Gene al5kht and uses thereof

Abstract

This invention discloses genes AL5 KHT Its applications relate to the field of plant genetic engineering technology, and the genes... AL5 KHT The encoded amino acid sequence is shown in SEQ ID NO. 6. This invention provides a near-isogenic rice line NIL-AL5. KHT This technology, along with its applications, solves the technical problem of the lack of publicly available genes regulating rice anther length and pollen quantity in existing technologies. This near-isogenic line exhibits a large anther phenotype, which can be used to breed large-anther paternal restorer lines or for hybrid seed production. The near-isogenic line NIL-AL5... KHT Corresponding wild-type rice AL5 HZ Genes and their proteins have the ability to negatively regulate anther epidermal cell length, anther length, and pollen viability, and their application in rice variety improvement is of great significance.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically to genes. AL5 KHT And its applications. Background Technology

[0002] The selection of restorer lines in hybrid seed production technology is crucial for creating strong hybrid combinations in hybrid breeding, and the cultivation cycle of restorer lines is relatively short. Therefore, the cultivation of restorer lines has always been one of the main contents of hybrid seed production research. For example, the breeding of the restorer line Fuhui 7076 is to improve the resistance of hybrid rice to rice blast. The restorer line Fuhui 7076 was obtained by improving the strong restorer line Hang 2. The hybrid combination of restorer line Fuhui 7076 increased the yield by 3.24% compared with the control variety, significantly increasing the resistance to rice blast (Chen Liping, Xie Hongguang, et al. (2020) Breeding and application of long-grain, disease-resistant hybrid rice restorer line Fuhui 7076. Fujian Journal of Agricultural Sciences 35(6): 576-581.). Therefore, we can improve restorer lines to increase seed production.

[0003] Rice is a self-pollinating crop with a naturally low cross-pollination seed set rate. Only by increasing the cross-pollination seed set rate can seed production yield be increased. The cross-pollination seed set rate can be divided into saturated seed set rate and unsaturated seed set rate. The saturated seed set rate depends on the cross-pollination ability of the female sterile line, while the level of the unsaturated seed set rate is mainly determined by the amount of pollen of the male restorer line. The unsaturated seed set rate is generally low in seed production. Under the condition that the flowering periods of the male and female parents coincide, the greater the amount of pollen of the male parent, the higher the cross-pollination seed set rate of the female parent, and the higher the seed production yield. The amount of pollen of the male parent and the seed set rate of the female parent are highly significantly positively correlated (r = 0.7932**) (Wang Xiaomin, Li Bo, et al. (2015). Factors affecting the cross-pollination seed set rate of the female parent in hybrid rice seed production. Crop Research, 29(3), 4.). Meanwhile, studies have shown a highly significant positive correlation between pollen production and anther length (r = 0.765**), with pollen production increasing by about 6% for every 0.1 mm increase in anther length (Huang Lühe, Mo Zhijun, et al. (2002). Study on pollen production of rice restorer lines. Seeds, 000(006), 78-79.). However, no genes regulating anther length and pollen production in rice have yet been cloned or studied.

[0004] Therefore, identifying genes that regulate anther length and pollen quantity, accelerating the creation of male restorer lines, and increasing the yield of hybrid seed production are technical problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0005] This invention provides genes AL5 KHT And its applications.

[0006] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0007] Gene AL5 KHT The gene AL5 KHT The encoded amino acid sequence is shown in SEQ ID NO.6.

[0008] As a preferred technical solution, the gene AL5 KHT The nucleotide sequence is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0009] Another object of the present invention is to provide protein AL5. KHT The protein AL5 KHT The amino acid sequence is shown in SEQ ID NO. 6.

[0010] Another object of the present invention is to provide a near-isogenic line NIL-AL5 KHT The application is for breeding large anther male parent restorer lines or hybrid seed production; The near-isogenic line NIL-AL5 KHT of AL5 KHT The amino acid sequence encoded by the gene is shown in SEQ ID NO.6.

[0011] As a preferred technical solution, when the application is to cultivate a large anther male restorer line, the method is to... AL5 KHT The gene was derived from the near-isogenic line NIL-AL5 KHT By introducing the paternal restorer line with small anthers but excellent phenotypic traits in other traits, a paternal restorer line with large anthers can be obtained. When the application is hybrid seed production, the method is to... AL5 KHT The gene was derived from the near-isogenic line NIL-AL5 KHT Large anther paternal restorer lines were obtained by introducing them into existing paternal restorer lines with small anthers but excellent phenotypic traits. These large anther paternal restorer lines were then crossed with maternal sterile lines to obtain hybrid seeds.

[0012] Another object of the present invention is to provide genes. AL5 HZ or protein AL5 HZ The application is one of the following: A. Negatively regulates the length of anther epidermal cells; B. Negative regulation of anther length; C. Negative regulation of pollen viability; D. Variety improvement; The gene AL5 HZ The encoded amino acid sequence is shown in SEQ ID NO.3, and the protein AL5 is described. HZ The amino acid sequence is shown in SEQ ID NO.3.

[0013] Another object of the present invention is to provide a reduction AL5 HZ Application of biomaterials for gene expression levels, wherein the biomaterial is any one of the following: a. capable of enabling AL5 HZ Gene repression expression cassette; b. A recombinant vector containing the expression cassette described in a; c. Recombinant microorganisms containing the expression cassette described in a or the recombinant vector described in b; The application is one of the following: A. Negatively regulates the length of anther epidermal cells; B. Negative regulation of anther length; C. Negative regulation of pollen viability; D. Variety improvement; The gene AL5 HZ The encoded amino acid sequence is shown in SEQ ID NO.3.

[0014] As a preferred technical solution, the gene AL5 HZ The nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a near-isogenic rice line NIL-AL5 KHT And its applications: This near-isogenic line possesses a large anther phenotype, which can be used to breed large-anther male restorer lines or for hybrid seed production. This near-isogenic line, NIL-AL5... KHT Corresponding wild-type rice AL5 HZ Genes and their proteins have the ability to negatively regulate anther epidermal cell length, anther length, and pollen viability, and their application in rice variety improvement is of great significance. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 for AL5 Gene localization. Where A is a histogram of anther length in a population; B is... AL5 A detailed schematic diagram of the located chromosomes.

[0018] Figure 2 This study investigated the anther phenotype and important agronomic traits of near-isogenic lines. AD (from left to right) represent the near-isogenic line NIL-AL5. HZ With NIL-AL5 KHT Phenotypic characteristics after heading, scale bar = 10 cm (A); Comparison of anther lengths before pollen shedding, scale bar = 2 mm (B); NIL-AL5 HZ Observation of I2-KI staining of mature pollen grains, scale bar = 0.1 mm (C); NIL-AL5 KHT I2-KI staining observation of mature pollen grains, scale bar = 0.1 mm (D). EF represents NIL-AL5. HZ With NIL-AL5 KHT Pollen germination in vitro, scale bar = 20 μm. GH represents NIL-AL5. HZ With NIL-AL5 KHT Statistical analysis of anther length and pollen count.

[0019] Figure 3 This is a cytological phenotypic examination of anthers in near-isogenic lines. AC (from left to right) represents NIL-AL5. HZ With NIL-AL5 KHT Comparative scanning electron microscopy analysis of mature anthers (AB), scale bar = 20 μm; statistical analysis of cell length of anther epidermal cells (C). DE represents NIL-AL5. HZ (Left) with NIL-AL5 KHT (Right) Transverse semi-thin section of pollen grains during the vacuole stage, observing cell wall thickness, scale bar = 20 μm (E: epidermis; En: endodermis; T: tapetum; Msp: microspore). FG represent NIL-AL5. HZ (Left) with NIL-AL5 KHT(Right) FDA fluorescent staining of mature pollen grains, scale bar = 0.1 mm. H represents the statistical analysis of relative fluorescence intensity of FDA fluorescent staining. Each data point was repeated >3 times, using Student's t-test. P<0.05; Significant difference analysis was performed with P<0.01.

[0020] Figure 4 for AL5 Genetic verification. Where A is... AL5 Gene structure diagram and mutation site—a single base variation at the signal peptide, where A is replaced by G, results in AL5. HZ Threonine becomes AL5 KHT Alanine. B is a near-isogenic line complementation verification, KHT type AL5 KHT Imported into Huazhan. C represents the anther length statistics of complementary positive plants. D represents the Nipponbare gene. AL5 Knockout and overexpression validation. E represents the statistical analysis of anther length in knockout and overexpression cases. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] Example 1: NIL-AL5, a near-isogenic rice line with large anthers kHT Construction and QTL positioning The male parent (male parent *Huazhan*) and the female parent (Kaluheenati (KHT)) F2 generation were backcrossed with the female parent *Huazhan* for four consecutive generations to obtain the BC4F2 population. QTL mapping (primer sequences SEQ ID NO.7~SEQ ID NO.20) was performed on 384 plants from the BC4F2 population. Based on the QTL results, five BC4F2 plants with heterozygous intervals between CHR5-25 and CHR5-23 markers were selected for self-pollination to obtain the BC4F2 population. 2：3 Group. Using 5 BC4Fs 2：3The AL5 locus was finely mapped in the population (SEQ ID NO.21~SEQ ID NO.26), and the near-isogenic line NIL-AL5 with large anthers was obtained. KHT .

[0024] Statistical analysis of anther length in the BC4F2 population revealed that the anther length ranged from 2.0 mm to 2.8 mm, and overparental segregation occurred. Histogram results showed that the average anther length was 2.42 mm, with anther length biased towards the KHT parent. The values ​​exhibited a continuous distribution trend, with a coefficient of variation of only 0.02. The absolute values ​​of skewness and kurtosis were both less than 0.5, at 0.06 and -0.26 respectively. Therefore, the anther length distribution trend of the BC4F2 population basically conforms to a normal distribution, allowing for further QTL analysis and localization. (See attached image) Figure 1 (A). Using WinQTLCart 2.5, the additive effect and phenotypic contribution rate of anther length were calculated. Between the CHR5-23 and CHR5-25 markers, the LOD value was 42.05, with a contribution rate reaching 47.32%. Finally, through five BC4F... 2：3 The group and markers narrowed the interval of AL5 to between markers CHR5-21 and HK007 (see appendix). Figure 1 (B) Finally, select BC4F, which is homogeneous in the target interval. 2：3 Individual plants were backcrossed with *Haloxylon ammodendron* and then self-crossed to construct recombinant inbred lines to verify the QTL mapping results and obtain the near-isogenic line NIL-AL5. HZ With NIL-AL5 KHT (See appendix) Figure 2 In step A), the genes within the interval between the CHR5-21 and HK007 markers were analyzed, and sequencing analysis was performed to select the candidate gene AL5.

[0025] AL5 KHT A nonsynonymous mutation from A to G occurred at 55 bp of the CDS, resulting in AL5 KHT The polar amino acid threonine (Thr) at position 19 is mutated to the nonpolar amino acid alanine (Ala) (see appendix). Figure 4 (A). Further protein structure analysis revealed that the mutation site was located at AL5. KHT On the signal peptide.

[0026] AL5 HZ Full-length gene sequence: AAAGAACCCCCTCCCCAAATTGCTCCAAACGCCAACCTCCCCAGCCCCCCACCACCTCGGAATTCGGCGTGAATCCATCCCATGGCTCGCCTCGGCATCGGCGTGGCGGCGGCGGCTGCGGCGGTGGTGGCGGCCACGCTGGTGGTCTCCTGCCTGCGCGCGTCGCCTGCCGAGGCCTACAGCGGCGGCGGCCTCGGCTACAGCCAGCTGCTCACCACGGCCCACCTCGGCGCCATCTCCTCCTCGTCGTGCGGCGGCAGGCTGGGGCGGCAGTGCTCAGCGGCGGTGGGGGCCGACGGCGGGCTGCTCCGGCGGGCGCTGGCGGCGAGGAAGCCGACGAACCGGTACGTCAGCTACTCGGCGCTGGACGCGAACAAGGTGCCGTGCAACAAGCGAGGCCAGACCTACTACCAGAACTGCGCGTCGCAGCAGGCGGCGAATCCCTACCGCCGCGGCTGCTCCGCCATTACCCGCTGCTCCCGCAACATGAACTGACGATGATGATGATGGTGATCACATCATTGTCCATCCAAATTCCCCAATTCCCAATTCGTCCAATCCATTGATTCGGTCGGTGAATGCATGGATGGATCGATTATATATATATATATATATATATATGTGTGTGTGATGCATTATTATTATATATTGGCTAATTATTAATCGATTAGTTGTATTTATATGCATGCTGCTGATGATGATGATTATATATTTATATATATGTGATGTGATGATGTTCTTGGATCTCCTTTTTTTTTCTTTTTGTTCGATCAGTCGATGAGTTTCGCGGAAGGATCATATGTGTGTGTTGCTGGTGATCGTTTCATGTGTAATTATGTAGTTTAATTTAATCCCCTCAAATTTGTTACTCTCTTCTATCTATGCGAACATTGCTACGTACATATGTATATATATATGTTCTTTA, SEQ ID NO.1.

[0027] AL5 HZ CDS sequence of ATGGCTCGCCTCGGCATCGGCGTGGCGGCGGCGGCTGCGGCGGTGGTGGCGGCCACGCTGGTGGTCTCCTGCCTGCGCGCGTCGCCTGCCGAGGCCTACAGCGGCGGCGGCCTCGGCTACAGCCAGCTGCTCACCACGGCCCACCTCGGCGCCATCTCCTCCTCGTCGTGCGGCGGCAGGCTGGGGCGGCAGTGCTCAGCGGCGGTGGGGGCCGACGGCGGGCTGCTCCGGCGGGCGCTGGCGGCGAGGAAGCCGACGAACCGGTACGTCAGCTACTCGGCGCTGGACGCGAACAAGGTGCCGTGCAACAAGCGAGGCCAGACCTACTACCAGAACTGCGCGTCGCAGCAGGCGGCGAATCCCTACCGCCGCGGCTGCTCCGCCATTACCCGCTGCTCCCGCAACATGAACTGA, SEQ ID NO.2。

[0028] AL5 HZ Amino acid sequence of: MARLGIGVAAAAAAVVAATLVVSCLRASPAEAYSGGGLGYSQLLTTAHLGAISSSSCGGRLGRQCSAAVGADGGLLRRALAARKPTNRYVSYSALDANKVPCNKRGQTYYQNCASQQAANPYRRGCSAITRCSRNMN, SEQ ID NO.3。

[0029] AL5 KHT Full-length gene sequence of: AAAGAACCCCCTCTCCAAATTGCTCCAAACGCCAACCTCCCCAGCCCCCCACCACCTCGGAATTCGGCGTGAATCCATCGCATGGCTCGCCTTGGCATCGGCGTGGCGGCGGCGGCCGCGGCGGTGGTGGCGGCCGCGCTGGTGGTCTCCTGCCTGCGCGCGTCGCCTGCCGAGGCCTACAGCGGCGGCGGCCTCGGCTACAGCCAGCTGCTCACCACGGCCCACCTCGGCGCCATCTCCTCCTCGTCATGCGGCGGCAGGCTGGGGCGGCAGTGCTCGGCGGCGGTGGGGGCCGACGGCGGGCTGCTCCGGCGGGCGCTGGCAGCGAGGAAGCCGACGAACCGGTACGTTAGCTACTCGGCGCTGGACGCGAACAAGGTGCCGTGCAACAAGCGAGGCCAGACCTACTACCAGAACTGCGCATCGCAGCAGGCGGCGAATCCCTACCGCCGCGGCTGCTCCGCCATTACCCGCTGCTCCCGCAACATGAACTGACGATGATGATGATGGTGATCACATCATTGTCCATCCAAATTCCCCAATTCCCAATTCGTCCAATCCATTGATTCGGTCGGCGAATGCATGGATGGATCGATTATATATATATATGTGTGTGTGATGCATTATTATTATATATTGGCTAATTATTAATCGATTAGTTGTATTTATATGCATGCTGCTGCTGATGATGATTATATATTTATATATATGTGATGTGATGATGTTCTTGGATCTCCTTTTTTTTTCTTTTTGTTCGATCAGTCGATGAGTTTCGCGGAAGGATCATATGTGTGTGTTGCTGGTGATCGTTTCATGTGTAATTATGTAGTTTAATTTAATCCCCTCAAATTTGTTACTCTCTTCTATCTATGCGAACATTGCTACGTACATATGTATATATATATGTTCTTTA, SEQ ID NO.4。

[0030] AL5 KHT CDS sequence of ATGGCTCGCCTTGGCATCGGCGTGGCGGCGGCGGCCGCGGCGGTGGTGGCGGCCGCGCTGGTGGTCTCCTGCCTGCGCGCGTCGCCTGCCGAGGCCTACAGCGGCGGCGGCCTCGGCTACAGCCAGCTGCTCACCACGGCCCACCTCGGCGCCATCTCCTCCTCGTCATGCGGCGGCAGGCTGGGGCGGCAGTGCTCGGCGGCGGTGGGGGCCGACGGCGGGCTGCTCCGGCGGGCGCTGGCAGCGAGGAAGCCGACGAACCGGTACGTTAGCTACTCGGCGCTGGACGCGAACAAGGTGCCGTGCAACAAGCGAGGCCAGACCTACTACCAGAACTGCGCATCGCAGCAGGCGGCGAATCCCTACCGCCGCGGCTGCTCCGCCATTACCCGCTGCTCCCGCAACATGAACTGA, SEQ ID NO.5.

[0031] AL5 KHT The amino acid sequence of : MARLGIGVAAAAAAVVAAALVVSCLRASPAEAYSGGGLGYSQLLTTAHLGAISSSSCGGRLGRQCSAAVGADGGLLRRALAARKPTNRYVSYSALDANKVPCNKRGQTYYQNCASQQAANPYRRGCSAITRCSRNMN, SEQ ID NO.6.

[0032] CHR5-9F: 5' TGGTTTTGCTTTGTTTGGTGA 3', SEQ ID NO.7; CHR5-9R: 5' TACACCGTAATCACTGAATGCTAAT 3', SEQ ID NO.8; CHR5-13F: 5' AGGAAAATATGCTGACATGGATCTC 3', SEQ ID NO.9; CHR5-13R: 5' GCAGTGGGGTTTATCTCTGACAA 3', SEQ ID NO.10; CHR5-22F: 5' GAAAATATGTCTTCATTGGCCTTT 3', SEQ ID NO.11; CHR5-22R:5' GCACTTGCACATCTTCATCGA 3',SEQ ID NO.12; CHR5-23F:5' TGTGCGAATCTAATGTTGCTT 3',SEQ ID NO.13; CHR5-23R:5' AATCAGGGGCTACACCATTATA 3',SEQ ID NO.14; CHR5-25F:5' GAAAATGCCGCCGAGAGA 3',SEQ ID NO.15; CHR5-25R:5' CCGCACAACTTCCCTAAACAC 3',SEQ ID NO.16; CHR5-27F:5' GTAAATCCACCCATGACCTCGTA 3',SEQ ID NO.17; CHR5-27R:5' GCCTTCAGAGTTTGGACGACAT 3',SEQ ID NO.18; CHR5-39F:5' TAGCAGTCGGTAATGGAAACAGCA 3',SEQ ID NO.19: CHR5-39R:5' TCCCTCTTTCACCTGTGTGTTCAGT 3',SEQ ID NO.20; CHR5-43F:5' ATGGTGATTCAGGAGTACGCAATA 3',SEQ ID NO.21; CHR5-43R:5' AGATGAAAAGACAAGAACAAGGGAC 3',SEQ ID NO.22; CHR5-21F:5' ACCATGACAAACAATAGATCAATCC 3',SEQ ID NO.23; CHR5-21R:5' ATGCCCATAAATTCATTGTGATT 3',SEQ ID NO.24; HK007F:5' GTTACAAAGGCTGGTGATGAAGTCC 3',SEQ ID NO.25; HK007R:5'TAGAATCAAAGCAGAACACCAGAGG 3',SEQ ID NO.26。

[0033] Example 2: NIL-AL5, a near-isogenic rice line with large anthers KHT Phenotypic and physiological analysis 1. Pollen activity and viability detection To detect pollen viability and activity, samples were taken from the near-isogenic line NIL-AL5. KHT With NIL-AL5 HZ Mature anther samples were placed on a glass slide and immersed in a 1% (w / v) I2-KI or 0.1 mg / ml FDA solution. The anthers were crushed with pointed tweezers to stain the pollen, and then observed and photographed using a Leica DM2500 microscope or a fluorescence microscope.

[0034] 2. Pollen germination outside the body Mature anthers were placed on a glass slide, and pollen grains were released using tweezers. An appropriate amount of germination solution (1 mM CaCl2, 1 mM KCl, 0.8 mM MgSO4, 1.6 mM H3BO3, 30 μM CaSO4, 0.3% 2-(N-morpholino)ethanesulfonic acid, 10% sucrose, and 12.5% ​​PEG-6000) was added. The mixture was placed in an environment with humidity greater than 90% and temperature of 37℃ for 1 hour, and observed and photographed using a Leica DM2500 microscope.

[0035] 3. Scanning electron microscopy observation of anther epidermal cells After fixing and dehydrating the samples, they were treated with a 1:1 mixture of ethanol and isoamyl acetate for 30 mins, or with pure isoamyl acetate for 1-2 h. After drying to the critical value, a coating was applied, and the samples were observed and photographed using a Hitachi TM-1000 scanning electron microscope.

[0036] 4. Transverse semi-thin slices of anthers After fixing and dehydrating mature anthers, they were embedded using the Heraeus Kulzer Technovit 7100 resin kit. The embedded anthers were then cut into 3 μm thick slices using a Leica RM2265 microtome and placed on glass slides. 0.1% toluidine blue staining solution was added, and the slices were washed with distilled water before being observed and photographed under a Leica DM2500 microscope.

[0037] The results showed that NIL-AL5 HZ The grain length is 9.5 mm, and the anther length is 2.0 mm; NIL-AL5 KHT Particle length and NIL-AL5 HZ The anther length is still approximately 9.5 mm, but it reaches 2.7 mm, representing a very significant increase of about 25% (see appendix). Figure 2(B and G). Statistical analysis of pollen counts showed NIL-AL5. KHT The number of pollen also increased significantly, by about 15% (see appendix). Figure 2 (H).

[0038] Further analysis of NIL-AL5 KHT The changes in anther length and pollen, as shown by scanning electron microscopy and semi-thin sections, compared to NIL-AL5 HZ NIL-AL5 KHT The number of epidermal cells in the anthers did not change significantly, but the length of the epidermal cells increased dramatically (see Appendix). Figure 3 (AE), indicating NIL-AL5 KHT The anther elongation is due to the increase in cell length. FDA fluorescent staining and in vitro pollen germination show NIL-AL5. KHT The pollen can germinate normally in vitro (see Appendix). Figure 2 In the middle CF), pollen viability also increased significantly (see appendix). Figure 3 (FH).

[0039] Example 3 Rice AL5 Genetic analysis of genes 1. Carrier Construction Complementary material construction: AL5 KHT The complete genome sequence of the gene (SEQ ID NO.4) was ligated into the pCAMBIA1300 expression vector using a double-digestion and double-ligation method. First, the pCAMBIA1300 expression vector was linearized by digestion with KpnI and PstI. Then, rice KHT genomic DNA was amplified by PCR using primers 1300AL5-1F (ACGAATTCGAGCTCGGTACCTGGGGAAAGAGATGGGAGAAAGGAA, SEQ ID NO.27) and 1300AL5-1R (GCTTGCATGCCTGCAGGGATGATTGTACTTTGTCAAGACCC, SEQ ID NO.28). After electrophoresis detection and gel recovery, the fragments were digested with KpnI and PstI, and the PCR product was ligated into the pCAMBIA1300 complementary vector using the T4 ligase system. Sequencing confirmed no base mutations. The constructed vector was then chemically transformed into Agrobacterium EH105 strain for genetic transformation in the Huazhan variety.

[0040] Knockout material construction: Based on the principles of CRISPR / Cas9 gene editing, the online gene knockout target site and primer design website CRISPR-GE (http: / / skl.scau.edu.cn / home / ) was used. AL5 A highly specific target site with a GC content of approximately 65% ​​(CAGGCAGGAGACCACCAGCG, SEQ ID NO.29) was selected genetically, and primers were designed: AL5-U3F (GGCACAGGCAGGAGACCACCAGCG, SEQ ID NO.30) and AL5-U3R (AAACCGCTGGTGGTCTCCTGCCTG, SEQ ID NO.31). Two rounds of nested PCR were used for specific amplification to construct an sgRNA expression cassette with an OsU3 promoter. Finally, using the BsaI enzyme, the cassette was ligated to the CRISPR / Cas9 binary vector pYLCRISPR / Cas9Pubi-HygB (Ma et al 2015) initiated by the maize ubiquitin promoter (UBI) via a cleavage-ligation method. The cassette was transformed into *E. coli*, and clones with correct ligation were identified and screened using colony PCR and sent to the company for sequencing. The sequencing primers were SP1 / SP2. After confirming correct sequencing results, positive strains were extracted for genetic transformation in a NIP variety.

[0041] Construction of overexpression material: Using wild-type Huazhan genomic cDNA (as shown in SEQ ID NO.2) as a template, homologous recombination primers were designed for amplification. AL5 The full-length CDS is 414 bp. The restriction sites are SmaⅠ and SalⅠ. Homologous recombination was used with the OneStep Cloning Kit to construct a pCAMBIA1300S binary complementary vector driven by a 35S promoter. The vector was transformed into E. coli. The correctly ligated clones were identified and screened by bacterial PCR and sent to the company for sequencing. After the sequencing results were confirmed to be correct, the plasmid of the positive strain was extracted and genetically transformed. The transformation background was NIP strain.

[0042] 2. Genetic transformation: (1) Selection of conversion receptors Wild-type Huazhan or NIP seeds were used to induce callus tissue. After growing in the induction medium for 2 weeks, the embryos were cut off and cultured for another week. Vigorous callus tissue was selected as the recipient for transformation.

[0043] (2) Genetic transformation Using Agrobacterium-mediated genetic transformation (Hiei et al. 1994), rice callus was infected with either pCAMBIA1300-AL5 or the pYLCRISPR / Cas9Pubi-HygB vector EHA105. After co-culturing in the dark at 25°C for 3 days, the callus was cultured on selection medium containing 120 mg / L hygromycin. Resistant callus was screened and cultured on pre-differentiation medium containing 120 mg / L for approximately 10 days. The pre-differentiated callus was then transferred to differentiation medium and cultured under light conditions. Hygromycin-resistant transgenic plants were obtained after approximately two months.

[0044] 3. Results To verify AL5 To determine if the gene was the target gene, we constructed a complementation vector for a backfilling experiment. Sequencing identified seven independently transformed positive plants, from which two were selected and named HCOM-1 and HCOM-2 for phenotypic evaluation. The results showed that the T2 generation stably inherited complementary transgenic plants HCOM-1 and HCOM-2 exhibited a highly significant increase in anther length, approximately 10%-15% longer than the control variety Huazhan (see Appendix). Figure 4 (B and C in the middle).

[0045] At the same time, in order to verify AL5 To verify the function of the overexpression, we conducted genetic validation and obtained a total of 23 independently transformed single plants. The two single plants with the highest expression levels were selected and named OE-AL5-7 and OE-AL5-8 for further phenotypic identification. Phenotypic examination of the T2 generation stable overexpression lines revealed that the anther length of OE-AL5-7 and OE-AL5-8 was significantly reduced, by approximately 11%-26% compared to the wild-type NIP (see Appendix). Figure 4 (D and E). We also knocked out NIP using the CRISPR-Cas9 editing system. AL5 Knockout lines were constructed, and two knockout lines with different mutation types were identified and named al5-cp1 and al5-cp2. Phenotypic examination of the T2 generation of stable knockout lines revealed that anther length was significantly increased in both al5-cp1 and al5-cp2, with an increase of approximately 7%-10% compared to the negative control NIP (see Appendix). Figure 4 (D and E in the middle), therefore AL5 It is a semi-dominant gene that negatively regulates anther length. Knocking out this gene is beneficial for creating rice with large anthers.

[0046] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0047] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Genes AL5 KHT Its characteristics are, The gene AL5 KHT The encoded amino acid sequence is shown as SEQ ID NO.

6.

2. The gene according to claim 1 AL5 KHT Its characteristics are, The gene AL5 KHT The nucleotide sequence of the gene is shown in SEQ ID NO. 4 or SEQ ID NO.

5.

3. Protein AL5 KHT characterized in that, The protein AL5 KHT The amino acid sequence of AL5 is shown as SEQ ID NO.

6.

4. Near-isogenic line NIL-AL5 KHT for use, characterized in that The application is for breeding large anther paternal restorer lines or hybrid seed production; The near-isogenic line NIL-AL5 KHT of AL5 KHT The amino acid sequence encoded by the gene is shown as SEQ ID NO.

6.

5. The application according to claim 4, characterized in that, When the application is a large anther male parent restorer line, the method is to AL5 KHT The gene is introduced into an existing male parent restorer line with small anthers but excellent phenotypes of other traits to obtain a large anther male parent restorer line. KHT The gene is introduced into an existing male parent restorer line with small anthers but excellent phenotypes of other traits to obtain a large anther male parent restorer line. When the application is hybrid seed production, the method is to... AL5 KHT The gene was derived from the near-isogenic line NIL-AL5 KHT Large anther paternal restorer lines were obtained by introducing them into existing paternal restorer lines with small anthers but excellent phenotypic traits. These large anther paternal restorer lines were then crossed with maternal sterile lines to obtain hybrid seeds.

6. Genes AL5 HZ or protein AL5 HZ The application is characterized by, The application is one of the following: A. Negatively regulates the length of anther epidermal cells; B. Negative regulation of anther length; C. Negative regulation of pollen viability; D. Variety improvement; The gene AL5 HZ The encoded amino acid sequence is shown in SEQ ID NO.3, and the protein AL5 is described. HZ The amino acid sequence is shown in SEQ ID NO.

3.

7. Reduce AL5 HZ The application of biomaterials for gene expression level is characterized by, The biomaterial is any one of the following: a. capable of enabling AL5 HZ Gene repression expression cassette; b. A recombinant vector containing the expression cassette described in a; c. Recombinant microorganisms containing the expression cassette described in a or the recombinant vector described in b; The application is one of the following: A. Negatively regulates the length of anther epidermal cells; B. Negative regulation of anther length; C. Negative regulation of pollen viability; D. Variety improvement; The gene AL5 HZ The encoded amino acid sequence is shown in SEQ ID NO.

3.

8. The application according to claim 7, characterized in that, The gene AL5 HZ The nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO.2.

Images