CN112175973A - Rice scab control gene SPL36 and application thereof - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to a method for cloning a rice SPL36 gene by using a map-based cloning technology, identifying the function of the gene by using a transgenic complementation test, and simultaneously, regulating and controlling the separation and the application of a rice cell programmed death gene SPL36 coding region. The invention discloses a rice scab gene SPL36 for regulating programmed cell death and disease resistance, wherein the amino acid sequence of the encoded protein is shown as SEQ ID NO: 4, respectively. The nucleotide sequence of the gene is shown in SEQ ID NO. 2. The invention also provides application of the rice disease spot gene SPL36 in improving disease resistance of plant varieties.

Description

Rice scab control gene SPL36 and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a method for cloning a rice SPL36 gene by using a map-based cloning technology, identifying the function of the gene by using a transgenic complementation test, and simultaneously, regulating and controlling the separation and the application of a rice cell programmed death gene SPL36 coding region.

Background

Plant scabs (vegetative scabs) are scabs that spontaneously develop in different sizes and shapes on leaves and leaf sheaths, even stalks and seeds, in the absence of both abiotic and biotic stresses. These lesions are caused by apoptosis due to allergic reaction (HR), a disease-resistant reaction typical of rapid necrosis of cells occurring after incompatible interaction between plants and pathogens, a reaction that is spontaneous to plants and involvesAnd Programmed Cell Death (PCD)^[1]. In addition, most of the defense genes in lesion spot mutants are expressed, so that the mutants show strong disease resistance to some plant pathogens. Therefore, the lesion-like mutant has important significance and value for researching plant defense stress response mechanism and PCD^[2]。

SPL7 is the starting point for the cloning of a lesion-like gene, which is the first lesion-like mutant gene successfully cloned in rice. HSFA4 is a heat shock protein transcription factor encoded by spl7, and plays a negative regulation role in apoptosis pathway^[3]. SPL7 is highly homologous with corn HSFb, tomato HSF8, Arabidopsis HSF21 and HSF1, and the mutants have the functions of regulating apoptosis of cells and showing the characteristics of scab-like spots in plants. The mutant can start a corresponding system to obtain the expression of related genes, and has certain influence on the disease resistance of the plant. Although the rice lesion spot mutants have the function of improving the disease resistance of plants, some mutations also have certain harm to the plants. For example, the leaves of the mutant lms1 begin to appear yellow brown spots in the jointing stage, the spots spread to the whole leaves and even stems along with the growth of the plants, and the symptoms of senescence appear after the plants grow to the heading stage, the stems, leaves and ears obviously dry up and die rapidly^[4]. In addition, partial lesions have been found to even cause death in rice^[5]. Li Xiulan et al^[6]Genetic analysis of C23 revealed a new gene, which was tentatively designated as spl29(t), and the mutant had an extended heading period, a reduced plant height, a reduced number of grains per panicle and effective tillers, a reduced thousand kernel weight and a reduced seed set. The disease-like spots of spl1, spl2 and spl5 are accompanied by plant development retardation, and sterility and yield reduction of spl3 and spl4 occur^[7]。

The generation mechanism of rice scabs is relatively complex, and the generation of the scabs is closely related to factors such as enzymes, signal molecules and PCD. The abnormal expression or mutation of disease-resistant genes in rice can cause the abnormal expression of defense-related genes, thereby causing the disorder of defense reaction signal paths and finally causing the death of cells on plantsDeath leaves a plaque-like lesion. For example, the appearance of the phenotype of spl18 is related to the insertion of a T-DNA activation tag, the insertion of the T-DNA activation tag has an enhancement effect on the expression of genes around an insertion site, a gene OsATL similar to an acyltransferase sequence inducing anaphylactic reaction in tobacco is arranged at about 500bp downstream of the inserted T-DNA activation tag, the expression level of the OsATL is lower in wild rice, but the expression level is higher in spl18^[8]This is the occurrence of disease-like spots caused by abnormal expression of typical rice disease-resistant genes. Zeng et al^[9]Through studies on the spl11 mutant, it was demonstrated that the protein of spl11 comprises a U-box and ARM (armadillo) repeat domain, which can ubiquitinate and interact with proteins in yeast and mammalian systems. Finally, comparison of the amino acid sequences indicated that a single base substitution was detected in the spl11 mutant gene, which resulted in premature termination of translation of the spl11 protein.

The previous research on rice scab-like mutants lays a foundation for a regulation and control network of scab-like formation mechanism and defense-related reaction, but the molecular mechanism of the rice scab-like mutants needs to be further researched,

the references referred to above are as follows:

1. xianheng swallow, the nature of the face, Chua Ying frequently, Wang Hui, Gong Yuhua, the research progress of programmed cell death of plants, biological technology communication, 2008,19(2): 296-;

matrix M N, Pandeya D, Baek K H, Dong S L, Lee J H, Kang H, Kang S G, photosynthetic and genetic analysis of rice deficiency mutants plant Pathol J,2010,26(2):159-169 (matrix M N, Pandeya D, Baek K H, Dong S L, Lee J H, Kang S G. phenotypic and genotypic analysis of rice lesion mutants plant journal, 2010,26(2): 159-169);

3. inspiring the application of the rice SPL7 gene in regulating plant type CN 106811471 A.2017;

4. forest shoujin, Chen Jie, Tianda gang, Yangman, Yanshaohua, Liuhuaqing, Chensong Biao, Wangfeng, identification of rice scab and presenility mutant lms1 and preliminary gene location, Fujian agricultural science, 2014,29(1): 29-34;

5. chenjian, Zhao Zeng Lin, Zhang Shihong, Panhong Yu, a preliminary study of rice T-DNA insertion lesion-like mutant, Gill agriculture university report, 2008,30(2): 133) 137;

6. li Xiulan, Wang Ping, Quzhi, Sun autumn, Wang Bing, Deng Xiao Jiang, Rice scab mutant C23 genetic analysis and gene fine location, Chinese agricultural science, 2010,43(18): 3691-;

yin Z, Chen J, Zeng L, Goh M, Leung H, Khush G S, Wang G L, Charactering rice deficiency mutants and identification a mutant with branched-span resistance to rice blast and bacterial leaf blight, 2000,13(8): 869-containing 876(Yin Z, Chen J, Zeng L, Goh M, Leung H, Khush G S, Wang G L. identification of a rice-like lesion mutant with broad-spectrum resistance to rice blast and bacterial leaf blight. Plant molecular microbial interaction, 2000,13(8): 869-containing 876);

mori M, Tomita C, Sugimoto K, Hasegawa M, Hayashi N, Dubouzet J G, Ochia H, Sekimoto H, Hirochika H, Kikuchi S.isolation and molecular characterization of a Spotted leaf 18 mutant by modified activation-tagging in rice plant Mol Biol 2007,63(6):847-860(Mori M, Tomita C, Sumogio K, Hasegawa M, Hayashi N, Dubouzet J G, Oai H, Sekimoto H, Hirochika H, Kikuchi S. rice leaf spot leaf 18 mutant isolation and molecular characterization studies, 2007, 847-847);

9. the induction and preliminary analysis of Wangzhong, Jiayanlin, rice lesion mutant lmm1, Nuclear agriculture report 2006,20(4): 255-258.

Disclosure of Invention

The invention aims to provide a rice scab control gene SPL36 and application thereof.

In order to solve the technical problems, the invention provides a rice scab gene SPL36 for regulating and controlling programmed cell death and disease resistance, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

Further, the nucleotide sequence also includes mutants, alleles or derivatives produced by adding, substituting, inserting or deleting one or more nucleotides.

The invention also provides a protein coded by the rice scab gene SPL36, and the amino acid sequence of the protein is shown as SEQ ID NO: 4, respectively.

Further, the above amino acid sequence also includes an amino acid sequence produced by adding, substituting, inserting or deleting one or more amino acids or homologous sequences of other species, or derivatives thereof.

The invention also provides a plant expression vector or a host cell line containing the rice scab gene SPL 36.

As an improvement of the host cell line of the present invention: the host cell line is an escherichia coli cell, an agrobacterium cell or a plant cell.

The invention also provides a pCAMBIA1300 complementary vector fused with the rice lesion control gene SPL36, and the sequence is shown as SEQ ID NO. 5. The gene is fused with a genome DNA segment containing a promoter of the SPL36 gene in wild type oryza sativa.

The invention also provides the application of the rice disease spot gene SPL36 in improving the disease resistance of plant varieties.

As an improvement of the application of the invention: the plant variety is rice, and the disease is bacterial disease (preferably rice bacterial blight). The breeding method is to inoculate the mutant spl36 by leaf cutting. The leaf-cutting inoculation is carried out at the tillering stage.

The amino acid sequence of the rice lesion spot mutant corresponding to the wild type is SEQ ID NO: 1.

the invention also provides a plasmid containing the gene, and engineering bacteria or host cells containing the gene or the vector.

The engineered bacteria and host cells are understood to be those used by the skilled person in the transgenic process. However, with the development of science and technology, the selection of the engineering bacteria and the host cells may be changed, or in the application field of non-transgenic purpose, the utilization of the vector and the engineering bacteria is also related, but the invention is within the protection scope as long as the gene or the vector of the invention is contained.

The invention also aims to provide the application of the gene, the protein and the recombinant vector in improving the disease resistance of rice. The mutation of the gene or the protein can regulate and control programmed death and disease-resistant defense reaction of rice cells, and the resistance of the rice to bacterial blight of rice can be improved by using the gene or the protein.

The preparation of the transgenic rice is a conventional technical means in the field, the invention is not limited, and the technical scheme of utilizing the gene to perform rice transgenosis is within the protection scope of the invention.

The invention relates to a rice scab control gene SPL36 and a molecular mechanism thereof for mediating cell death and disease-resistant defense reaction. In order to deeply analyze the molecular mechanism of HR mediated cell death and disease-resistant defense reaction of a plant body, the inventor separates and identifies a new rice lesion mutant from an EMS mutagenesis mutant library of a rice variety Yundio and the new rice lesion mutant is named as spl36(spotted leaf 36). The mutant exhibits spotted leaves and increased resistance to bacterial blight. The SPL36 gene is obtained by map-based cloning, and encodes a receptor-like protein kinase 2 precursor. The inventor utilizes a transgenic complementation test to identify the functions of genes and finds that the genes related to the induced defense reaction are obviously up-regulated after the mutant generates the scab-like spots, namely the defense reaction of the plant is activated, and further, the resistance to pathogenic bacteria is obviously enhanced.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

the invention identifies a novel scab-like mutant from EMS mutagenesis mutation population of wild type oryza sativa and is named as spl 36. The mutant begins from the tillering stage, leaves begin to have a scab-like phenotype and gradually expand to the whole leaves, and the programmed cell death characteristics are presented. The expression of disease defense related genes such as MAPK12, WRXY53, BIMK2, AOS2, ASP90, LYP6, PR2, PR1a, PR1b and the like in the mutant spl36 is obviously up-regulated, and the resistance to bacterial blight is obviously enhanced. LOC _ Os12g08180 of the gene for controlling the phenotype of the scab is separated by using a map-based cloning method, and the LOC _ Os12g08180 is proved to be the target gene SPL36 for controlling the formation of the scab by a functional complementation experiment. The mutant spl36 and the target mutant gene thereof are beneficial to cultivating a new rice material and a new variety with broad-spectrum and lasting disease resistance.

The invention has the following beneficial effects:

the invention preliminarily analyzes the biological function of the SPL36 gene and provides a theoretical basis for clarifying the programmed cell death mechanism of plants. The mutant gene of the SPL36 is introduced into rice by means of conventional hybridization, molecular marker-assisted selection or genetic engineering and the like by utilizing the rice lesion mutant SPL36 and SPL36 mutant genes, so that the resistance of the rice to bacterial diseases (such as bacterial blight of rice and the like) can be obviously enhanced. The invention has important theoretical and application values for further clarifying the molecular mechanism of plant PCD and disease-resistant defense reaction and cultivating broad-spectrum durable disease-resistant varieties.

In conclusion, the gene SPL36 for controlling the scab is separated and cloned by a map locus technology, the gene codes a receptor-like protein kinase precursor, the mutation of the receptor-like protein kinase precursor causes cell programmed death to cause the formation of the scab, the resistance to bacterial diseases (bacterial blight of rice) is obviously enhanced, and the function of the gene is identified by a transgenic function complementation experiment.

Drawings

FIG. 1 shows a comparison of the wild type and mutant spl36 phenotypes;

wherein A is the field phenotype of wild type and mutant in tillering stage; b is upper-table contrast of wild type and mutant leaves; c is the phenotype 5 and 10 days after the mutant was untreated and shaded with tinfoil.

FIG. 2 shows Tunel experiment and physiological and biochemical index measurement of wild type and mutant leaf;

wherein A, B, C and D are fluorescence results of wild type and mutant leaf Tunel experiments respectively; e is hydrogen peroxide (H) from wild type and mutant leaves₂O₂) Content (c); f is the Malondialdehyde (MDA) content of wild type and mutant leaves; g is wild-type and mutant leaf Peroxidase (POD) activity; h is the activity of wild type and mutant leaf superoxide dismutase (SOD).

FIG. 3 is analysis of expression of defense-associated genes and identification of disease resistance;

wherein, A, B, C and D are phenotypes of the wild type and the mutant at 5 days and 10 days after inoculation of the bacterial strain; e is the average lesion length of wild type and mutant after 5 and 10 days after B.subtilis inoculation, data represent the average of 10 biological replicates. + -. standard deviation SD (. P <0.05,. P < 0.01; Student's t test); f is qRT PCR analysis of defense-related genes.

FIG. 4 is a map of the isolation and identification of the plaque-like gene SPL36 by using map-based cloning.

FIG. 5 shows the plant transformation and complementation function verification;

wherein A is a phenotype map and a leaf map of a wild type Yunnan rice, a scab-like mutant spl36 and a transgenic plant pGSPL36 transformed into a complementary vector at a tillering stage; b is the result of identifying transgenic plant pGSPL36 transferred with complementary vector and transgenic plant pEmv hygromycin transferred with empty vector.

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

Detailed Description

The invention is further described below with reference to specific examples. The following examples are intended to further illustrate the invention and are not to be construed as limiting the invention. In the examples, unless otherwise specified, all experimental methods used were conventional methods; the experimental materials, reagents and the like used are commercially available.

Example 1: acquisition of mutants and their phenotypes

Through EMS chemical mutagenesis of japonica rice variety Yundio, soaking the Yundio seeds to be treated in clear water for 8 hours, drying in the air at room temperature, completely soaking the seeds to be treated in 1% EMS solution, treating at 28 ℃ for 10 hours, washing the treated seeds with tap water for 10-12 hours, finally performing germination accelerating seeding, and identifying a scab-like mutant in a mutagenesis mutant library and naming as spl 36. When the mutant is in a tillering stage, a small amount of irregular reddish brown spots appear on leaves, and then the spots gradually increase and the area becomes larger and the spots are spread on the whole leaves, so that the mutant presents a scab-like phenotype. As the plants grew, reddish-brown spots appeared on each leaf (fig. 1A, fig. 1B). When a small amount of rufous spots appear on the leaf of the mutant spl36, the rufous spots on the leaf can continuously spread without treatment; after a small amount of reddish brown spots are generated on the leaves, the leaves are covered by tinfoil paper, and the size and the density of the spots at the covered parts cannot be increased;

even when the red brown spots are full of the whole leaf, the shading part is yellow due to lack of photosynthetic products, but the red brown spots are not generated, which shows that the generation of the disease spots is regulated by illumination (figure 1C), and all the rice materials are planted in the test fields of the biochemical institute of Zhejiang university and Hangzhou China rice research institute in Zhejiang province and are managed conventionally.

Example 2: agronomic traits of wild type and mutants

Wild type Yunya rice and the mutant spl36 were planted in Hangzhou season, and agronomic character examination found that the plant height and thousand seed weight of the mutant were significantly reduced and the number of seeds per ear was significantly reduced compared to the wild type (Table 1).

TABLE 1 comparison of major agronomic traits in wild type oryza sativa and mutant spl36

And indicate the significance of differences in the mutant compared to the wild type at P <0.05 and P <0.01 levels, respectively.

Example 3: cell death and related physio-biochemical indicator assays for mutants and wild-type

The principle of the TUNEL (TdT-mediated dUTP Nick-End Labeling) method for detecting apoptosis is that cells can activate some DNA endonucleases which cut off genome DNA between nucleosomes when apoptosis occurs. When the genomic DNA is fragmented, the exposed 3' -OH can be detected by fluorescence microscopy by adding fluorescein-dUTP (fluorescein-dUTP) labeled with Fluorescein (FITC) under the catalysis of Terminal Deoxynucleotidyl Transferase (TdT).

The mutant spl36 leaf had more positive fluorescence results compared to the wild type, indicating that the mutant leaf had more cells that had died or are dying in progress, while the wild type leaf wasThe positive fluorescence results in the tablets were less, indicating that essentially no cell death occurred in the wild type (FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D). By means of H₂O₂The kit, the MDA kit, the POD kit and the SOD kit are used for measuring corresponding indexes in the mutant spl36 and wild type Yunnan rice leaves, and the results show that hydrogen peroxide (H) in the mutant spl36 leaves₂O₂) The content and Malondialdehyde (MDA) content were significantly increased (fig. 2E, fig. 2F), while Peroxidase (POD) and Superoxide Dismutase (SOD) activities were significantly decreased in the mutant spl36 leaf (fig. 2G, fig. 2H), indicating that the mutant spl36 leaf had more active oxygen accumulation and cell death than the wild type.

Example 4: expression and resistance identification of defense-related genes

To test whether the mutants activated the defence response, the expression levels of genes on several different resistance signalling pathways were tested using the qRT-PCR method. Total RNAs of leaves at the tiller stage wild type and at the time of the emergence of a lesion-like lesion were extracted using the RNAprep Pure Plant Kit (DP441) from Tiangen Biochemical company, and the first strand cDNA was synthesized using ReverTra qPCR RT Master Mix gDNA remover FSQ-301 (TOYOBO). The rice OsActin gene is used as an internal reference, and qRT-PCR primers are shown in Table 2. As a result, the mutant remarkably induces the expression of resistance related genes after generating the scab-like spots, and the expressions of MAPK12, WRXY53, BIMK2, AOS2, ASP90, LYP6, PR2, PR1a and PR1b are all remarkably up-regulated (FIG. 3F), which indicates that the generation of the scab-like spots activates the defense response of the plant per se.

The leaf cutting inoculation of the rice bacterial leaf blight strain HM73 (from China Rice research institute) on the wild type and the mutant results in obviously shortened lesion length of the mutant leaf (FIG. 3E) and obviously better resistance than the wild type (FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D). These results indicate that the mutant has significantly improved resistance to bacterial diseases and shows broad-spectrum resistance characteristics.

TABLE 2 defense-related genes qRT-PCR primers

Example 5: identification and isolation of the SPL36 Gene

Genetic analysis was performed on the mutant spl36 obtained in example 1, and the mutant spl36 and conventional indica TN1 and ZF802 were respectively subjected to cross-breeding, F₁All plants showed normal wild type phenotype, indicating that spl36 is controlled by a recessive nuclear gene. Statistics F₂Segregating population segregation ratios (table 3) indicate that the segregation ratio for plants of the normal phenotype and plants of the mutant phenotype approaches 3:1 segregation by chi-square test, indicating that the lesion-like phenotype of spl36 is controlled by 1 pair of single recessive nuclear genes.

TABLE 3 genetic analysis of mutant spl36

The 262 pairs of SSR primer pair mutants uniformly distributed on 12 rice chromosomes and TN1 stored in the laboratory are utilized to carry out polymorphism screening, and 126 pairs of SSR primers are screened to have polymorphism. Then using 21 spl36/TN₁Middle F₂And (4) carrying out linkage analysis on the individual plant with the similar lesion spots to preliminarily confirm the chromosome position of the target gene. The genomic DNA was extracted by the CTAB method. The method comprises the following specific steps:

0.1g of rice leaves are weighed and ground into powder by using liquid nitrogen, then DNA extraction buffer solution prepared by 600 mul of CTAB solution (2% (m/V) CTAB, 100mmol/L Tris-Cl, 20mmol/L EDTA, 1.4mol/L NaCl, pH8.0) is added, and water bath at 65 ℃ is carried out for 40 minutes. Then 600. mu.l of chloroform/isoamyl alcohol (24:1 by volume) was added thereto and mixed well. Centrifuge at 10,000rpm for 5 minutes and transfer the supernatant to a new centrifuge tube.

And secondly, adding 2/3-1 times volume of precooled (to 4 ℃) isopropanol into the supernatant obtained after centrifugation in the step I, and lightly and uniformly mixing until DNA precipitates. Centrifuge at 13,000rpm for 8 minutes and pour the supernatant.

③ washing the DNA precipitate obtained in the step (c) with 200. mu.l of 70% (volume concentration) hexanol.

The washed DNA was air-dried and dissolved in 100. mu.l of TE buffer or pure water.

Fifth, ultraviolet spectrophotometry detects the concentration of the DNA sample obtained in the fourth step, and 0.7% agarose gel electrophoresis detects the integrity of the DNA. The intact DNA was used for PCR amplification and the incomplete DNA was re-extracted until the intact DNA was obtained.

The PCR reaction system adopts a10 mu L system: DNA template 1. mu.L, 10 XPCR buffer 1. mu.L, forward and reverse primers (10. mu. mol/L) each 0.5. mu.L, dNTPs 1. mu.L, rTaq enzyme 0.2. mu.L, add ddH₂O make up to 10. mu.L. The PCR amplification procedure was as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 55-60 ℃ for 30s (the temperature is different according to different primers), and extension at 72 ℃ for 30s for 40 cycles; and finally extension at 72 ℃ for 10 min. The PCR product was electrophoresed on a 4% agarose gel, and after the electrophoresis was completed, the gel was photographed and read on a gel imager. SPL36 gene linkage analysis was performed on SSR primers using 126 selected above, and these 21 individuals were used to lock the gene region of interest between molecular markers B12-5 and B12-6, where new molecular markers were developed, and 148F were used₂The individual locates the gene in JHL-3 and JHL-7 intervals, and new molecular markers are designed in the interval and 554F genes are used₂The individual finally mapped the gene within an interval of approximately 60kb between Indel1 and Indel2 (FIG. 4). The primer sequences are shown in Table 4.

TABLE 4 molecular markers for Gene mapping

A total of 11 Open Reading Frames (ORFs) were found based on the data information of the rice genomic database (http:// rice. plant biology. msu. edu /). Among them, 7 expression proteins and 4 functional proteins are included. The genome sequence of mutant and wild type was amplified by PCR, and sequence analysis showed that LOC _ Os12g08180 had a mutation, and nucleotide C at 1462 of the coding region of the gene was replaced with T, resulting in the change of the encoded amino acid from arginine to cysteine (FIG. 4).

The nucleotide sequence of the rice scab gene SPL36 is SEQ ID NO: 2, the nucleotide sequence of the wild type Yunnan rice corresponding to the rice lesion spot mutant is SEQ ID NO: 1.

the amino acid sequence of the protein coded by the rice lesion mutant gene SPL36 is shown as SEQ ID NO: 4, the method is described in the specification. The amino acid sequence of the protein coded by the wild type Yunnan rice is shown as SEQ ID NO: 3, the preparation method is as follows.

Description of the drawings: SEQ ID No: 1 and SEQ ID No: 2 is a gene sequence comprising an exon and an intron; while only the exon regions of the encoded protein.

Example 6 plant transformation and complementation function verification

Amplifying a genomic DNA fragment containing a promoter of the SPL36 gene in the Oryza sativa, wherein the sequence is shown as SEQ ID NO: 5. then, the DNA fragment was ligated into pEASY-Blunt Cloning Vector (TransGen Biotech Co.), followed by ligation into pCAMBIA1300 complementary Vector. This plasmid was transformed into rice by electric shock method into Agrobacterium tumefaciens strain EHA 105. Callus induced by SPL36 mature embryo is cultured in inducing culture medium for 2 weeks, and then selected as transformation receptor. The rice calli were infected with EHA105 strain containing binary plasmid vector (pCAMBIA 1300-SPL 36), co-cultured in the dark at 25 ℃ for 2 days, and then cultured in screening medium containing 50mg/L of Hygromycin for about 14 days (light intensity 13200LX, temperature 32 ℃). Transferring the pre-differentiated callus to a differentiation culture medium, and culturing for about one month under the illumination condition (the illumination intensity is 13200LX, the temperature is 32 ℃) to obtain a resistant transgenic plant. The phenotype of the complementary seedling plant is observed and analyzed, the hygromycin resistance is verified to be the accuracy of the complementary seedling, and finally, the leaves of the complementary seedling transferred into the complementary vector are found to be restored to be normal without a scab-like phenotype (figure 5), so that the gene LOC _ Os12g08180, namely the target gene, is verified.

And (4) verifying hygromycin resistance, wherein if the plant is alive, the plant is proved to have hygromycin resistance, and if the plant is dead, the plant is not proved to have hygromycin resistance.

Description of the drawings: each of the media mentioned above (induction medium, screening medium, differentiation medium) is a conventional medium.

Example 7 application of Rice Patch Gene SPL36 in Rice Breeding

In production practice, the above genes can be used to transform plant cells, and the transformed plant cells can be cultivated into plants. By the transgenic method, the plant expression vector is used for transforming plant cells to increase the disease resistance of rice and improve rice varieties.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

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atagtatcca tacctctgca attgatttcg tgaaagcaat ggaagcagtc tccaatcaca 2280

gcaacatttt tctgaaaaca aggttctgca cttactataa ggctgtgatg cctaatggct 2340

caacctattc tttaaaacag attaattgta gtgacaagat attccaaatt gggagccaag 2400

ggaaggttgc tcatgaactt gaggtacttg ggaagttgag caattccaat gtaatggtgc 2460

cattggctta tgtgttgaca gaagacaatg catacatcat ctatgagcat gtgcacaagg 2520

gcacggtgtt cgatttcctt catgctggaa gatcagatgt tctggactgg ccttcacggt 2580

atagcatagc ttttgggcta gcccaagggc tgacatttct tcatgggtgc actcagccag 2640

ttctgcttct tgatctgtca acaaggactg tccacttgaa gtcaatgaat gagcctcaga 2700

ttggagatgt tgaactttac aaaattgttg atactttgaa gagcagtggg agcctttcga 2760

ccattgctgg tacagttggt tatattccac caggtaatgg actgttctgg ttcaatttga 2820

gtgcataatt atcaacttaa gcagttaaac tgaaactgct gatagtggtt atttactgtt 2880

ctctgttgat ttgatctatt tatagttcat atctatgttt tcaagtaatt tacttgaggc 2940

aattctgttt gcattacctg catacttcaa ttgaccacaa tcctgtctac actcctatgc 3000

tagttttgtt ttcagaattt ctggagaaaa catatttgct ttgtttccca tgggttgtcg 3060

tctactacat ttgtagtatc atccaaaaaa ggggctttta actatttgcc actcttacgg 3120

atgttgctta atgatttacc actaggccca catgtcatag acacatgaga gcccacatgt 3180

cattgacagg gtgtggcaaa tcgttaactg ccatgtcaca aaagtggcaa atagttaaat 3240

ttccctccaa aataagcaca tctcctactt ttgacaaatc taataaaaaa cctagcttct 3300

atgatcttgt ccttcagtac actagcacaa aatgatttga cgctttccga gatctatatg 3360

agatgagatt tatactgaag ctaaattttg ttgaactatg ttgcagagta tgcatacacg 3420

atgaggttga cgatggctgg caacgtttac agctttggag taatcttact ggagcttttg 3480

actgggaaac catcggtcag tgatggcatc gagttagcca agtgggctct gagtctttca 3540

ggcagccctg atcagaggga gcagatcctt gacaccaggg tctcaagaac ttcagctgct 3600

gttcacagcc agatgttgtc ggtcctgaac atcgccctcg cttgtgttgc gctctctcca 3660

gatgctcgac caaagatgcg caccgtcttg aggatgctat tcaacgcaaa gtgatctggc 3720

atgatatgtt ttggtagctt aggtaggttc tgaatggagt ttgatcattt tgcttagttt 3780

gtggatgtca gcaatcagta tacggggtcc ttgtatagta ccagcaccat agttcaaggt 3840

agagtagtag attatgatat ggctgatcaa tttagtattc agatatctat tttggaatga 3900

atgaccaatg ggtttatcta tgctgtatca tcatatattc acacatggtt attagcatac 3960

cttgtacacg gcaattccat ataacaatag tagtgaaata gagccatagt taaggtagaa 4020

cagtagatag actatgatat atatccctga acaatttaga agccagatat ctgttttgga 4080

atgaatcagg aattgtgttc ttctatgcta tatgatcata tttgtaaatg gttggttgtg 4140

tacatccatg tgggatgtcg aggccggatt ggcatccatt atctaacaaa catatatata 4200

aattgttatt acttattagc 4220

<210> 2

<211> 4220

<212> DNA

<213> Rice (Oryza sativa. L)

<400> 2

actgggagtc tgggaggagc aagaagagga gagaagagag ccatggccaa gaacaccgag 60

agccccaaaa cctagggaaa gagaggaaaa aatcgatttt cttgggtggt ggttgagcct 120

gcagtgaagt tcatcaggga ggaaagggag gggattaagg gaaggagata caggaatcaa 180

tgggaggcca agaacaggtg gccatggcgg agaggaggcc ttgttactgt cgcaacaatg 240

gcttcttgct gctgctggta caagttctcc tcttcttgtt cttgtttctt gattcgtcca 300

tggtcgccac tgccgcgtcg gcgccggcac cgctaaatac gactcaggta tcgatcatga 360

aggagctttc tggcttggtt actgccagtg ccaagtggaa cacgagcgat tcgaatccat 420

gccgatggga tggagtgagt tgctcttcta gctccaattc catatcagtt gtgaccaacc 480

ttactttgtc tgggtatggt ttgtccaact ccaccatatt tgcaaccata tgctctcttg 540

acaccttgca aatccttgat ctctctaaga attccttcac caattcgata gagcaattct 600

tcacctcttc ttgctccatg aaggctggat tgcggtcact taatctcagc agcagccagc 660

tatccatgcc tcttagtaat ttctctggtt ttccccttct tgaggttctt gacttgtcct 720

tcaattcttt cagtggtgat gtcagaaccc agttgagttc tttgctcaag ctgagaagct 780

tgaatcttag tagcaataac ttggctggtg atgttcctac aagcatgacc ccgtctttgg 840

aggaattggt gttgtccatc aacaatttca gtggtagcat tccaatagct ttgttcaatt 900

accaaaatct tactatgctg gatcttagtc agaacaatct aaatggtgat gtaccggatg 960

agttcttaaa gttacccaag ctcaagactt tgctcttatc aggtaatcaa ctgagtggca 1020

atatacctgt gagtgtgtca aatgttgcaa gccttgctcg gtttgcagct aatcagaaca 1080

attttaccgg tttcatccct agtggtatca ccaagaatgt gaagatgctg gatctgagtt 1140

acaacgaact tagtggagtg attccctctg atattcttag tcctgtggga ctgtggactg 1200

ttgatctcac tcacaataag ctcgaagggc ccatccctag cagcttgtct ccgaccctct 1260

atcggttgag gcttggtgga ggcaactctc tcaatggaac catcccggcc accattggtg 1320

atgcatcgac cttggcttat cttgagctgg atagcaatca gttgacggga agcataccat 1380

tggaacttgg cagatgcaag agtttgtctt tgctgaatct ggcatcaaat aagtttcagg 1440

gtccagtgcc tgatgcaatc agcagccttg acaaactggt agttcttaaa ctccaaatga 1500

acaatctgga tggacctatc ccaagtgtat tttctaattt gacaagcctg atcacattga 1560

atcttagtgg taattcattc actggaggga taccaagaga aatcggcaag ctgccaaagc 1620

tttccatttt gaatttgcaa tgcaacaaga tcagcggcac cattccagat tcactccatt 1680

tattaacttc tctaattgag ctcaatctgg ggaataatat cttgactggt accatcccaa 1740

caatgccgac caaattgagt actgttctta atctaagcca caacaatctc agcggatcta 1800

ttccttcaaa tattgactta ttgagtgatc tagagattct tgatctttca tacaacaact 1860

tgtatggtga ggtgccggcc tcacttgcaa agctagaaag cttgacgcaa ctagtgcttt 1920

cttataatca cctttctggg tccattccta tatttcgtca acatgttgat attgctacca 1980

atggaaatcc cgatcttaca aatggtacaa gaaattatga caacgcccct acaagtggta 2040

agagaaggac acacaataca gtcatcatcg tcgttgccat tactggtgct cttgttggat 2100

tgtgcttgct tgctgctatt gttacaattt catattctaa gagaatttat cgtgttgaag 2160

atgaaggacc atcaactgag gatgttgctc gaatcatcaa tggtcacctc ataactatga 2220

atagtatcca tacctctgca attgatttcg tgaaagcaat ggaagcagtc tccaatcaca 2280

gcaacatttt tctgaaaaca aggttctgca cttactataa ggctgtgatg cctaatggct 2340

caacctattc tttaaaacag attaattgta gtgacaagat attccaaatt gggagccaag 2400

ggaaggttgc tcatgaactt gaggtacttg ggaagttgag caattccaat gtaatggtgc 2460

cattggctta tgtgttgaca gaagacaatg catacatcat ctatgagcat gtgcacaagg 2520

gcacggtgtt cgatttcctt catgctggaa gatcagatgt tctggactgg ccttcacggt 2580

atagcatagc ttttgggcta gcccaagggc tgacatttct tcatgggtgc actcagccag 2640

ttctgcttct tgatctgtca acaaggactg tccacttgaa gtcaatgaat gagcctcaga 2700

ttggagatgt tgaactttac aaaattgttg atactttgaa gagcagtggg agcctttcga 2760

ccattgctgg tacagttggt tatattccac caggtaatgg actgttctgg ttcaatttga 2820

gtgcataatt atcaacttaa gcagttaaac tgaaactgct gatagtggtt atttactgtt 2880

ctctgttgat ttgatctatt tatagttcat atctatgttt tcaagtaatt tacttgaggc 2940

aattctgttt gcattacctg catacttcaa ttgaccacaa tcctgtctac actcctatgc 3000

tagttttgtt ttcagaattt ctggagaaaa catatttgct ttgtttccca tgggttgtcg 3060

tctactacat ttgtagtatc atccaaaaaa ggggctttta actatttgcc actcttacgg 3120

atgttgctta atgatttacc actaggccca catgtcatag acacatgaga gcccacatgt 3180

cattgacagg gtgtggcaaa tcgttaactg ccatgtcaca aaagtggcaa atagttaaat 3240

ttccctccaa aataagcaca tctcctactt ttgacaaatc taataaaaaa cctagcttct 3300

atgatcttgt ccttcagtac actagcacaa aatgatttga cgctttccga gatctatatg 3360

agatgagatt tatactgaag ctaaattttg ttgaactatg ttgcagagta tgcatacacg 3420

atgaggttga cgatggctgg caacgtttac agctttggag taatcttact ggagcttttg 3480

actgggaaac catcggtcag tgatggcatc gagttagcca agtgggctct gagtctttca 3540

ggcagccctg atcagaggga gcagatcctt gacaccaggg tctcaagaac ttcagctgct 3600

gttcacagcc agatgttgtc ggtcctgaac atcgccctcg cttgtgttgc gctctctcca 3660

gatgctcgac caaagatgcg caccgtcttg aggatgctat tcaacgcaaa gtgatctggc 3720

atgatatgtt ttggtagctt aggtaggttc tgaatggagt ttgatcattt tgcttagttt 3780

gtggatgtca gcaatcagta tacggggtcc ttgtatagta ccagcaccat agttcaaggt 3840

agagtagtag attatgatat ggctgatcaa tttagtattc agatatctat tttggaatga 3900

atgaccaatg ggtttatcta tgctgtatca tcatatattc acacatggtt attagcatac 3960

cttgtacacg gcaattccat ataacaatag tagtgaaata gagccatagt taaggtagaa 4020

cagtagatag actatgatat atatccctga acaatttaga agccagatat ctgttttgga 4080

atgaatcagg aattgtgttc ttctatgcta tatgatcata tttgtaaatg gttggttgtg 4140

tacatccatg tgggatgtcg aggccggatt ggcatccatt atctaacaaa catatatata 4200

aattgttatt acttattagc 4220

<210> 3

<211> 973

<212> PRT

<213> Rice (Oryza sativa. L)

<400> 3

Met Gly Gly Gln Glu Gln Val Ala Met Ala Glu Arg Arg Pro Cys Tyr

1 5 10 15

Cys Arg Asn Asn Gly Phe Leu Leu Leu Leu Val Gln Val Leu Leu Phe

20 25 30

Leu Phe Leu Phe Leu Asp Ser Ser Met Val Ala Thr Ala Ala Ser Ala

35 40 45

Pro Ala Pro Leu Asn Thr Thr Gln Val Ser Ile Met Lys Glu Leu Ser

50 55 60

Gly Leu Val Thr Ala Ser Ala Lys Trp Asn Thr Ser Asp Ser Asn Pro

65 70 75 80

Cys Arg Trp Asp Gly Val Ser Cys Ser Ser Ser Ser Asn Ser Ile Ser

85 90 95

Val Val Thr Asn Leu Thr Leu Ser Gly Tyr Gly Leu Ser Asn Ser Thr

100 105 110

Ile Phe Ala Thr Ile Cys Ser Leu Asp Thr Leu Gln Ile Leu Asp Leu

115 120 125

Ser Lys Asn Ser Phe Thr Asn Ser Ile Glu Gln Phe Phe Thr Ser Ser

130 135 140

Cys Ser Met Lys Ala Gly Leu Arg Ser Leu Asn Leu Ser Ser Ser Gln

145 150 155 160

Leu Ser Met Pro Leu Ser Asn Phe Ser Gly Phe Pro Leu Leu Glu Val

165 170 175

Leu Asp Leu Ser Phe Asn Ser Phe Ser Gly Asp Val Arg Thr Gln Leu

180 185 190

Ser Ser Leu Leu Lys Leu Arg Ser Leu Asn Leu Ser Ser Asn Asn Leu

195 200 205

Ala Gly Asp Val Pro Thr Ser Met Thr Pro Ser Leu Glu Glu Leu Val

210 215 220

Leu Ser Ile Asn Asn Phe Ser Gly Ser Ile Pro Ile Ala Leu Phe Asn

225 230 235 240

Tyr Gln Asn Leu Thr Met Leu Asp Leu Ser Gln Asn Asn Leu Asn Gly

245 250 255

Asp Val Pro Asp Glu Phe Leu Lys Leu Pro Lys Leu Lys Thr Leu Leu

260 265 270

Leu Ser Gly Asn Gln Leu Ser Gly Asn Ile Pro Val Ser Val Ser Asn

275 280 285

Val Ala Ser Leu Ala Arg Phe Ala Ala Asn Gln Asn Asn Phe Thr Gly

290 295 300

Phe Ile Pro Ser Gly Ile Thr Lys Asn Val Lys Met Leu Asp Leu Ser

305 310 315 320

Tyr Asn Glu Leu Ser Gly Val Ile Pro Ser Asp Ile Leu Ser Pro Val

325 330 335

Gly Leu Trp Thr Val Asp Leu Thr His Asn Lys Leu Glu Gly Pro Ile

340 345 350

Pro Ser Ser Leu Ser Pro Thr Leu Tyr Arg Leu Arg Leu Gly Gly Gly

355 360 365

Asn Ser Leu Asn Gly Thr Ile Pro Ala Thr Ile Gly Asp Ala Ser Thr

370 375 380

Leu Ala Tyr Leu Glu Leu Asp Ser Asn Gln Leu Thr Gly Ser Ile Pro

385 390 395 400

Leu Glu Leu Gly Arg Cys Lys Ser Leu Ser Leu Leu Asn Leu Ala Ser

405 410 415

Asn Lys Phe Gln Gly Pro Val Pro Asp Ala Ile Ser Ser Leu Asp Lys

420 425 430

Leu Val Val Leu Lys Leu Gln Met Asn Asn Leu Asp Gly Pro Ile Pro

435 440 445

Ser Val Phe Ser Asn Leu Thr Ser Leu Ile Thr Leu Asn Leu Ser Gly

450 455 460

Asn Ser Phe Thr Gly Gly Ile Pro Arg Glu Ile Gly Lys Leu Pro Lys

465 470 475 480

Leu Ser Ile Leu Asn Leu Gln Arg Asn Lys Ile Ser Gly Thr Ile Pro

485 490 495

Asp Ser Leu His Leu Leu Thr Ser Leu Ile Glu Leu Asn Leu Gly Asn

500 505 510

Asn Ile Leu Thr Gly Thr Ile Pro Thr Met Pro Thr Lys Leu Ser Thr

515 520 525

Val Leu Asn Leu Ser His Asn Asn Leu Ser Gly Ser Ile Pro Ser Asn

530 535 540

Ile Asp Leu Leu Ser Asp Leu Glu Ile Leu Asp Leu Ser Tyr Asn Asn

545 550 555 560

Leu Tyr Gly Glu Val Pro Ala Ser Leu Ala Lys Leu Glu Ser Leu Thr

565 570 575

Gln Leu Val Leu Ser Tyr Asn His Leu Ser Gly Ser Ile Pro Ile Phe

580 585 590

Arg Gln His Val Asp Ile Ala Thr Asn Gly Asn Pro Asp Leu Thr Asn

595 600 605

Gly Thr Arg Asn Tyr Asp Asn Ala Pro Thr Ser Gly Lys Arg Arg Thr

610 615 620

His Asn Thr Val Ile Ile Val Val Ala Ile Thr Gly Ala Leu Val Gly

625 630 635 640

Leu Cys Leu Leu Ala Ala Ile Val Thr Ile Ser Tyr Ser Lys Arg Ile

645 650 655

Tyr Arg Val Glu Asp Glu Gly Pro Ser Thr Glu Asp Val Ala Arg Ile

660 665 670

Ile Asn Gly His Leu Ile Thr Met Asn Ser Ile His Thr Ser Ala Ile

675 680 685

Asp Phe Val Lys Ala Met Glu Ala Val Ser Asn His Ser Asn Ile Phe

690 695 700

Leu Lys Thr Arg Phe Cys Thr Tyr Tyr Lys Ala Val Met Pro Asn Gly

705 710 715 720

Ser Thr Tyr Ser Leu Lys Gln Ile Asn Cys Ser Asp Lys Ile Phe Gln

725 730 735

Ile Gly Ser Gln Gly Lys Val Ala His Glu Leu Glu Val Leu Gly Lys

740 745 750

Leu Ser Asn Ser Asn Val Met Val Pro Leu Ala Tyr Val Leu Thr Glu

755 760 765

Asp Asn Ala Tyr Ile Ile Tyr Glu His Val His Lys Gly Thr Val Phe

770 775 780

Asp Phe Leu His Ala Gly Arg Ser Asp Val Leu Asp Trp Pro Ser Arg

785 790 795 800

Tyr Ser Ile Ala Phe Gly Leu Ala Gln Gly Leu Thr Phe Leu His Gly

805 810 815

Cys Thr Gln Pro Val Leu Leu Leu Asp Leu Ser Thr Arg Thr Val His

820 825 830

Leu Lys Ser Met Asn Glu Pro Gln Ile Gly Asp Val Glu Leu Tyr Lys

835 840 845

Ile Val Asp Thr Leu Lys Ser Ser Gly Ser Leu Ser Thr Ile Ala Gly

850 855 860

Thr Val Gly Tyr Ile Pro Pro Glu Tyr Ala Tyr Thr Met Arg Leu Thr

865 870 875 880

Met Ala Gly Asn Val Tyr Ser Phe Gly Val Ile Leu Leu Glu Leu Leu

885 890 895

Thr Gly Lys Pro Ser Val Ser Asp Gly Ile Glu Leu Ala Lys Trp Ala

900 905 910

Leu Ser Leu Ser Gly Ser Pro Asp Gln Arg Glu Gln Ile Leu Asp Thr

915 920 925

Arg Val Ser Arg Thr Ser Ala Ala Val His Ser Gln Met Leu Ser Val

930 935 940

Leu Asn Ile Ala Leu Ala Cys Val Ala Leu Ser Pro Asp Ala Arg Pro

945 950 955 960

Lys Met Arg Thr Val Leu Arg Met Leu Phe Asn Ala Lys

965 970

<210> 4

<211> 973

<212> PRT

<213> Rice (Oryza sativa. L)

<400> 4

Met Gly Gly Gln Glu Gln Val Ala Met Ala Glu Arg Arg Pro Cys Tyr

1 5 10 15

Cys Arg Asn Asn Gly Phe Leu Leu Leu Leu Val Gln Val Leu Leu Phe

20 25 30

Leu Phe Leu Phe Leu Asp Ser Ser Met Val Ala Thr Ala Ala Ser Ala

35 40 45

Pro Ala Pro Leu Asn Thr Thr Gln Val Ser Ile Met Lys Glu Leu Ser

50 55 60

Gly Leu Val Thr Ala Ser Ala Lys Trp Asn Thr Ser Asp Ser Asn Pro

65 70 75 80

Cys Arg Trp Asp Gly Val Ser Cys Ser Ser Ser Ser Asn Ser Ile Ser

85 90 95

Val Val Thr Asn Leu Thr Leu Ser Gly Tyr Gly Leu Ser Asn Ser Thr

100 105 110

Ile Phe Ala Thr Ile Cys Ser Leu Asp Thr Leu Gln Ile Leu Asp Leu

115 120 125

Ser Lys Asn Ser Phe Thr Asn Ser Ile Glu Gln Phe Phe Thr Ser Ser

130 135 140

Cys Ser Met Lys Ala Gly Leu Arg Ser Leu Asn Leu Ser Ser Ser Gln

145 150 155 160

Leu Ser Met Pro Leu Ser Asn Phe Ser Gly Phe Pro Leu Leu Glu Val

165 170 175

Leu Asp Leu Ser Phe Asn Ser Phe Ser Gly Asp Val Arg Thr Gln Leu

180 185 190

Ser Ser Leu Leu Lys Leu Arg Ser Leu Asn Leu Ser Ser Asn Asn Leu

195 200 205

Ala Gly Asp Val Pro Thr Ser Met Thr Pro Ser Leu Glu Glu Leu Val

210 215 220

Leu Ser Ile Asn Asn Phe Ser Gly Ser Ile Pro Ile Ala Leu Phe Asn

225 230 235 240

Tyr Gln Asn Leu Thr Met Leu Asp Leu Ser Gln Asn Asn Leu Asn Gly

245 250 255

Asp Val Pro Asp Glu Phe Leu Lys Leu Pro Lys Leu Lys Thr Leu Leu

260 265 270

Leu Ser Gly Asn Gln Leu Ser Gly Asn Ile Pro Val Ser Val Ser Asn

275 280 285

Val Ala Ser Leu Ala Arg Phe Ala Ala Asn Gln Asn Asn Phe Thr Gly

290 295 300

Phe Ile Pro Ser Gly Ile Thr Lys Asn Val Lys Met Leu Asp Leu Ser

305 310 315 320

Tyr Asn Glu Leu Ser Gly Val Ile Pro Ser Asp Ile Leu Ser Pro Val

325 330 335

Gly Leu Trp Thr Val Asp Leu Thr His Asn Lys Leu Glu Gly Pro Ile

340 345 350

Pro Ser Ser Leu Ser Pro Thr Leu Tyr Arg Leu Arg Leu Gly Gly Gly

355 360 365

Asn Ser Leu Asn Gly Thr Ile Pro Ala Thr Ile Gly Asp Ala Ser Thr

370 375 380

Leu Ala Tyr Leu Glu Leu Asp Ser Asn Gln Leu Thr Gly Ser Ile Pro

385 390 395 400

Leu Glu Leu Gly Arg Cys Lys Ser Leu Ser Leu Leu Asn Leu Ala Ser

405 410 415

Asn Lys Phe Gln Gly Pro Val Pro Asp Ala Ile Ser Ser Leu Asp Lys

420 425 430

Leu Val Val Leu Lys Leu Gln Met Asn Asn Leu Asp Gly Pro Ile Pro

435 440 445

Ser Val Phe Ser Asn Leu Thr Ser Leu Ile Thr Leu Asn Leu Ser Gly

450 455 460

Asn Ser Phe Thr Gly Gly Ile Pro Arg Glu Ile Gly Lys Leu Pro Lys

465 470 475 480

Leu Ser Ile Leu Asn Leu Gln Cys Asn Lys Ile Ser Gly Thr Ile Pro

485 490 495

Asp Ser Leu His Leu Leu Thr Ser Leu Ile Glu Leu Asn Leu Gly Asn

500 505 510

Asn Ile Leu Thr Gly Thr Ile Pro Thr Met Pro Thr Lys Leu Ser Thr

515 520 525

Val Leu Asn Leu Ser His Asn Asn Leu Ser Gly Ser Ile Pro Ser Asn

530 535 540

Ile Asp Leu Leu Ser Asp Leu Glu Ile Leu Asp Leu Ser Tyr Asn Asn

545 550 555 560

Leu Tyr Gly Glu Val Pro Ala Ser Leu Ala Lys Leu Glu Ser Leu Thr

565 570 575

Gln Leu Val Leu Ser Tyr Asn His Leu Ser Gly Ser Ile Pro Ile Phe

580 585 590

Arg Gln His Val Asp Ile Ala Thr Asn Gly Asn Pro Asp Leu Thr Asn

595 600 605

Gly Thr Arg Asn Tyr Asp Asn Ala Pro Thr Ser Gly Lys Arg Arg Thr

610 615 620

His Asn Thr Val Ile Ile Val Val Ala Ile Thr Gly Ala Leu Val Gly

625 630 635 640

Leu Cys Leu Leu Ala Ala Ile Val Thr Ile Ser Tyr Ser Lys Arg Ile

645 650 655

Tyr Arg Val Glu Asp Glu Gly Pro Ser Thr Glu Asp Val Ala Arg Ile

660 665 670

Ile Asn Gly His Leu Ile Thr Met Asn Ser Ile His Thr Ser Ala Ile

675 680 685

Asp Phe Val Lys Ala Met Glu Ala Val Ser Asn His Ser Asn Ile Phe

690 695 700

Leu Lys Thr Arg Phe Cys Thr Tyr Tyr Lys Ala Val Met Pro Asn Gly

705 710 715 720

Ser Thr Tyr Ser Leu Lys Gln Ile Asn Cys Ser Asp Lys Ile Phe Gln

725 730 735

Ile Gly Ser Gln Gly Lys Val Ala His Glu Leu Glu Val Leu Gly Lys

740 745 750

Leu Ser Asn Ser Asn Val Met Val Pro Leu Ala Tyr Val Leu Thr Glu

755 760 765

Asp Asn Ala Tyr Ile Ile Tyr Glu His Val His Lys Gly Thr Val Phe

770 775 780

Asp Phe Leu His Ala Gly Arg Ser Asp Val Leu Asp Trp Pro Ser Arg

785 790 795 800

Tyr Ser Ile Ala Phe Gly Leu Ala Gln Gly Leu Thr Phe Leu His Gly

805 810 815

Cys Thr Gln Pro Val Leu Leu Leu Asp Leu Ser Thr Arg Thr Val His

820 825 830

Leu Lys Ser Met Asn Glu Pro Gln Ile Gly Asp Val Glu Leu Tyr Lys

835 840 845

Ile Val Asp Thr Leu Lys Ser Ser Gly Ser Leu Ser Thr Ile Ala Gly

850 855 860

Thr Val Gly Tyr Ile Pro Pro Glu Tyr Ala Tyr Thr Met Arg Leu Thr

865 870 875 880

Met Ala Gly Asn Val Tyr Ser Phe Gly Val Ile Leu Leu Glu Leu Leu

885 890 895

Thr Gly Lys Pro Ser Val Ser Asp Gly Ile Glu Leu Ala Lys Trp Ala

900 905 910

Leu Ser Leu Ser Gly Ser Pro Asp Gln Arg Glu Gln Ile Leu Asp Thr

915 920 925

Arg Val Ser Arg Thr Ser Ala Ala Val His Ser Gln Met Leu Ser Val

930 935 940

Leu Asn Ile Ala Leu Ala Cys Val Ala Leu Ser Pro Asp Ala Arg Pro

945 950 955 960

Lys Met Arg Thr Val Leu Arg Met Leu Phe Asn Ala Lys

965 970

<210> 5

<211> 7123

<212> DNA

<213> Rice (Oryza sativa. L)

<400> 5

aaaatttcag aagaattaat tctgcagcaa ataggtgtaa aagtattgga ctgaactctt 60

ggtgcaagca aacacaatgg accagtccaa gtctgaaact gatgcagatg caacccagag 120

agggctccag gacctgaatg agaagctgca ggtaaacttc agaacacagg tatggacaga 180

agaaatagat cttggaacga gtgagaggca aatggaggtg agctgaagtc cattcaaagt 240

ctcctcgtca agtgtgtggg gtaggccaga tgagatgaaa ggaacagcat aggatgagat 300

gtgatcattc actcccaatt ccaaccttcc actttcctaa cctccaaatt ccacactaag 360

cacagctttt atttgtttta cattcttttg attcaggggt gttggtaatg gacggctaaa 420

gcgtccacat gaaacatttg aaaattttcc attcatggtt tatttggaac acatgatttt 480

ttttcttatt ttttctttag aaaatgaaaa tgtaaaattt gatggaaatt atgtaaaatt 540

cgcacttctc ttcaattgta tataataatg tgaaatttga aggaaattat ataaagtttg 600

cacatctctt caattgtata tatattaaat caaataagaa aacaatgctt gattattcaa 660

atacatatat cctcagttgt atggagtcaa atagaatacc aaggcgttcg tgatgactta 720

cagcaattcg aatgtacaat ttaattgtgg aatagtatga aatatcaatc tttgttaatc 780

tagtgaatca agaattatta ttctgaaaca caccacacca aaaacttcta tccgcatatc 840

tggtgaaaag cccacttgga tgaaatttct gaacctgtga attcctttaa tcttctgttt 900

gatcaaccag tacacttgaa gacaatttca ttctacatgg gcttgtatgc tcctaactat 960

ttctctccct gcctgtatgt gtttgaggta aagctagata agccaaagcc ttcgtttggt 1020

caagctcctc tgtcatttcc aacacgtcgt ccttttcccg ggcaaaggcc ccacgattgt 1080

tacgtagact aactttaatc actttggcac atttcaacaa tctcgattac ctttcctact 1140

tgaatctgtt gatcataatt ttggctagca ctagctagtc cttttcagaa tttgtagaag 1200

cttttcttct tcagggaaaa ggtaagaatt tacagatggt tttggtgtag ctggtgagct 1260

ccagcttctt tgacccggca tctacaaatg gtaattaata catttatttt tcaagtctca 1320

gtttgatggt ctgggaggga tcctctattt cgtctgcatt aatttcagag aatgacatgt 1380

ctgcattagt aatagagaat ggcatgtgtt acttcagaat aattagctga aattgaaagt 1440

gacagttttt agattgtagt attagtgtat tactagtata ctattctgca agatgcaatg 1500

tgtttctgga tactaactaa tctactagta gttttagtga aacagctttg cagatgttga 1560

atggtgccct cacattgctc aacggcatag gattctcttg gtacactaat aaagcgaagg 1620

ctagttgcct cttaataaaa atgctaagct tttcttttga aaaggtactc agaagcaaga 1680

acttgttcca gaggcagttc aaatactcaa aaagactatt tataatttca tgtaccatta 1740

tcttttttcc tgaacattac tgtgtttttt taagttgatt ttaaatactc ctaaatcacc 1800

aaaaacaaat tggacagcaa gtatattcca gtcagtttgc atatatgaaa aattggcatc 1860

catggatata actgagaaat taccaacaga aaagatcatt ttttttcttt ggaatagtaa 1920

cataaagacc attcttttgt tgtttgaatg gtaacagaaa gaccattctt caaagctgcc 1980

aaagtttccc taaaactcag cctaaatgca ggttctcatc atcactgttc tcaacctgct 2040

ttcttcagtg ttttcagctc ccctctctca caggttttgg tcactcacac aagatatacg 2100

tttcagtcag tcaaccctgg accccaagag gtgtatgtgt gcgagagaaa aaaaaaatcc 2160

tcctttcctt ctctctaaaa gactggactc ccaacgccga agcactggga gtctgggagg 2220

agcaagaaga ggagagaaga gagccatggc caagaacacc gagagcccca aaacctaggg 2280

aaagagagga aaaaatcgat tttcttgggt ggtggttgag cctgcagtga agttcatcag 2340

ggaggaaagg gaggggatta agggaaggag atacaggaat caatgggagg ccaagaacag 2400

gtggccatgg cggagaggag gccttgttac tgtcgcaaca atggcttctt gctgctgctg 2460

gtacaagttc tcctcttctt gttcttgttt cttgattcgt ccatggtcgc cactgccgcg 2520

tcggcgccgg caccgctaaa tacgactcag gtatcgatca tgaaggagct ttctggcttg 2580

gttactgcca gtgccaagtg gaacacgagc gattcgaatc catgccgatg ggatggagtg 2640

agttgctctt ctagctccaa ttccatatca gttgtgacca accttacttt gtctgggtat 2700

ggtttgtcca actccaccat atttgcaacc atatgctctc ttgacacctt gcaaatcctt 2760

gatctctcta agaattcctt caccaattcg atagagcaat tcttcacctc ttcttgctcc 2820

atgaaggctg gattgcggtc acttaatctc agcagcagcc agctatccat gcctcttagt 2880

aatttctctg gttttcccct tcttgaggtt cttgacttgt ccttcaattc tttcagtggt 2940

gatgtcagaa cccagttgag ttctttgctc aagctgagaa gcttgaatct tagtagcaat 3000

aacttggctg gtgatgttcc tacaagcatg accccgtctt tggaggaatt ggtgttgtcc 3060

atcaacaatt tcagtggtag cattccaata gctttgttca attaccaaaa tcttactatg 3120

ctggatctta gtcagaacaa tctaaatggt gatgtaccgg atgagttctt aaagttaccc 3180

aagctcaaga ctttgctctt atcaggtaat caactgagtg gcaatatacc tgtgagtgtg 3240

tcaaatgttg caagccttgc tcggtttgca gctaatcaga acaattttac cggtttcatc 3300

cctagtggta tcaccaagaa tgtgaagatg ctggatctga gttacaacga acttagtgga 3360

gtgattccct ctgatattct tagtcctgtg ggactgtgga ctgttgatct cactcacaat 3420

aagctcgaag ggcccatccc tagcagcttg tctccgaccc tctatcggtt gaggcttggt 3480

ggaggcaact ctctcaatgg aaccatcccg gccaccattg gtgatgcatc gaccttggct 3540

tatcttgagc tggatagcaa tcagttgacg ggaagcatac cattggaact tggcagatgc 3600

aagagtttgt ctttgctgaa tctggcatca aataagtttc agggtccagt gcctgatgca 3660

atcagcagcc ttgacaaact ggtagttctt aaactccaaa tgaacaatct ggatggacct 3720

atcccaagtg tattttctaa tttgacaagc ctgatcacat tgaatcttag tggtaattca 3780

ttcactggag ggataccaag agaaatcggc aagctgccaa agctttccat tttgaatttg 3840

caacgcaaca agatcagcgg caccattcca gattcactcc atttattaac ttctctaatt 3900

gagctcaatc tggggaataa tatcttgact ggtaccatcc caacaatgcc gaccaaattg 3960

agtactgttc ttaatctaag ccacaacaat ctcagcggat ctattccttc aaatattgac 4020

ttattgagtg atctagagat tcttgatctt tcatacaaca acttgtatgg tgaggtgccg 4080

gcctcacttg caaagctaga aagcttgacg caactagtgc tttcttataa tcacctttct 4140

gggtccattc ctatatttcg tcaacatgtt gatattgcta ccaatggaaa tcccgatctt 4200

acaaatggta caagaaatta tgacaacgcc cctacaagtg gtaagagaag gacacacaat 4260

acagtcatca tcgtcgttgc cattactggt gctcttgttg gattgtgctt gcttgctgct 4320

attgttacaa tttcatattc taagagaatt tatcgtgttg aagatgaagg accatcaact 4380

gaggatgttg ctcgaatcat caatggtcac ctcataacta tgaatagtat ccatacctct 4440

gcaattgatt tcgtgaaagc aatggaagca gtctccaatc acagcaacat ttttctgaaa 4500

acaaggttct gcacttacta taaggctgtg atgcctaatg gctcaaccta ttctttaaaa 4560

cagattaatt gtagtgacaa gatattccaa attgggagcc aagggaaggt tgctcatgaa 4620

cttgaggtac ttgggaagtt gagcaattcc aatgtaatgg tgccattggc ttatgtgttg 4680

acagaagaca atgcatacat catctatgag catgtgcaca agggcacggt gttcgatttc 4740

cttcatgctg gaagatcaga tgttctggac tggccttcac ggtatagcat agcttttggg 4800

ctagcccaag ggctgacatt tcttcatggg tgcactcagc cagttctgct tcttgatctg 4860

tcaacaagga ctgtccactt gaagtcaatg aatgagcctc agattggaga tgttgaactt 4920

tacaaaattg ttgatacttt gaagagcagt gggagccttt cgaccattgc tggtacagtt 4980

ggttatattc caccaggtaa tggactgttc tggttcaatt tgagtgcata attatcaact 5040

taagcagtta aactgaaact gctgatagtg gttatttact gttctctgtt gatttgatct 5100

atttatagtt catatctatg ttttcaagta atttacttga ggcaattctg tttgcattac 5160

ctgcatactt caattgacca caatcctgtc tacactccta tgctagtttt gttttcagaa 5220

tttctggaga aaacatattt gctttgtttc ccatgggttg tcgtctacta catttgtagt 5280

atcatccaaa aaaggggctt ttaactattt gccactctta cggatgttgc ttaatgattt 5340

accactaggc ccacatgtca tagacacatg agagcccaca tgtcattgac agggtgtggc 5400

aaatcgttaa ctgccatgtc acaaaagtgg caaatagtta aatttccctc caaaataagc 5460

acatctccta cttttgacaa atctaataaa aaacctagct tctatgatct tgtccttcag 5520

tacactagca caaaatgatt tgacgctttc cgagatctat atgagatgag atttatactg 5580

aagctaaatt ttgttgaact atgttgcaga gtatgcatac acgatgaggt tgacgatggc 5640

tggcaacgtt tacagctttg gagtaatctt actggagctt ttgactggga aaccatcggt 5700

cagtgatggc atcgagttag ccaagtgggc tctgagtctt tcaggcagcc ctgatcagag 5760

ggagcagatc cttgacacca gggtctcaag aacttcagct gctgttcaca gccagatgtt 5820

gtcggtcctg aacatcgccc tcgcttgtgt tgcgctctct ccagatgctc gaccaaagat 5880

gcgcaccgtc ttgaggatgc tattcaacgc aaagtgatct ggcatgatat gttttggtag 5940

cttaggtagg ttctgaatgg agtttgatca ttttgcttag tttgtggatg tcagcaatca 6000

gtatacgggg tccttgtata gtaccagcac catagttcaa ggtagagtag tagattatga 6060

tatggctgat caatttagta ttcagatatc tattttggaa tgaatgacca atgggtttat 6120

ctatgctgta tcatcatata ttcacacatg gttattagca taccttgtac acggcaattc 6180

catataacaa tagtagtgaa atagagccat agttaaggta gaacagtaga tagactatga 6240

tatatatccc tgaacaattt agaagccaga tatctgtttt ggaatgaatc aggaattgtg 6300

ttcttctatg ctatatgatc atatttgtaa atggttggtt gtgtacatcc atgtgggatg 6360

tcgaggccgg attggcatcc attatctaac aaacatatat ataaattgtt attacttatt 6420

agcatacctt ttgcatgtca gttcaaagtt ttgtattttt ttttgtgcca gtgttattca 6480

gtattaacaa agagccactg atctttcttt tttaagggaa ctagagccag agccactgac 6540

ctaagtttga gtgaaacatg caatgaggaa tttagctccc attgttttgc ttttaaacct 6600

ttctgaaact tgctttctct aaccacttca acacttcagt atgtatgttc acttcctagt 6660

agtaggaatt ataggtcttg aagaaactcc cagccaaaaa ataagaaaaa attgcatcaa 6720

catgaactat accattactt ttgacatgaa tttagtgagg ctagagatta actaccggat 6780

gcatttcttt gaacatgtgt taacaaggtt tactcacagg catgcaggat ttaccctttt 6840

cgttttcaaa gtgaaaaccg ctcccaccga tttactcaaa accggttaaa gaaaaacagt 6900

aaggctgtgt ttgcaatccc atgttcccaa cccctctcca tcgttttccg tgcgcacgct 6960

tttcaaacta ctaaacggtg cgctttttac aataagtttt tatatgaaag ttgcttaaaa 7020

aaatcatatt gatacatttt tgaaaaaaaa ttagcaaata cttaattaat cacacgctaa 7080

tgtaccactc cgttttccgt gcggggagtt tagttcccaa ctt 7123

Claims (9)

1. A rice scab gene SPL36 for regulating programmed cell death and disease resistance is characterized in that: the amino acid sequence of the encoded protein is shown as SEQ ID NO: 4, respectively.

2. The rice lesion gene SPL36 according to claim 1, wherein: the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

3. A plant expression vector or a host cell line comprising the rice lesion gene SPL36 according to claim 1.

4. The host cell line of claim 3, wherein: the host cell line is an escherichia coli cell, an agrobacterium cell or a plant cell.

5. The pCAMBIA1300 complementary vector fused with the rice lesion control gene SPL36 is characterized in that: the sequence is shown as SEQ ID NO. 5.

6. The application of the rice disease-like spot gene SPL36 in improving disease resistance of plant varieties according to claim 1.

7. Use according to claim 6, characterized in that: the plant variety is rice, and the disease is bacterial disease.

8. The use of claim 6, wherein: the breeding method is to inoculate the mutant spl36 by leaf cutting.

9. The use of claim 6, wherein: the leaf-cutting inoculation is carried out at the tillering stage.

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