Comprehensive cultivation method of heat-resistant transgenic rice for extreme high-temperature environment

Abstract

The invention discloses a heat-resistant transgenic rice comprehensive cultivation method oriented to an extreme high-temperature environment, and relates to the technical field of agricultural biology. A core heat-resistant gene is screened from an extreme high-temperature biological resource, a transgenic expression vector is constructed by combining a monocotyledonous plant specific regulation element, abnormal cells are eliminated through detection such as imprinting hybridization on a plant cell level and multi-level identification, efficient and directional expression of a target gene in rice is ensured, and the gene expression efficiency is improved. Based on heat resistance and agronomic traits, differentiated high-temperature stress parameters are set according to the key growth stage of rice in a simulated extreme high-temperature environment, the survival rate and photosynthetic efficiency of plants at high temperature are guaranteed, and meanwhile it is guaranteed that the agronomic traits are not sacrificed through yield detection; the cultivated strain is suitable for planting in different high-temperature areas, reference is provided for forest breeding and seedling raising industries, and agronomic traits are guaranteed while heat resistance is improved.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a comprehensive breeding method for heat-resistant transgenic rice designed for extreme high-temperature environments. Background Technology

[0002] In existing rice heat resistance improvement technologies, traditional hybridization breeding relies on limited heat-resistant germplasm resources, has a long breeding cycle, and offers limited improvement in heat resistance. For example, Chinese patent application CN113229077A discloses a method for increasing rice and wheat yields year-round in the Jianghuai region. The method includes the following steps: selecting late-maturing, high-yielding rice varieties with long growth periods; using a tray-seedling machine transplanting method to obtain basic rice seedlings; transplanting the basic rice seedlings after wheat harvest using a machine transplanting method; and selecting early-maturing spring wheat varieties with short growth periods, tolerance to waterlogging, and resistance to Fusarium head blight. This patent application's breeding method can optimize resource allocation between seasons, effectively alleviate wheat waterlogging damage and high-temperature heat damage during rice flowering, and achieve simultaneous improvement in year-round yield and resource utilization efficiency.

[0003] However, while the aforementioned patents have achieved a passive mitigation of high-temperature heat damage to rice, the following problems still exist:

[0004] Existing technologies alleviate heat damage by avoiding the overlap between rice flowering and high-temperature periods, but they do not enhance the physiological tolerance of rice to extreme high temperatures. When faced with continuous extreme high-temperature weather, yield reduction still occurs. They cannot fundamentally overcome the limitation of the scarcity of heat-resistant germplasm resources in traditional varieties, and it is difficult to achieve synergistic improvement of heat resistance and agronomic traits such as high yield and quality. Summary of the Invention

[0005] The purpose of this invention is to provide a comprehensive breeding method for heat-resistant transgenic rice for extreme high-temperature environments. By accurately screening core heat-resistant genes, constructing specific expression vectors, and optimizing the transgenic identification and breeding process, the invention enhances the physiological tolerance of rice under extreme high temperatures, thereby simultaneously improving the heat resistance of rice and its agronomic traits such as high yield and high quality, thus solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A comprehensive breeding method for heat-resistant transgenic rice designed for extreme high-temperature environments, characterized by the following steps:

[0008] Based on public genome databases and bioinformatics analysis, candidate genes for extreme heat resistance were obtained, optimized and screened to obtain target heat-resistant genes, and transgenic expression vectors were constructed.

[0009] Rice callus tissue was obtained as rice receptor material, and the rice receptor material was pretreated. A gene expression vector was introduced into the pretreated rice receptor material to obtain transgenic rice cells.

[0010] The transgenic rice cells were screened and identified. The qualified transgenic rice cells were induced and cultured to regenerate into complete plants, thereby obtaining transgenic rice plants.

[0011] The heat resistance of the transgenic rice plants was tested and evaluated under simulated extreme high-temperature conditions. The differences in heat resistance performance between the transgenic rice plants and the non-transgenic control plants were compared to screen out transgenic rice lines with excellent heat resistance performance.

[0012] Furthermore, the acquisition of data related to heat-resistant biological resources and the screening of core heat-resistant genes specifically include:

[0013] Genome data of plants or microorganisms grown under extreme high-temperature environments are obtained from public genome databases. Sequence alignment and evolutionary conservation analysis are performed using bioinformatics analysis tools to initially screen candidate genes and establish an initial candidate gene library.

[0014] Based on the physiological needs of rice to adapt to extreme high temperatures, functional pathway analysis was performed on candidate genes in the initial candidate gene library. The candidate genes were mapped to the high-temperature response metabolic pathways of rice, and functional correlation analysis and optimization were carried out to obtain a candidate gene population adapted to the rice expression system.

[0015] Prokaryotic expression vectors for candidate genes were constructed, and candidate gene populations were inserted into the prokaryotic expression vectors. The high-temperature tolerance of candidate genes that could be expressed normally was verified, and the growth rate and stress damage of yeast were measured under extreme high-temperature stress to narrow down the candidate gene range.

[0016] Data on the protein stability and cell protection of candidate genes under high temperature conditions were obtained, the actual heat resistance regulation effect of candidate genes was evaluated, core genes with efficient heat resistance function were screened, and no fewer than three core heat-resistant genes were identified as target heat-resistant genes.

[0017] Furthermore, the construction of transgenic expression vectors also includes:

[0018] Based on the sequence characteristics and expression requirements of the target heat-resistant gene, regulatory elements that can drive the efficient and specific expression of the target gene in rice are screened as regulatory modules for vector construction.

[0019] Based on the target heat-resistant gene and regulatory module, the obtained vector backbone is assembled into vector fragments to form a complete expression cassette. The assembled vector fragments are then ligated using ligase to obtain the initially constructed vector.

[0020] The preliminarily constructed vectors were subjected to full-sequence detection. The detection results were compared with the preset vector sequence map. Based on the comparison results, vectors with sequence consistency higher than the preset consistency threshold were selected and identified as transgenic expression vectors to be functionally tested.

[0021] Functional testing was conducted on the transgenic expression vectors to be tested, the effectiveness of the transgenic expression vectors in driving the expression of the target gene was evaluated, and qualified vectors that could achieve the expression of the target gene were screened out as transgenic expression vectors for introduction into rice recipient materials.

[0022] Furthermore, the control module specifically includes:

[0023] Based on public genome databases, regulatory elements suitable for monocotyledonous plants were screened. Combined with bioinformatics analysis, sequence complementarity analysis was performed on the promoter binding regions of the screened regulatory elements and the target heat-resistant genes.

[0024] Based on the analysis results, the regulatory elements were initially linked to the core coding region of the target heat-resistant gene. The sequence integrity of the splicing site between the regulatory elements and the target heat-resistant gene was detected. Combinations of regulatory elements with fitness higher than the preset fitness threshold were screened to determine the regulatory module used for vector construction.

[0025] Furthermore, the process of obtaining rice receptor material and pretreating the rice receptor material specifically includes:

[0026] Select mature rice seeds and dehull them to obtain initial seed material. Place the initial seed material in a sterile container for soaking and disinfection. After disinfection, wash and dry the seeds with sterile distilled water to obtain sterile seeds.

[0027] Sterile seeds were inoculated into callus induction medium. After inoculation, the culture dish was sealed with sealing film and placed in a light incubator for culture. The physiological state of the seeds was monitored in real time during the culture process.

[0028] Target embryogenic callus tissue was selected as preliminary rice recipient material and placed in subculture medium. It was then placed in a light incubator for subculture, and the hormone composition and ratio in the subculture medium were adjusted to optimize the growth status of the target embryogenic callus tissue.

[0029] Real-time monitoring of the physiological state of preliminary rice recipient materials after subculture, assessment of the transformation potential of preliminary rice recipient materials, and determination of rice recipient materials suitable for gene delivery based on the assessment results.

[0030] Furthermore, the specific process for screening and identifying the transgenic rice cells includes:

[0031] Genomic DNA was extracted from transgenic rice cells, and PCR amplification was performed based on the specific sequence of the target heat-resistant gene. The presence of the target heat-resistant gene in the transgenic rice cells was detected, and PCR-positive cells were screened based on the detection results.

[0032] Genomic DNA was extracted from PCR-positive cells, and the genomic DNA was digested with restriction endonucleases to obtain the digested genomic DNA fragments. The genomic DNA fragments were then hybridized with the sequence characteristics of the target thermostable gene to form a hybridization complex.

[0033] The hybridization signal generated by the hybridization complex was detected, the hybridization signal map was obtained, the integration site and copy number of the target thermostable gene in PCR-positive cells were analyzed, and cells with single-copy integration and no functional conflict at the integration site were screened out.

[0034] Total RNA was extracted from the screened cells, and cDNA was obtained by reverse transcription. The transcriptional expression level of the target heat-resistant gene was detected and compared with the expression level of non-transgenic rice cells to screen out rice cells with normal transcriptional expression levels.

[0035] Total protein was extracted from rice cells with normal transcriptional expression. Based on a protein-specific antibody targeting the core coding region of the target heat-resistant gene, the expression level and molecular weight of the target protein were detected, and cells that correctly expressed the target protein were screened out.

[0036] Cells that correctly express the target protein are subjected to high-temperature stress treatment. The cell survival rate, reactive oxygen species accumulation, and co-expression of heat-response-related genes under high-temperature conditions are detected to evaluate the functional effectiveness of the target heat-resistant gene and screen out qualified transgenic rice cells.

[0037] Furthermore, the cells in which the target protein is correctly expressed are obtained based on the results of Western blot detection. Based on the matching between the band and the theoretical molecular weight of the target protein, combined with the analysis results of the band gray value, it is determined whether the target protein has reached a detectable level and has no obvious degradation.

[0038] Furthermore, the specific process for obtaining transgenic rice plants includes:

[0039] Transgenic rice cells that passed the identification were transferred to a differentiation medium, the hormone ratio in the medium was adjusted, and the cells were culturing under preset environmental conditions to induce cell differentiation and form adventitious shoots.

[0040] The growth of adventitious buds is observed regularly. When the adventitious buds reach the preset length, they are transferred to a rooting medium, and the auxin ratio in the medium is adjusted to induce the adventitious buds to root and form complete seedlings.

[0041] During the rooting culture process, data on the root development status of seedlings are obtained. Seedlings with poor root development or whitening are removed. Seedlings with strong growth and complete root systems are selected and transferred to a sterile substrate for hardening treatment.

[0042] After the seedling hardening process, the seedlings are transplanted to a greenhouse for routine water and fertilizer management. Stable plants are obtained to determine whether they meet the standards for transgenic rice plants and are then identified as transgenic rice plants.

[0043] Furthermore, the simulated extreme high-temperature environment specifically includes:

[0044] A controlled environment growth chamber was selected. Based on natural climate data from extreme high-temperature regions, high-temperature stress parameters were set to obtain data on different growth stages of transgenic rice plants. Corresponding high-temperature stress strategies were then matched based on the data from different growth stages.

[0045] Based on the goals of promoting heat tolerance in seedlings and ensuring fruit set during the grain-filling stage, high temperature parameters were set for the seedling and grain-filling stages. During the implementation of the high temperature stress strategy, the temperature, humidity, and light intensity in the growth chamber were monitored in real time to determine whether temperature fluctuations exceeded the limits, and environmental control equipment was used to maintain parameter stability.

[0046] Furthermore, the simulation of extreme high-temperature environment also includes obtaining non-transgenic rice plants as a control group, placing the control group and transgenic rice plants in the same high-temperature environment, and cultivating them together based on consistent stress conditions. At the same time, during the high-temperature stress treatment, environmental parameter changes are recorded at preset time intervals to generate an environmental regulation log.

[0047] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention uses recombinant DNA technology as its core, screening core heat-resistant genes from biological resources exposed to extreme high temperatures, constructing transgenic expression vectors by combining them with monocotyledonous plant-specific regulatory elements, and eliminating abnormal cells through multi-level identification and detection such as imprinting hybridization at the plant cell level, ensuring efficient and targeted expression of the target gene in rice, improving the accuracy of heat resistance improvement in transgenic rice, and solving the problems of blind gene selection and low expression efficiency in traditional methods; Based on heat resistance and agronomic traits, differentiated high-temperature stress parameters are set for key growth stages of rice in simulated extreme high-temperature environments, ensuring plant survival rate and photosynthetic efficiency under high temperatures, while ensuring that agronomic traits are not sacrificed through yield testing, making the cultivated lines suitable for planting in different high-temperature regions, providing a reference for forestry breeding and seedling industry, ensuring that agronomic traits are maintained while heat resistance is improved; By providing a standardized path, combined with environmental logs and control group settings, this invention ensures the reproducibility of experimental results, facilitating the promotion and application by research institutions and breeding companies, and providing a reliable technical solution for stable rice yield in extreme high-temperature regions. Attached Figure Description

[0048] Figure 1 This is a flowchart of the integrated breeding method for heat-resistant transgenic rice for extreme high-temperature environments according to the present invention. Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0050] Please see Figure 1 A comprehensive breeding method for heat-resistant transgenic rice for extreme high-temperature environments, characterized by the following steps:

[0051] Based on public genome databases and bioinformatics analysis, candidate genes for extreme heat resistance were obtained, optimized and screened to obtain target heat-resistant genes, and transgenic expression vectors were constructed.

[0052] Rice callus tissue was obtained as rice receptor material, and the rice receptor material was pretreated to improve its transformation efficiency, such as hormone ratio adjustment and sterile environment optimization. Gene expression vector was introduced into the pretreated rice receptor material to obtain transgenic rice cells.

[0053] The transgenic rice cells were screened and identified to confirm the stable integration and expression of candidate genes. The qualified transgenic rice cells after screening and identification were induced and cultured to regenerate into complete plants, thereby obtaining transgenic rice plants.

[0054] The heat resistance of the transgenic rice plants was tested and evaluated under simulated extreme high-temperature conditions. The differences in heat resistance performance between the transgenic rice plants and the non-transgenic control plants were compared to screen out transgenic rice lines with excellent heat resistance performance.

[0055] In this embodiment, heat resistance testing and evaluation are carried out, including testing the morphological indicators of the two types of plants, obtaining data on plant height, effective tiller number, flag leaf length and fruit set rate, and recording the specific values ​​of each indicator; testing the physiological and biochemical indicators of the two types of plants, extracting chlorophyll from leaves, measuring chlorophyll content and chlorophyll fluorescence parameters, analyzing the degree of damage to the photosynthetic system, and establishing a heat resistance evaluation system based on the obtained morphological and physiological and biochemical indicator data to quantify the differences in heat resistance performance between the two types of plants;

[0056] In this embodiment, plants with superior morphological indicators, lower degree of damage to physiological and biochemical indicators, and satisfactory heat resistance among transgenic rice plants are selected and identified as transgenic rice lines with excellent heat resistance.

[0057] In this embodiment, the constructed vector can efficiently drive the expression of target genes, achieving synergistic adaptation between gene screening and vector construction. Callus tissue is obtained and pretreated with hormone regulation and aseptic optimization to improve transformation efficiency. The state of rice recipient materials is specifically optimized to solve the problem of poor material compatibility in traditional transformation, providing high-potential recipients for gene introduction, ensuring the efficiency of transgenic cell acquisition, establishing a systematic screening and identification system to avoid the limitations of single detection, screening out cells with stable integrated expression, laying the foundation for plant regeneration, simulating extreme high temperatures and evaluating multiple indicators to screen superior lines, developing detection strategies based on plant growth stages, comprehensively evaluating heat resistance, and cultivating rice lines adapted to extreme high temperatures to address the impact of high temperatures on rice production.

[0058] In this embodiment, the acquisition of data related to heat-resistant biological resources and the screening of core heat-resistant genes specifically include:

[0059] Genome data of plants or microorganisms growing under extreme high-temperature environments were obtained from public genome databases, including desert plants, heat-resistant microorganisms, and heat-resistant species closely related to rice. The focus was on acquiring gene sequence data related to stress response and protein protection. Bioinformatics analysis tools were used for sequence alignment and evolutionary conservation analysis to initially screen candidate genes related to heat shock proteins, antioxidant enzymes, or membrane stability. An initial candidate gene library was established, containing multi-dimensional heat-resistant biological resources with gene sequences, expression levels, and functional annotations. Some information is shown in Table 1.

[0060] Table 1 Core Information of the Initial Candidate Gene Library

[0061] Gene number Source organisms Functional annotations Expression level under high temperature stress (FPKM) Evolutionary conservation score G1 Sea buckthorn Heat shock protein 70 (HSP70) 186.3 0.92 G6 Thermophilic Bacillus stearothermophilus Superoxide dismutase (SOD) 215.7 0.88 G12 common wild rice Membrane transport proteins 153.2 0.90 G28 Camel thorn Catalase (CAT) 178.5 0.85 G35 thermophilic fungi Heat shock protein 90 (HSP90) 201.4 0.91

[0062] Based on the physiological needs of rice to adapt to extreme high temperatures, functional pathway analysis was performed on candidate genes in the initial candidate gene library. The candidate genes were mapped to the high-temperature response metabolic pathways of rice, and functional correlation analysis and optimization were carried out to obtain a candidate gene population adapted to the rice expression system.

[0063] In vitro functional validation experiments were conducted to construct prokaryotic expression vectors for candidate genes. The candidate gene population was inserted into the prokaryotic expression vectors to verify the high temperature tolerance of the candidate genes that could be expressed normally. The growth rate and stress damage of yeast were measured under extreme high temperature stress to narrow down the candidate gene range.

[0064] Data on the protein stability and cell protection of candidate genes under high temperature conditions were obtained, the actual heat resistance regulation effect of candidate genes was evaluated, core genes with efficient heat resistance function were screened, and no fewer than three core heat-resistant genes were identified as target heat-resistant genes.

[0065] In this embodiment, constructing the transgenic expression vector further includes:

[0066] Based on public genome databases, regulatory elements suitable for monocotyledonous plants were screened. Combined with bioinformatics analysis, sequence complementarity analysis was performed on the binding regions of the screened regulatory elements and the promoters of the target heat-resistant genes to exclude regulatory elements with base conflicts or low binding efficiency.

[0067] Based on the analysis results, the regulatory elements were initially linked to the core coding region of the target heat-resistant gene, the sequence integrity of the splicing site between the regulatory elements and the target heat-resistant gene was detected, and combinations of regulatory elements with fitness higher than the preset fitness threshold were screened to determine the regulatory modules used for vector construction.

[0068] Based on the target heat-resistant gene and regulatory module, the obtained vector backbone is assembled into vector fragments to form a complete expression cassette. The assembled vector fragments are then ligated using ligase to obtain the initially constructed vector.

[0069] The preliminarily constructed vectors were subjected to full-sequence detection. The detection results were compared with the preset vector sequence map. Based on the comparison results, vectors with sequence consistency higher than the preset consistency threshold were selected and identified as transgenic expression vectors to be functionally tested.

[0070] Functional testing was conducted on the transgenic expression vectors to be tested, the effectiveness of the transgenic expression vectors in driving the expression of the target gene was evaluated, and qualified vectors that could achieve the expression of the target gene were screened out as transgenic expression vectors for introduction into rice recipient materials.

[0071] In this embodiment, multi-source high-temperature organism genomic data are integrated, combined with bioinformatics analysis and in vitro functional verification, to adapt to the physiological needs of rice, precisely identify highly efficient heat-resistant genes, provide core genes with strong adaptability for rice transgenics, avoid the introduction of ineffective genes, improve breeding efficiency, and selectively screen monocotyledonous regulatory elements. Through sequence complementarity analysis and functional testing, the vector is optimized to achieve precise matching between regulatory elements and target genes, ensuring the integrity and functional effectiveness of the vector sequence. The constructed vector can efficiently drive gene expression, providing a reliable tool for subsequent recipient material transformation and reducing breeding losses caused by vector construction failures.

[0072] In this embodiment, the process of obtaining rice receptor material and pretreating the rice receptor material specifically includes:

[0073] Select mature rice seeds and dehull them to obtain initial seed material. Place the initial seed material in a sterile container for soaking and disinfection. After disinfection, wash and dry the seeds with sterile distilled water to obtain sterile seeds.

[0074] Sterile seeds were inoculated into a callus induction medium containing plant hormones, sucrose and agar. After inoculation, the culture dish was sealed with sealing film and placed in a light incubator for cultivation. The physiological state of the seeds was monitored in real time during the cultivation process.

[0075] Naturally divided, pale yellow, and densely spherical target embryogenic callus tissues were selected as preliminary rice recipient materials and placed in a subculture medium. The subculture medium was then placed in a light incubator for subculture, and the hormone composition and ratio in the subculture medium were adjusted to optimize the growth status of the target embryogenic callus tissues.

[0076] Real-time monitoring of the physiological state of preliminary rice recipient materials after subculture, assessment of the transformation potential of preliminary rice recipient materials, and determination of rice recipient materials suitable for gene delivery based on the assessment results.

[0077] In this embodiment, the selected seeds are placed in a sterile container and soaked in 70%-75% alcohol for 1-3 minutes, then the alcohol is discarded. A 15%-25% sodium hypochlorite solution is added to the container along with a few drops of surfactant, and the seeds are soaked for 20-40 minutes, shaking occasionally during soaking. The sodium hypochlorite solution is discarded, and the seeds are washed 4-6 times with sterile distilled water and then soaked for 20-40 minutes. The washed seeds are placed on sterile filter paper to absorb excess water and then inoculated into the induction medium, 10-15 seeds per dish. After the operation is completed, the culture dishes are sealed with sealing film and cultured in a 26-30℃ light incubator for 2-4 weeks. Culture is continued in a 26-30℃ light incubator for 1 week. If not used immediately, the culture is transferred to a dark place and cultured at 22-24℃ for another week to provide basic materials for subsequent transgenic operations.

[0078] In this embodiment, the specific process for screening and identifying the transgenic rice cells includes:

[0079] Genomic DNA was extracted from transgenic rice cells, and polymerase chain reaction (PCR) technology was used to amplify the target heat-resistant gene based on its specific sequence. The presence of the target heat-resistant gene in the transgenic rice cells was detected, and PCR-positive cells were screened based on the detection results.

[0080] Genomic DNA was extracted from PCR-positive cells, and the genomic DNA was digested with restriction endonucleases to obtain the digested genomic DNA fragments. The genomic DNA fragments were then hybridized with the sequence characteristics of the target thermostable gene to form a hybridization complex.

[0081] The hybridization signal generated by the hybridization complex was detected, the hybridization signal map was obtained, the integration site and copy number of the target thermostable gene in PCR-positive cells were analyzed, and cells with single-copy integration and no functional conflict at the integration site were screened out.

[0082] Total RNA was extracted from the screened cells, and cDNA was obtained by reverse transcription. Real-time quantitative PCR was used to detect the transcriptional expression level of the target heat-resistant gene and compared with the expression level of non-transgenic rice cells to screen out rice cells with normal transcriptional expression levels.

[0083] Total protein was extracted from rice cells with normal transcriptional expression. Based on a protein-specific antibody targeting the core coding region of the heat-resistant gene, Western blotting was used to detect the expression level and molecular weight of the target protein. Cells that correctly expressed the target protein were screened out. Based on the Western blotting results, the matching between the band and the theoretical molecular weight of the target protein, combined with the band gray value analysis, was used to determine whether the target protein reached a detectable level and had no obvious degradation.

[0084] Cells that correctly express the target protein are subjected to high-temperature stress treatment. The cell survival rate, reactive oxygen species accumulation, and co-expression of heat-response-related genes under high-temperature conditions are detected to evaluate the functional effectiveness of the target heat-resistant gene and screen out qualified transgenic rice cells.

[0085] In this embodiment, the criteria for determining PCR-positive cells are: the PCR amplification product, when detected by gel electrophoresis, shows a specific band consistent with the theoretical molecular weight of the target thermostable gene, and the negative control shows no corresponding band.

[0086] In this embodiment, cells that correctly expressed the target proteins were subjected to a high-temperature stress of 45°C for 12 hours. Cell viability, reactive oxygen species accumulation, and the co-expression of heat-response-related genes were detected. The results are shown in Table 2.

[0087] Table 2 Results of transgenic cell functional effectiveness test

[0088] Cell numbering Survival rate (%) Reactive oxygen species accumulation Expression levels of heat-responsive genes (fold) Test results C3 89 1.32 6.8 qualified C7 92 1.25 7.3 qualified C12 87 1.38 6.5 qualified C18 90 1.29 7.1 qualified C23 85 1.42 6.3 qualified

[0089] In this embodiment, standardized dehulling and disinfection, targeted callus induction, and dynamic subculture optimization are used to precisely regulate the physiological state of callus tissue, obtaining recipient materials with high transformation potential. This provides a high-quality foundation for subsequent gene introduction, reduces the risk of transformation failure, and constructs a progressive screening system from multi-dimensional detection of gene presence, integration, transcription, protein expression to functional verification. This ensures that the target gene is stably integrated and functionally effective in the cells. After screening out qualified transgenic cells, a reliable source is provided for plant regeneration, ensuring the quality of the transgenic rice subsequently cultivated.

[0090] The specific process for obtaining transgenic rice plants in this embodiment includes:

[0091] Transgenic rice cells that have passed the identification process are transferred to a differentiation medium, the hormone ratio in the medium is adjusted (e.g., the proportion of cytokinin is increased), and differentiation culture is carried out under preset environmental conditions to induce cell differentiation and the formation of adventitious shoots.

[0092] The growth of adventitious buds is observed regularly. When the adventitious buds reach the preset length, they are transferred to a rooting medium, and the auxin ratio in the medium is adjusted to induce the adventitious buds to root and form complete seedlings.

[0093] During the rooting culture process, data on the root development status of seedlings, such as root length and number of roots, are obtained. Seedlings with poor root development or whitening are removed, and seedlings with strong growth and complete root systems are selected and transferred to a sterile substrate for hardening-off treatment to adapt to the external environment.

[0094] After the seedling hardening process, the seedlings were transplanted to a greenhouse for routine water and fertilizer management to obtain stable plants. The plants were then assessed to determine if they met the standards for transgenic rice plants and were identified as transgenic rice plants, thus providing complete plant material for subsequent heat resistance testing.

[0095] In this embodiment, the simulated extreme high-temperature environment specifically includes:

[0096] A controlled environment growth chamber was selected, and high temperature stress parameters were set based on natural climate data from extreme high-temperature regions, including daytime high temperature, nighttime high temperature, duration of high temperature, and light intensity. Data on different growth stages of transgenic rice plants, such as seedling stage, tillering stage, and grain-filling stage, were obtained, and corresponding high temperature stress strategies were matched based on the data of different growth stages. High temperature parameters were set at the seedling stage to promote the formation of heat tolerance in plants, and high temperature parameters were adjusted at the grain-filling stage to ensure the seed setting rate.

[0097] Based on the goals of promoting heat tolerance in seedlings and ensuring fruit set during grain filling, high temperature parameters were set for the seedling and grain filling stages. During the implementation of the high temperature stress strategy, the temperature, humidity and light intensity in the growth chamber were monitored in real time to determine whether temperature fluctuations exceeded the limits. Environmental control equipment was used to maintain parameter stability and avoid excessive temperature fluctuations.

[0098] Non-GMO rice plants were used as a control group. The control group and GMO rice plants were placed in the same high-temperature environment and co-cultured under the same stress conditions to provide a fair reference for subsequent heat resistance comparison. At the same time, during the high-temperature stress treatment, changes in environmental parameters were recorded at preset time intervals to generate a complete environmental regulation log and ensure the reproducibility of heat resistance test results.

[0099] In this embodiment, targeted hormone regulation is used to induce cell differentiation and adventitious bud formation, and to induce the adventitious buds to root. Combined with dynamic observation, robust seedlings with well-developed root systems and no albinism are selected. After hardening treatment, the seedlings are helped to adapt to the external environment, establishing a complete plant regeneration system covering cell differentiation, seedling selection and environmental adaptation, and accurately controlling the physiological state of each link in the regeneration process. High temperature stress parameters are set based on natural climate data of extreme high temperature regions. Corresponding high temperature stress strategies are matched according to different growth stages of transgenic rice plants, such as seedling stage, tillering stage and grain filling stage. At the same time, the indoor environment of the growth chamber is monitored in real time and the parameters are kept stable through equipment. The synergy of parameter setting, strategy implementation and environmental stabilization is realized, providing a real and controllable extreme high temperature scenario for heat resistance testing. In addition, by setting control plants and recording environmental logs, the reliability and comparability of the test results are ensured.

[0100] In this embodiment, after the high-temperature stress ended, the morphological, physiological, and yield-related indicators of the transgenic plants and the control group were measured, as shown in Table 3:

[0101] Table 3. Results of heat tolerance assessment of transgenic rice plants

[0102] Plant number Plant height reduction (%) Photosynthetic rate (μmol / m²·s) Fruit setting rate (%) 1000-grain weight (g) Overall heat resistance score T3 8.2 18.6 85.3 27.8 0.92 T7 7.5 19.3 87.6 28.5 0.95 T12 9.1 17.8 83.5 26.9 0.89 T18 7.8 19.0 86.4 28.1 0.93 control group 18.5 12.3 52.7 22.3 0.65

[0103] Based on the evaluation results, three plants, T7, T18, and T3, with a comprehensive heat resistance score ≥0.92, a seed setting rate ≥85%, and a thousand-grain weight ≥27g, were selected and identified as transgenic rice lines with excellent heat resistance, thus fulfilling the purpose of screening transgenic rice lines with excellent heat resistance.

[0104] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A comprehensive breeding method for heat-resistant transgenic rice for extreme high-temperature environments, characterized in that, Includes the following steps: Based on public genome databases and bioinformatics analysis, candidate genes for extreme heat resistance were obtained, optimized and screened to obtain target heat-resistant genes, and transgenic expression vectors were constructed. Rice callus tissue was obtained as rice receptor material, and the rice receptor material was pretreated. A gene expression vector was introduced into the pretreated rice receptor material to obtain transgenic rice cells. The transgenic rice cells were screened and identified. The qualified transgenic rice cells were induced and cultured to regenerate into complete plants, thereby obtaining transgenic rice plants. The heat resistance of the transgenic rice plants was tested and evaluated under simulated extreme high-temperature conditions. The differences in heat resistance performance between the transgenic rice plants and the non-transgenic control plants were compared to screen out transgenic rice lines with excellent heat resistance performance.

2. The integrated breeding method for heat-resistant transgenic rice for extreme high-temperature environments according to claim 1, characterized in that, The acquisition of data related to heat-resistant biological resources and the screening of core heat-resistant genes specifically include: Genome data of plants or microorganisms grown under extreme high-temperature environments are obtained from public genome databases. Sequence alignment and evolutionary conservation analysis are performed using bioinformatics analysis tools to preliminarily screen candidate genes and establish an initial candidate gene library. Based on the physiological needs of rice to adapt to extreme high temperatures, functional pathway analysis was performed on candidate genes in the initial candidate gene library. The candidate genes were mapped to the high-temperature response metabolic pathways of rice, and functional correlation analysis and optimization were carried out to obtain a candidate gene population adapted to the rice expression system. Prokaryotic expression vectors for candidate genes were constructed, and candidate gene populations were inserted into the prokaryotic expression vectors. The high-temperature tolerance of candidate genes that could be expressed normally was verified, and the growth rate and stress damage of yeast were measured under extreme high-temperature stress to narrow down the candidate gene range. Data on the protein stability and cell protection of candidate genes under high temperature conditions were obtained, the actual heat resistance regulation effect of candidate genes was evaluated, core genes with efficient heat resistance function were screened, and no fewer than three core heat-resistant genes were identified as target heat-resistant genes.

3. The integrated breeding method for heat-resistant transgenic rice for extreme high-temperature environments according to claim 1, characterized in that, Constructing transgenic expression vectors also includes: Based on the sequence characteristics and expression requirements of the target heat-resistant gene, regulatory elements that can drive the efficient and specific expression of the target gene in rice are screened as regulatory modules for vector construction. Based on the target heat-resistant gene and regulatory module, the obtained vector backbone is assembled into vector fragments to form a complete expression cassette. The assembled vector fragments are then ligated using ligase to obtain the initially constructed vector. The preliminarily constructed vectors were subjected to full-sequence detection. The detection results were compared with the preset vector sequence map. Based on the comparison results, vectors with sequence consistency higher than the preset consistency threshold were selected and identified as transgenic expression vectors to be functionally tested. Functional testing was conducted on the transgenic expression vectors to be tested, the effectiveness of the transgenic expression vectors in driving the expression of the target gene was evaluated, and qualified vectors that could achieve the expression of the target gene were screened out as transgenic expression vectors for introduction into rice recipient materials.

4. The integrated breeding method for heat-resistant transgenic rice for extreme high-temperature environments according to claim 3, characterized in that, The control module specifically includes: Based on public genome databases, regulatory elements suitable for monocotyledonous plants were screened. Combined with bioinformatics analysis, sequence complementarity analysis was performed on the promoter binding regions of the screened regulatory elements and the target heat-resistant genes. Based on the analysis results, the regulatory elements were initially linked to the core coding region of the target heat-resistant gene. The sequence integrity of the splicing site between the regulatory elements and the target heat-resistant gene was detected. Combinations of regulatory elements with fitness higher than the preset fitness threshold were screened to determine the regulatory module used for vector construction.

5. The integrated breeding method for heat-resistant transgenic rice for extreme high-temperature environments according to claim 1, characterized in that, The process of obtaining rice receptor materials and pretreating the rice receptor materials specifically includes: Select mature rice seeds and dehull them to obtain initial seed material. Place the initial seed material in a sterile container for soaking and disinfection. After disinfection, wash and dry the seeds with sterile distilled water to obtain sterile seeds. Sterile seeds were inoculated into callus induction medium. After inoculation, the culture dish was sealed with sealing film and placed in a light incubator for culture. The physiological state of the seeds was monitored in real time during the culture process. Target embryogenic callus tissue was selected as preliminary rice recipient material and placed in subculture medium. It was then placed in a light incubator for subculture, and the hormone composition and ratio in the subculture medium were adjusted to optimize the growth status of the target embryogenic callus tissue. Real-time monitoring of the physiological state of preliminary rice recipient materials after subculture, assessment of the transformation potential of preliminary rice recipient materials, and determination of rice recipient materials suitable for gene delivery based on the assessment results.

6. The integrated breeding method for heat-resistant transgenic rice for extreme high-temperature environments according to claim 1, characterized in that, The specific process for screening and identifying the transgenic rice cells includes: Genomic DNA was extracted from transgenic rice cells, and PCR amplification was performed based on the specific sequence of the target heat-resistant gene. The presence of the target heat-resistant gene in the transgenic rice cells was detected, and PCR-positive cells were screened based on the detection results. Genomic DNA was extracted from PCR-positive cells, and the genomic DNA was digested with restriction endonucleases to obtain the digested genomic DNA fragments. The genomic DNA fragments were then hybridized with the sequence characteristics of the target thermostable gene to form a hybridization complex. The hybridization signal generated by the hybridization complex was detected, the hybridization signal map was obtained, the integration site and copy number of the target thermostable gene in PCR-positive cells were analyzed, and cells with single-copy integration and no functional conflict at the integration site were screened out. Total RNA was extracted from the screened cells, and cDNA was obtained by reverse transcription. The transcriptional expression level of the target heat-resistant gene was detected and compared with the expression level of non-transgenic rice cells to screen out rice cells with normal transcriptional expression levels. Total protein was extracted from rice cells with normal transcriptional expression. Based on a protein-specific antibody targeting the core coding region of the target heat-resistant gene, the expression level and molecular weight of the target protein were detected, and cells that correctly expressed the target protein were screened out. Cells that correctly express the target protein are subjected to high-temperature stress treatment. The cell survival rate, reactive oxygen species accumulation, and co-expression of heat-response-related genes under high-temperature conditions are detected to evaluate the functional effectiveness of the target heat-resistant gene and screen out qualified transgenic rice cells.

7. The integrated breeding method for heat-resistant transgenic rice for extreme high-temperature environments according to claim 6, characterized in that, Cells in which the target protein is correctly expressed are obtained based on the results of Western blot analysis. Based on the matching between the band and the theoretical molecular weight of the target protein, combined with the analysis of the band gray value, it is determined whether the target protein has reached a detectable level and has no obvious degradation.

8. The integrated breeding method for heat-resistant transgenic rice for extreme high-temperature environments according to claim 1, characterized in that, The specific process of obtaining transgenic rice plants includes: Transgenic rice cells that passed the identification were transferred to a differentiation medium, the hormone ratio in the medium was adjusted, and the cells were culturing under preset environmental conditions to induce cell differentiation and form adventitious shoots. The growth of adventitious buds is observed regularly. When the adventitious buds reach the preset length, they are transferred to a rooting medium, and the auxin ratio in the medium is adjusted to induce the adventitious buds to root and form complete seedlings. During the rooting culture process, data on the root development status of seedlings are obtained. Seedlings with poor root development or whitening are removed. Seedlings with strong growth and complete root systems are selected and transferred to a sterile substrate for hardening treatment. After the seedling hardening process, the seedlings are transplanted to a greenhouse for routine water and fertilizer management. Stable plants are obtained to determine whether they meet the standards for transgenic rice plants and are then identified as transgenic rice plants.

9. The integrated breeding method for heat-resistant transgenic rice for extreme high-temperature environments according to claim 1, characterized in that, The simulated extreme high-temperature environment specifically includes: A controlled environment growth chamber was selected. Based on natural climate data from extreme high-temperature regions, high-temperature stress parameters were set to obtain data on different growth stages of transgenic rice plants. Corresponding high-temperature stress strategies were then matched based on the data from different growth stages. Based on the goals of promoting heat tolerance in seedlings and ensuring fruit set during the grain-filling stage, high temperature parameters were set for the seedling and grain-filling stages. During the implementation of the high temperature stress strategy, the temperature, humidity, and light intensity in the growth chamber were monitored in real time to determine whether temperature fluctuations exceeded the limits, and environmental control equipment was used to maintain parameter stability.

10. The integrated breeding method for heat-resistant transgenic rice for extreme high-temperature environments according to claim 9, characterized in that, The simulated extreme high-temperature environment also includes obtaining non-transgenic rice plants as a control group, placing the control group and transgenic rice plants in the same high-temperature environment, and co-cultivating them under consistent stress conditions. At the same time, during the high-temperature stress treatment, environmental parameter changes are recorded at preset time intervals to generate an environmental regulation log.

Images