Rice hybrid male sterility genes sc-p1 and sc-p3, the encoded proteins and applications thereof

Abstract

This invention discloses two rice hybrid sterility-related genes, Sc-P1 and Sc-P3, their encoded proteins, and their applications. This invention utilizes genetic analysis methods to identify the hybrid sterility locus Sc. i The constituent genes Sc-P1 and Sc-P3 were identified, and suitable gene editing sites were selected for Sc-P1 and Sc-P3. Spectral knockout of Sc-P1 and Sc-P3 was then performed using genetic engineering techniques. Based on the demonstration that knockout of Sc-P1 or Sc-P3 does not affect pollen fertility, the pollen fertility of F1 hybrids obtained by crossing sc-p1 and sc-p3 mutant lines with japonica rice was normal, indicating that the knockout of Sc-P1 or Sc-P3 had been eliminated. i The genetic effects of hybrid sterility in rice. The rice hybrid sterility-related genes Sc-P1 and Sc-P3 provided in this invention can effectively eliminate Sc... i Seat-mediated distant hybrid reproductive barriers have significant application value for overcoming the bottleneck in utilizing the heterosis of distant hybrids in rice.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and crop genetics and breeding, specifically to two genes controlling hybrid sterility in rice, Sc-P1 and Sc-P3, their encoded proteins, and their applications. Background Technology

[0002] Rice is one of my country's main staple crops. Utilizing heterosis in hybrid rice is an important means to increase rice yield. Currently, hybrid rice mainly utilizes heterosis from inter-subspecies hybridization (between indica and japonica varieties), but its yield-increasing potential is approaching saturation. Therefore, utilizing distant heterosis between indica and japonica subspecies is an essential path for the development of hybrid rice. However, distant hybrids suffer from severe reproductive isolation, primarily hybrid sterility, leading to infertility, which limits the utilization of distant heterosis. Therefore, overcoming distant hybrid sterility in rice is of great significance for the utilization of distant heterosis.

[0003] At least 50 hybrid sterility loci in the genus *Osmanthus* have been identified in different hybridization combinations, but only 12 genes associated with these loci have been cloned, of which only 4 control indica-japonica hybrids (Hou et al. 2019; Koide et al. 2018; Kubo et al. 2016; Long et al. 2008; Mizuta et al. 2010; Nguyen et al. 2017; Shen et al. 2017; Xie et al. 2017a; Xie et al. 2019b; Yamagata et al. 2010; Yang et al. 2012; Yu et al. 2018; Yu et al. 2016). The problem of hybrid sterility cannot be completely overcome at present. Therefore, it is necessary to clone more hybrid sterility genes and combine genetic engineering techniques and molecular design breeding to create and screen broadly compatible materials, thereby enabling the application of distant hybridization in superior rice varieties. Summary of the Invention

[0004] The purpose of this invention is to overcome the serious hybrid sterility problem and lack of hybrid sterility genes in the existing technology of rice distant hybridization, and to provide a key rice hybrid sterility related gene Sc-P1 and Sc-P3 and its application in rice distant hybrid vigor.

[0005] The first objective of this invention is to provide rice hybrid sterility-related genes Sc-P1 and Sc-P3.

[0006] A second objective of this invention is to provide rice hybrid sterility-related proteins Sc-P1 and Sc-P3.

[0007] A third objective of this invention is to provide the rice hybrid sterility-related genes Sc-P1 and Sc-P3 in the breeding of distantly hybridized indica rice lines and their application in distant hybridization breeding.

[0008] The objective of this invention is achieved through the following technical solutions:

[0009] A rice hybrid sterility-related gene, comprising Sc-P1 and / or Sc-P3, wherein the nucleotide sequence of Sc-P1 is as shown in SEQ ID NO. As shown in NO:1, the promoter region (1-2034): 2034bp, exon 1 region (2035-2388): 354bp, exon 2 region (4204-4265): 62bp, exon 3 region (5177-5247): 71bp, exon 4 region (5360-5448): 89bp, exon 5 region (5507-5764): 258bp, exon 6 region (5837-6598): 762bp, exon 7 region (6689-6823): 135bp, exon 8 region (6900-6965): 66bp, and terminator region (6966-8000): 1035bp; the nucleotide sequence of Sc-P3 is as shown in SEQ ID. As shown in NO:2, the promoter region (1-2345): 2090bp, exon 1 region (2346-2709): 364bp, exon 2 region (5593-5653): 61bp, exon 3 region (7375-7445): 71bp, exon 4 region (7591-7673): 83bp, exon 5 region (2755-2985): 231bp, exon 6 region (8058-8816): 759bp, exon 7 region (8906-9042): 137bp, exon 8 region (9126-9192): 67bp, and the terminator region (9193-10336): 1144bp.

[0010] A rice hybrid sterility-related protein, comprising Sc-P1 and / or Sc-P3, wherein the amino acid sequence of Sc-P1 is shown in SEQ ID NO: 3 and the amino acid sequence of Sc-P3 is shown in SEQ ID NO: 4.

[0011] This invention utilizes genetic analysis to identify a locus Sc located on chromosome 3 of indica rice that controls male sterility in indica-japonica hybrids. i Regarding this Sc of indica rice i Genomic sequence analysis of the seats revealed that Sc i The locus consists of three homologous repeat segments (28.2kb or 28.4kb) arranged in the same direction, and each homologous repeat segment contains four genes: Sc-P1, Sc-P2, Sc-P3, and Sc-P4; while the typical japonica rice variety's Scj The locus does not contain this structural variation. This invention further uses site-directed gene editing technology to knock out the Sc-P1 or Sc-P3 gene (the nucleotide sequences of Sc-P1 and Sc-P3 are shown in SEQ ID No. 1-2, and the amino acid sequences of their encoded proteins Sc-P1 and Sc-P3 are shown in SEQ ID No. 3-4), demonstrating that the loss-of-function mutation of Sc-P1 or Sc-P3 does not affect the pollen fertility of the mutant line, but the pollen fertility of the hybrids produced by crossing this mutant line with japonica rice is normal. That is, this mutant line is the Sc gene created in this invention. i Seat affinity can be used to eliminate Sc i Seat-mediated reproductive isolation in distant hybrids of rice.

[0012] Therefore, this application also provides the following applications of Sc-P1 and Sc-P3 and their encoded proteins Sc-P1 and Sc-P3:

[0013] The application of the above-mentioned rice heterosis-related genes or rice heterosis-related proteins, or their knockout reagents or expression inhibitors, in overcoming inter-subspecies heterosis in indica and japonica rice (specifically, in eliminating Sc... i The application of seat-mediated hybrid sterility between indica and japonica rice subspecies involves knocking out or inhibiting the expression of Sc-P1 and / or Sc-P3 genes in rice, preventing them from producing functional Sc-P1 and / or Sc-P3 proteins, thus preventing the hybrid sterility effect and restoring hybrid fertility.

[0014] The aforementioned rice heterosis-related genes or proteins, their knockout reagents, or their expression inhibitors are used to create compatible rice lines that overcome heterosis between indica and japonica subspecies (i.e., heterosis-resistant SC). i The application of the Sc-P1 and / or Sc-P3 gene in rice (a compatible line) involves knocking out or inhibiting the expression of the gene, preventing the production of functional Sc-P1 and / or Sc-P3 proteins, thus preventing hybrid sterility and restoring hybrid fertility.

[0015] This invention does not limit the methods of functional knockout or expression inhibition of Sc-P1 and / or Sc-P3 genes. Any biotechnology that prevents Sc-P1 and / or Sc-P3 from producing functional Sc-P1 and / or Sc-P3 proteins can achieve the objectives of this invention. For example, antisense RNA technology or RNA interference technology can inhibit the expression of Sc-P1 or Sc-P3, allowing for the cultivation of Sc-P1 and / or Sc-P3 proteins. i Sequence affinity lines. Methods for knocking out Sc-P1 or Sc-P3 include, but are not limited to, CRISPR / Cas gene editing systems, single-base editing systems, or guided editing systems.

[0016] This invention also provides a rice hybrid compatibility line that overcomes inter-subspecies hybrid sterility in indica and japonica rice (i.e., hybrid sterility Sc). i The method for creating a hybrid sterility line (HS-P1) involves knocking out or suppressing the expression of the Sc-P3 gene, which is related to hybrid sterility, in an indica rice variety.

[0017] Furthermore, the techniques employed for functional knockout or expression inhibition include sequence-specific nuclease gene editing, antisense gene technology, or RNA interference technology.

[0018] Furthermore, this study aims to utilize CRISPR / Cas, single-base editing systems, or guided editing systems to functionally knock out the Sc-P1 and / or Sc-P3 genes in rice.

[0019] Furthermore, the nucleotide sequence of the CRISPR / Cas9 gene editing target of Sc-P1 is shown in SEQ ID NO: 5, and the nucleotide sequence of the CRISPR / Cas9 gene editing target of Sc-P3 is shown in SEQ ID NO: 6.

[0020] Furthermore, to ligate the adapter nucleotide sequences of the CRISPR / Cas9 gene editing target sites of Sc-P1 and / or Sc-P3 into the intermediate vector of the guide RNA expression cassette, and to amplify the sgRNA expression cassette, which is then ligated into a CRISPR / Cas9 binary vector, Agrobacterium-mediated transformation is performed on rice lines containing the Sc-P1 and / or Sc-P3 genes; plants with knockout of Sc-P1 and / or Sc-P3 function are then screened, and the Sc-P1 and / or Sc-P3 knockout lines are the rice hybrid compatible lines.

[0021] As a preferred embodiment, the present invention also provides a hybrid sterility Sc i Methods for preparing seat affinity systems:

[0022] S1. Targeting hybrid sterility Sc i The sequences of the locus-related genes Sc-P1 and Sc-P3 are used to select one or more target sites based on a gene editing technology, and to construct the expression vector of the gene editing system used or to synthesize the relevant gene editing components.

[0023] Preferably, the gene editing technology described in S1 is CRISPR / Cas9; on the other hand, one or more target sites at any position in the Sc-P1 or Sc-P3 gene can be used for functional knockout, so the selected target site sequence is, but is not limited to, the one shown in SEQ ID NO: 5-6.

[0024] S2. The gene editing vector or gene editing component is introduced into rice varieties containing Sc-P1 and / or Sc-P3 using a suitable method to obtain single-gene knockout plants of Sc-P1 or Sc-P3 respectively; the target fragment containing the target site is amplified and sequenced using a DNA sequencing method, such as PCR, to analyze the mutation status of Sc-P1 and Sc-P3, thereby obtaining the corresponding knockout mutants sc-p1 and sc-p3, and the pollen and female gamete fertility of the mutants are examined to obtain plants with normal pollen and female gamete fertility;

[0025] S3. Perform PCR-based transgene identification on the self-pollinated progeny of fertile plants containing the knockout mutants sc-p1 or sc-p3 obtained using the gene-editing expression vector. Obtain transgene-free segregating sc-p1 or sc-p3 mutants, i.e., obtain the Sc mutants. i The seat is compatible with indica rice.

[0026] Preferably, gene editing technology is used to knock out the Sc-P1 or Sc-P3 gene at specific sites.

[0027] More preferably, the gene editing technology includes, but is not limited to, CRISPR / Cas9, or CRISPR / Cpf1, or single-base gene editing, or guided editing system for targeted knockout of Sc-P1 or Sc-P3 genes.

[0028] More preferably, a CRISPR / Cas9 vector containing one or more specific targets of the Sc-P1 or Sc-P3 gene is constructed and introduced into the vector containing Sc-P1 or Sc-P3 gene. i In rice loci, the Sc-P1 or Sc-P3 gene is knocked out by expressing the Cas9 / sgRNA nuclease complex to target the cleavage site.

[0029] More preferably, the nucleotide sequence of the specific target is shown in SEQ ID NO: 5-6, and its complementary sequence is shown in SEQ ID NO: 7-8.

[0030] More preferably, the specific target is ligated into the sgRNA expression cassette intermediate vector via the target adapter sequence in Table 1, and the sgRNA expression cassette is amplified using the nucleotide sequence shown in SEQ ID NO: 9-10. This cassette is then ligated into a CRISPR / Cas9 vector, and the constructed CRISPR / Cas9 vector is transformed into Agrobacterium, infecting bacteria containing Sc... i Among near-isogenic rice lines at the locus.

[0031] More preferably, primers with nucleotide sequences as shown in SEQ ID NO: 11-12 are used to screen for site-specific knockout of Sc-P1 positive plants; primers with nucleotide sequences as shown in SEQ ID NO: 13-14 are used to screen for site-specific knockout of Sc-P3 positive plants.

[0032] Most preferably, the target adapter sequence is denatured using the method described in Table 1 and then ligated into the intermediate vector pLYsgRNA-OsU3 of the sgRNA expression cassette; the expression cassette is then amplified using primers with nucleotide sequences as shown in SEQ ID NO: 9-10 and ligated into a CRISPR / Cas9 vector; the constructed CRISPR / Cas9 vector is then transformed into Agrobacterium and infected with Sc... i Among near-isogenic rice lines at the locus.

[0033] This invention also provides the application of the above-mentioned method for creating hybrid compatible rice lines in overcoming hybrid sterility between indica and japonica subspecies. Specifically, the method involves hybridizing the rice hybrid compatible lines obtained by the above-mentioned method with japonica rice lines to produce fertile hybrids. That is, the above-mentioned sc-p1 or sc-p3 compatible lines are hybridized with japonica rice to obtain mutant hybrid F1 (mF1). The fertility of mF1 is restored to normal, and the segregation ratio of the genotype at this locus in its self-pollinated offspring (mF2) is restored to the Mendelian segregation ratio (1:2:1).

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] This invention identifies rice hybrid sterility Sc i The key genes Sc-P1 and Sc-P3 were knocked out to obtain sc-p1 and sc-p3 mutants. It was found that their fertility was normal. This was the first time that Sc-P1 and Sc-P3 were not essential genes for the normal development of male and female gametes and could be used as parents for hybridization breeding. (2) The hybrids obtained by crossing sc-p1 and sc-p3 mutants with japonica rice no longer produced male gamete abortion. This was the first time that Sc-P1 and Sc-P3 were the hybrid sterility Sc genes of rice. i (2) Essential key genes for the locus, knocking out the function of any one of them can produce hybrid compatibility; (3) This invention provides for the first time a method for rapidly creating hybrid compatible lines using gene-targeted editing technology, which can be used to overcome hybrid sterility in rice, and is beneficial for utilizing the heterosis of distant hybrids in rice and comprehensively utilizing the advantageous traits of both parents. This invention provides a technical method for efficiently creating indica rice lines with hybrid compatibility by knocking out Sc-P1 and Sc-P3 genes using genetic engineering, which provides potential application value for molecular breeding to utilize the heterosis of distant hybrids and improve crop yield. Attached Figure Description

[0036] Figure 1 For Sc i Genomic structure analysis of the loci and construction of site-directed knockout vectors; Figure 1 A represents Sc located on chromosome 3 of indica rice. i The genomic structure of the seat shows Sc i The locus consists of three homologous repeat segments (28.2kb or 28.4kb) arranged in the same direction, and each homologous repeat segment contains four genes. Figure 1 B represents the gene structure of Sc-P1 and Sc-P3. The small black box indicates the exon, * indicates the editing target of the CRISPR / Cas9 system, and the bases in the dashed box indicate the editing target sequence of Sc-P1 and Sc-P3.

[0037] Figure 2 To investigate near-isogenic lines (NIL-Sc) containing Sc-P1 and Sc-P3 i Gene knockout was performed, and mutants sc-p1 and sc-p3 were obtained through sequencing screening. PCR amplification products containing the target fragments sc-p1 and sc-p3 were directly sequenced, and mutants sc-p1 and sc-p3 with 4 bases missing in each of their three copies (i.e., frameshift and premature stop codon) were obtained. The reference was the wild-type reference target sequence.

[0038] Figure 3 The fertility phenotypes of sc-p1 and sc-p3 functional knockout mutants were observed. In the figure, FF (Full Fertility) indicates that both pollen and spikelet are fully fertile. The pollen scale bar is 100 μm; the spikelet scale bar is 5 cm.

[0039] Figure 4 The pollen fertility and allele segregation in self-crossed offspring of mutant hybrids (mF1) produced by crossing gene-edited mutants sc-p1 and sc-p3 with the japonica rice parent (T65). Figure 4 A and B represent T65 and its near-isogenic line (NIL-Sc), respectively. i The pollen fertility of hybrid F1 and sc-p1 and sc-p3 (containing wild-type Sc-P1 and Sc-P3) and hybrid mF1 of T65; SS (Semi Sterility) indicates semi-sterile (small arrow points to sterile pollen), and FF indicates fully fertile; Figure 4 C is the Sc of F2 and mF2 i Locus genotype segregation ratio; χ² 2 The (1:2:1) test indicates that F2 produces japonica rice-type Sc. j Selective male gamete abortion leads to severe skewed segregation, while mF2 does not produce japonica rice-type Sc. jSelective sterility of male and female gametes (without skewed segregation) indicates that Sc-P1 and Sc-P3 are essential genes for producing hybrid male sterility at this locus. Knocking out the function of either of these genes can create Sc-P3. i Seating hybrid affinity system. *** indicates a p-value less than 0.001, showing a highly significant difference; i / i, i / j, j / j represent Sc i / Sc i homozygous genotype, Sc i / Sc j Heterozygous genotype, and Sc j / Sc j Homozygous genotype. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, and conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. For example, one or more editing targets other than those described in this specification can be designed for the Sc-P1 and Sc-P3 genes (including their promoters). Effective functional knockout can also be achieved using different gene-specific editing systems (such as TALLEN, CRISPR / Cpf1 systems, single-base editing systems, or guided editing systems), including the deletion of the entire Sc-P1 or / and Sc-P3 gene (including its promoter) sequence, all of which can achieve the same purpose. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods and techniques well known to those skilled in the art, and the reagents or materials used are all commercially available.

[0041] Example 1: Construction of Sc-P1 and Sc-P3 functional knockout vectors

[0042] This invention utilizes genetic analysis to identify a locus Sc located on chromosome 3 of indica rice that controls male sterility in indica-japonica hybrids. i Regarding this Sc of indica rice i Genomic sequence analysis of the seats revealed that Sc i The locus consists of three homologous repeat segments (28.2kb or 28.4kb) arranged in the same direction, and each homologous repeat segment contains four genes: Sc-P1, Sc-P2, Sc-P3, and Sc-P4 (e.g., ...). Figure 1 A). Target sequences (SEQ ID NO: 5-6) were designed targeting the coding sequences of the rice hybrid sterility genes Sc-P1 and Sc-P3 (shown in SEQ ID NO: 1-2) (the amino acid sequences of their encoded proteins Sc-P1 and Sc-P3 are shown in SEQ ID No. 3-4). Figure 1B), whose complementary sequences are shown in SEQ ID NO: 7-8. Using the CRISPR / Cas9 gene editing vector system and construction method created in the inventors' laboratory (Ma et al. 2015, Molecular Plant, 8, 1274-1284), two pairs of complementary primers (U3-Sc-P1-F / Sc-P1-R and U3-Sc-P3-F / Sc-P3-R) were synthesized to form a target double linker (Table 1), which was ligated into the expression cassette of sgRNA driven by the rice OsU3 promoter. The PCR-amplified sgRNA expression cassette fragment (amplified using primers SEQ ID No. 9-10) was cloned into the gene editing binary vector pYLCRISPR / Cas9Pubi-H (containing the T-DNA region), generating KO-Sc-P1 and KO-Sc-P3, respectively.

[0043] Table 1 Primer sequences for Sc-P1 and Sc-P3 target linkers

[0044]

[0045] Example 2: Screening and identification of Sc-P1 and Sc-P3 gene knockout lines sc-p1 and sc-p3

[0046] The binary transformation vectors KO-Sc-P1 and KO-Sc-P3 from Example 1 were transformed with Agrobacterium-mediated transformation into cells containing Sc. i near-isogenic line NIL-Sc i Transformants were obtained. Target fragments of the Sc-P1 and Sc-P3 genes from multiple T1 transformants were amplified by PCR (using primers shown in SEQ ID NO: 11-12 to screen for site-specific knockout of Sc-P1 positive plants; using primers shown in SEQ ID NO: 13-14 to screen for site-specific knockout of Sc-P3 positive plants). The PCR products were directly sequenced, and mutants with only a single mutation peak (i.e., homozygous frameshift mutations with 4 bases deleted from all 3 copies, such as...) were obtained. Figure 2 As shown), they are named sc-p1 and sc-p3.

[0047] Example 3: Phenotypic observation of sc-p1 and sc-p3 mutants

[0048] The fertility of the sc-p1 and sc-p3 functional knockout mutants obtained in the examples was observed, and it was found that the pollen and spikelets of these mutants were fully fertile. Figure 3 This indicates that loss-of-function mutations in Sc-P1 or Sc-P3 do not affect pollen fertility in mutant lines.

[0049] Example 4: Fertility performance of hybrids of gene-edited sc-p1 and sc-p3 mutants with japonica rice

[0050] The sc-p1 and sc-p3 mutant lines were crossed with the japonica rice parent (T65) to obtain hybrid mF1. Observation of the fertility of mF1 revealed that its pollen fertility was normal. Figure 4 B), while the near-isogenic line (NIL-Sc) containing wild-type Sc-P1 and Sc-P3. i The pollen of the F1 hybrid of T65 is semi-sterile. Figure 4 A). Further testing was conducted on the mF2 lines produced by self-pollination of mF1. i and Sc j Segregation ratio analysis of genotype combinations showed that their segregation ratios recovered Mendelian segregation ratios. Figure 4 C). That is, the sc-p1 and sc-p3 mutants can be used as indica-japonica hybrid compatible lines for crossbreeding with japonica rice.

Claims

1. A method for creating hybrid compatible rice lines that overcome inter-subspecies hybrid sterility in indica and japonica rice, characterized in that, Genes related to hybrid sterility in indica rice varieties Sc - P1 Genes are functionally knocked out, thus obtaining the desired result; Sc - P1 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The creation method according to claim 1, characterized in that, Using CRISPR / Cas9 for indica rice Sc - P1 Genes are knocked out of their function.

3. The creation method according to claim 2, characterized in that, Sc - P1 The nucleotide sequence of the CRISPR / Cas9 gene editing target site is shown in SEQ ID NO:

5.

4. The creation method according to claim 3, characterized in that, Will Sc - P1 The adapter nucleotide sequence of the CRISPR / Cas9 gene editing target site was ligated into an intermediate vector containing a guide RNA expression cassette. The sgRNA expression cassette was then amplified and ligated into a CRISPR / Cas9 binary vector. This CRISPR / Cas9 binary vector was then transformed into Agrobacterium-mediated transformation containing… Sc - P1 Indica rice varieties with gene knockout; further selection and knockout Sc - P1 Functional indica rice, the knockout Sc - P1 Functional indica rice varieties are rice hybrid compatible lines.

5. The application of the creation method according to any one of claims 1 to 4 in overcoming hybrid sterility between indica and japonica subspecies, characterized in that, The rice hybrid compatible line obtained by any one of the creation methods described in claims 1 to 4 is crossed with a japonica rice line to produce a fertile hybrid.

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