Application of ZEP1 gene in creating rice recessive genic male sterility line

Abstract

The invention discloses application of a ZEP1 gene to creation of a rice recessive genic male sterility line, and belongs to the field of rice molecular breeding. A nucleotide sequence of the ZEP1 gene is shown as SEQ ID NO: 1, a nucleotide sequence of a coding region of the ZEP1 gene is shown as SEQ ID NO: 2, an amino acid sequence of a protein coded by the nucleotide sequence of the coding region of the ZEP1 gene is shown as SEQ ID NO: 3, and a rice recessive genic male sterile plant can be obtained by knocking out a basic group in the sequence of the coding region of the ZEP1 gene by utilizing a CRISPR / Cas9 gene editing technology; an ms4 sterile plant is obtained on the basis of Yuanjiang common wild rice YJWT and Yunjing No.1 YD1, further fine positioning is performed, the ZEP1 gene is screened out as the sterile gene, and it is verified that a rice recessive genic male sterile plant can be obtained by knocking out a base in a ZEP1 gene coding region through a CRISPR / Cas9 gene editing technology.

Description

Technical Field

[0001] The present invention belongs to the field of rice molecular breeding, and specifically relates to a ZEP1 Application of genes in creating recessive nuclear male sterile lines in rice. Background Art

[0002] Rice ( Oryza sativa L. ) is one of the important food crops in my country and the world. Rice hybrids are significantly superior to their parents in growth vitality, adverse and non-adverse stress, yield and quality, and are therefore widely used in rice production; wild rice is widely distributed in many regions of my country and has many excellent traits such as drought and flood resistance, disease and pest resistance, and stress resistance; in 2018, the team of Academician Wan Jianmin revealed the cause of hybrid sterility and found that the probability of wild rice containing sterility genes is much greater than that of cultivated rice. Therefore, hybrid rice obtained from wild rice based on intelligent sterile rice technology will have the excellent qualities of wild rice on the basis of possessing the excellent traits of cultivated rice parents.

[0003] The discovery of male sterile materials is a key link in the breeding of hybrid rice. The concept of one-line hybrid rice not only breaks away from the constraints of restoration and protection relationships of three-line hybrid rice, but also breaks away from the influence of the environment of two-line hybrid rice, and proposes the possibility of large-scale preparation of male nuclear sterile materials; therefore, the discovery of complete male nuclear sterile materials and the enrichment of sterile gene resources have important application prospects for hybrid rice breeding; for this reason, the creation of rice recessive nuclear male sterile lines is crucial for the breeding of hybrid rice. Summary of the Invention

[0004] The purpose of the present invention is to provide a ZEP1 The application of genes in creating recessive nuclear male sterile lines of rice, thereby obtaining recessive nuclear male sterile rice plants.

[0005] The purpose of the present invention is achieved in that ZEP1 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, ZEP1 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 2. ZEP1 The amino acid sequence of the protein encoded by the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 3, and the CRISPR / Cas9 gene editing technology was used to knock out ZEP1 The bases in the gene coding region sequence can produce recessive nuclear male sterile rice plants.

[0006] The present invention is based on Yuanjiang common wild rice ( YJCWR, Oryza rufipogon Griff. ) on the basis of hybridization and other means to obtain a small number of sterile plants, and further locate the factors that cause plant sterility. ZEP1 Gene, study finds, can be knocked out using CRISPR / Cas9 gene editing technology ZEP1Mutations in the gene coding region can produce recessive nuclear male sterile rice plants. ZEP1 The discovery and functional identification of genes provide new materials for exploring the molecular mechanism of recessive nuclear male sterility in rice and its breeding application, and also lay the foundation for gene function exploration and molecular mechanism research. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 WT and WT of fertile plants under normal field management in Experimental Examples 1 and 2 ms4 Phenotypic comparison of sterile plants, where a is the fertile plant WT and ms4 Plant morphology of sterile plants during reproductive growth period, scale bar is 6 cm; b, g, h are fertile plants WT and ms4 Spikelets of sterile plants at the heading stage, c and d are fertile plants WT and ms4 Anthers of sterile plants at heading stage. The scale bar in h is 3 cm, the scale bars in c and g are 1 cm, and the scale bars in b and d are 2 mm. The scale bars in e and f are WT and fertile plants stained with potassium iodide and aniline blue, respectively. ms4 sterile plants, scale bar is 100 μm; Figure 2 The fertile plants WT and ms4 Phenotype and fruiting rate of sterile plants, scale bar is 6 cm, red indicates fertile and blue indicates sterile; Figure 3 The fertile plants WT and ms4 Hybridization experiment diagram of sterile plants, where a and b are fertile plants WT and ms4 Anthers of sterile plants, scale bar is 2 mm; c, d are fertile plants WT and ms4 Pollen grains of sterile plants, scale bar is 100 μm; e, f, g are pollen grains of fertile plants WT and ms4 Spikelets of sterile plants and their hybrid progeny, scale bar is 3 cm; Figure 4 The fertile plants WT and ms4 Scanning electron micrographs of sterile plants, where a and f are fertile plants WT and ms4 Anthers of sterile plants, scale bar is 200 μm; b, g are anthers of fertile plants WT and ms4 The anther surface of the sterile plant, the arrow indicates the anther epidermal cells, the scale bar is 10 μm; c, h are the fertile plants WT and ms4 Pollen grains of sterile plants, scale bar is 30 μm; d, i are pollen grains of fertile plants WT and ms4 Pollen surface of sterile plants, GP represents germination pore, scale bar is 4 μm; e, j are fertile plants WT and ms4 Surface of pollen grains of sterile plants, scale bar is 2 μm; Figure 5 The fertile plants WT and ms4 Aniline blue staining of sterile plants, scale bar is 10 μm, where: a, c are fertile plants WT and ms4 Sterile plants in the dichotomy stage; b and f are fertile plants WT and ms4 Sterile plant tetrad stage; d, e are ms4 Abnormal tetrad period of sterile plants; Figure 6 The fertile plants WT and ms4 Paraffin sections of anthers of sterile plants, where a~e are paraffin sections of WT anthers of fertile plants at stages 6, 7, 8a, 8b, and 9; f~j are ms4 Paraffin sections of sterile plant anthers at stages 6, 7, 8a, 8b, and 9; k~o are paraffin sections of fertile plant WT anthers at stages 10, 11, 12, 13, and 14; p~t are ms4 Paraffin sections of anthers of sterile plants at stages 10, 11, 12, 13, and 14; E is epidermis, En is endodermis, ML is middle layer, T is tapetum, M is middle layer, T is tapetum, MMC is microspore mother cell, Tds is tetrad, Msp is microspore, BP is bicellular pollen, MP is mature pollen, scale bar is 10 μm; Figure 7 For Example 3 ms4 Fine-scale mapping of the sterility gene in sterile plants on rice chromosome 4, where a is the rough QTL location, the ordinate represents the threshold range, the abscissa represents the chromosome position, and the threshold standard (LOD value) = 3; b and c are the linkage map and fine-scale mapping of the candidate interval on chromosome 4, the abscissa represents the physical distance between different KASP markers and their adjacent markers, and the ordinate represents 33 groups of recombinant plants. For example, L1-2 indicates that two recombinant plants exhibited the first crossover phenotype. The green marker F (Fertile) indicates a fertile phenotype, the yellow marker S (Sertile) indicates a sterile phenotype, and the gray marker indicates a heterozygous genotype. Figure 8 This is a diagram showing the genotyping results of markers KASP9 and KASP10 in Example 3. Red and blue icons represent different homozygous genotypes, green icons represent heterozygous genotypes, and black icons represent DNA samples that have not undergone any amplification; Figure 9 For example 3 Chinese National Rice Database ZEP1 Relative expression level graph of genes; Figure 10 For Example 3 ZEP1 qPCR graphs of genes and related genes in different plants or tissues, where: a is ZEP1The relative expression levels of the gene in the roots, stems, leaves, panicles, and sheaths of sterile and fertile plants; b is ZEP1 The relative expression levels of genes in spikelets of sterile and fertile plants at different developmental stages. S6 to S14 represent the period from microspore mother cell generation to mature pollen grain release; c is PAIR2 The relative expression level of the gene in sterile and fertile plants; d is PAIR3 The relative expression level of the gene in sterile and fertile plants; e is CRC1 The relative expression level of the gene in sterile and fertile plants; f is ZIP4 The relative expression level of the gene in sterile and fertile plants; g is HOP2 The relative expression levels of genes in sterile and fertile plants; UBQ is the internal reference gene, green indicates fertile, yellow indicates sterile, ns indicates no significance, *** indicates extremely significant; Figure 11 For Example 4 ZEP1 Genes and zep1-1, zep1-2 The gene structure diagram of the mutant, where a is ZEP1 The structure of the gene; b is after CRISPR / Cas9 editing zep1-1, zep1-2 Comparison of the gene structure of the mutant and ZH11; Figure 12 ZH11 and zep1-1 、 zep1-2 Phenotypic diagram of mutants, where a, b, and c are ZH11, zep1-1 、 zep1-2 Photos of spikelets at the heading stage, scale bar is 2 mm; d, e, f are ZH11, zep1-1 、 zep1-2 Photos of anthers at the heading stage, scale bar is 200 μm; g, h, i are ZH11, zep1-1 、 zep1-2 The images were stained with 1% potassium iodide. The scale bar is 100 μm. DETAILED DESCRIPTION

[0008] The present invention is further described in detail below with reference to the accompanying drawings and test examples, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the scope of protection of the present invention. The reagents required for the present invention are conventional experimental reagents purchased from commercial channels. The experimental methods not mentioned are conventional experimental methods and will not be described in detail.

[0009] The present invention provides a ZEP1 Application of gene in creating recessive nuclear male sterile rice line, ZEP1The nucleotide sequence of the gene is shown in SEQ ID NO: 1, ZEP1 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 2. ZEP1 The amino acid sequence of the protein encoded by the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 3, and the CRISPR / Cas9 gene editing technology was used to knock out ZEP1 The bases in the gene coding region sequence can produce recessive nuclear male sterile rice plants.

[0010] Further, knockout ZEP1 6 or 10 bases in the gene coding region sequence.

[0011] The present invention is based on Yuanjiang common wild rice ( YJCWR, Oryza rufipogon Griff. ) was found based on ZEP1 genes, and identified through ZEP1 Loss of gene function can produce recessive nuclear male sterile rice plants, as follows: Experimental Example 1: Discovery of sterile rice plants and preliminary localization of the sterile phenotype gene 1. Experimental Materials Yuanjiang common wild rice ( YJCWR, Oryza rufipogon Griff. ) was collected in Yuanjiang County, Yunnan Province in June 2018 (collection number YJCWR-7). The japonica rice variety Yundao No. 1 (YD1) was bred by the Yunnan Academy of Agricultural Sciences and was obtained from the Yunnan Academy of Agricultural Sciences in this study.

[0012] 2. Test process Using Yuanjiang wild rice as the donor, a series of backcross introgression lines (ILs, BC4) were constructed in the genetic background of the japonica rice variety Yundao 1. Two introgression lines, U99 and J186 (BC4), were hybridized and self-pollinated to obtain fertile segregating populations. In the F2 generation population, one plant was observed to exhibit sterility. The gene controlling the sterility phenotype was initially located on chromosome 4 through positional cloning, and the sterile mutant was named ms4 Sterile plants, fertile plants are WT; compared with WT, ms4 The anthers of the sterile plants are shriveled and wrinkled, no pollen grains are formed, the outer wall of the pollen is tightly attached, there is irregular sporopollenin deposition, and irregular triplets are produced, affecting the development of microspores; since the sterile plants do not form pollen grains during the reproductive development process, seeds cannot be obtained by self-pollination. The fine-positioning population will continue to plant heterozygous fertile plants of the segregating population to obtain a single-plant genetic population until the fine positioning is completed; the introgression lines J186, U99, the recurrent parent Yundao No. 1 and the genetic segregating population are all planted in the Crop Protection and Breeding Base of the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. The experimental materials are planted twice a year and managed normally in the field.

[0013] Experimental Example 2: Mutant phenotypic identification and cytological observation Phenotypic identification and hybridization experiments were carried out at the heading stage of rice. ms4 The anthers and pollen of sterile plants were compared, and important agronomic traits such as plant height, panicle type, panicle length and fruit setting rate were investigated. ms4 The spikelets of sterile plants were stained with aniline blue and analyzed by scanning electron microscopy. The specific steps included FAA sample fixation, different alcohol gradient dehydration treatment (30%, 50%, 70%, 90%, 100%, 100%, 100%), and finally electron microscopy observation. After dehydration, the scanning electron microscope also needed to be dried and coated, and finally the magnification was adjusted to take pictures. Cytological observation was based on WT and ms4 Anthers from sterile plants at different developmental stages of spikelets were used as materials and preserved with FAA fixative. The fixed tissue material was dehydrated, transparentized, wax-impregnated, and embedded. The embedded wax blocks were sliced ​​and adhered to slides and dried. They were then stained and mounted with resin, allowed to air dry, and then observed and photographed under a microscope. The results are as follows: At the heading stage of rice, WT and ms4 There were no significant differences in plant type, ear shape, and ear length between sterile plants (see Figure 1 a, b, h), but ms4 The fruiting rate of sterile plants was significantly lower than that of fertile plants (see Figure 2 Compared with WT, ms4 The anthers of sterile plants are short and thin, with a light yellow color. The pollen grains of sterile plants are unevenly stained and have different shapes (see Figure 1 c, d, e, f, g); There were 1814 segregating populations in the BC5F3 population. The chi-square test showed that the total fecundity of the plants met the segregation ratio of 3:1, indicating that the sterile phenotype may be controlled by a pair of recessive single genes in the nucleus (see Table 1); The fertile plants in the F3 generation population were WT as the male parent and ms4 Sterile plants can bear fruit normally after hybridization (see Figure 3 (g) ms4 The pistil of the sterile plant develops normally, but the pollen grains of the stamens are abnormal, which causes its sterile phenotype. The sterile phenotype is controlled by a single recessive nuclear gene, and fertility is a dominant trait.

[0014] Table 1 Genetic analysis of BC5F3 Note: The segregation ratio of fertile and sterile plants in the BC5F3 generation was 3:1; *** indicates extremely significant (P < 0.001).

[0015] To determine ms4 The period of abnormal anther development in sterile plants ms4The spikelets of sterile and fertile WT plants were analyzed by electron microscopy, stained and sectioned. Electron microscopy showed that compared with the fertile WT plants, ms4 The anthers of sterile plants are wrinkled, the intercellular spaces between the anther epidermis are narrow, the pollen is underdeveloped and has different shapes, and the surface is covered with an irregular layer of pollen grains (see Figure 4 After aniline blue staining ms4 The sterile plants had abnormal meiosis, producing irregular triplets and affecting the development of microspores (see Figure 5); paraffin sections showed that at the S10 stage, ms4 The cytoplasm of the microspores of the sterile strains was wrinkled, vacuolation was not obvious, and as mitosis proceeded, ms4 The microspores of sterile plants have irregular morphology, no sickle-shaped structure, no obvious accumulation of nutrients such as starch, and are lightly stained (see Figure 6 q, r), so that in the subsequent development of pollen grains, the pollen grains shrink and cannot form mature pollen grains (see Figure 6 (in Chinese, s, t).

[0016] Test Example 3 ms4 Localization and functional analysis of sterility genes in sterile plants The method adopted was to use a large genetic mapping population obtained by hybridizing the introgression lines U99 and J186 as the research object, and to extract the genomes of the population plants and the parents using the CTAB method; by finding high-density SNP sites between the parents, a large number of KASP molecular markers were designed, and the sample genotypes were analyzed using the KASP high-throughput genotyping technology. Finally, 16 KASP markers were used to locate the gene causing sterility in the genome between the KASP9 and KASP10 markers (see Table 2), with a physical distance of 49.8 kb (see Figure 7 and Figure 8 ), thereby narrowing down the candidate intervals of the genome; combined with the functional annotation of the National Rice Database, sequencing analysis and qPCR experiments to confirm the target gene that causes sterility, the results are as follows: A total of 7 candidate genes were found by quantitative query and sequencing analysis of genes in the candidate interval of the genome, including STP17 and ZEP1 The gene underwent a single base change; however, the base change in STP17 did not result in an amino acid change, only ZEP1 The base changes in the promoter region of the gene resulted in amino acid changes, and its function was annotated as being involved in meiosis as a central component of the synaptonemal complex (see Table 3). The highest expression level was found in the anther of rice (see Figure 9 ), which is consistent with the results of qPCR experiments (see Figure 10 Therefore, the genomes of the population plants and their parents were preliminarily determined. ZEP1 Genes are the cause ms4 The target gene for sterility of sterile plants; after sequencing, ZEP1The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and further sequencing confirmed ZEP1 The nucleotide sequence of the gene coding region is shown in SEQ ID NO: 2, ZEP1 The amino acid sequence of the protein encoded by the nucleotide sequence of the gene coding region is shown in SEQ ID NO: 3.

[0017] Table 2 KASP molecular marker primer sequences used in the present invention Table 3 Functional annotation of candidate genes Test Example 4 ZEP1 Application and functional verification of genes The rice variety Zhonghua 11 (ZH11) was used as the rice transgenic material. Zhonghua 11 is a domestically cultivated variety. Zhonghua 11 was obtained and propagated by the Xishuangbanna Tropical Botanical Garden of the Chinese Academy of Sciences and is preserved in the germplasm resource bank of the Xishuangbanna Tropical Botanical Garden of the Chinese Academy of Sciences. The Zhonghua 11 used in this study was obtained from the germplasm resource bank. Although Experiment 3 found ZEP1 The base changes in the gene promoter region lead to amino acid changes, but in order to facilitate production and application, we try to ZEP1 Gene editing was performed on the gene coding region, that is, CRISPR / Cas9 gene editing technology was used to knock out the gene coding region in the context of Zhonghua 11. ZEP1 Two strains were constructed by 6 and 10 bases of the gene coding region. ZEP1 Mutants: zep1-1 and zep1-2 Mutants (see Figure 11 );Will zep1-1 and zep1-2 The mutant and Zhonghua 11 were planted together in a constant temperature rice culture room. When the rice was heading, the rice spikelets were phenotypically observed and stained with iodine solution. The results showed that the pollen grains of Zhonghua 11 were plump, evenly stained and spherical; unlike Zhonghua 11, zep1-1 and zep1-2 The pollen grains of the mutants are of different sizes and irregular shapes, and lack nutrients such as starch, resulting in uneven staining of the pollen grains (see Figure 12 ); Obviously, this experiment further proves ZEP1 The loss-of-function mutation caused by the knockout of some bases in the gene coding region will also affect the pollen fertility of the plant and lead to rice sterility; therefore, the CRISPR / Cas9 gene editing technology can be used to knock out ZEP1 The bases in the gene coding region can produce recessive nuclear male sterile rice plants, such as knocking out ZEP1 The gene coding region is 6 or 10 bases.

[0018] In summary, the present invention is ms4 The precise positioning and functional verification of the genes that cause sterility in sterile plants have confirmed ZEP1 The gene can be used as a target gene for creating recessive nuclear male sterile lines in rice; the discovery of this gene not only provides new mutation sites and germplasm resources for the creation of recessive nuclear male sterile lines, but also provides an effective functional marker for the breeding application of this site.

Claims

1. A ZEP1 The application of the gene in creating recessive nuclear male sterile rice lines is characterized by: described ZEP1 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, ZEP1 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO:

2. ZEP1 The amino acid sequence of the protein encoded by the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 3, and the CRISPR / Cas9 gene editing technology was used to knock out ZEP1 The bases in the gene coding region sequence can produce recessive nuclear male sterile rice plants.

2. The use according to claim 1, characterized in that Knockout ZEP1 6 or 10 bases in the gene coding region sequence.

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