Breeding method for novel lodging-resistant rice variety

Abstract

The invention relates to the technical field of rice seed selection, and particularly discloses a rice lodging-resistant new variety breeding method which comprises the following steps: determining a target gene; constructing a corresponding gene editing carrier; introducing the constructed gene editing vector into rice callus cells by using an agrobacterium-mediated method; screening out successfully transformed calluses through antibiotic screening markers, and inducing and differentiating transgenic plants; carrying out molecular identification on the transgenic plant by adopting PCR (Polymerase Chain Reaction) to confirm whether the target gene is successfully edited or not; after the target gene is successfully edited, hybridizing the target gene with a high-yield and high-quality rice variety with good comprehensive characters to form a hybridized combination; and carrying out multi-generation screening and identification on the hybridized combination, and selecting excellent single plants meeting the breeding target for seed reservation. Editing is higher in rice breeding efficiency, target genes can be edited quickly, applicability is high, and using flexibility is high.

Description

Technical Field

[0001] The present invention relates to the technical field of rice seed selection, and particularly relates to a method for breeding a new rice variety with lodging resistance. Background Art

[0002] At present, in order to reduce production costs, the demand for technologies such as high-quality, high-yield, mechanization adaptation, machine transplanting, mechanical harvesting, light simplification, throwing seedlings, and direct seeding no-tillage technology production technology for rice varieties is very urgent, and all require rice to have stronger lodging resistance.

[0003] The prior art CN112262766B provides a method for breeding a new rice variety with lodging resistance based on phenotype. Specifically, first, two varieties of rice are sown and then transplanted; then, a high-yield and lodging-resistant variety is used as the female parent, and a conventional rice variety is used as the male parent for hybridization to obtain F1-generation seeds; the F1-generation seeds are sown, and the lodging resistance-related traits are measured 20 days after full heading, and then the F2-generation seeds are harvested; all the F2-generation seeds are sown to obtain an F2 population. From F2 to F5 generations, the single-seed descent method is adopted, and only one seed is taken from each plant. After the F6 generation, the traits of the population are basically stable, and excellent individual plants that meet the breeding objectives are selected and reserved for seed production.

[0004] However, in the prior art, the breeding method of obtaining seeds by hybrid sowing takes a long time, resulting in a significant reduction in the breeding efficiency of rice. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for breeding a new rice variety with lodging resistance, aiming to solve the technical problem that the breeding method of obtaining seeds by hybrid sowing in the prior art takes a long time, resulting in a significant reduction in the breeding efficiency of rice.

[0006] To achieve the above purpose, a method for breeding a new rice variety with lodging resistance adopted by the present invention includes the following steps:

[0007] Determine the key genes of the stem strength, plant height, and root system of rice with lodging resistance to determine the target genes;

[0008] Select the CRISPR / Cas9 gene editing technology to construct a corresponding gene editing vector;

[0009] Use the Agrobacterium-mediated method to introduce the constructed gene editing vector into rice callus cells;

[0010] Through antibiotic screening markers, screen out the successfully transformed callus and induce the differentiation of transgenic plants;

[0011] Use PCR to perform molecular identification on the transgenic plants to confirm whether the target genes are successfully edited;

[0012] After the target gene is successfully edited, it is hybridized with a rice variety with good comprehensive traits of high yield and good quality to form a hybrid combination;

[0013] The hybrid combination is subjected to multiple generations of screening and identification, and the excellent individual plants that meet the breeding objectives are selected for seed retention.

[0014] Among them, samples are collected from rice seeds of different regions and different qualities, including lodging-resistant and lodging-susceptible varieties. RNA of different tissues of rice is extracted, and high-throughput sequencing is carried out to analyze the gene expression profile. The differences in gene expression between lodging-resistant and lodging-susceptible varieties are compared, and candidate genes related to the lodging-resistant trait are screened out. Combining genomic sequencing data and phenotypic data, genome-wide association analysis is carried out using statistical methods to locate the gene loci significantly related to the lodging-resistant trait, clone the candidate genes, analyze their expression patterns in different tissues and different treatment conditions by real-time fluorescence quantitative PCR technology, use the CRISPR / Cas9 gene editing technology to knockout or overexpress the candidate genes, observe the changes in the lodging resistance of transgenic plants, verify the function of the genes, and finally determine the target gene.

[0015] Among them, using the sgRNA tool, select a target sequence with strong specificity and high efficiency in the target gene, design the corresponding sgRNA sequence, usually select a sequence of about 20pb to ensure its uniqueness in the genome to reduce the off-target effect, select a CRISPR / Cas9 vector backbone suitable for plants, and carry out homologous recombination or restriction enzyme digestion ligation of the designed sgRNA sequence with the sgRNA backbone in the vector to construct an sgRNA expression cassette. Insert the Cas9 gene into the corresponding position of the vector to ensure its efficient expression in plant cells. Connect the constructed sgRNA expression cassette and Cas9 expression cassette into the vector backbone to form a complete gene editing vector, and verify the vector by enzyme digestion to ensure its correctness.

[0016] Among them, the target gene sequence is obtained by gene synthesis technology, cloned onto the constructed plasmid vector to form a complete expression vector. Select an Agrobacterium strain, culture it in an LB or YEB medium containing antibiotics, shake-culture at 28°C until the logarithmic growth phase, centrifuge to collect Agrobacterium cells, wash the cells with a pre-cooled calcium- and magnesium-free buffer to remove the components in the medium, and then resuspend the cells in an appropriate amount of buffer to prepare competent cells. Use the heat shock method or electroporation method to introduce the constructed plasmid DNA into the competent Agrobacterium cells. The transformed Agrobacterium cells are placed on a selective medium containing the corresponding antibiotics and cultured at 28°C for recovery to screen out the successfully transformed Agrobacterium clones. Transfer the screened callus to a pre-culture medium to allow the callus to resume growth and improve its transformation efficiency.

[0017] Among them, the cultured callus is taken out, washed repeatedly with sterile water and then added to sterile water and soaked for 10 - 20 min to free the bacteria inside the callus. The washing solution is poured off, and then soaked in sterile water containing 400 mg / L cefotaxime for 20 min, rinsed once with sterile water containing 400 mg / L cefotaxime, and finally placed on sterile paper to drain. The selected resistant callus is transferred to the incineration medium until small seedlings are differentiated. When the buds grow to 1 - 2 cm in height, the bud seedlings are transferred to the rooting medium for rooting culture. After a complete plant is formed, the lid is opened for acclimatization for 1 - 2 d to induce the differentiation of transgenic plants.

[0018] Among them, samples are taken from different tissues of the transgenic plants to ensure that the extracted DNA is representative. The genomic DNA is extracted using a plant genomic DNA extraction kit. According to the sequence information of the target gene, a pair of specific primers is designed, and the designed primers are verified by BLAST to ensure their uniqueness in the rice genome and avoid non-specific amplification. The PCR reaction system is prepared for PCR amplification to ensure the complete extension of the DNA strand. A 1 - 2% agarose gel is prepared, EB dye is added, and the electrophoresis tank is set up. The PCR product is mixed with the loading buffer and prepared for loading. The mixed PCR product is added to the gel wells, and electrophoresis is carried out for 30 - 60 min to separate the DNA fragments in the gel. After electrophoresis, the DNA bands in the gel are observed under ultraviolet light. When specific bands of the expected size appear, it indicates that the target gene has been successfully amplified.

[0019] A method for breeding a new rice variety with lodging resistance of the present invention can accurately edit specific genes of rice through the CRISPR / Cas9 gene editing technology, thereby effectively improving the lodging resistance traits of rice. Gene editing has a higher breeding efficiency for rice, can quickly achieve the editing of target genes, has strong applicability and high flexibility in use. The plants transformed by the Agrobacterium-mediated method have good genetic stability, which is beneficial for subsequent screening and identification. The operation is simple and easy to start, reducing the technical threshold of breeding. After successful gene editing, it is hybridized with rice varieties with good comprehensive traits of high yield and high quality, combining the advantages of traditional breeding methods, which helps to cultivate excellent varieties that better meet the production requirements. Through multi-generation screening and identification, the stable inheritance of the target gene and the continuous manifestation of excellent traits can be ensured, improving the success rate of breeding. While improving the yield and quality of crops, the gene editing technology reduces the use of pesticides and environmental pollution. Description of the Drawings

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is a step flowchart of a method for breeding a new rice variety with lodging resistance of the present invention. Specific embodiments

[0022] Please refer to Figure 1 The present invention provides a method for breeding a new rice variety with lodging resistance, including the following steps:

[0023] S1: Determine the key genes of the stem strength, plant height and root system of rice with lodging resistance, and thus determine the target genes;

[0024] S2: Select the CRISPR / Cas9 gene editing technology and construct the corresponding gene editing vector;

[0025] S3: Use the Agrobacterium-mediated method to introduce the constructed gene editing vector into rice callus cells;

[0026] S4: Through the antibiotic screening marker, screen out the successfully transformed callus and induce the differentiation of transgenic plants;

[0027] S5: Use PCR to perform molecular identification on the transgenic plants to confirm whether the target genes are successfully edited;

[0028] S6: After the target genes are successfully edited, hybridize with rice varieties with good comprehensive traits of high yield and good quality to form a hybrid combination;

[0029] S7: Perform multi-generation screening and identification on the hybrid combination, and select excellent individual plants that meet the breeding objectives for seed saving.

[0030] In this embodiment, through CRISPR / Cas9 gene editing technology, specific genes of rice can be accurately edited, thereby effectively improving the lodging resistance of rice. Gene editing has higher efficiency in rice breeding, can quickly realize the editing of target genes, has strong applicability, and high flexibility in use. Plants transformed by Agrobacterium-mediated method have good genetic stability, which is conducive to subsequent screening and identification. The operation is simple and easy to use, which lowers the threshold of breeding technology. After successful gene editing, hybridization with high-yield, high-quality rice varieties with good comprehensive traits is combined with the advantages of traditional breeding methods, which helps to cultivate better varieties that better meet production needs. Through multi-generation screening and identification, the stable inheritance of target genes and the continuous expression of excellent traits can be ensured, thereby improving the success rate of breeding. Gene editing technology reduces the use of pesticides and reduces environmental pollution while increasing crop yield and quality.

[0031] Furthermore, samples were collected from rice varieties of different regions and qualities, including lodging-resistant and lodging-prone varieties. RNA was extracted from different rice tissues for high-throughput sequencing, and gene expression profiles were analyzed. The differences in gene expression between lodging-resistant and lodging-prone varieties were compared, and candidate genes related to the lodging resistance trait were screened out. Genome sequencing data and phenotypic data were combined, and whole-genome association analysis was performed using statistical methods to locate gene loci significantly associated with the lodging resistance trait, and candidate genes were cloned. Real-time fluorescence quantitative PCR technology was used to analyze their expression patterns in different tissues and under different treatment conditions. CRISPR / Cas9 gene editing technology was used to knock out or overexpress candidate genes, and the changes in lodging resistance of transgenic plants were observed. The functions of genes were verified, and finally the target genes were determined.

[0032] In this embodiment, rice seeds from different regions and with different qualities are used as collected samples, which can increase genetic diversity, improve the construction quality of core germplasm, screen excellent traits, explore excellent genes, improve the quality of rice, enhance its applicability, enable it to quickly adapt to different ecological environments, face different climate changes, and effectively improve breeding efficiency.

[0033] Furthermore, using the sgRNA tool, select a target sequence with high specificity and efficiency in the target gene, design the corresponding sgRNA sequence. Usually, a sequence of about 20 pb is selected to ensure its uniqueness in the genome to reduce off-target effects. Select a CRISPR / Cas9 vector backbone suitable for plants, and perform homologous recombination or restriction enzyme digestion ligation between the designed sgRNA sequence and the sgRNA backbone in the vector to construct an sgRNA expression cassette. Insert the Cas9 gene into the corresponding position of the vector to ensure its efficient expression in plant cells. Connect the constructed sgRNA expression cassette and Cas9 expression cassette into the vector backbone to form a complete gene editing vector, and verify the vector by enzymatic digestion to ensure its correctness.

[0034] In this embodiment, using the sgRNA tool can help accurately select a target sequence with high specificity and efficiency in the target gene, ensuring that the Cas9 protein can accurately recognize and bind to the target site, thereby improving the success rate of gene editing. The stable expression of the complete gene editing vector in plant cells can ensure the persistence and stability of the gene editing effect, accelerate the cultivation of excellent varieties, and improve the breeding technology level.

[0035] Furthermore, obtain the target gene sequence through gene synthesis technology, clone it onto the constructed plasmid vector to form a complete expression vector. Select an Agrobacterium strain, culture it in LB or YEB medium containing antibiotics, shake culture at 28 °C until the logarithmic growth phase, centrifuge to collect Agrobacterium cells, wash the cells with pre-cooled calcium- and magnesium-free buffer to remove the components in the medium, and then resuspend the cells in an appropriate amount of buffer to prepare competent cells. Use the heat shock method or electroporation method to introduce the constructed plasmid DNA into the competent Agrobacterium cells. The transformed Agrobacterium cells are placed on a selective medium containing the corresponding antibiotics and cultured at 28 °C for recovery to screen out successfully transformed Agrobacterium clones. Transfer the selected callus to the pre-culture medium to allow the callus to resume growth and improve its transformation efficiency.

[0036] In this embodiment, gene synthesis technology can accurately obtain the target gene sequence, providing precise materials for subsequent gene editing. The Agrobacterium-mediated transformation method has the advantages of simple operation, high transformation efficiency, and good genetic stability. The pre-culture medium usually contains appropriate plant hormones and other nutrients, which can provide a good environment for the growth and differentiation of callus, promote the vitality and transformability of callus, and lay a foundation for subsequent gene transformation and plant regeneration.

[0037] Furthermore, take out the cultured callus, wash it several times with sterile water and then add it to sterile water and soak for 10 - 20 min to free the bacteria inside the callus. Pour out the washing solution, then soak it in sterile water containing 400 mg / L cefotaxime for 20 min, rinse it once with sterile water containing 400 mg / L cefotaxime, and finally place it on sterile paper to drain. Transfer the selected resistant callus into the regeneration medium until small seedlings are differentiated. When the buds grow to 1 - 2 cm in height, transfer the bud seedlings into the rooting medium for rooting culture. After forming a complete plant, open the lid and harden the seedlings for 1 - 2 d to induce and differentiate transgenic plants in this way.

[0038] In this embodiment, by washing the callus with sterile water multiple times and adding cephalosporin antibiotics during the soaking process, it is possible to effectively kill or inhibit the possible microorganisms and reduce the risk of microbial contamination. By culturing the callus in a specific selection medium, resistant callus can be screened out. These callus are more likely to survive and differentiate into transgenic plants during the subsequent culture process. The selected resistant callus has better adaptability and growth ability, which can improve the success rate of inducing and differentiating transgenic plants, thereby increasing the probability of obtaining transgenic plants.

[0039] Furthermore, sample from different tissues of the transgenic plant to ensure that the extracted DNA is representative. Use a plant genomic DNA extraction kit to extract genomic DNA. According to the sequence information of the target gene, design a pair of specific primers, and verify the designed primers through BLAST to ensure their uniqueness in the rice genome and avoid non-specific amplification. Configure the PCR reaction system and perform PCR amplification to ensure the complete extension of the DNA strand. Prepare a 1 - 2% agarose gel, add EB dye, prepare the electrophoresis tank, mix the PCR product with the loading buffer, prepare for loading, add the mixed PCR product into the gel wells, and perform electrophoresis for 30 - 60 min to separate the DNA fragments in the gel. After electrophoresis, observe the DNA bands in the gel under ultraviolet light. When specific bands of the expected size appear, it indicates that the target gene has been successfully amplified.

[0040] In this embodiment, using a plant genomic DNA extraction kit can quickly and efficiently extract genomic DNA, and the reagents and operation steps in the kit have been optimized, which can effectively remove impurities and improve the purity of DNA, providing a high-quality template for subsequent PCR amplification. Sampling from different tissues of the transgenic plant can ensure that the extracted DNA has wide representativeness, covering the genetic information of all parts of the plant, thus more comprehensively reflecting the genomic situation of the transgenic plant.

[0041] Furthermore, when introducing the constructed plasmid DNA into competent Agrobacterium cells using the heat shock method, the competent Agrobacterium cells are in a state that is prone to accepting foreign DNA. Then, the foreign DNA is mixed with the competent Agrobacterium cells, and heat shock treatment is carried out at a temperature of 26 - 31°C. After heat shock treatment, the cells are transferred to an appropriate medium for recovery culture to promote the growth and reproduction of the transformed cells.

[0042] In this embodiment, the heat shock method is adopted, and the operation is relatively simple and the cost is relatively low. Heat shock treatment can induce cells to produce heat shock proteins, and these proteins have the effect of protecting cells from damage by high temperature, oxidation, and environmental toxins, enhancing the survival ability of cells under adverse environments.

[0043] Furthermore, when introducing the constructed plasmid DNA into competent Agrobacterium cells using the electroporation method, plant cells are prepared into a suspension, the foreign DNA is mixed with the cell suspension, and it is placed between the electrodes of an electroporator for electroporation treatment. After electroporation treatment, the cells are transferred to a medium for recovery culture to promote the growth and reproduction of the transformed cells.

[0044] In this embodiment, the transformation efficiency is relatively high, and foreign genes can be effectively introduced into various plant cells. The electroporation method causes relatively less damage to cells, and the operation process is relatively fast.

[0045] What is disclosed above is only a preferred embodiment of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Those of ordinary skill in the art can understand all or part of the processes of implementing the above embodiments, and the equivalent changes made according to the claims of the present invention still fall within the scope covered by the invention.

Claims

1. A method for breeding a new rice variety with lodging resistance, characterized in that, It includes the following steps: Determine the key genes of stem strength, plant height and root system for lodging resistance in rice, and thereby determine the target genes; Select the CRISPR / Cas9 gene editing technology and construct the corresponding gene editing vector; Use the Agrobacterium-mediated method to introduce the constructed gene editing vector into rice callus cells; Through antibiotic screening markers, screen out the successfully transformed callus and induce the differentiation of transgenic plants; Use PCR to conduct molecular identification of the transgenic plants to confirm whether the target genes are successfully edited; After the target genes are successfully edited, cross them with rice varieties with good comprehensive traits of high yield and good quality to form a hybrid combination; Carry out multi-generation screening and identification of the hybrid combination, and select excellent individual plants that meet the breeding objectives for seed saving.

2. A method for breeding a new rice variety with lodging resistance as described in claim 1, which determines the key genes of stem strength, plant height and root system for lodging resistance in rice, and thereby determines the target genes. It is characterized in that Collect samples from rice varieties in different regions and with different qualities. The collected samples include lodging-resistant and lodging-susceptible varieties. Extract the RNA of different tissues of rice, conduct high-throughput sequencing, analyze the gene expression profile, compare the differences in gene expression between lodging-resistant and lodging-susceptible varieties, screen out candidate genes related to the lodging resistance trait, combine the genomic sequencing data and phenotypic data, use statistical methods for genome-wide association analysis, locate the gene loci significantly related to the lodging resistance trait, clone the candidate genes, analyze their expression patterns in different tissues and under different treatment conditions through real-time fluorescence quantitative PCR technology, use the CRISPR / Cas9 gene editing technology to knockout or overexpress the candidate genes, observe the changes in the lodging resistance of transgenic plants, verify the functions of the genes, and finally determine the target genes.

3. A method for breeding a new rice variety with lodging resistance as described in claim 1, which selects the CRISPR / Cas9 gene editing technology and constructs the corresponding gene editing vector. It is characterized in that Use the sgRNA tool to select a target sequence with strong specificity and high efficiency in the target gene, design the corresponding sgRNA sequence. Usually select a sequence of about 20pb to ensure its uniqueness in the genome to reduce the off-target effect. Select a CRISPR / Cas9 vector backbone suitable for plants, perform homologous recombination or restriction enzyme digestion ligation on the designed sgRNA sequence and the sgRNA backbone in the vector to construct the sgRNA expression cassette, insert the Cas9 gene into the corresponding position of the vector to ensure its efficient expression in plant cells, connect the constructed sgRNA expression cassette and Cas9 expression cassette to the vector backbone to form a complete gene editing vector, and verify the vector by enzyme digestion to ensure its correctness.

4. A method for breeding a new rice variety with lodging resistance as described in claim 1, which uses the Agrobacterium-mediated method to introduce the constructed gene editing vector into rice callus cells. It is characterized in that The target gene sequence is obtained through gene synthesis technology and cloned onto the constructed plasmid vector to form a complete expression vector. An Agrobacterium strain is selected and cultured in LB or YEB medium containing antibiotics. It is cultured with shaking at 28 °C until the logarithmic growth phase, and the Agrobacterium cells are collected by centrifugation. The cells are washed with a pre-cooled calcium- and magnesium-free buffer to remove the components in the medium. After that, the cells are resuspended in an appropriate amount of buffer to prepare competent cells. The constructed plasmid DNA is introduced into the competent Agrobacterium cells by heat shock method or electroporation method. The transformed Agrobacterium cells are placed on a selective medium containing the corresponding antibiotics and cultured at 28 °C for recovery. The successfully transformed Agrobacterium clones are screened out. The screened callus is transferred to a pre-culture medium to allow the callus to resume growth and improve its transformation efficiency.

5. A method for breeding a new rice variety with lodging resistance as described in claim 1, wherein the successfully transformed callus is screened out through an antibiotic selection marker and transgenic plants are induced and differentiated, characterized in that The cultured callus is taken out, washed several times with sterile water and then soaked in sterile water for 10 - 20 min to free the bacteria inside the callus. The washing solution is poured off, and then soaked in sterile water containing 400 mg / L cefotaxime for 20 min, rinsed once with sterile water containing 400 mg / L cefotaxime, and finally drained on sterile paper. The screened resistant callus is transferred to an induction medium until small seedlings are differentiated. When the buds grow to 1 - 2 cm in height, the bud seedlings are transferred to a rooting medium for rooting culture. After forming complete plants, they are acclimatized for 1 - 2 d with the lid open to induce and differentiate transgenic plants.

6. A method for breeding a new rice variety with lodging resistance as described in claim 1, wherein PCR is used to perform molecular identification on the transgenic plants to confirm whether the target gene has been successfully edited, characterized in that Samples are taken from different tissues of the transgenic plants to ensure that the extracted DNA is representative. The genomic DNA is extracted using a plant genomic DNA extraction kit. According to the sequence information of the target gene, a pair of specific primers is designed and verified by BLAST to ensure its uniqueness in the rice genome and avoid non-specific amplification. The PCR reaction system is prepared and PCR amplification is carried out to ensure the complete extension of the DNA strand. A 1 - 2% agarose gel is prepared, EB dye is added, and an electrophoresis tank is set up. The PCR product is mixed with the loading buffer and prepared for loading. The mixed PCR product is added to the gel wells and electrophoresed for 30 - 60 min to separate the DNA fragments in the gel. After electrophoresis, the DNA bands in the gel are observed under ultraviolet light. When specific bands of the expected size appear, it indicates that the target gene has been successfully amplified.

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