Rice ltpl28 mutant protein, encoding gene and application thereof
By using radiation-induced mutagenesis screening and gene editing technology, stable rice completely male-sterile materials were created, solving the problem of unstable pollen maturation process and realizing the creation of sterile lines with a single genetic background, which is suitable for rice anther development research and germplasm improvement.
Patent Information
- Application Number
- CN202611499123.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-24
- Publication Date
- 2026-11-06
AI Technical Summary
Current research on rice anther development lacks functional regulation of LTPL28, leading to instability in the pollen maturation process, affecting the reliability and yield of hybrid seed production. Furthermore, existing sterile lines are susceptible to environmental influences and lack sterile materials with a single genetic background.
The LTPL28 mutant was screened by 60Co-γ ray radiation mutagenesis, the LTPL28 gene was located by map-based cloning, and CRISPR/Cas9 knockout vector and genetic complementation vector were constructed to achieve OsLTPL28 gene function loss and create stable completely male-sterile materials.
Genetically stable, completely male-sterile rice material was obtained, with stable pollen fertility unaffected by the environment, suitable for basic research on anther development and the creation of new sterile germplasm.
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Figure CN123043079A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of agricultural biotechnology and rice developmental molecular biology, particularly to the rice LTPL28 mutant protein and its encoding gene and applications. Specifically, it relates to the rice LTPL28 mutant protein, the mutant gene encoding the mutant protein, gene editing vectors, genetic complementation vectors, and the applications of the mutant protein and mutant gene in creating completely male-sterile rice materials and elucidating the developmental mechanisms of the anther tapetum and pollen exine. Background Technology
[0002] Suitable temperature and light conditions, along with normal anther development, are fundamental for rice to complete grain filling and ensure yield. The tapetum is a key tissue supplying lipids and sporophytin precursors to microspores. Imbalance in the programmed cell death sequence of the tapetum can directly block the pollen maturation process, leading to complete pollen abortion. Lipid transporters are responsible for the transport of intercellular lipids and are key functional proteins regulating tapetum degradation and pollen exwall formation.
[0003] Rice hybrid seed production highly relies on stable genetic male-sterile germplasm resources. Currently used photoperiod-temperature-sensitive genic male-sterile lines are susceptible to seasonal and climatic disturbances in fertility, and the germplasm pool for cytoplasmic male-sterile lines is narrow. Furthermore, basic research on anther development lacks materials on completely sterile models controlled by a single recessive nuclear gene with a single genetic background. Existing research on the function of lipid transporters mainly focuses on abiotic stress and seed development processes; there are no reports on LTPL28's specific regulation of the temporal degradation of the rice anther tapetum and pollen formation, nor have there been any public reports on LTPL28 loss-of-function naturally occurring point mutant proteins, their accompanying gene editing, and genetic complementation verification systems. Summary of the Invention
[0004] The purpose of this invention is to provide the rice LTPL28 mutant protein, its encoding gene, and its applications, in order to solve the problems existing in the prior art.
[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is knockout. OsLTPL28 The application of genes in the creation of completely male-sterile rice materials, the aforementioned OsLTPL28 The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0006] The second technical solution of the present invention is a method for knocking out OsLTPL28 The sgRNA of the gene, OsLTPL28 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.5.
[0007] The third technical solution of the present invention is a gene editing vector, wherein the gene editing vector contains the sgRNA.
[0008] The fourth technical solution of the present invention is a recombinant bacterium containing the aforementioned gene editing vector.
[0009] The fifth technical solution of the present invention is the application of the sgRNA, the gene editing vector, or the recombinant bacteria in the creation of completely male-sterile rice materials.
[0010] The sixth technical solution of this invention is a method for creating completely male-sterile rice materials, comprising the following steps: Using gene editing technology to knock out rice OsLTPL28 Genes, making the OsLTPL28 The gene function loss; OsLTPL28 The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0011] The seventh technical solution of the present invention is a genetic complementation vector, wherein the genetic complementation vector comprises... OsLTPL28 Promoter fragments and complete coding sequences of genes; The promoter segment is OsLTPL28 The gene contains a 2.0 kb promoter fragment upstream; the backbone vector of the genetic complementation vector is the pCAMBIA1300 vector.
[0012] The eighth technical solution of the present invention is the application of the genetic complementation vector in restoring male fertility in rice.
[0013] Based on the above technical solution, the present invention has the following technical effects: (1) This invention uses radiation-induced mutation screening. ltpl28 The sterile mutant was used to locate the LTPL encoding gene that regulates rice anther development for the first time using map-based cloning, and the single base substitution mutation site was identified, filling the gap in research on lipid transporter regulation of male reproductive development in rice.
[0014] (2) This invention uses three genetic evidences: natural mutant phenotype, CRISPR knockout reverse verification, and genetic complementation positive reinstatement, to fully confirm gene function, and the experimental evidence chain is complete and reliable.
[0015] (3) This invention is equipped with two types of standardized molecular vectors: VK005 knockout vector for rapid creation of sterile materials and pCAMBIA1300 complementary vector for gene function verification, forming a complete experimental toolkit.
[0016] (4) The sterile material obtained by the present invention only specifically damages the anther and pollen development, does not interfere with vegetative growth, has stable sterility, and does not have temperature and light sensitivity defects. Attached Figure Description
[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0018] Figure 1 Wild type and ltpl28 Phenotypic comparison of mutant strains; where A and B represent wild type, ltpl28 Phenotypic characteristics of the mutant at the heading stage, scale bar 20 cm; C: Ear morphology of wild type and mutant, scale bar 3 cm; D, E: Anther morphology after removal of palea and lemma, scale bar 2 mm; F, G: Observation images of mature pollen stained with I2-KI, scale bar 200 μm; Figure 2 Wild type and ltpl28 Paraffin sections of anthers at different developmental stages of the mutant strain; labeled: E epidermis, En anther inner wall, ML mesophyll, T tapetum, MC meiotic cells, Tds tetrads, Msp microspores, Mp mature pollen, dT degraded tapetum, dMsp degenerate microspores, dMp degenerate mature spores, AT abnormal tapetum; all sections are scaled at 20 μm, showing the anther cell structure at each stage from S7 to S12. Figure 3 for ltpl28 Fine mapping and sequencing peak diagrams of mutant candidate genes; including peak diagrams comparing sequencing of genes from molecular marker regions, chromosomal segments, and wild-type and mutant strains, used to show mutagenic single-base substitution sites; Figure 4 Phenotypic images of anthers and pollen from wild-type, complementary, and CRISPR knockout strains; AC: anther morphology of different strains, scale bar 2 mm; DF: staining results of mature pollen of the corresponding strains, scale bar 200 μm. Detailed Implementation
[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.
[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0024] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.
[0025] To address the technical shortcomings of existing rice male-sterile germplasm, such as environmental sensitivity, complex genetic background, and lack of standardized experimental materials for lipid transporter protein-regulated anther development, this invention relies on... 60 Co-γ ray radiation-induced mutagenesis ltpl28 Stable male-sterile mutants were developed, and key fertility genes were located through map-based cloning. Natural single-base substitution mutation sites were identified, and novel LTPL28 mutant proteins and encoding mutant genes were provided. CRISPR knockout vectors and genetic complementation vectors were also developed to create stable, completely male-sterile rice materials. These materials can be used for research on the basic mechanisms of rice anther development and for the creation of nuclear male-sterile germplasm.
[0026] This invention provides a knockout method. OsLTPL28 The application of genes in the creation of completely male-sterile rice materials, the aforementioned OsLTPL28 The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0027] This invention also provides a method for knocking out OsLTPL28 The sgRNA of the gene, OsLTPL28 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.5.
[0028] This invention also provides a gene editing vector comprising the sgRNA.
[0029] In some specific implementations, the backbone vector of the gene editing vector is the VK005 vector.
[0030] This invention also provides a recombinant bacterium containing the aforementioned gene-editing vector.
[0031] The present invention also provides the application of the sgRNA, the gene editing vector, or the recombinant bacteria in the creation of completely male-sterile rice materials.
[0032] This invention also provides a method for creating completely male-sterile rice materials, comprising the following steps: Using gene editing technology to knock out rice OsLTPL28 Genes, making the OsLTPL28 The gene function loss; OsLTPL28 The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0033] In some specific implementations, the gene editing technology is CRISPR / Cas9 technology; The knockout of rice OsLTPL28 The gene editing method involves constructing the gene editing vector or recombinant bacteria from the sgRNA, transforming it into rice, and then screening. OsLTPL28 Homozygous mutant strains with gene knockout.
[0034] This invention also provides a genetic complementation vector, the genetic complementation vector comprising... OsLTPL28 Promoter fragments and complete coding sequences of genes; The promoter segment is OsLTPL28 The gene contains a 2.0 kb promoter fragment upstream; the backbone vector of the genetic complementation vector is the pCAMBIA1300 vector.
[0035] This invention also provides the application of the genetic complementation vector in restoring male fertility in rice.
[0036] Example 1 rice ltpl28 Mutant screening and genetic segregation identification Wuyunjing 7 was used as the experimental material (preserved by the Key Laboratory of Crop Genetics and Breeding of the Ministry of Education, Jiangxi Agricultural University). 60 Co-γ ray radiation mutagenesis was used to construct a mutant library, and stable male-sterile single plants were screened. ltpl28 The mutant was crossed with the wild type to obtain the F1 generation. The F1 generation was then self-crossed to construct the F2 segregating population. Population fertility was analyzed, revealing 127 fertile plants and 43 sterile plants, with a segregation ratio of 3:1. (χ²) 2 The test result P>0.05 confirms that this infertility trait is controlled by a single recessive nuclear gene.
[0037] Cultivation of wild-type Wuyunjing No. 7 and ltpl28 For mutant strains, spikelets, anthers, and mature pollen were collected during the heading stage. Overall morphology and pollen staining were then photographed. Corresponding images are attached. Figure 1 Anthers were collected from stages S7 to S12 to prepare paraffin sections. The differences in tapetum and microspore development were observed under an optical microscope. The results are shown in the appendix. Figure 2 .
[0038] During the vegetative growth period: Compared with the wild-type Wuyunjing 7, the mutant strain, the CRISPR-edited strain, and the wild-type Wuyunjing 7 showed no significant differences in plant height, tillering, leaf morphology, and growth rate that could be identified by the naked eye.
[0039] Reproductive growth period: After the mutant plant emerges, the spike remains green for a long time and cannot mature normally; the anthers are short, wrinkled, and light yellow in color.
[0040] Pollen fertility test: Staining with 1% I2-KI solution showed that wild-type pollen was plump, dark black, and round. ltpl28 The mutant strain has transparent, irregularly shaped pollen, no starch accumulation, and zero pollen fertility; (attached) Figure 1 Figures F and G show pollen staining observations.
[0041] Anther cytological characteristics: In stage S7, there was no structural difference between wild and mutant anthers; in stage S8, the tapetum of the mutant prematurely became concentrated and vacuolated; from S9 to S10, the tapetum of the mutant remained abnormally swollen; from S11 to S12, the tapetum of the wild type completely degraded and disappeared, while a large amount of the tapetum remained in the mutant, with microspores collapsing and degenerating inwards; (Appendix) Figure 2 Wild type and ltpl28 Comparison of paraffin sections of anthers at different developmental stages of the mutant.
[0042] Genetic characteristics: ltpl28 The sterility trait is controlled by a single recessive nuclear gene. All F1 generation obtained by crossing the mutant with the wild type are fertile. The ratio of fertile to sterile plants in the F2 population follows Mendelian inheritance law of 3:1.
[0043] Example 2 OsLTPL28Map-based cloning and mutation site identification The mutant was crossed with Nipponbare (preserved by the Key Laboratory of Crop Genetics and Breeding, Ministry of Education, Jiangxi Agricultural University) to construct an F2 fine-mapping population. Initial molecular marker mapping narrowed down candidate gene regions, and fine mapping further reduced the region to a 68kb segment on chromosome 3. Only the single exon gene LOC_Os03g59380 was found within this segment. Sequence amplification and sequencing comparison of the wild-type and mutant gene sequences confirmed that the G mutation at exon 236 was replaced by an A mutation. This base substitution caused the arginine at position 79 to be replaced by histidine, which is the mutagenic site. The fine-mapping sequencing results are as follows: Figure 3 As shown.
[0044] Example 3 Construction of genetic complementation vectors and verification of fertility reinstatement Amplification OsLTPL28 Upstream 2.0 kb promoter fragment and complete coding sequence (SEQ ID NO.1).
[0045] SEQ ID NO.1: ATGGCGAGCGCTCGTCGCAGCCGCGGCGTGGCGGCGGTGGGGATCGCGGTGTTTGTGGCTGTCGTGGTGGTGGCGGCGGGCGCCGGGCGCGGCGGCGGTGTCGTGCGGCGACG CGGTGAGCGCGCTGGCGCCGTGCGGGCCGTTCCTGCTGGGCGGCGCGGCGCGGCCGGGCGATCGGTGCTGCGGCGGCGCGAGGGCCGCTGCGCGGGATGGCGGGCACGGCGGAGGCGCGGC G CGCGCTGTGCAGGTGCCTGGAGCAGTCCGGGCCGTCGTTCGGCGTGCTCCCCGACCGCGCGCGGGCCTCCCGGCGCTCTGCAAGCTCGGACTCGCCATCCCCGTCGGCGCCGCCACCGACTGCAGCAAG.
[0046] Note: The underlined positions are mutation sites.
[0047] OsLTPL28 The protein sequence encoded by the gene is as follows: SEQ ID NO.2: MASARRSRGVAAVGIAVFVAVVVVAAGAGGAAAVSCGDAVSALAPCGPFLLGGAARPGDRCCGGARALRGMAGTAEAR RALCRCLEQSGPSFGVLPDRARRLPALCKLGLAIPVGAATDCSK.
[0048] OsLTPL28 The sequence after gene mutation is as follows: SEQ ID NO.3: ATGGCGAGCGCTCGTCGCAGCCGCGGCGTGGCGGCGGTGGGGATCGCGGTGTTTGTGGCTGTCGTGGTGGTGGCGGCGGGCGCCGGGCGCGGCGGCGGTGTCGTGCGGCGACG CGGTGAGCGCGCTGGCGCCGTGCGGGCCGTTCCTGCTGGGCGGCGCGGCGCGGCCGGGCGATCGGTGCTGCGGCGGCGCGAGGGCCGCTGCGCGGGATGGCGGGCACGGCGGAGGCGCGGC A CGCGCTGTGCAGGTGCCTGGAGCAGTCCGGGCCGTCGTTCGGCGTGCTCCCCGACCGCGCGCGGGCCTCCCGGCGCTCTGCAAGCTCGGACTCGCCATCCCCGTCGGCGCCGCCACCGACTGCAGCAAG.
[0049] The mutated protein sequence is as follows: SEQ ID NO.4: MASARRSRGVAAVGIAVFVAVVVVAAGAGGAAAVSCGDAVSALAPCGPFLLGGAARPGDRCCGGARALRGMAGTAEAR H ALCRCLEQSGPSFGVLPDRARRLPALCKLGLAIPVGAATDCSK.
[0050] Insert the pCAMBIA1300 vector to construct the complementary vector pCAMBIA1300-LTPL28; transform the recombinant plasmid into Agrobacterium and infect it. ltpl28 Complementary transgenic plants were obtained from mutant callus; field observations and pollen staining results are attached. Figure 4 The complementary plants recovered to the wild-type level in terms of plant type, anthers, and pollen fertility.
[0051] Example 4 OsLTPL28 CRISPR knockout vector construction and mutant creation Design Targets OsLTPL28The coding region sgRNA (SEQ ID NO.5: TGCGGGCCGTTCCTGCTGGGCGG) was homologously recombinated with the linear VK005 vector to obtain VK005-LTPL28. This vector was transformed into Agrobacterium tumefaciens EHA105 and used to infect mature embryo callus of Wuyunjing 7 rice. After resistance selection and sequencing, two homozygous frameshift mutants with single-base T insertion and single-base G insertion were obtained. Both mutations induced premature termination of protein translation, reproducing the complete male sterility phenotype of rice. Homozygous knockout plants exhibited anther and pollen sterility phenotypes consistent with natural male sterility. ltpl28 The mutants are identical, see appendix. Figure 4 .
[0052] In summary, this invention achieves... 60 Co radiation-induced mutagenesis ltpl28 A stable male-sterile mutant was developed, and the functional gene LOC_Os03g59380 was located using map-based cloning. The natural mutant gene exhibits a G263A base substitution, resulting in a mutation of arginine to histidine at position 79, leading to protein loss and complete pollen abortion. This invention constructs the VK005-LTPL28 knockout vector and transforms it into wild rice, obtaining two types of frameshift homozygous plants that reproduce the sterility phenotype. Constructing a complementary vector containing the natural promoter and transforming the mutant completely restores normal anther and pollen development. Cytological observations show that gene loss causes premature vacuolation of the tapetum at S8 stage, persistent tapetum residue in later development, microspore collapse, lack of starch accumulation in pollen, and complete abortion. This invention provides the LTPL28 mutant protein, mutant gene, and a complete set of knockout and complementary vectors to obtain genetically stable monorecessive male-sterile rice, suitable for basic research on the mechanism of lipid transport in rice anthers and programmed tapetum death, as well as the creation of novel sterile germplasm.
[0053] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. Knockout OsLTPL28 The application of genes in the creation of completely male-sterile rice materials is characterized by, The OsLTPL28 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. A method for knocking out OsLTPL28 The sgRNA of a gene is characterized by, The OsLTPL28 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.
5.
3. A gene editing vector, characterized in that, The gene editing vector comprises the sgRNA as described in claim 2.
4. The gene editing vector according to claim 3, characterized in that, The backbone vector of the gene editing vector is the VK005 vector.
5. A recombinant bacterium, characterized in that, It includes the gene editing vector as described in claim 4.
6. The application of the sgRNA as described in claim 2, the gene editing vector as described in claim 3, or the recombinant bacteria as described in claim 5 in the creation of completely male-sterile rice materials.
7. A method for creating completely male-sterile rice materials, characterized in that, Includes the following steps: Using gene editing technology to knock out rice OsLTPL28 Genes, making the OsLTPL28 The gene function loss; OsLTPL28 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
8. The method according to claim 7, characterized in that, The gene editing technology is CRISPR / Cas9 technology; The knockout of rice OsLTPL28 The gene editing method is as follows: the sgRNA described in claim 2 is used to construct the gene editing vector described in claim 3 or the recombinant bacteria described in claim 5, which is then transformed into rice and screened. OsLTPL28 Homozygous mutant strains with gene knockout.
9. A genetic complementation vector, characterized in that, The genetic complementation vector contains OsLTPL28 Promoter fragments and complete coding sequences of genes; The promoter segment is OsLTPL28 The gene contains a 2.0 kb promoter fragment upstream; the backbone vector of the genetic complementation vector is the pCAMBIA1300 vector.
10. The application of the genetic complementation vector as described in claim 9 in restoring male fertility in rice.