Rice lesion-like mutant lrs3 and application thereof

Abstract

The invention belongs to agricultural biotechnology engineering, and particularly relates to a rice scab-like gene LRS3 and application thereof in rice breeding. The invention discloses a rice scab-like gene LRS3, which has a DNA (deoxyribonucleic acid) sequence as shown in SEQ ID NO: 1 and a cDNA (complementary deoxyribonucleic acid) sequence as shown in SEQ ID NO: 2. The invention also provides a rice scab-like lrs3 mutant at the same time, and the rice scab-like lrs3 mutant is a mutant of the LOCOs03g06410 gene; the mutation is characterized in that the structure of the protein translated by the LRS3 gene is changed through replacement of a single basic group, so that the function of the protein is changed. The rice lesion-like mutant affects the growth and development of rice, changes the agronomic traits of the rice, and can be applied to rice breeding.

Description

Technical Field

[0001] This invention belongs to agricultural biotechnology engineering, specifically relating to a gene for rice lesions. LRS3 And its application in rice breeding. Background Technology

[0002] Rice lesion mimics refer to necrotic or chlorotic spots that appear on leaves and other organs of rice when there is no obvious pathogen infection. Their phenotypes are diverse: according to color, they can be brown, white, red, etc.; according to shape, they can be dotted, streaked, or irregular; and their size varies significantly. Furthermore, some lesion mimics gradually enlarge or merge as the plant grows. [1] These lesion-like spots mostly occur during the tillering to grain-filling stage of rice, initially appearing on the lower functional leaves and spreading upwards as the plant grows. Their occurrence exhibits spatiotemporal specificity; some mutant lesion-like spots are only expressed under specific temperature (25-30℃) or light conditions (12-14h light exposure), and the formation of these spots is accompanied by typical characteristics of programmed cell death (PCD), such as nuclear pyknosis and cytoplasmic degradation. [2] Conversely, precise control of rice lesions can effectively ensure the photosynthetic efficiency and yield stability of rice, thereby safeguarding the economic and ecological benefits of grain production.

[0003] Leaves are the core organs of rice for photosynthesis and the accumulation of photosynthetic products. They are also the key parts of the plant that sense external stress and initiate defense responses. Their integrity and physiological functions directly determine the material production capacity of rice. [3] As lesions appear and expand on leaves, the chloroplast structure inside the leaf is damaged first, leading to thylakoid membrane disintegration and disordered grana arrangement. This is accompanied by the degradation of photosynthetic pigments and the abnormal decomposition of macromolecules such as proteins and nucleic acids. Furthermore, the lesion area triggers an excessive accumulation of reactive oxygen species (ROS), disrupting the intracellular redox balance and further exacerbating cell damage. [4] During key growth stages of rice, such as tillering, jointing, and grain filling, leaves play a crucial role in providing carbohydrates and nutrients for root growth, panicle development, and grain filling. These substances are transported through the phloem to sink organs such as young panicles and grains, directly affecting the seed setting rate and thousand-grain weight. [5] Therefore, the occurrence and distribution characteristics of leaf lesions can generally serve as important indicators for assessing rice health and predicting yield potential. The formation of rice lesions is not a random process but is regulated by multiple intrinsic factors, such as lesion-related genes (…). OsLSD1 , OsPAL The expression of hormones (such as α, β, γ) regulates the signal balance of hormones and is also significantly affected by external environmental factors, such as pathogen infection, extreme temperature and light conditions, soil nutrient imbalance, and heavy metal stress. [6][7].

[0004] It is noteworthy that some rice lesion-like mutants or lesion-like induced plants exhibit enhanced resistance to bacterial leaf streak (Xanthomonas oryzae pv. oryzicola, induced by Xoc), a phenomenon closely related to the activation of the defense response accompanying lesion formation. Physiologically, during lesion formation, the plant initiates local and systemic acquired resistance-related physiological metabolic changes: on the one hand, the lesion area and surrounding tissues accumulate defense hormones such as salicylic acid (SA) and jasmonic acid (JA). SA can regulate PR (pathogenesis-related protein) genes (such as...) OsPR1 , OsPR5 The expression of Xoc enhances the plant's early recognition and response to Xoc infection. [8] On the other hand, the local accumulation of reactive oxygen species (ROS) induced by lesion-like spots, while causing programmed cell death (PCD) and lesion formation, can also act as signaling molecules, activating the activity of downstream antioxidant enzyme systems (such as SOD and POD), enhancing the plant's ability to scavenge excess ROS produced after Xoc infection, and reducing oxidative damage caused by pathogens. [9] .

[0005] From a molecular perspective, some cloned lesion-like genes can directly or indirectly participate in the regulation of resistance to bacterial blight and leaf streak. For example, lesion-like genes... OsLSD1 While forming lesion-like structures, mutants in vivo exhibit changes to disease-resistance-related MAPK signaling pathways (such as...). OsMAPK3 / 6 This pathway is continuously activated and can phosphorylate downstream transcription factors (such as...). OsWRKY45 This process regulates the synthesis of antimicrobial substances (such as phytoalexins and phenolic compounds), thereby inhibiting the colonization and spread of Xoc in rice. Furthermore, during the formation of lesion-like spots, the plant cell walls undergo structural modifications such as lignification and pectin cross-linking, enhancing the physical barrier function of the cell walls against Xoc infection and reducing the spread of pathogens through the vascular bundle system. Field planting trial data show that, compared to wild-type rice, OsLSD1 The mutant exhibits 30%-40% increased resistance to Xoc, significantly shortened lesion length, and reduced bacterial load by 1-2 orders of magnitude. [6] This further confirms the positive correlation between lesion-like spots and resistance to bacterial blight and fine streak, providing a theoretical basis for breeding rice varieties resistant to bacterial blight and fine streak by regulating lesion-like spot-related genes.

[0006] As one of the world's most important food crops, the stability of rice yield and its quality directly affect food security and agricultural economic development, and have irreplaceable strategic significance for ensuring the stability of human society.

[10] By precisely regulating the expression of lesion-related genes to inhibit the formation of non-functional lesions, or by utilizing disease resistance mechanisms mediated by lesion-related genes, it is possible to reduce yield loss while enhancing rice's resistance to pathogens. Therefore, conducting research on the cloning, functional analysis, and genetic regulation mechanisms of rice lesion-related genes not only provides theoretical support for elucidating the molecular mechanisms of lesion formation, but also lays the foundation for breeding new rice varieties with synergistic optimization of "low lesion incidence, high disease resistance, and high yield," and has significant practical guiding value for achieving high, stable, and sustainable rice production.

[0007] LOC_Os03g06410 The gene sequence has been published in the China Rice Data Center (https: / / www.ricedata.cn / gene / ). LOC_Os03g06410 The gene is known to encode a mitogen-activated protein kinase kinase and act as a positive regulator of ethylene synthesis in rice.

[0008] The references mentioned above are as follows: [1] Kang SG, Lee KE, Singh M, Kumar P, Matin MN. Rice Lesion MimicMutants (LMM): The Current Understanding of Genetic Mutations in the Failure of ROS Scavenging during Lesion Formation. Plants (Basel). 2021 Aug 4;10(8):1598; [2] Zhang A, Jiang H, Chu H, Cao L, Chen J. Rice Lesion Mimic GeneCloning and Association Analysis for Disease Resistance. Curr Issues MolBiol. 2022 May 22;44(5):2350-2361; [3] Chen X, Ma J, Wang X, Lu K, Liu Y, Zhang L, Peng J, Chen L, YangM, Li Y, Cheng Z, Xiao S, Yu J, Zou S, Liang Y, Zhang M, Yang Y, Ding X, DongH. Functional modulation of an aquaporin to intensify photosynthesis underbrogates bacterial virulence in rice. Plant J. 2021 Oct;108(2):330-346; [4] Li W, Cheng W, Jiang H, Fang C, Peng L, Tao L, Zhan Y, Huang X,Ma B, Chen X, Wu Y, Liu B, Fu X, Wu K, Ye Y. Mutation of rice EARLY LEAFLESION AND SENESCENCE 1 (ELS1), which encodes an anthranilate synthase, induces ROS-α subunit synthesis and accumulation through activating thetryptophan synthesis pathway in rice. Plant J. 2024 Dec;120(6):2723-2737; [5] Zhao Z, Wang C, Yu X, Tian Y, Wang W, Zhang Y, Bai W, Yang N,Zhang T, Zheng H, Wang Q, Lu J, Lei D, He X, Chen K, Gao J, Liu X, Liu S,Jiang L, Wang H, Wan J. Auxin regulates source-sink carbohydrate partitioning in reproducing organ development. Proc Natl Acad Sci US A. 2022Sep 6;119(36):e2121671119: [6] Xiang J, Qian K, Zhang Y, Chew J, Liang J, Zhu J, Zhang Y, Fan X.OsLSD1.1 is involved in the photosystem II reaction and affects nitrogenallocation in rice. Plant Physiol Biochem. 2021 Sep;166:246-257; [7] Su P, Kang H, Peng Q, Wicaksono WA, Berg G, Liu Z, Ma J, Zhang D,Cernava T, Liu Y. Microbiome homeostasis on rice leaves is regulated by aprecursor molecule of lignin biosynthesis. Nat Commun. 2024 Jan 2;15(1):23; [8] Wang M, Li S, Li H, Song C, Xie W, Zuo S, Zhou X, Zhou C, Ji Z,Zhou H. Genome editing of a dominant resistance gene for broad-spectrumresistance to bacterial diseases in rice without growth penalty. PlantBiotechnol J. 2024 Mar;22(3):529-531; [9] Wei Q, Yan Z, Xiong Y, Fang Z. Altered Expression of OsAAP3Influences Rice Lesion Mimic and Leaf Senescence by Regulating ArginineTransport and Nitric Oxide Pathway. Int J Mol Sci. 2021 Feb 22;22(4):2181;

[10] Danso Ofori A, Zheng T, Titriku JK, Appiah C, Xiang X, KandhroAG, Ahmed MI, Zheng A. The Role of Genetic Resistance in Rice DiseaseManagement. Int J Mol Sci. 2025 Jan 23;26(3):956. Summary of the Invention

[0009] The problem to be solved by this invention is to provide a rice lesion gene. LRS3 And its applications.

[0010] To address the above problems, this invention provides a rice lesion gene. LRS3 Rice lesion gene LRS3 The DNA sequence is as described in SEQ ID NO:1.

[0011] As the rice lesion gene of the present invention LRS3 Improvement: Rice lesion gene LRS3 The cDNA sequence is as described in SEQ ID NO:2.

[0012] This invention also provides the above-mentioned rice lesion gene. LRS3 The encoded protein has the amino acid sequence as described in SEQ ID NO:3.

[0013] This invention also provides mutant materials. lrs3 The gene: the mutant DNA sequence is as described in SEQ ID NO:4, and the cDNA sequence is as described in SEQ ID NO:5.

[0014] This invention also provides the above-mentioned rice lesion gene. LRS3, Mutant materials lrs3 The purpose of this gene: to influence the growth and development of rice.

[0015] An improvement to the use of this invention: altering agronomic traits for application in rice breeding.

[0016] As a further improvement to the use of the present invention: it affects the plant height, number of tillers, panicle length, seed setting rate, and grain size of rice.

[0017] As a further improvement to the use of the invention: the agronomic trait includes reddish-brown rust-like lesions.

[0018] This invention also provides a method for regulating the growth and development of rice and altering agronomic traits: utilizing the rice lesion gene described above. LRS3,Mutant materials lrs3 The genes.

[0019] The purpose of this invention is to provide LRS3 Application of genes in rice breeding, rice lesions lrs3 The mutant is LOC_Os03g06410 Mutants of the gene; the above mutations are caused by the substitution of a single base. LRS3 Changes in the structure of proteins during gene translation lead to changes in their function. The aforementioned rice lesion mutants affect rice growth and development, altering its agronomic traits, and can be applied in rice breeding.

[0020] The above-mentioned objective of the present invention is achieved by the following method: Rice lesion mutant of the present invention lrs3 It was obtained from the EMS mutant library of the japonica rice variety Yundao. (Mutant) lrs3 During the peak tillering stage, reddish-brown rust-like lesions appear on older leaves. From the heading to maturity stage, the lesions gradually spread to all leaves. During this stage, the spread becomes more severe, appearing on all leaves and gradually increasing in size. Shading experiments showed that lesions covered with aluminum foil did not spread, indicating that this mutant... lrs3 The lesions are induced by light, making it a light-dependent rice lesion mutant. Furthermore, due to the spread of the lesions, the mutant... lrs3 Most agronomic traits were significantly affected compared to wild-type Yunnan rice, especially plant height, number of tillers, panicle length and seed setting rate were significantly reduced.

[0021] This invention uses map-based cloning to clone the lesion-like mutant gene. LRS3 . LRS3 Genes are composed of LOC_ Os03g06410 The gene underwent a single base substitution, specifically the change from G to A at nucleotide position 5870 of Seq ID NO:1. This resulted in an aberration in the splicing of the 11th intron of the gene, leading to two transcripts. Transcript 1 partially retained the 11th intron, only the first 5 bp; while transcript 2 retained the 11th intron completely. Both splicing methods prematurely introduced the same stop codon, causing premature termination of translation, as shown in SEQ ID NO:6. LRS3 Abnormal gene function.

[0022] Explanation: The premature introduction of a stop codon by the LRS3 single-base mutation is the cause of the two abnormal transcripts. These are homologous abnormal transcripts resulting from differences in splicing efficiency caused by the same single-base mutation. This single-base mutation, by introducing a stop codon, disrupts the splicing regulatory elements corresponding to the downstream intron, interfering with the spliceosome's recognition and cleavage efficiency of the splice site. Furthermore, it allows the abnormal transcript to escape nonsense-mediated mRNA degradation by altering the structure of the mRNA reading frame or 3' untranslated region, thus avoiding degradation by cellular surveillance mechanisms. Due to differences in the spliceosome's cleavage efficiency of the target intron, two abnormal transcripts are ultimately formed: when the splicing efficiency is slightly low, most of the intron is cleaved, leaving only a small fragment (partial retention); when the splicing efficiency is extremely low, the intron cannot be cleaved and remains completely in the transcript (complete retention). Both are products of splicing abnormality, differing only in the degree of downstream intron retention, and both produce non-functional truncated proteins after translation.

[0023] The rice lesion gene of the present invention LRS3 Genetic engineering methods can be used to improve the disease resistance and physiological characteristics of rice, such as enhancing resistance to diseases like bacterial leaf streak and bacterial blight, regulating the intensity of plant defense responses, and improving adaptability to adversity. In-depth functional analysis of lesion-like genes further elucidates the molecular mechanisms of lesion formation in rice, the activation mechanisms of defense signaling pathways, and their synergistic regulatory relationship with photosynthetic physiology, laying a foundation for production practices. With the frequent occurrence of diseases in my country's main rice-producing areas and the increasing ecological pressure caused by the excessive use of chemical pesticides, disease-resistant and high-quality breeding has become an important direction in rice research. Studying the genetic mechanisms of lesion formation and their association with disease resistance can provide a good theoretical foundation for creating breeding materials or varieties with strong disease resistance and excellent agronomic traits, and has important guiding significance for green disease-resistant rice breeding, which is also a key focus of rice breeding under the current background of green agriculture. Therefore, this invention is of great significance for improving rice disease resistance, promoting ecological planting, and enhancing yield and quality.

[0024] In summary, in order to discover new genes related to rice lesion-like spots, this invention screened a lesion-like mutant from an EMS-induced variant library based on the japonica rice variety Yundao, and named it... lrs3 ( Leaf rust spots 3 ).right lrs3 Phenotypic analysis, photosynthetic efficiency, resistance, and cell structure analysis were performed. This invention... LRS3 Genes have broad application prospects in influencing the disease resistance of rice. Attached Figure Description

[0025] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0026] Figure 1Wild-type cloud rice and mutant lrs3 Phenotypic diagram; Figure 1 middle: A: Wild-type rice and mutants lrs3 Phenotypic diagram of tillering stage, scale bar is 10cm; B: Wild-type Yun rice and mutants lrs3 Phenotypic diagram at the heading stage, scale bar is 10cm; C: Wild type and mutant lrs3 Leaf phenotypic diagram during tillering stage, scale bar is 2cm.

[0027] Figure 2 Wild-type cloud rice and mutant lrs3 Comparison of major agronomic traits.

[0028] Figure 3 Wild-type cloud rice and mutant lrs3 Analysis of chlorophyll content and expression of genes related to chlorophyll synthesis and chloroplast development in leaves during the tillering stage; Figure 3 middle: A: Wild type and mutant lrs3 SPAD value of relative chlorophyll content during the tillering peak period; B: Wild type and mutants lrs3 Chlorophyll content during the peak tillering period; C: Wild type and mutant lrs3 Maximum photosynthetic rate during the tillering peak period; D: Wild type and mutant lrs3 Quantitative analysis of genes related to chlorophyll synthesis during the tillering peak stage; E: Wild type and mutant lrs3 Quantitative analysis of genes related to chloroplast development during the tillering stage.

[0029] Figure 4 Wild-type cloud rice and mutant lrs3 Blade surface structure; Figure 4 middle: A: Scanning electron microscope image of wild-type leaf, scale bar is 200 μm; B: Scanning electron microscope image of wild-type leaves, scale bar 100 μm; C: Scanning electron microscope image of wild-type leaves, scale bar is 10 μm; D: Mutant lrs3 Leaf scanning electron microscope, scale bar 200 μm; E: Mutant lrs3 Leaf scanning electron microscope, scale bar 100 μm; F: Mutant lrs3 Leaf scanning electron microscope, scale bar 10 μm.

[0030] Figure 5 Wild-type cloud rice and mutant lrs3 Chloroplast ultrastructure diagram; Figure 5 middle: A: Transmission electron microscope image of wild-type leaves, scale bar 5 μm; B: Transmission electron microscope image of wild-type leaves, scale bar 1 μm; C: Mutant lrs3 Transmission electron microscope image of the leaf, scale bar 5 μm; D: Mutant lrs3 Transmission electron microscope image of the blade, scale bar 1 μm; C represents chloroplast; Og represents osmophilic granules; Sg represents starch granules; Thy represents thylakoids.

[0031] Figure 6 Wild-type cloud rice and mutant lrs3 Phenotypic diagram of lesion length after inoculation with Bacillus subtilis and analysis of expression of defense-related genes; Figure 6 middle: A: Wild type and mutant lrs3 Phenotype of leaves 14 days after inoculation with Bacillus subtilis; B: Wild type and mutants lrs3 Length of lesions on leaves 14 days after inoculation with Bacillus subtilis; C: Wild type and mutant lrs3 Quantitative analysis of the expression levels of defense-related genes during the tillering peak period.

[0032] Figure 7 for lrs3 Map-based cloning of genes; Figure 7 middle: A: LRS3 Initial positioning; B~C: LRS3 Precise positioning; D: LRS3 Gene structure and mutation sites; E~F: LRS3 Single-base mutation sites and amino acid changes in genes. Detailed Implementation

[0033] To more fully explain the implementation of this invention, rice lesion mutants are provided below. lrs3 Examples of gene implementation. The described embodiments are merely some, not all, embodiments of the present invention, and are for illustrative purposes only, not for limiting the scope of the invention. All raw materials used are commercially available.

[0034] Example 1: Obtaining mutant materials The japonica rice variety Yundao was subjected to EMS chemical mutagenesis: seeds were soaked in water for 8 hours; then drained, and soaked in a pre-prepared 1% EMS buffer solution for another 8 hours, stirring frequently with a small wooden stick to ensure thorough mixing. After mutagenesis, the toxic 1% EMS buffer solution was rinsed off with running water, and the seeds were germinated at 25℃ before sowing. Single-plant transplanting and single-plant harvesting were performed, and the growing period was managed according to conventional field practices. A lesion-like mutant was obtained through screening. lrs3 .

[0035] The mutant's traits have been stably inherited through multiple generations of self-pollination. All rice materials were planted in the experimental field of the College of Biochemistry, Zhejiang Normal University, Jinhua City, Zhejiang Province, and were managed under routine conditions.

[0036] According to the above embodiment 1, Extracting wild-type rice DNA and amplifying it LRS3 The gene sequence was obtained and sequenced to obtain SEQ ID NO:1; RNA was extracted from wild-type rice, reverse transcribed to obtain cDNA, and amplified. LRS3 The cDNA sequence of the gene was obtained as SEQ ID NO:2; Extract mutants lrs3 DNA, amplification LRS3 The gene sequence was obtained and sequenced to obtain SEQ ID NO:4; Extract mutants lrs3 RNA is reverse transcribed to obtain cDNA, which is then amplified. LRS3 The cDNA sequence of the gene was obtained as SEQ ID NO:5.

[0037] Example 2: Phenotypic Analysis of Plants Under field planting conditions, mutant lrs3 It exhibits unique phenotypic characteristics: from the peak tillering stage, reddish-brown rust-like lesions begin to appear on older leaves, which continue to spread to the entire leaf as the plant grows, with the lesions spreading more severely. A small number of lesions are also distributed on the stems. Simultaneously, this mutant differs significantly from wild-type Yunnan rice in agronomic traits. At maturity, its plant height, panicle length, number of tillers, seed setting rate, and thousand-grain weight are all significantly reduced, and the grains are also significantly smaller than those of the wild type (e.g., ...). Figure 1 , 2 ).

[0038] Example 3: Chlorophyll content determination and expression analysis of genes related to chlorophyll synthesis and chloroplast development In field cultivation, the mutant material exhibited persistent lesions on its leaves from the peak tillering stage to maturity. Therefore, leaves from wild-type rice and the mutant material were collected at the peak tillering stage to determine chlorophyll content.

[0039] To analyze mutants lrs3To investigate the effects on the expression of genes related to chlorophyll synthesis and chloroplast development, RNA was extracted from wild-type rice and mutant rice during the tillering peak period, and the expression of genes related to these pathways was analyzed using RT-qPCR technology.

[0040] Total RNA extraction was performed strictly following the procedures outlined in the RNeasy Plant Mini Kit (QIAGEN) Total RNA Extraction Kit. Total RNA was isolated from leaf samples taken at the tillering stage of both mutant and wild-type rice. Subsequently, it was reverse transcribed into cDNA using the ReverTra Ace® qPCR RT Kit (TOYOBO) reverse transcription kit. Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of each gene in wild-type rice and mutants. OsActin was used as an internal control gene, and each reaction was performed in triplicate. -ΔΔCt Relative quantitative analysis was performed, and each experiment was independently repeated three times. Real-time PCR experiments were conducted using a 7500 real-time PCR system (Applied Biosystems, Life Technologies). Experimental data were statistically analyzed using Excel and SPSS 19.0 software, and differences between groups were compared using t-tests. The qRT-PCR reaction system (10 μL) consisted of: 1 μL cDNA template, 6 μL SYBR qPCR Mix (TOYOBO), 1 μL each of forward and reverse primers (10 μmol / L), and ddH2O to a final volume of 10 μL. The qRT-PCR amplification program was set as follows: 95℃ pre-denaturation for 30 s; followed by 40 cycles, each cycle consisting of 95℃ denaturation for 5 s, 55℃ annealing for 10 s, and 72℃ extension for 15 s.

[0041] Table 1. Primer sequence list for expression analysis of genes related to chlorophyll synthesis and chloroplast development

[0042] The results are as follows Figure 3 As shown, the mutant material exhibited significantly lower levels of chlorophyll a, chlorophyll b, and carotenoids compared to the wild-type rice. lrs3 Most genes involved in chlorophyll synthesis and chloroplast development showed downregulated expression, indicating that... lrs3 The mutation affected chlorophyll synthesis and chloroplast development.

[0043] Example 4: Scanning Electron Microscopy Observation Stomatal pore size is an important factor affecting photosynthesis. Scanning electron microscopy was used to study the effects of scanning electron microscopy on wild-type rice and mutant materials during their tillering peak. lrs3 The leaves were observed. The results are as follows: Figure 4As shown, the mutant leaves have a smoother surface, fewer barbs on the surface hairs, and the number of siliceous papillae around the stomata of the mutant is significantly less than that of wild-type rice.

[0044] Example 5: Observation using transmission electron microscopy To further investigate mutants lrs3 The decrease in chlorophyll content is due to the fact that during the peak tillering stage, wild-type rice and mutant rice... lrs3 Transmission electron microscopy was performed. The results are as follows: Figure 5 As shown, mutant lrs3 The chloroplasts shrink, the internal lamellar structure becomes sparse, and the thylakoids are poorly developed. More noticeably, in the mutant... lrs3 The number of osmophilic granules and starch granules in chloroplasts increased significantly, and their volume increased.

[0045] Example 6: Inoculation with Blight Coptis chinensis and expression analysis of defense-related genes Experiment with inoculation of Blightococcus pyogenes: Wild-type rice and rice in the field during their peak tillering stage were selected. lrs3 Ten plants of each pathogen were inoculated with *Bacillus thuringiensis* using the leaf-cutting method. All other culture conditions were the same (inoculation method and culture conditions refer to "Cultivation and Inoculation of *Bacillus thuringiensis* and *Bacterial Leaf Streak* of Rice," specifically, at inoculation, scissors were dipped in the bacterial solution, and the tip of the flag leaf was cut off approximately 2 cm. At least 5 flag leaves were inoculated from each plant. An investigation was conducted 14 days after inoculation, with at least 5 flag leaves from each plant investigated). After 14 days of treatment, wild-type Yun rice and... lrs3 The length of white leaf blight spots. Additionally, samples were extracted from wild-type rice and the mutant. lrs3 RNA from the peak tillering stage was reverse transcribed into cDNA, and the expression levels of defense-related genes were analyzed.

[0046] The results are as follows Figure 6 According to Figure 6 It can be seen that after inoculation with Bacillus subtilis, the length of bacterial blight lesions in wild-type rice was significantly longer than that in the mutant. lrs3 The expression levels of defense-related genes were detected by RT-qPCR, and it was found that in the mutant... lrs3 The expression levels of most defense-related genes in the brain were significantly increased, indicating that... lrs3 Mutations in this substance can lead to increased disease resistance.

[0047] Table 2. Primer sequence list for defense-related gene expression analysis

[0048] Example 7: Population Construction and Genetic Analysis mutant lrs3 Reciprocal crosses were performed with conventional indica rice varieties TN1 and 9311, and the F1 plants all exhibited the normal wild-type phenotype, indicating that...lrs3 Controlled by recessive nuclear genes. The segregation ratio of the F2 segregating population was statistically analyzed (Table 3). The results showed that the segregation ratio of plants with the normal phenotype to those with the mutant phenotype was approximately 3:1 after a chi-square test, indicating that... lrs3 The lesion-like phenotype is controlled by a pair of single recessive nuclear genes.

[0049] Table 3. Lesion Mutants lrs3 Genetic analysis

[0050] Example 8 lrs3 Fine mapping of genes Using SSR primer pairs evenly distributed across the 12 chromosomes of rice preserved in our laboratory, mutants lrs3 Polymorphic screening was performed on the background rice varieties Yun rice and 9311. Then, linkage analysis was performed on 21 lesion-like single plants from the F2 generation to preliminarily confirm the chromosomal location of the target gene. Genomic DNA was extracted using the CTAB method. The specific steps are as follows: ① Weigh 0.1 g of rice leaves and grind them into powder using liquid nitrogen. Then add 500 ml of DNA extraction buffer prepared with CTAB solution (2% m / V), 100 mmol / L Tris-HCl, 20 mmol / L EDTA, and 1.4 mol / L NaCl (pH 8.0), and incubate at 65°C for 40 minutes. Add 500 ml of chloroform:isoamyl alcohol (24:1 volume ratio) and mix well. Centrifuge at 10,000 rpm for 10 minutes and transfer the supernatant to a new centrifuge tube.

[0051] ② Add 2 / 3 to 1 volume of pre-cooled (to 4°C) isopropanol to the supernatant obtained after centrifugation in step ① above, and gently mix until DNA precipitates. Centrifuge at 13,000 rpm for 10 minutes and discard the supernatant.

[0052] ③ Wash the DNA precipitate obtained in step ② with 200 ml of 75% (volume concentration) ethanol.

[0053] ④ Dry the washed DNA and dissolve it in 100ml of TE buffer or pure water.

[0054] ⑤ The concentration of the DNA sample obtained in step ④ above was determined by ultraviolet spectrophotometry, and the integrity of the DNA was detected by 0.8% agarose gel electrophoresis. Complete and suitable DNA was used for PCR amplification, while incomplete DNA was extracted again until complete DNA was obtained.

[0055] The PCR reaction system used was a 10 μL system: 1 μL DNA template, 0.5 μL each of forward and reverse primers (10 μmol / L), 5 μL Taq enzyme, and ddH2O to make up to 10 μL. The PCR amplification program was as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 30 s, annealing at 55℃~60℃ for 30 s (temperature varies depending on the primers), extension at 72℃ for 30 s, for 35 cycles; and final extension at 72℃ for 10 min.

[0056] PCR products were subjected to 4% agarose gel electrophoresis. After electrophoresis, images were taken using a gel imaging system and the gel was read. The 120 pairs of SSR primers selected above were then used for... lrs3 Gene linkage analysis revealed linkage at B3-4 and B3-7 on chromosome 3. New Indel markers were designed upstream and downstream of these linkage markers, and these 21 single plants were used to locate the target gene region between molecular markers M2 and M10. New molecular markers were then designed again within this region, and 224 F2 single plants were used to finally locate the gene within a 78.249 kb region between M4 and M5. Primer sequences are shown in Table 4.

[0057] Table 4 Molecular markers used for fine localization

[0058] According to data from the Rice Genome Database (http: / / rice.plantbiology.msu.edu / ), the accession number is... LOC_Os03g06410 This indicates that the gene is a candidate gene. Primers covering the gene region were used to amplify wild-type rice and the mutant, respectively. lrs3 DNA sequencing results showed that lrs3 A mutation in the first bit of the 11th intron of a gene, from G to A, leads to abnormal splicing of that intron, prematurely introducing a stop codon and causing a mutation in gene function. (e.g.) Figure 7 ).

[0059] Rice lesion gene LRS3 The DNA sequence of wild-type Osmanthus fragrans is as described in SEQ ID NO:1, the cDNA sequence is as described in SEQ ID NO:2, and the amino acid sequence is as described in SEQ ID NO:3. Mutant material. lrs3 The coding region of this gene has a mutation at position 5870, where a G is replaced by an A. The mutated DNA sequence is shown in SEQ ID NO:4, and the cDNA sequence is shown in SEQ ID NO:5. This mutation results in a change in the structure of the encoded protein, and the mutated amino acid sequence is shown in SEQ ID NO:6. The present invention obtains… LRS3 Base substitutions in the gene cause a phenotype of reddish-brown rust-like spots on the leaves, stems, and grains of rice plants.

[0060] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. Finally, it should be noted that the above are merely several specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. Rice lesion gene LRS3 Its features are: Rice lesion gene LRS3 The DNA sequence is as described in SEQ ID NO:

1.

2. The rice lesion gene according to claim 1 LRS3 Its features are: Rice lesion gene LRS3 The cDNA sequence is as described in SEQ ID NO:

2.

3. The rice lesion gene as described in claim 1 or 2 LRS3 The encoded protein is characterized by: The amino acid sequence is as described in SEQ ID NO:

3.

4. Mutant materials lrs3 The gene is characterized by: The mutant DNA sequence is as described in SEQ ID NO:4, and the cDNA sequence is as described in SEQ ID NO:

5.

5. Rice lesion genes LRS3, Mutant materials lrs3 The use of the gene is characterized by: It affects the growth and development of rice.

6. Rice lesion genes LRS3, Mutant materials lrs3 The use of the gene is characterized by: Modifying agronomic traits and applying them to rice breeding.

7. The rice lesion gene according to claim 5 or 6 LRS3, Mutant materials lrs3 The use of the gene is characterized by: It affects the plant height, number of tillers, panicle length, seed setting rate, and grain size of rice.

8. The rice lesion gene according to claim 5 or 6 LRS3, Mutant materials lrs3 The use of the gene is characterized by: The agronomic traits include reddish-brown rust-like lesions.

9. A method for regulating the growth and development of rice and altering its agronomic traits, characterized in that: Using the rice lesion gene as described in claim 1 or 2 LRS3, The mutant material as described in claim 4 lrs3 The genes.

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