Mutant gene and protein causing rice flag leaf to become smaller and application of mutant gene and protein

Abstract

The invention relates to the technical field of gene engineering, in particular to a mutant gene and protein for causing rice flag leaves to become smaller and application of the mutant gene and protein. According to the mutant gene capable of causing the rice flag leaf to become smaller, the nucleotide sequence of the mutant gene is a nucleotide sequence shown as SEQ ID NO.1 inserted into the 153rd site of the nucleotide sequence of a wild rice OsGATA15 gene from an initiation codon ATG. According to the invention, an indica type rice line 93-11 is subjected to < 60 > Co-gamma radiation treatment to obtain a new flag-leaf-reduced mutant ym66, and the ym66 mutant is hybridized with the indica type rice line YHSM to obtain an excellent flag-leaf-reduced restorer line NP27. In the seed production process, the seed production yield is not reduced under the condition that flag leaves of NP27 are not cut, and in addition, the F1 generation shows excellent characters in the aspects of plant type and thousand seed weight.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a mutant gene and protein causing rice flag leaves to become smaller, and applications thereof. Background Art

[0002] Rice is one of the world's major food crops. With a growing population and shrinking arable land, increasing rice yields is essential for ensuring food security. By effectively utilizing heterosis, hybrid rice has become an important way to increase yield per unit area.

[0003] The goal of hybrid rice is to leverage the genetic differences between different rice varieties and artificially control the hybridization process, allowing the hybrid offspring to combine the beneficial traits of both parents, thereby exhibiting hybrid vigor. This advantage is primarily manifested in greater growth potential, higher yields, and improved stress tolerance. During the hybridization process, a male sterile line (incapable of self-reproduction) is typically used as the female parent and crossed with a restorer line (the male parent) with beneficial traits, resulting in the first generation of hybrids (F1 generation) with hybrid vigor. These F1 hybrids can significantly improve rice yield and quality during cultivation.

[0004] Simplified and efficient F1 seed production methods are a development trend in hybrid rice. Years of hybrid rice seed production practice have shown that smaller flag leaves maximize sunlight exposure throughout the plant, thereby increasing leaf photosynthetic area and light energy utilization efficiency. This trait improves rice yield and seed set, reduces pollination barriers during seed production, and effectively prevents some pests and diseases caused by insufficient light and ventilation. Therefore, it is often necessary to manually remove the top third to half of the flag leaf at appropriate times. However, this method is not only labor-intensive and requires high operator skill, but also causes some damage to the emerging panicles, reducing seed yield.

[0005] Therefore, cultivating varieties with smaller flag leaves and avoiding artificial leaf cutting are important directions for the future development of hybrid rice seed production. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a mutant gene and protein causing rice flag leaves to become smaller and their applications. 60 Co-γ irradiation of the indica rice line 93-11 generated a new flag leaf reduction mutant, ym66. Hybridization of the ym66 mutant with the indica rice line YHSM yielded an excellent flag leaf reduction restorer line, NP27. During seed production, NP27 maintained its yield even when its flag leaves were not trimmed. Furthermore, its F1 generation exhibited superior plant architecture and 1000-grain weight.

[0007] To this end, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides, in an optional embodiment, a mutant gene that causes rice flag leaves to become smaller, wherein the nucleotide sequence of the mutant gene is a nucleotide sequence as shown in SEQ ID NO.1 inserted into the nucleotide sequence of the wild-type rice OsGATA15 gene at position 153 starting from the start codon ATG.

[0009] Preferably, the nucleotide sequence of the mutant gene is shown as SEQ ID NO.2.

[0010] In a second aspect, the present invention provides, in an optional embodiment, a mutant protein that causes rice flag leaves to become smaller, wherein the amino acid sequence of the mutant protein is an amino acid sequence as shown in SEQ ID NO.3 inserted starting from position 51 of the amino acid sequence of the wild-type rice OsGATA15 protein.

[0011] Preferably, the amino acid sequence of the mutant protein is shown in SEQ ID NO.4.

[0012] In a third aspect, the present invention provides, in an optional embodiment, a mutant ym66 that causes the flag leaves of rice to become smaller, wherein the nucleotide sequence of the mutant ym66 contains the above-mentioned mutant gene that causes the flag leaves of rice to become smaller; or, the amino acid sequence of the mutant ym66 contains the above-mentioned mutant protein that causes the flag leaves of rice to become smaller.

[0013] In a fourth aspect, the present invention provides an expression cassette in an optional embodiment, comprising the aforementioned mutant gene causing the reduction of rice flag leaves or the aforementioned mutant protein causing the reduction of rice flag leaves.

[0014] In a fifth aspect, the present invention provides, in an optional embodiment, a recombinant vector comprising the aforementioned mutant gene causing the rice flag leaf to become smaller or the aforementioned mutant protein causing the rice flag leaf to become smaller.

[0015] In a sixth aspect, the present invention provides, in an optional embodiment, a recombinant cell comprising the aforementioned mutant gene causing the reduction of rice flag leaves or the aforementioned mutant protein causing the reduction of rice flag leaves.

[0016] In the seventh aspect, the present invention provides, in an optional embodiment, the use of rice containing the above-mentioned mutant gene that causes the rice flag leaf to become smaller, rice expressing the above-mentioned mutant protein that causes the rice flag leaf to become smaller, or the above-mentioned mutant ym66 that causes the rice flag leaf to become smaller in cultivating restorer hybrid rice.

[0017] Preferably, the application includes hybridizing rice containing the above-mentioned mutant gene that causes the rice flag leaf to become smaller, rice expressing the above-mentioned mutant protein that causes the rice flag leaf to become smaller, or the above-mentioned mutant ym66 that causes the rice flag leaf to become smaller and YHSM rice to obtain the hybrid rice NP27, a restorer line for the reduction of the flag leaf.

[0018] The nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0019] GCCGCG.

[0020] The nucleotide sequence shown in SEQ ID NO.2 is as follows:

[0021] GGTAGAGTGTTGTGAATAGCCAGTTCAGTTCATCAGTTGATAGGAGCCATGCTTCACCATTACTACAGCGGCGGCGCCGGCCATCATCAGGACGTCGCTGCAGCTGGTAGCCCCGGCGACATGGCTTCCTCCACCTTCTCGCTCTTCTTCCCGATGTCCAATGGGCAGTGTTGGCCGCCGTCGACGGTGGAGGAGTCCGCGGCCGCGGCCTACGACGACCACAGCACCGTCACCACCTCTCCTTCCTCGCCTTCGTCGTCCTCCACCGGCTCCGTCGACTGCACGCTCTCGCTCGGCACGCCGTCGTCTCGCCGCGCCGAGCCCGTCGCGGCGGCGGCGCCAGCGGCAAACCATGGGGCGCCCGTGCCGGCGCATTATCCGTCGCTGTCAGCGGCGACCGTGTCCTGGGACGCGACTGCCGAGTCGTACTATTGTGGCCAGCAGGGGAGGCCGGCCACCGGCGCCGCCAAGTGCGCCGCCGGCGCCGGGCACGACGCGCTCCTCGACCGCCGCTGCGCCAACTGCGGCACCGCGTCCACGCCGCTCTGGAGGAACGGCCCTCGCGGACCCAAGGTTCGTCGCCGGCGACGCTCGCTCTCGCCGTCGCTGTTCTCTATCTCTGCAGTTCACTTCGCGGTTTCGCCGACGTACGTCGTGTCGCTGACATTGCCGCCGTCGTACGTTTCTCTGCAGTCGCTGTGCAACGCGTGCGGGATCAGGTACAAGAAGGAGGAGCGGCGCGCGGCGGCGACGACGACGACGGCCGACGGCGCCGCCGGATGCGGCTTCATCACCGCGCAGCGTGGACGCGGGTCGACCGCGGCCAAGGCGGCGCCCGCCGTGACGACGTGCGGCGAGGAGACGTCACCGTACGTCGTCGGCGGCGGCGGCGGCGGCGGCGAGGTCGCGGACGCGGCGTATCTCGCCTGGCGGCTCAACGTCGTCCCACCGGCGGCGACGGCGACGGCGTTCTCGGTGTGGCCGGAGCGAGCTAGCCTCTACCACTACAACTAGCTATAGGCGATTAACTAGTGTAGCATTAGCGTTGGGGAGAAGGGAAGCTGCACCGTTTTTTTGGGGAACATTTTGTACGGTCGCACGCTCCCGGATTGATCGAACTGTATGCATCTGCTGTGCTGTGTTGCACGCTAGCTAGCTAGATGACTCTGATCTTGAAACGAACCGTGATCATTTCATTCATTTGGAATGAACATGTAGCTGTGCAAGGTCATCTTGCTCGG。

[0022] The amino acid sequence shown in SEQ ID NO.3 is as follows:

[0023] AA.

[0024] The amino acid sequence shown in SEQ ID NO.4 is as follows:

[0025] MLHHYYSGGAGHHQDVAAAGSPGDMASSTFSLFFPMSNGQCWPPSTVEESAAAAYDDHSTVTTSPSSSSSSTGSVDCTLSLGTPSSRRAEPVAAAAPAANHGAPVPAHYPSLSAATVSWDATAESYYCGQQGRPATGAAK CAAGAGHDALLDRRCANCGTASTPLWRNGPRGPKSLCNACGIRYKKEERRAAATTTTADGAAGCGFITAQRGRGSTAAKAAPAVTTCGEETSPYVVGGGGGGGEVADAAYLAWRLNVVPPAATATAFSVWPERASLYHYN.

[0026] Compared with the prior art, the present invention has one of the following beneficial effects:

[0027] 1. The present invention is 60 Co-γ irradiation of the indica rice line 93-11 generated a new flag leaf reduction mutant, ym66. Hybridization of the ym66 mutant with the indica rice line YHSM yielded an excellent flag leaf reduction restorer line, NP27. During seed production, NP27 maintained its yield even when its flag leaves were not trimmed. Furthermore, its F1 generation exhibited superior plant architecture and 1000-grain weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The phenotypic characteristics and positional cloning of the ym66 mutant in Example 2 of the present invention are shown. Figure 1 A is the phenotypic characteristics of wild-type 93-11 and ym66 mutant, among which, Figure 1 Aa is the plant type at the end of heading stage, scale = 10 cm, Figure 1 Ab is the panicle shape before flowering, scale = 1 cm, Figure 1 Ac is the flag leaf type before flowering, scale = 1 cm, Figure 1 Ad is the stem of wild type 93-11 (left) and mutant ym66 (right) before flowering; the nutrient leaves are removed, and the horizontal lines represent the nodes separating the continuously elongated upper internodes (UPIs). UPI-I to UPI-VIII represent different UPIs from the uppermost to the base of the nutrient node. Scale bar = 5 cm. Figure 1 Ae- Figure 1 Ah is the TUNEL test of flag leaves before flowering, Figure 1 Ae / Figure 1 Af is the center of the longitudinal axis of the flag leaf of the 93-11 / mutant ym66 plant, Figure 1 Ag / Figure 1Ah is the longitudinal side of the flag leaf of the 93-11 / mutant ym66 plant. VS denotes vascular bundle, MSC denotes bundle sheath cell, VE denotes vessel, PH denotes phloem, MT denotes mesophyll tissue, MC denotes mother cell, AC denotes air cavity, and BSC denotes bundle sheath cell. Scale bar = 100 μm. Figure 1 B is the agronomic traits of wild type 93-11 and ym66 mutant, among which, Figure 1 Ba is plant height, Figure 1 Bb is the ear length, Figure 1 Bc is the number of particles, Figure 1 Bd is the number of branches, Figure 1 Be is the thousand-grain weight, Figure 1 Bf is the sword leaf length, Figure 1 Bg is the sword leaf width, Figure 1 Bh is the sword leaf angle; Figure 1 C is the map-based cloning of ym66 and the gene structure of OsGATA15, among which, Figure 1 Ca is the fine mapping of ym66. The molecular markers are marked above the black line. The black line represents chromosome 5. The corresponding number of recombinants is marked below the black line. The candidate open reading frame (OFR) is highlighted in red. Figure 1 Cb is the exon-intron structure of the OsGATA15 gene. Black rectangles represent different exons, black lines represent introns, and white rectangles represent 5' and 3' untranslated regions (UTRs). The mutation site in ym66 is located in exon 1. The specific sequence is listed below. Figure 1 D is transgenic verification by map-based cloning, where Figure 1 Da / Figure 1 Db is the genetic verification of OsGATA15 by transgenic knockout (cas9-1 and cas9-2) and overexpression (oe-1 and oe-2). The flag leaf phenotype is shown in the lower left corner of the image, scale bar = 1 cm, Figure 1 Dc is the sequence information of two knockout transgenic lines, and the specific positions are marked at both ends. Figure 1 Dd is the relative expression level of OsGATA15 in the overexpression transgenic lines, p(ab)<0.05;

[0029] Figure 2 To screen target genes related to leaf size regulated by OsGATA15 in Example 2 of the present invention, Figure 2 A is a preliminary screening of target genes using RNA-Seq and rice gene expression database, where Figure 2 The Venn diagram of Aa shows the overlap of RNA-Seq data of 151 leaf size-related genes and 2958 differentially expressed genes compared with 93-11. Figure 2The heat map of Ab shows the expression patterns of OsGATA15 and candidate genes based on the RiceXPro database. OsGATA15 highly expressed in inflorescence is marked in red, and candidate genes with similar expression to OsGATA15 in inflorescence are marked in blue; Figure 2 B is the expression pattern of OsGATA15, where Figure 2 Ba is the relative expression level of OsGATA15 in different tissues (including aerial parts, roots, stems, leaf sheaths and panicles) of 93-11 before flowering, Figure 2 Bb- Figure 2 Bd is 93-11 spike ( Figure 2 Bb), stem ( Figure 2 Bc) and leaf sheath ( Figure 2 Bd) Relative expression levels of OsGATA15 in different reproductive development stages (ear length 1-3; 3-5; 5-8; 8-10; 10-15; >15 cm); Figure 2 C is the qRT-PCR expression verification analysis of leaf size candidate genes in 93-11 and ym66 mutants, where Figure 2 Ca- Figure 2 Ch represents Os10g0403000, Os12g0555600, Os03g0640800, Os05g0389000, Os03g0308200, Os10g0324900, Os09g0531600, and Os07g0659800; Figure 2 D is the yeast one-hybrid interaction verification of OsGATA15, pHIS2-candidate gene + pGADT7 represents the negative control, pGAD53m + pHIS2-p53 represents the positive control, SD-TL represents -trp and -leuYPDA medium, SD-TLH represents -trp, -leu and -hisYPDA medium, yeast one-hybrid interaction analysis was performed in 3-ATSD-TLHYPDA medium with different concentrations, where Figure 2 Da- Figure 2 Dd represent the interactions between OsGATA15 and Os09g0531600, Os10g0403000, Os07g0659800, and Os03g0308200, respectively;

[0030] Figure 3 The phenotypic characteristics and agronomic traits of the short sword leaf restorer line NP27 and the F1 generation hybridized with the sterile lines 1892s and 20y394 in Example 2 of the present invention are as follows: Figure 3 A represents the phenotypic characteristics and agronomic traits of YHSM and NP27, among which, Figure 3 Aa represents the plant, ear, and flag leaf types at the end of heading stage, with scale bars of 10 cm, 1 cm, and 1 cm, respectively. Figure 3Ab is plant height, Figure 3 Ac is the spike length, Figure 3 Ad is the thousand-grain weight, Figure 3 Ae is the stem thickness, Figure 3 Af is the sword leaf length, Figure 3 Ag is the width of the sword leaf, Figure 3 Ah is the sword blade angle, Figure 3 Ai is the length of the upper internode; Figure 3 B is the plant type and agronomic traits of the F1 generation of the restorer lines YHSM and NP27 hybridized with 1892s, among which, Figure 3 Ba is the plant type, Figure 3 Bb is the seed production yield with the sword leaves cut off and the sword leaves retained, Figure 3 Bc is plant height, Figure 3 Bd is the spike length, Figure 3 Be is the plant shape angle, Figure 3 Bf is the number of grains per spike, Figure 3 Bg is the number of branches; Figure 3 C represents the plant type and agronomic traits of the F1 generation of the restorer lines YHSM and NP27 hybridized with 20y394, among which, Figure 3 Ca is the plant type, Figure 3 Cb is the seed production yield of cutting off the flag leaves and keeping the flag leaves, Figure 3 Cc is plant height, Figure 3 Cd is the spike length, Figure 3 Ce is the plant type angle, Figure 3 Cf is the number of grains per spike, Figure 3 Cg is the number of branches;

[0031] Figure 4 This is a diagram of the seed production model of the small-leaf restorer hybrid rice in Example 2 of the present invention, wherein the yellow arrows represent the pollination process;

[0032] Figure 5 are other different phenotypic characteristics between the wild type 93-11 and the ym66 mutant in Example 2 of the present invention, wherein, Figure 5 A / B shows the number of branches at maturity of wild type 93-11 and ym66 mutant, scale bar = 2 cm, Figure 5 C is the spikelet type at the top of the spike, scale bar = 1 cm, Figure 5 D is the spike neck type, scale bar = 2 cm, Figure 5 E is the stem diameter, Figure 5 F is the length of the sword leaf sheath, Figure 5 G is the length of the second leaf, Figure 5 HH is the width of the second leaf, Figure 5 I is the length of the inverted trefoil, Figure 5 J is the width of the inverted trefoil, Figure 5 K is the length of unpulled ears;

[0033] Figure 6 The safranin O-fast green staining and microscopic observation of the flag leaves of 93-11 and mutant ym66 after flowering in Example 2 of the present invention are shown, wherein: Figure 6 A- Figure 6 D is Safranin O-Fast Green staining detection, VS represents vascular bundle, MSC represents bundle sheath cell, VE represents vessel, PH represents phloem, MT represents mesophyll tissue, MC represents mother cell, AC represents air cavity, and BSC represents bundle sheath cell; scale bars = 500 μm, 500 μm, 100 μm and 50 μm, Figure 6 E- Figure 6 H is the microscope observation showing that the wild type 93-11 ( Figure 6 E and Figure 6 G) and mutant ym66 ( Figure 6 F and Figure 6 H) Normal and irregular cell morphology in plants, scale bars = 5 μm, 5 μm, 2 μm, and 1 μm;

[0034] Figure 7 This is a sequence alignment of the amino acid mutation sites of the GATA-B family member ym66 in Arabidopsis and rice in Example 2 of the present invention, wherein the red line indicates the mutation site in the ym66 mutant, and the red box shows the highly conserved characteristics of the amino acid site;

[0035] Figure 8 This is the GO and KEGG enrichment analysis of the RNA-Seq data of the flag leaf primordium of the wild type 93-11 and ym66 mutant in Example 2 of the present invention, wherein, Figure 8 A is GO term enrichment analysis, Figure 8 B is KEGG pathway enrichment analysis;

[0036] Figure 9 This is an analysis of the plant hormone content in the flag leaves of the wild type 93-11 and ym66 mutant in Example 2 of the present invention, wherein: Figure 9 A is a chord diagram. The thickness of the line connecting the hormones indicates the magnitude of the correlation between the two hormones. The blue line indicates a negative correlation, and the red line indicates a positive correlation. Figure 9 B is the correlation analysis. When the linear relationship between the two hormones is enhanced, the positive correlation approaches 1 and the negative correlation approaches -1. Figure 9 C- Figure 9 H represents the phytohormone content of indole-3-carboxaldehyde, isopentenyl adenosine, N-jasmonic acid isoleucine, 1-aminocyclopropanecarboxylic acid, (±)-jasmonic acid, and abscisic acid;

[0037] Figure 10 This is an analysis of nutrient elements in the flag leaves of the wild type 93-11 and ym66 mutant in Example 2 of the present invention, wherein: Figure 10 A is calcium, Figure 10B is magnesium, Figure 10 C is manganese, Figure 10 D is zinc. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0041] Example 1

[0042] Acquisition of ym66 mutants

[0043] use 60 The seeds of the indica rice line 93-11 were treated with Co-γ radiation and then sown. During the growth process of 93-11, a mutant with a short flag leaf and smaller second and third leaves was found. This mutant was named ym66 mutant.

[0044] Example 2

[0045] Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay

[0046] Flag leaves from wild-type 93-11 and mutant ym66 plants were paraffin-embedded. After pretreatment, such as dehydration and washing, the rTdT incubation solution from the TUNEL apoptosis detection kit was added to the tissue sections. The mixture was incubated at 37°C for 1 hour. The slides were then immersed in sodium citrate buffer (SSc solution) and incubated at room temperature for 15 minutes to terminate the reaction. Subsequently, the slides were immersed in 1 μg / mL propidium iodide and incubated at room temperature in the dark for 15 minutes, followed by a 5-minute rinse with deionized water. After mounting, the slides were examined using a laser confocal microscope. The green and red fluorescence emitted by propidium iodide were detected at wavelengths of 520 nm and 610 nm, respectively, with an excitation wavelength of 488 nm. Images were then acquired and saved.

[0047] Transmission electron microscopy (TEM) observation

[0048] Flag leaves of wild-type 93-11 and mutant ym66 plants were collected and cut into small pieces. The samples were placed in a 2.5% by volume glutaraldehyde fixative, evacuated with a vacuum pump for 30 minutes, centrifuged at 12,000 rpm for 5 minutes, and left at room temperature for more than 24 hours. All samples were rinsed three times with 0.1 mol / L phosphate buffer, each rinse lasting 10 minutes. The samples were then fixed in a 1% by volume osmium solution for 4 hours. The fixed samples were dehydrated in a gradient of 30%, 50%, 70%, 90%, or 100% ethanol, each dehydration lasting 10 minutes. The samples were then embedded in epoxy resin and polymerized for more than 48 hours. The samples were sectioned using an ultrathin microtome, stained with uranyl acetate and lead citrate, and then imaged using a JEM transmission electron microscope.

[0049] Safranin fast green staining

[0050] Flag leaf tissue sections from wild-type 93-11 and mutant ym66 plants were removed at 70°C and dewaxed in xylene for 20 minutes, then washed out with ethanol. The samples were then hydrated with 95%, 85%, or 75% ethanol for 5 minutes. The samples were then stained with safranin solution for 1 hour, rinsed with alcohol to remove excess dye, and then stained with Fast Green solution for 30 seconds. The tissues were rinsed with 95% and 70% ethanol, and the samples were then dried. Finally, neutral gum was added to the samples, covered with coverslips, and observed and photographed under a light microscope.

[0051] Positional cloning

[0052] In passing 60 Mutant ym66 was isolated from wild-type rice line 93-11 after Co-γ irradiation. An F2 mapping population was derived from a cross between ym66 and YHSM (Yuehe Si Miao, indica rice). Over 1,500 individuals exhibiting a short sword-leaf phenotype were selected from this population. Linkage analysis and individual exchange detection were performed using simple sequence repeat (SSR) and insertion-deletion (Indel) markers, listed in Table 1.

[0053] Table 1 Markers and primers

[0054] Primers Sequences(5'-3') RM274-F TAGATATGCAGACGGGCAGATCG RM274-R GGTCCACCTCTTCCTGCTCTTCC RM480-F TGGTACTCACCATGCAAGTAGAACG RM480-R ATGCTCAAGCATTCTGCAGTTGG RM26-F ACCTCACCCGAATTACTGTGATATGC RM26-R GTTGAATTGCACGCGACTCTGG IR7-F TGGTGGTTATCAATGTTTGTGGTTTGC IR7-R CCCTTGTTTCATCTGGTCCAATCGTA IL4-F GTACGAGTACACTCCTACG IL4-R CGACTAGATGGACTGACTG IL16-F AAAGTAGATGGAGGGAGTAC IL16-R GCTGGATGCTTATGTGTT IL28-F GCAGGGTTCAGAGGAGAA IL28-R CAGCTTTCGCTTTGGATTT IL30-F CACCGCCAATAAATCTGAC IL30-R ATATGCCCTTGTAGTTCCG IR2-F TGCGAGCCTCAGCCTAGACTTG IR2-R CGCTTCTGCTGTTGTGGTGTGG IR11-F CGGGCAAATGTGCGGACGATAT IR11-R ACACTTAGGTGAAAGCCGTGAAAGG IR19-F CTCATATTGGATTATGCAGCTCTCA IR19-R TTTACTTTCCCTTGTTTCATCTGGTCC OsGATA15-qPCR-F ATGTCCAATGGGCAGTGT OsGATA15-qPCR-R AGGAAGGAGAGGTGGTGA Actin-qPCR-F GACCCAGATCATGTTTGAGACCT Actin-qPCR-R CAGTGTGGCTGACACCATCAC OsGATA15-RT-PCR-F GCCAGTTCAGTTCATCAGT OsGATA15-RT-PCR-R CCAACGCTAATGCTACACT

[0055] Construction of OsGATA15 overexpression vector and CRISPR plasmid

[0056] Overexpression vectors and CRISPR plasmids for candidate gene testing were constructed using the pCAMBIA1301 and Pyl-hu vectors. For transgene overexpression, the coding sequence (CDS) region of OsGATA15, coupled with the ubiquitin (UBI) promoter, was amplified by polymerase chain reaction (PCR) and inserted into the pCAMBIA1301 vector using the restriction endonuclease KpnI. For gene knockout analysis, CATCAGGACGTCGCTGCAGC was selected as the target sequence for the single-stranded guide RNA (sgRNA) and inserted into the Pyl-hu vector using the HindIII restriction site. The primers used are listed in Table 1. Subsequently, the recombinant plasmids were introduced into the ym66 and wild-type 93-11 lines using Agrobacterium-mediated transformation.

[0057] RNA extraction, complementary deoxyribonucleic acid (cDNA) synthesis, and real-time quantitative polymerase chain reaction (qRT-PCR)

[0058] Total RNA was extracted from rice using TRIzol reagent, and genomic DNA was removed using deoxyribonuclease I (DNase I). For cDNA synthesis, 1000 ng of total RNA was reverse transcribed according to the manufacturer's instructions. OsGATA15 transcript levels were measured by real-time quantitative polymerase chain reaction (qRT-PCR) using Power SYBR Green PCR Master Mix. The cycling conditions were as follows: denaturation at 95°C for 2 minutes, followed by 40 cycles in a qRT-PCR system, each consisting of 10 seconds, 30 seconds at 95°C, and 30 seconds at 72°C. The primers used are listed in Table S1.

[0059] Yeast one-hybrid assay

[0060] The target gene cloned into the pHIS2 vector and the OsGATA15 gene cloned into pGADT7 were co-transformed into the yeast strain Y187 according to the method described in the yeast one-hybrid library screening system kit (Clontech, California, USA) to verify the interaction between the pHIS2 target gene and pGADT7-OsGATA15. Single colonies of the experimental group pHIS2-target gene + pGADT7-OsGATA15, the control group pHIS2-target gene + pGADT7, and the positive control group pGAD53m + pHIS2-p53 were verified. The samples were then resuspended in 2 mL of double-distilled water (ddH2O), adjusted to OD600 = 0.002, and set to 10 0 , 10 -1 and 10 -2Three concentration gradients were used. A total of 10 μL was plated onto SD-TL, SD-TLH, or SD-TLH + 130 mM 3AT medium, with three samples per plate. The samples were incubated at 30°C for 3-5 days.

[0061] in conclusion

[0062] To obtain small flag leaf mutants and explore the regulatory mechanism of flag leaf development in rice, we used 60 A stable small-flag leaf mutant, ym66, was obtained by mutagenizing indica rice 93-11 with Co-γ radiation. The yield of the ym66 mutant was always slightly higher than that of the wild type 93-11 from the seedling stage to the heading stage (see Figure 1 Aa and Figure 1 Ba). During the vegetative growth stage, the increase in rice plant height is due to the continuous growth of upper internodes (UPIs). Compared with 93-11, the ym66 mutant has an additional upper internode (see Figure 1 Ad). The basal internodes (UPI-V to UPI-VII) are longer than those of the wild type, while the uppermost internode (UPI-I) is significantly shorter than that of 93-11 (see Figure 5 These findings suggest that the initiation and elongation patterns of the upper internodes in the ym66 mutant are altered. Before flowering, the panicles of ym66 are also shorter than those of 93-11 (see Figure 1 Ab and Figure 1 Bb). This phenomenon occurs because the number of primary branches in ym66 is less than that in 93-11, resulting in a decrease in the number of spikelets (see Figure 1 Bd and Figure 5 In addition, no awns and deformed panicles were observed in the ym66 mutant (see Figure 5 The most notable phenotype of ym66 is that its flag leaves are extremely short, narrow, and hard (see Figure 1 Ac). The average sword leaf length of 93-11 was 29.5 cm, and the average sword leaf width was 2.36 cm. Compared with 93-11, the average sword leaf length of ym66 was 6.1 cm, which was greatly reduced by 79.32%, and the average sword leaf width was 0.36 cm, which was reduced by 84.75% (see Figure 1 Bf and Figure 1 Bg), the average sword leaf angle also decreased significantly (see Figure 1 Similarly, the average lengths of the second and third lobes were slightly reduced in ym66, while the average widths of the second and third lobes were not significantly different (see Figure 5 In addition, the ym66 mutant exhibits various abnormal phenotypes, such as excessive glumes, thickened stems, delayed flowering, and heavier grains.

[0063] To further investigate the cellular changes associated with the small flag leaf phenotype of ym66, Safranin fast green staining and transmission electron microscopy (TEM) analysis were performed on the middle part of the flag leaf of ym66 before anthesis. Figure 6 As shown, the mutant ym66 has fewer veins than 93-11. Given that the vascular system in the veins is thought to be directly related to leaf size, this finding may be the reason why the mutant flag leaf is narrower. In addition, the darker red staining indicates severe lignification, which surrounds the mutant flag leaf, especially at the edge of the ym66 cross-section. Structurally, the small air cavity (AC) in the mutant flag leaf is unable to support microstructures. This problem causes some important structures to be squeezed together, such as bundle sheath cells (MSCs), bundle sheath cells (BSCs) and mesophyll tissue (MT). TEM analysis of the vascular system showed that the cell morphology and organelles of the ym66 mutant were abnormal (see Figure 6 These structures may block the transport of some essential nutrients and induce cell death. Therefore, the degenerating cells in the flag leaf of the mutant ym66 were evaluated by TUNEL assay. A strong fluorescent signal (green fluorescence) was observed in ym66, but no obvious signs of apoptosis were observed in 93-11 ( Figure 1 Ae- Figure 1 Ah). These experimental results confirmed that cell death occurred in the flag leaves of the ym66 mutant.

[0064] To further investigate the causative gene for the short and narrow flag leaf phenotype of ym66, an F2 mapping population was constructed by crossing the ym66 mutant with the indica rice variety YHSM. In the F2 population, approximately one-quarter of the individuals (983 / 4128) exhibited a small flag leaf phenotype, suggesting that this abnormal flag leaf phenotype is regulated by a single recessive gene (Chi-square test: χ 2 =2.327<χ 2 0.05 =3.841). Then, these 983 mutant individuals were used for mapping. The ym66 locus was initially located between markers RM26 and IR-7 on the long arm of chromosome 5 (see Figure 1 Ca). After fine mapping, the mapping interval was further narrowed to a 94 kb genomic region between the markers IL-28 and IR-2, in which 17 open reading frames (ORFs) were annotated (see Figure 1 To further identify the target gene, the genome of these 17 ORFs was sequenced individually and then compared with the sequence of 93-11. Sequence comparison revealed that in the ym66 mutant, the six-base sequence GCCGCG was inserted between nucleotides 153 and 154 of the first exon of LOC_Os05g50270 (OsGATA15), along with two corresponding amino acids AA.

[0065] Transgenic knockout and overexpression experiments were performed to verify whether the OsGATA15 insertion mutation caused the short, narrow flag leaf phenotype of the ym66 mutant. In the transgenic knockout analysis, CATCAGGACGTCGCTGCAGC was selected as the sgRNA target sequence, and the CRISPR / Cas9 recombinant plasmid was introduced into the 93-11 line. Two transgenic knockout lines (cas9-1 and cas9-2) were obtained, and all individuals showed a small flag leaf phenotype similar to ym66 (see Figure 1 Sequence comparison of cas9-1, cas9-2, and 93-11 revealed that dozens of nucleotides were missing near the sgRNA (see Figure 1 Dc). In the transgenic overexpression analysis, the CDS region of OsGATA15 plus the UBI promoter was cloned and inserted into pCAMBIA1300, and the recombinant plasmid was then introduced into the mutant ym66. Two transgenic overexpression lines (oe-1 and oe-2) were obtained, and the small flag leaf phenotype was significantly restored (see Figure 1 Db). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that the relative expression level of OsGATA15 in the transgenic overexpression line was higher than that in ym66 (p value < 0.05) (see Figure 1 These results suggest that the short and narrow flag leaf is attributed to the variation in OsGATA15.

[0066] GATA factors are transcriptional regulators in animals, plants, and fungi that recognize WGATA-RDNA sequences through a single type IV zinc finger structure. GATA factors are crucial in various developmental processes in plants, including the regulation of metal binding complexes, nitrogen metabolism, carbon metabolism, and light response. To further identify downstream target genes associated with flag leaf size regulated by OsGATA15, RNA sequencing analysis was performed on leaf primordia of 93-11 and the mutant ym66 at the early stages of panicle development. The results showed that compared with 93-11, 1341 differentially expressed genes (DEGs) were upregulated and 1617 differentially expressed genes were downregulated in ym66 (see Figure 2 A total of 2958 differentially expressed genes were enriched in non-membrane-bound organelles according to Gene Ontology (GO) terms and in ribosome-related metabolism according to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (see Figure 8). Subsequently, 151 genes reported to be associated with leaf size (length and width) were identified, and 2958 differentially expressed genes were screened. Subsequently, 21 down-regulated genes and 4 up-regulated genes were identified in the ym66 mutant. Previous studies have shown that OsGATA15 is most highly expressed in the early stages of panicle development. Therefore, the expression patterns of these 25 differentially expressed genes associated with leaf size were analyzed using the RiceXPro database (https: / / ricexpro.dna.affrc.go.jp / index.html) to investigate their consistency with OsGATA15 expression. The results are shown in Figure 2. Figure 2 As shown in Ab, the high expression period and tissue expression of OsGATA15 are marked in red. Except for Os01g0360200 and Os01g0659400, other differentially expressed genes are expressed in the early stage of panicle development. However, considering the degree of consistency, that is, these genes are expressed at the highest level in the early stage of panicle development, but not at high levels in other tissues or stages, 8 differentially expressed genes were selected for verification. These genes are Figure 2 Ab is marked in blue.

[0067] First, the expression pattern of OsGATA15 in 93-11 was studied by real-time fluorescence quantitative polymerase chain reaction. Figure 2 As shown in Figure B, OsGATA15 is highly expressed in the panicle and stem before flowering and slightly expressed in the leaf sheath. In addition, OsGATA15 is also expressed in the seedling stage. In the specific expression analysis of panicle and stem, OsGATA15 is highly expressed in the early stage of panicle development (1-3 cm in length) and its expression gradually decreases with reproductive growth (see Figure 2 Bb and Figure 2 Bc). However, this pattern was not observed in the leaf sheaths (cf. Figure 2 Bd). These results differ slightly from previous reports, in which OsGATA15 was only highly expressed during the early stages of panicle development. This finding may be due to their different genetic backgrounds, resulting in slight changes in the expression pattern of OsGATA15. The expression of these candidate genes during the early stages of panicle development was also verified by qRT-PCR. All candidate target genes were significantly downregulated in the ym66 mutant (see Figure 2 C) To further identify genes that affect leaf size and are regulated by OsGATA15, yeast one-hybrid cross-talk was performed. Figure 2As shown in D, four pHIS2 plasmids, namely Os09g0531600, Os10g0403000, Os07g0659800, and Os03g0308200, can grow together with the pGADT7-OsGATA15 plasmid in a triple-deficient medium containing 3-aminotriazole (3-AT), indicating that these genes are directly regulated by the transcription factor OsGATA15. In addition, the plant hormone and nutrient content in the flag leaves of 93-11 and ym66 plants was measured. In addition to 1-aminocyclopropanecarboxylic acid, the levels of various plant hormones, such as indole-3-carboxaldehyde and jasmonic acid, were increased in the flag leaves of the mutants (see Figure 9 In terms of nutrients, the contents of calcium, magnesium, and zinc in the mutant's flag leaves were significantly reduced, while the content of manganese increased (see Figure 10 These results indicate that OsGATA15 is a ubiquitously expressed functional transcription factor in rice. It regulates a range of genes during early panicle development to control rice growth and development, including hormone signaling and ion transport. Multiple factors may contribute to the short, narrow flag leaf phenotype of the mutant.

[0068] Previous phenotypic investigations have shown that the sword leaves of the ym66 mutant are shorter and narrower than those of 93-11, the second and third leaves are shortened, and the lengths of the other leaves remain unchanged (see Figure 1 ). These leaf distribution characteristics are very suitable for simplifying seed production and help avoid the leaf clipping process in two-line hybrid rice seed production. Therefore, individual plants with short sword leaves and excellent agronomic traits were screened from the YHSM / ym66 population. After years of continuous backcrossing and breeding, the excellent small-flag-leaf restorer line NP27 was cultivated. Compared with YHSM, NP27 has an increased plant height, a more compact plant shape, and significantly improved grain weight and stem thickness (see Figure 3 A). The length, width, and base angle of the NP27 flag leaf are similar to those of the ym66 mutant. Although the panicle length (19.36 cm) and branch number of the short-flag leaf restorer NP27 are significantly lower than those of the YHSM (23.86 cm), the pollen content is sufficient to ensure seed production (see Figure 4 ).

[0069] Subsequently, hybrid rice seed production experiments were conducted using the sterile lines 1892s and 20y394. The sword-leaf pruned 1892s / YHSM and 20y394 / YHSM combinations were used as blank controls. The sword-leaf-unpruned 1892s / YHSM and 20y394 / YHSM combinations were used as negative controls. The experimental groups included the sword-leaf-unpruned 1892s / NP27 and 20y394 / NP27 combinations. The results showed that, in both 1892s and 20y394 lines, the unpruned sword-leaf combination with the short sword-leaf restorer line NP27 as the male parent had a higher seed yield than the unpruned sword-leaf combination with YHSM as the male parent, and was similar to the yield of the pruned sword-leaf hybrid combination with YHSM as the male parent (see Figure 3 Bahe Figure 3 Ca). These results indicate that when hybrid rice seed production is carried out using the short-leaf restorer line NP27 as the male parent, the flag leaf pruning step is not required, which can simplify the seed production process. In addition, the agronomic traits of the F1 hybrids of 1892s and 20y394 with YHSM and NP27 were also studied. Regardless of whether the female parent is 1892s or 20y394, the hybrids with NP27 as the male parent have a better plant shape than YHSM, which is manifested by a more compact plant shape and the panicles are at the same level (see Figure 3 B and Figure 3 C). Furthermore, except for the potentially higher yield of the NP27 combination, there are few differences in other agronomic traits between the YHSM and NP27 combinations. Overall, NP27, with its smaller flag leaves, is an excellent restorer line for hybrid rice.

[0070] In the present invention, the mutation of OsGATA15 results in the ym66 mutant having short and narrow flag leaves, with the second and third leaves also shortened to varying degrees. This leaf shape distribution fully meets the ideal plant type requirements for determining rice leaf length. Therefore, this trait was introduced into the restorer line to cultivate the short flag leaf restorer line NP27, which does not require flag leaf pruning during hybrid rice seed production (see Figure 3 Bb and Figure 3 Cb). Compared with the negative control combination with untrimmed leaves, the seed yield of the short-leaf combination was significantly increased, and the agronomic traits were also excellent. This method provides a good strategy for simplifying hybrid rice seed production. However, the disadvantage is that the panicle length of the restorer line NP27 is short, a phenomenon also found in the ym66 mutant (see Figure 1Ab and 3Aa). Based on mapping population analysis and backcross breeding results, this short panicle trait is tightly linked to the short sword-shaped leaf trait. This finding suggests that mutations in OsGATA15 also reduce panicle branching. OsSPL14 is an ideal plant architecture gene in rice. Point mutations in OsSPL14 interfere with the regulation of OsmiR156 on OsSPL14, resulting in an ideal rice plant architecture with reduced tiller number, lodging resistance, and increased grain yield. High expression of OsSPL14 during the reproductive period promotes panicle branching and increases grain yield. Chromatin immunoprecipitation sequencing (ChIP-seq) identified downstream genes regulated by OsSPL14, showing that OsSPL14 can directly bind to the OsTB1 promoter and affect panicle length. Furthermore, previous studies have shown that OsSPL14 interacts with OsGATA15 and influences its expression. Therefore, given its high expression during early panicle development, OsGATA15 may regulate the OsSPL14-OsTB1 pathway and promote panicle branching. Furthermore, the ym66 mutant exhibits numerous other phenotypes, including altered flowering habit, persistent glumes, increased 1000-grain weight, and loss of the awn at the top of the panicle. These results suggest that OsGATA15 regulates a range of downstream genes involved in rice growth and development.

[0071] In addition, the initiation and elongation pattern of the upper internode (UPI) is altered in the ym66 mutant. Compared with the wild type, the mutant has an additional upper internode (see Figure 1 Ad and Figure 5 K). The basal internodes (UPI-V to UPI-VII) are longer than those of the wild type, while the uppermost internode (UPI-I) is significantly shorter than that of 93-11. Normally, elongation starts from the uppermost internode at the base and extends upward synchronously with the development of the floral organs of the panicle. The shorter length of the upper internodes is often due to gibberellin (GA) signaling defects. Therefore, it is speculated that the shortening of the upper internodes of this mutant may be caused by GA deficiency. First, the GA content in the upper internodes, flag leaves and sheaths was detected, and it was found that there were significant differences in the GA content in these mutant tissues compared with the wild type (the GA content in the upper internodes and flag leaves was lower than that in the wild type, while the GA content in the mutant sheaths was higher than that in the wild type sheaths). Subsequently, exogenous GA3 was added to the mutant in the early stage of panicle development, and it was found that all upper internodes elongated rapidly, and the plant height was much higher than that of 93-11 treated with the same amount of GA3. Similar experiments were also carried out at the seedling stage. Five days after seed germination, 10 -8MGA3. The results showed that the mutant seedlings grew rapidly and the length of their aboveground parts was significantly greater than that of the wild type. In addition, many differentially expressed genes (DEGs) related to GA synthesis and signal transduction, such as GA20ox, GID1 and GRAS2, were found in the RNA-Seq data. These findings indicate that the OsGATA15 gene mutation increases the sensitivity of rice to GA. Euil is a mutant that accumulates a large amount of bioactive GA in the upper internodes. In addition, a previous study constructed a double mutant by hybridizing the OsGATA15 mutant with eui1 and reported an additive effect, that is, the length of all upper internodes increased, but the equal length pattern was maintained. These findings suggest that OsGATA15 may be involved in a new GA metabolic pathway to regulate the sensitivity of rice to GA.

[0072] GATA transcription factors are common in plants and play an important role in regulating flowering time, leaf growth, flower development, photoperiod phenomena and light signal transduction. Generally speaking, plant GATA factors contain a zinc finger structure with two CX2C motifs, which are connected by a 17-20 amino acid long loop (CX2CX 17-20 CX2C), followed by a highly alkaline region. So far, four classes of GATA factors have been identified in the Arabidopsis and rice genomes. OsGATA15 belongs to class B GATA factors, which contain only one conserved DNA binding motif. In the ym66 mutant, the 6bp insertion site in the first exon is not located within the conserved domain of the DNA binding motif, but its phenotype is consistent with that of the complete knockout of OsGATA15, indicating that this insertion mutation site is also important for the function of OsGATA15 (see Figure 1 Cb and Figure 7 ). Transcription factors are usually composed of four functional regions: a DNA binding domain, a transcriptional regulatory domain, an oligomerization site, and a nuclear localization signal. Therefore, based on the sequence conservation of these four functional regions, it is inferred that the mutation site may be located in the transcriptional regulatory domain of OsGATA15. B-GATA factors contain a zinc finger structure with 18 amino acids in its zinc finger ring. In angiosperms, the B class can be subdivided into B-GATA factors containing a HAN domain or a LLM domain. OsGATA15 is the first GATA factor containing a HAN domain to be studied in rice. In Arabidopsis, the function of the B-class GATA factor HANABATARANU (HAN) was studied, and it was found that it plays an important role in the development of floral organs and shoot apical meristems. In contrast, loss of OsGATA15 function leads to shortening of flag leaves and panicles, and changes in the elongation pattern of the upper internodes (see Figure 1A). Therefore, although HAN and OsGATA15 share conserved protein domains, they should have functional differences and participate in different developmental processes in dicots and monocots, respectively.

[0073] Although the principles of the present invention have been described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Without departing from the spirit and scope of the present invention, any obvious changes such as equivalent transformations and simple substitutions based on the technical solution of the present invention fall within the scope of protection of the present invention.

Claims

1. A mutant gene that causes rice flag leaves to become smaller, characterized in that: The nucleotide sequence of the mutant gene is a nucleotide sequence as shown in SEQ ID NO. 1 inserted into the 153rd position starting from the start codon ATG of the nucleotide sequence of the wild-type rice OsGATA15 gene.

2. The mutant gene causing the rice flag leaf to become smaller according to claim 1, characterized in that The nucleotide sequence of the mutant gene is shown in SEQ ID NO.

2.

3. A mutant protein that causes rice flag leaves to become smaller, characterized in that: The amino acid sequence of the mutant protein is obtained by inserting the amino acid sequence shown in SEQ ID NO. 3 starting from position 51 of the amino acid sequence of the wild-type rice OsGATA15 protein.

4. The mutant protein causing the rice flag leaf to become smaller according to claim 3, characterized in that The amino acid sequence of the mutant protein is shown in SEQ ID NO.

4.

5. A mutant ym66 causing a reduction in flag leaf size in rice, characterized in that: The nucleotide sequence of the mutant ym66 comprises the mutant gene causing the rice flag leaf to become smaller according to claim 1 or 2; or The amino acid sequence of the mutant ym66 comprises the mutant protein causing the rice flag leaf to become smaller as described in claim 3 or 4.

6. An expression cassette, characterized in that The invention comprises the mutant gene causing the rice flag leaf to become smaller as described in claim 1 or 2 or the mutant protein causing the rice flag leaf to become smaller as described in claim 3 or 4.

7. A recombinant vector, characterized in that The invention comprises the mutant gene causing the rice flag leaf to become smaller as described in claim 1 or 2 or the mutant protein causing the rice flag leaf to become smaller as described in claim 3 or 4.

8. A recombinant cell, characterized in that The invention comprises the mutant gene causing the rice flag leaf to become smaller as described in claim 1 or 2 or the mutant protein causing the rice flag leaf to become smaller as described in claim 3 or 4.

9. Use of a rice plant containing the mutant gene causing the reduction of rice flag leaves according to claim 1 or 2, a rice plant expressing the mutant protein causing the reduction of rice flag leaves according to claim 3 or 4, or the mutant ym66 causing the reduction of rice flag leaves according to claim 5 in cultivating restorer hybrid rice.

10. The use according to claim 9, characterized in that The application includes hybridizing rice containing the mutant gene causing the rice flag leaf to become smaller as described in claim 1 or 2, rice expressing the mutant protein causing the rice flag leaf to become smaller as described in claim 3 or 4, or the mutant ym66 causing the rice flag leaf to become smaller as described in claim 5 with YHSM rice to obtain the hybrid rice NP27, which is a restorer line for the reduced flag leaf.

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