7) a recombinant microorganism containing the recombinant vector described in (a3); (a8) A recombinant microorganism containing the recombinant vector described in (a4).

3. Use of target protein-related biological materials in the preparation of transgenic rice; compared with recipient rice, the transgenic rice has an increased fruit set rate under high temperature stress; the target protein-related biological materials are as described in claim 2.

4. A method for preparing transgenic rice with improved seed setting rate under high temperature stress environment, comprising the following steps: introducing and expressing a gene encoding a target protein into a starting rice plant to obtain transgenic rice with improved seed setting rate under high temperature stress environment; the target protein is shown in SEQ ID NO:

1.

5. A method for preparing transgenic rice resistant to high temperature stress, comprising the following steps: introducing a gene encoding a target protein into a starting rice plant and expressing the gene to obtain transgenic rice resistant to high temperature stress; the target protein is shown in SEQ ID NO:

1.

6. Use of a target protein in improving the stress tolerance of rice to high temperature stress; the target protein is shown in SEQ ID NO:

1.

7. Use of a target protein-related biological material in improving the stress tolerance of rice to high temperature stress; the target protein-related biological material is as described in claim 2.

Images