Application of rice male sterility gene OsGLOX5 in rice breeding
Through gene editing technology, the rice male sterile gene OsGLOX5 was targeted to be edited, and a rice male sterile line was created, which solved the problems of low breeding efficiency and low accuracy in the existing technology, and achieved the improvement of rice yield and stress resistance.
Patent Information
- Application Number
- CN202510282970.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-11
- Publication Date
- 2025-05-30
AI Technical Summary
There are complex supporting technologies and management measures for the application of existing rice male sterile genes in hybrid breeding. The seed production safety is low, and the breeding efficiency and accuracy are not high, which affects rice yield and stress resistance.
Through gene editing technology, the rice male sterile gene OsGLOX5 was targeted to be edited, functional deletion mutations were introduced, and the rice male sterile line was obtained, and a new rice breeding system was created using this genetic resource.
The stable creation of male sterile lines in rice has been achieved, breeding efficiency and accuracy have been improved, the stress resistance and adaptability of rice has been enhanced, and it has important application value in the third generation hybrid breeding technology.
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Figure CN120060347A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the fields of plant genetic engineering technology and molecular biology, and particularly relates to the application of the rice male sterility gene OsGLOX5 in rice breeding. Background Art
[0002] As one of the most important food crops in the world, the yield and quality of rice are directly related to global food security. Scientists have been constantly exploring new breeding technologies to improve the yield and stress resistance of rice. The discovery and application of male sterility genes have brought revolutionary breakthroughs to hybrid rice breeding.
[0003] Male sterility refers to the inability of plants to produce pollen with normal functions, but the female reproductive organs are normal. This characteristic provides an important tool for cross-breeding. Utilizing male sterility genes can efficiently produce hybrid seeds, thereby significantly increasing the yield and resistance of rice. However, the existing hybrid breeding systems based on male sterility genes (such as the "three-line method" and the "two-line method") require complex supporting technologies and management measures. For example, the "three-line method" needs to cultivate male sterile lines, maintainer lines, and restorer lines simultaneously, which is restricted by the restorer-maintainer relationship, while the two-line method is greatly affected by temperature and has low seed production safety. Therefore, exploring rice male sterility genes is of great significance for promoting the development of hybrid rice breeding technology, improving breeding efficiency and accuracy, enhancing the stress resistance and adaptability of rice, promoting sustainable agricultural development, driving innovation in agricultural biotechnology, and solving global food security problems. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide the application of the rice male sterility gene OsGLOX5 in rice breeding.
[0005] To achieve the above purpose, the present invention provides the following technical solutions:
[0006] The present invention provides an application of a rice male sterility gene OsGLOX5 in creating a rice male sterile line, and the amino acid sequence of the encoded protein of the rice male sterility gene OsGLOX5 is as shown in SEQ ID No.2.
[0007] Preferably, the CDS sequence of the rice male sterility gene OsGLOX5 is as shown in SEQ ID No.1.
[0008] Preferably, the rice male sterility gene OsGLOX5 is the rice LOC_Os11g06870 gene.
[0009] Preferably, the application is to perform targeted editing on the rice male sterility gene OsGLOX5 using gene editing technology to introduce loss-of-function mutations and obtain a rice male sterile line.
[0010] The present invention also provides a mutant of rice male sterility gene OsGLOX5, which is a loss-of-function mutant of rice male sterility gene OsGLOX5. The amino acid sequence of the encoded protein of the mutant is as shown in SEQ ID No.4, and the mutant makes rice exhibit male sterility traits.
[0011] Preferably, the mutant is a loss-of-function mutant of rice LOC_Os11g06870 gene. Compared with the rice LOC_Os11g06870 gene sequence, it only has a point mutation from G to T at the 289th amino acid of the exon of LOC_Os11g06870 gene. The CDS sequence of the mutant is as shown in SEQ ID No.3.
[0012] The present invention also provides the application of the above mutant of rice male sterility gene OsGLOX5 in creating rice male sterile lines.
[0013] Preferably, the application is to transfer the mutant of rice male sterility gene OsGLOX5 into wild-type rice to obtain a rice male sterile line.
[0014] The present invention also provides the application of a rice sterile line in rice breeding. The application is to use the rice male sterile line obtained by the above application as the female parent, cooperate with a male parent with heterosis, produce hybrid F1 generation, and conduct cross breeding.
[0015] The invention process of the present invention is as follows: The present invention identified a genetically stable male sterile mutant osglox5 from the ethyl methanesulfonate (EMS)-induced mutant library of Wuyunjing 21. The osglox5 mutant showed no abnormality in vegetative growth, but had defects in reproductive growth, pollen abortion, and no seed setting, showing complete male sterility. Genetic analysis found that the sterility trait of the osglox5 mutant was controlled by a pair of recessive nuclear genes OsGLOX5. Through BSA genome resequencing, sequencing alignment, and complementary verification, the results showed that LOC_Os11g06870 was the target gene of OsGLOX5, and the osglox5 mutant had a point mutation from G to T at the 289th amino acid of the exon of LOC_Os11g06870, changing proline to isoleucine, resulting in the loss of function of the encoded protein. Analysis of its sterile phenotype showed that the degradation of the tapetum in osglox5 was delayed, ultimately leading to pollen abortion and male sterility.
[0016] Advantages of the present invention: From an ethyl methanesulfonate (EMS)-induced mutant library of Wuyunjing 21, a genetically stable male sterile mutant, osglox5, was identified. It showed normal vegetative growth, defective reproductive growth, pollen abortion, sterility, and complete male sterility. Its mutant phenotype was controlled by a recessive nuclear gene. By bulked segregant analysis (BSA) genome resequencing and sequencing alignment, the gene OsGLOX5 controlling this trait was obtained, with the gene number LOC_Os11g06870. The mutant (the CDS sequence is as shown in SEQ ID No. 3, and the amino acid sequence of the encoded protein is as shown in SEQ ID No. 4) only had a point mutation from G to T at the 289th amino acid of the exon of LOC_Os11g06870 (the CDS sequence is as shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is as shown in SEQ ID No. 2), changing valine to phenylalanine. After the mutation, the degradation of the tapetum was delayed, ultimately leading to pollen abortion and male sterility. The rice male sterile gene OsGLOX5 disclosed in the present invention is a brand-new rice male sterile gene, greatly enriching the rice male sterile gene pool, providing new gene resources for creating rice male sterile lines and rice breeding. At the same time, its sterile trait is stable and the seed production is safe, having great application value in hybrid breeding, and can be applied to the third-generation hybrid breeding technology, with broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 For the determination and complementary verification of the candidate gene of osglox5; wherein, A: Gene mutation site; B, C: Anther stereomicroscopy and pollen iodine staining results of the wild type (WT); D, E: Anther stereomicroscopy and pollen iodine staining results of the osglox5 mutant; F, G: Anther stereomicroscopy and pollen iodine staining results of the complementary transgenic plants.
[0018] Figure 2 Scanning electron microscopy observation and semi-thin section analysis of anthers of the wild type and the osglox5 mutant; wherein, A - H are scanning electron microscopy observations of anthers of the wild type and the osglox5 mutant; A, B: Anthers of the wild type and the osglox5 mutant as a whole; C, F: Anther exine of the wild type and the osglox5 mutant; D, G: Anther intine of the wild type and the osglox5 mutant; E, H: Morphology of pollen grains of the wild type and the osglox5 mutant; I: Semi-thin section analysis of anthers at each stage of the wild type and the osglox5 mutant.
[0019] Figure 3 For the expression pattern analysis of OsGLOX5; wherein, A: Expression of OsGLOX5 in various parts of rice; B: Expression of OsGLOX5 at each stage of anthers; C: In-situ hybridization analysis of OsGLOX5. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0020] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for exemplarily illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. It should be noted that the experimental materials not specified in the experimental material sources in the embodiments of the present invention can all be commercially obtained, and the experimental methods not specified in the specific conditions in the embodiments of the present invention are usually carried out according to the conventional experimental methods in the art or according to the methods recommended by the experimental material manufacturers. The male sterile mutant osglox5 of the present invention was obtained by EMS mutagenesis of Wuyunjing 21. For the specific information of Wuyunjing 21, please refer to the National Rice Data Center (http: / / www.ricedata.cn).
[0021] Example 1 Obtaining and Genetic Analysis of osglox5 Mutant
[0022] The rice male sterile mutant osglox5 was identified from the mutant library of Wuyunjing 21 mutagenized by ethyl methanesulfonate (EMS) (derived from the School of Life Science and Engineering, Southwest University of Science and Technology). The osglox5 mutant showed no abnormality in vegetative growth, but had defects in reproductive growth, pollen abortion, infertility, and complete male sterility.
[0023] Using Wuyunjing 21 as the male parent and the osglox5 mutant as the female parent for hybridization, all the individual plants in the F1 generation were fertile. The F1 generation population was self-crossed to obtain the F2 generation population. The pollen fertility of the individual plants in the F2 generation population was detected and counted. The results showed that trait segregation occurred. Using the chi-square test method for analysis, it was found that the segregation ratio of fertile plants and sterile plants conforms to the Mendelian genetic segregation ratio (χ2 = 2.745, P > 0.05), indicating that the sterile trait of the osglox5 mutant is controlled by a pair of recessive nuclear genes.
[0024] Example 2 Determination and Complementary Verification of Candidate Gene OsGLOX5
[0025] The whole genome of the gene pool composed of wild type and 30 male sterile plants selected from the F2 population was re-sequenced using NextSeq 500 (Illumina). Then MutMap and SNP / INDEL-index analysis were carried out, and the short reads were aligned with the reference genome (Nipponbare) to obtain a certain number of F2-specific SNPs and INDELs. For each SNP / INDEL, its index was calculated, where the index refers to the ratio between the read count of the SNP / INDEL and the total read count. SNPs / INDELs with index = 1 were selected for subsequent sequence alignment. The sequencing and alignment results showed that the mutant only had a point mutation from G to T at the 289th amino acid of the exon of LOC_Os11g06870, changing proline to isoleucine. Therefore, LOC_Os11g06870 was taken as the candidate gene of OsGLOX5( Figure 1 A).
[0026] To verify whether LOC_Os11g06870 is the target gene, a complementary vector pOsGLOX5:OsGLOX5-pCAM1301 was constructed, and positive complementary transgenic plants osglox5-COM of the osglox5 mutant were obtained. Through iodine staining and stereomicroscopic observation, it was found that compared with the wild type and the osglox5 mutant, the pollen amount and starch filling of osglox5-COM were indistinguishable from those of the wild type, and the plants showed normal spikelet morphology and anther structure, and the fertility was restored( Figure 1 B-G). This result confirmed that LOC_Os11g06870 is the target gene of OsGLOX5.
[0027] Example 3 Functional Analysis
[0028] (1) Sterility phenotype analysis
[0029] Observation by scanning electron microscopy showed that there were no obvious changes in the inner and outer walls of the anthers of the mutant compared with the wild type( Figure 2 A, B, C, F, G); however, the observation of pollen grains showed that wild type pollen was round and plump, while osglox5 pollen was shrunken and wrinkled on the surface( Figure 2 E, H). The results of semi-thin sections showed that there were no obvious differences between the mutant osglox5 and the wild type during the St7-St11 stages of anther development, but when microspores were formed at the St12 stage, the tapetum in the wild type anthers was completely degraded and mature pollen grains were formed, while in osglox5, the degradation of the tapetum was delayed, and abnormal collapse and shrinkage of microspores occurred( Figure 2 I). The results showed that the degradation of the tapetum in osglox5 was delayed, ultimately leading to pollen abortion and male sterility.
[0030] (2) Expression analysis
[0031] The expression of OsGLOX5 in various tissues of rice was analyzed by qRT-PCR. The results showed that its expression mainly occurred in the spikelets, and the expression levels in other parts were extremely low ( Figure 3 A).
[0032] To further explore the expression of OsGLOX5 in the spike, qRT-PCR analysis was performed according to the 14 developmental stages of anthers divided by Zhang Dabing. The results showed that the expression level of OsGLOX5 was the highest at St8 and St9 of anther development, and the expression levels in other developmental stages were extremely low ( Figure 3 B).
[0033] Furthermore, the spatial expression pattern of OsGLOX5 at the wild-type anther development stage was detected by mRNA in situ hybridization technology. The results showed that at the 7th, 8a, 8b, and 9th stages, OsGLOX5 was expressed in both the tapetum and microspores of the anthers ( Figure 3 C).
[0034] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. All any modifications, equivalent replacements, improvements, etc. within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. Application of rice male sterility gene OsGLOX5 in creating rice male sterility lines, characterized in that: The amino acid sequence of the protein encoded by the rice male sterility gene OsGLOX5 is shown in SEQ ID No.
2.
2. The use according to claim 1, characterized in that: The CDS sequence of the rice male sterility gene OsGLOX5 is shown in SEQ ID No.
1.
3. The use according to claim 2, characterized in that: The rice male sterility gene OsGLOX5 is the rice LOC_Os11g06870 gene.
4. The use according to any one of claims 1 to 3, characterized in that: The application is to use gene editing technology to perform targeted editing on the rice male sterility gene OsGLOX5 to introduce a loss-of-function mutation and obtain a rice male sterile line.
5. A mutant of the rice male sterility gene OsGLOX5, characterized in that: The mutant is a functional mutant of the rice male sterility gene OsGLOX5. The amino acid sequence of the protein encoded by the mutant is shown in SEQ ID No.
4. The mutant causes rice to show male sterility.
6. The mutant according to claim 5, characterized in that The mutant is a loss-of-function mutant of the rice LOC_Os11g06870 gene. Compared with the rice LOC_Os11g06870 gene sequence, it only has a point mutation from G to T at the 289th amino acid in the exon of the LOC_Os11g06870 gene. The CDS sequence of the mutant is shown in SEQ ID No.
3.
7. Use of the mutant of the rice male sterility gene OsGLOX5 according to any one of claims 5 or 6 in creating rice male sterile lines.
8. The use according to claim 7, characterized in that: The application is to transfer the mutant of the rice male sterility gene OsGLOX5 into wild-type rice to obtain a rice male sterility line.
9. Application of a rice sterile line in rice breeding, characterized in that: The application is to use the rice male sterile line obtained in claim 4 or claim 8 as a female parent, in combination with a male parent having hybrid vigor, to produce hybrid F1 generation and perform hybrid breeding.