CN118956894A

CN118956894A - A rice fertility regulating gene OsRAD9 and its application

Info

Publication number: CN118956894A
Application number: CN202411173697.2A
Authority: CN
Inventors: 于恒秀; 冯为轩; 程祝宽; 张超; 刘康伟; 王孝峰; 李颖颖
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2024-08-26
Filing date: 2024-08-26
Publication date: 2024-11-15

Abstract

The present invention relates to a rice fertility regulating gene OsRAD9 in the field of rice molecular genetic breeding. The gene nucleotide sequence is shown in SEQ ID No.1, and the amino acid sequence of the protein encoded by the rice fertility regulating gene OsRAD9 is shown in SEQ ID No.2. The present invention clones the OsRAD9 gene from a rice sterile mutant by Mutmap+ technology, and verifies the function of the gene by using CRISPR/Cas9 gene editing technology. After the OsRAD9 mutation, chromosome bridges and fragments appear in the meiosis process, resulting in plant sterility. The above-mentioned OsRAD9 gene of the present invention can be applied to rice fertility regulation, and has important application value in the field of rice genetic breeding.

Description

Rice fertility regulation gene OsRAD9 and application thereof

Technical Field

The invention relates to the fields of plant genetic engineering and rice molecular genetic breeding, in particular to a rice fertility regulating gene OsRAD9 and application thereof.

Background

Meiosis is an important process in reproductive development in rice. During meiosis, the sex parent cell homologous chromosome and sister chromosome are separated in sequence, ultimately resulting in halving of the number of chromosomes in the daughter cell. By fertilization, male and female gametes combine to form a zygote, and the chromosome number is restored, thereby maintaining the stability of the chromosome number between species generations. Meanwhile, random combination among non-homologous chromosomes in the meiosis prophase I and crossover among non-sister chromatids of the homologous chromosomes lead to recombination of genetic materials, generate new variation, enrich the diversity of the genetic materials and promote the evolution of organisms. During meiosis, homologous recombination begins with the formation of a Double-Strand Break (DSB) in DNA, with repair of the DSB completing homologous recombination.

RAD9 exists conservatively in different organisms, can form a cyclic 9-1-1 complex with HUS1 and RAD1, regulates the cell cycle and participates in the DNA damage repair process. In rice, the mutation of OsHUS and OsRAD1 genes can lead to sterile phenotype, and the function of OsRAD9 in rice fertility regulation is not yet reported. The research on the function of the OsRAD9 gene is helpful for deep understanding of the rice fertility regulation mechanism, and provides a new way for rice fertility regulation and molecular breeding.

Disclosure of Invention

The invention aims to provide a rice fertility regulating gene OsRAD9 and application thereof, which are applied to rice fertility regulation and screening and breeding of high-quality rice.

The invention firstly provides a rice fertility regulating gene OsRAD9, which is characterized in that the nucleotide sequence of the gene is shown as SEQ ID No. 1.

Further, the rice fertility regulating gene OsRAD9 is characterized in that the amino acid sequence of the encoding protein of the rice fertility regulating gene OsRAD9 is shown as SEQ ID No. 2.

Still further, it also includes substitution, insertion or deletion of one or more nucleotides in the nucleotide sequence shown as SEQ ID No. 1.

Still further, sequences resulting from substitution, insertion or deletion of one or more amino acids in the amino acid sequences shown are also included.

The invention also provides application of the gene OsRAD9 in regulation and control of rice fertility.

Furthermore, the application of the invention is to reduce or destroy the activity of the protein encoded by OsRAD9 by means of genetic engineering, so that the rice meiosis chromosome is abnormal to generate an sterile phenotype.

Compared with the prior art, the invention has the beneficial effects that:

The invention discloses a rice fertility regulating gene OsRAD9 and biological functions thereof for the first time. The rice sterile mutant Osrad related by the invention is generated by EMS mutagenesis of japonica rice variety Japanese sunny. Osrad 9A shows abnormal adhesion of non-homologous chromosomes during the final and metaphase I of meiosis, and chromosome bridges and fragments are produced during the late I phase. Targeting LOC_Os03g22450 (OsRAD 9) by MutMap + technology is a candidate gene. Knocking out the wild-type OsRAD9 gene using CRISPR/Cas9 gene editing techniques, resulting in a chromosomal abnormal phenotype similar to Osrad. Osrad9 the pair1 double mutant showed a similar phenotype to pair1, indicating that OsRAD9 is involved in the repair process of rice meiosis DSB. Immunostaining experiments using OsRAD9 antibodies showed that OsRAD9 localizes to the chromosome of meiosis I. The functional analysis of OsRAD9 is helpful for understanding the fertility regulation mechanism of rice, and provides a new way for fertility regulation and molecular breeding of rice.

Drawings

FIG. 1A is a plant phenotype comparison of wild type and Osrad9 mutants; FIG. 1B is a schematic diagram of a method for screening mutation sites using Mutmap +; wherein grey bands represent homologous chromosomes, red and yellow tags represent wild type alleles and mutated alleles; FIG. 1C is a screening process for mutation sites, wherein numbers indicate the number of SNPs/indels after each filtration, and screening parameters are indicated above the arrows; panels D-G are anther iodination of wild type and Osrad mutant.

FIG. 2 shows chromosome morphology at various stages of wild-type meiosis; a is even line period; b is a thick line period; c is the final phase; d is a middle stage I; e is the later stage I; f is the end stage I; g is a middle stage II; h is the later stage II; i is tetrad.

FIG. 3 is a diagram showing chromosome morphology at various stages of meiosis of Osrad mutants; a is even line period; b is a thick line period; c is the final phase; d is a middle stage I; e is the later stage I; f is the end stage I; g is a middle stage II; h is tetrad.

FIG. 4A shows CRISPR/Cas9 gene knockout target sequence and OsRAD9 gene knockout to obtain two mutation types Osrad-1 and Osrad-2; osrad 9A is inserted into a second exon of the OsRAD9 gene, and Osrad A9-2 is inserted into a base T at the second exon of the OsRAD9 gene; panels B-E are the chromosomal behavior of Osrad-1 knockout mutants during meiosis: b is middle stage I; c is the later stage I; d is the end stage I; e is tetrad.

FIG. 5 is a chromosomal behavior of the pair1 Osrad double mutant during meiosis; a is even line period; b is a rough line period; c is the final phase; d is the later stage I.

FIG. 6 shows the location of OsRAD9 immunostaining in wild type pollen mother cells (Pollen mother cell, PMC); red indicates OsRAD9 signal, green indicates OsREC signal, and blue indicates DAPI signal.

Detailed Description

The invention is further described below by means of specific examples. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified.

Example 1: osrad9 phenotypic identification of 9

In the Nipponbare EMS mutant population of the japonica rice variety, materials were found that could isolate sterile individuals. The mutant showed normal vegetative growth compared to the wild type, but the mutant appeared completely sterile (figure 1A). The high-throughput sequencing-based Mutmap + method is adopted to separate the sterile gene, the amino acid sequence of the code protein of the gene is shown as SEQ ID No. 1, and the amino acid sequence of the code protein of the gene is shown as SEQ ID No. 2. 24 candidate mutation sites were obtained by pool-resequencing and mutation screening rules (FIGS. 1B, 1C). Combining with the gene annotation information, the fourth exon of LOC_Os03g22450 gene (OsRAD 9) with candidate mutation site located on chromosome 3 12868159 bp is obtained. The SNP is a C to T substitution resulting in the substitution of threonine (Thr) to methionine (Met) at amino acid 216 encoded. After heading, we observed that pollen grains in the Osrad mutant shrank and could not be stained by pollen iodination, indicating that their pollen was inactive (FIGS. 1D-G).

Example 2: osrad9 mutant meiosis chromosome behavior abnormality

In this example, the chromosomal behavior of Wild Type (WT) and Osrad PMC at different stages of meiosis was observed by DAPI staining. In wild type, chromosomes begin to meet at even line phase. In the thick line period, the chromosome association is completed. In the final phase, 12 highly aggregated bivalent bodies can be clearly observed. In mid-term I, the bivalent bodies are arranged on the equatorial plate. At the later stage I, homologous chromosomes separate from each other, and at the end stage I a bipartite is formed. During the second meiosis, sister chromatids segregate, eventually forming tetrads (fig. 2).

In the Osrad9 mutant, there was no significant difference in chromosome morphology from the wild type prior to the pachytene period. However, during the final phase, chromosomes adhere to each other to form a multivalent body. In metaphase I, the chromosomes are arranged on the equatorial plate but still exhibit abnormal adhesion between the chromosomes. In the late stage I, significant chromosome bridges and fragments were observed when homologous chromosomes separated from each other and migrated to both poles. At end stage I, both chromosomes reach the cell's dipoles, but a large number of chromosome fragments remain near the equatorial plate. During the second meiosis, chromosomal bridges and fragments were also observed and eventually micronuclei were formed during the tetrad period (fig. 3). It is shown that OsRAD9 plays a role in the meiosis process of rice.

Example 3: knockout of the OsRAD9 Gene leads to abnormal meiotic chromosomal behavior

The present example further utilizes CRISPR/Cas9 technology to obtain single base inserted knockout mutants Osrad-1 and Osrad-2 at the second exon of the coding region of the OsRAD9 gene. During the knockout mutant meiosis, chromosomal adhesion, chromosomal bridging and fragmentation were observed as in the phenotype of mutant Osrad (fig. 4). These results indicate that mutation of OsRAD9 is responsible for the meiosis of the mutant and sterility of the plant.

Example 4: osRAD9 is involved in rice meiosis double-strand break repair process

In this example, to investigate the role of OsRAD9 in meiosis, pair1 Osrad9 double mutants were constructed. The double mutant in pair1 Osrad showed a phenotype similar to that of the single mutant in pair1, i.e., homologous chromosomes could not pair normally, 24 monovalent bodies were formed in the final phase and the chromosome I was unevenly transferred to the two poles in the later phase (FIG. 5). No abnormal chromosomal adhesions and fragments were found in the pair1 Osrad double mutant. This result suggests that OsRAD9 is involved in DSB repair processes, likely to play a role downstream of PAIR1 regulatory homology.

In this example, in order to clarify the localization pattern of OsRAD9 in rice meiosis, polyclonal antibodies (rabbit antibodies) to OsRAD9 were prepared using full-length proteins as antigens. The antibodies were used to immunolocalize OsRAD 9. No OsRAD9 antibody signal was detected in the wild-type fine line phase, a small amount of punctate signal was observed in the even line phase, and then the punctate signal gradually increased in the early-thick line phase, and reached the peak in the thick line phase. During this process, osRAD9 was always located on the chromosome (fig. 6). No OsRAD9 antibody signal was detected in Osrad mutant, both even and thick phase, indicating the specificity of the OsRAD9 antibody.

The above list is only a few specific embodiments of the present invention, but the present invention is not limited to the above-described embodiments. Modifications and improvements made by those skilled in the art from the present invention are intended to be included within the scope of protection.

Claims

1. A rice fertility regulating gene OsRAD9, characterized in that the gene nucleotide sequence is shown in SEQ ID No. 1.

2. The rice fertility regulating gene OsRAD9 according to claim 1, characterized in that the amino acid sequence of the protein encoded by the rice fertility regulating gene OsRAD9 is shown in SEQ ID No. 2.

3. The rice fertility regulating gene OsRAD9 according to claim 1, characterized in that it further comprises replacing, inserting or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID No. 1.

4. The rice fertility regulating gene OsRAD9 according to claim 2, characterized in that it also includes a sequence generated by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown.

5. Use of the gene OsRAD9 according to claim 1 or 2 in regulating rice fertility.

6. The use according to claim 5, characterized in that the activity of the protein encoded by OsRAD9 is reduced or destroyed by interference or knockout through genetic engineering means, so that the rice meiotic chromosomes behave abnormally and produce a sterile phenotype.