Application of the lipoxygenase gene LOX6 in regulating rice spikelet development

Abstract

The present invention discloses the use of a lipoxygenase gene LOX6 in regulating the development of rice spikelets. The present invention obtains a rice spikelet development-abnormal mutant through EMS mutagenesis, and isolates the spikelet development-control gene LOX6 through the MutMap+ method. This gene encodes a lipoxygenase. Mutation of this gene causes the 917th amino acid of LOX6 to change from proline to leucine, resulting in abnormal development of rice spikelets. The present invention further obtains a LOX6 mutant through CRISPR / Cas9 knockout, and finds that the spikelets of the knockout plants develop abnormally, that is, mutations in the lipoxygenase gene LOX6 affect the development of rice spikelets. The research on the lipoxygenase gene LOX6 in the present invention provides a theoretical basis for the cultivation of transgenic rice such as multi-flowered spikelets.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and particularly relates to the application of a lipoxygenase gene LOX6 in regulating the development of rice spikelets. Background Art

[0002] "The people are the foundation of the country, and grain is the life of the people." Food security is "the most important thing for the country." However, in recent years, global food security has faced severe challenges due to the impact of multiple factors such as the COVID-19 pandemic, climate change, and geopolitical conflicts. According to reports from authoritative organizations such as the Food and Agriculture Organization of the United Nations, the number of people affected by hunger in the world reached 828 million in 2021, and ensuring food security is urgent. Rice is one of the staple foods on which most of the world's population depends for survival, and increasing its yield is an effective way to ensure food security. The rice flower organ is both a reproductive organ and the basis for grain formation. Its development directly affects rice yield and rice quality. Therefore, studying the morphological construction and regulatory mechanism of rice flower organs is not only instructive for other crops, but also crucial to food production.

[0003] In a typical dicot, the four floral organs, from the outside in, are sepals, petals, stamens, and carpels. As early as 1991, scientists proposed the "ABC" model to explain the mechanisms regulating the development of the four floral organs in Arabidopsis thaliana. This model was later developed into the "ABCDE" model, which includes D and E genes. Research has shown that the "ABCDE" model of dicots also partially applies to monocots. Rice, an important model crop for monocots, has the spikelet as the basic structural unit of its floral organs. Each spikelet contains a single floret, which, from the outside in, consists of a lemma, a palea, two lodicules, six stamens, and a pistil. Variation in rice floral organs involves not only homeotic conversion of individual whorl structures but also changes in the number of structures within each whorl, and even the generation of novel whorl structures.

[0004] In recent years, numerous researchers, both domestic and international, have systematically investigated the molecular mechanisms of rice floral organ development. Several genes controlling floral organ development have been cloned, most of which belong to the MADS-box gene family. For example, the A-class genes OsMADS14, OsMADS15, OsMADS18, and OsMADS20 primarily regulate the development of the palea, lemma, and lodicule; the B-class genes OsMADS2, OsMADS4, and OsMADS16 primarily control the development of the lodicule and stamen; and the C-class genes OsMADS3 and OsMADS58 primarily regulate the development of the stamen and pistil. In addition to these MADS-box family genes, several other types of genes are also involved in rice floral organ development. For example, the C-class gene DROOPING LEAF belongs to the YABBY gene family and controls the development of the lemma and carpel.

[0005] Plant hormones are synthesized during specific developmental stages in response to genetic and environmental cues. They serve as a crucial signaling source for normal plant growth and development, as well as for adaptation and survival. While the "ABCDE" model partially explains the morphogenesis of rice floral organs, a deeper understanding remains regarding how key regulatory genes in this model sense growth and developmental signals and participate in the later morphogenesis of floral organs. Jasmonic acid (JA) and its derivatives (collectively referred to as JAs), as important lipid-derived hormones, are widely present in young plant tissues, including early-stage florets and developing reproductive organs. JA synthesis and signaling pathways are known to regulate the development of rice floral organs. For example, Arabidopsis DEFECTIVE ANTHER DEHISCENCE1 (DAD1) encodes a chloroplast phospholipase A1 protein that catalyzes the initial steps of JA biosynthesis. Mutations in DAD1 lead to anther dehiscence, pollen grain maturation, and poor silique development. The rice gene EG1 is a homologous gene of DAD1. The number of guard glumes in eg1 mutants increases, and the lodicules develop into structures similar to guard glumes, or repeated structures similar to glumes or new floral organ primordia grow in the carpels.

[0006] Lipoxygenases (LOXs) are a family of fatty acid dioxygenases containing non-heme iron proteins. Commonly known as lipoxygenases, lipoxygenases, or carotenoid oxidases, they are widely distributed in plants, animals, and microorganisms. Lipoxygenases (LOXs) can participate in multiple metabolic pathways. Studies have shown that LOXs can catalyze the oxidation of polyunsaturated fatty acids (PUFAs) to form hydroperoxides (HPOs), thereby initiating downstream reactions. Among the many metabolic pathways of the LOX pathway, the AOS pathway, or jasmonic acid synthesis pathway, is currently the most clearly elucidated. LOXs can catalyze the biosynthesis of JA, and LOX activity directly affects JA levels in plants. Summary of the Invention

[0007] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an application of the lipoxygenase gene LOX6 in regulating the development of rice spikelets.

[0008] Another object of the present invention is to provide a mutant of the lipoxygenase gene LOX6.

[0009] Another object of the present invention is to provide an application of the mutant of the lipoxygenase gene LOX6.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] Application of the lipoxygenase gene LOX6 in regulating rice spikelet development.

[0012] The lipoxygenase gene LOX6 is located in NCBI (National Center for Biotechnology The gene ID (accession number) is LOC9267158 (https: / / www.ncbi.nlm.nih.gov / gene / 9267158) on the National Natural Science Foundation of China (NCBI). This gene is located on chromosome 4 in rice. Mutations in this gene can cause abnormal spikelet development in plants carrying the mutation, resulting in the simultaneous appearance of different types of spikelets on the same panicle. Based on the lemma morphology of the mutants, the spikelets can be divided into four types: normal (no difference between the mutant and wild type), degenerate (with varying degrees of palea degeneration or absence), cleavage (with two lemmas with hooked tips that cannot close), and multiple lemmas (compared to the wild type, with an additional lemma-like structure). The number of stamens in degenerate and cleavage spikelets is altered. Compared with the wild type (6 stamens), degenerate spikelets have ≤6 stamens, while cleavage spikelets have ≥6 stamens. The pistils of cleavage spikelets are also abnormal, and the mutants have multiple ovaries.

[0013] The regulation of rice spikelet development causes abnormal development of the spikelets of rice plants by mutation of the lipoxygenase gene LOX6.

[0014] The abnormal development of spikelets includes changes in the shape of the spikelets, changes in the number of stamens and pistils, and the like.

[0015] The lipoxygenase gene LOX6 mutation can be achieved by adding, substituting, inserting or deleting one or more nucleotides in the lipoxygenase gene LOX6 sequence, or adding, substituting, inserting or deleting one or more amino acids in the lipoxygenase gene LOX6 encoding protein sequence through ethyl methanesulfonate (EMS) mutagenesis and / or gene editing (such as CRISPR / Cas9, etc.), so as to inhibit the expression of the lipoxygenase gene LOX6 or its encoding protein, thereby affecting the development of rice spikelets; preferably, it is achieved by any of the following methods:

[0016] (a) The C at position 735 in the seventh exon of the lipoxygenase gene LOX6 was replaced by a T.

[0017] (b) A T was inserted between positions 11 and 12 of the first exon of the lipoxygenase gene LOX6;

[0018] (c) A G base was inserted between positions 729 and 730 of the seventh exon of the lipoxygenase gene LOX6;

[0019] (d) The 4-base AGTT deletion occurs at positions 14 to 17 of the first exon of the lipoxygenase gene LOX6.

[0020] A mutant of the lipoxygenase gene LOX6, wherein the nucleotide sequence is any one of the following:

[0021] (a) The C at position 735 in the seventh exon of the lipoxygenase gene LOX6 was replaced by a T.

[0022] (b) A T was inserted between positions 11 and 12 of the first exon of the lipoxygenase gene LOX6;

[0023] (c) A G base was inserted between positions 729 and 730 of the seventh exon of the lipoxygenase gene LOX6;

[0024] (d) The 4-base AGTT deletion occurs at positions 14 to 17 of the first exon of the lipoxygenase gene LOX6.

[0025] An expression vector, a recombinant microorganism or a transgenic cell line containing the mutant of the lipoxygenase gene LOX6.

[0026] The host cells include Escherichia coli cells, Agrobacterium cells or plant cells.

[0027] The plant is preferably rice.

[0028] The application of the mutant of the lipoxygenase gene LOX6 in regulating the development of rice spikelets.

[0029] The invention relates to an application of the mutant of the lipoxygenase gene LOX6 in the preparation of transgenic rice.

[0030] The transgenic rice includes multi-flowered spikelet rice and the like.

[0031] An application of a gRNA target sequence for editing the lipoxygenase gene LOX6 in regulating rice spikelet development, wherein the gRNA target sequence of the lipoxygenase gene LOX6 is as follows:

[0032] (1) located at positions 1 to 23 of the first exon of the lipoxygenase gene LOX6, and its nucleotide sequence is shown in SEQ ID NO. 3;

[0033] (2) Located at positions 720 to 741 of the seventh exon of the lipoxygenase gene LOX6, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0034] The present invention has the following advantages and effects compared to the prior art:

[0035] (1) The present invention obtained a rice spikelet developmental abnormality mutant through EMS mutagenesis and isolated the spikelet development control gene LOX6 by the MutMap+ method. This gene encodes a lipoxygenase. After the gene mutates, the 917th amino acid of LOX6 changes from proline to leucine (i.e., the 735th base C of the seventh exon of the lipoxygenase gene LOX6 is replaced by base T), resulting in abnormal rice spikelet development, providing a theoretical basis for the cultivation of multi-flowered spikelets.

[0036] (2) The spikelet development mutant lox6 obtained in the present invention causes abnormal development of rice spikelets, indicating that the normal function of the lipoxygenase gene LOX6 is very important for the development of rice spikelets. The study of the lipoxygenase gene LOX6 provides a new gene resource for subsequent research on rice spikelet development.

[0037] (3) The present invention can use the CRISPR / Cas9 system to edit the rice lipoxygenase gene LOX6, providing a feasible gRNA target sequence.

[0038] (4) The present invention obtained a mutant of the lipoxygenase gene LOX6 by CRISPR / Cas9 knockout, and found that the spikelet development of the knockout plant was abnormal, that is, the mutation of the lipoxygenase gene LOX6 would affect the development of rice spikelets. Therefore, the study of the lipoxygenase gene LOX6 helps to clarify and enrich the molecular mechanism of rice spikelet development. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 These are the phenotypic diagrams of the mutant lox6; (A) is the panicle phenotype of the wild-type WT and the mutant lox6 (the scale bar is 5 cm); (B) is the spikelet of the wild-type WT and the mutant lox6 (the phenotypes of the mutant lox6 are normal, degenerate, non-closed, and multi-lemma from top to bottom; the scale bar is 1 cm); (C) is the stamen of the wild-type WT and the mutant lox6 (the number of stamens in the mutant is 5, 6, 7, etc.; the scale bar is 5 mm); (D) is the pistil of the wild-type WT and the mutant lox6 (the mutant stamens have two ovaries, and the scale bar is 2.5 mm).

[0040] Figure 2 This is a diagram of the mature grain phenotype of the lox6 mutant (double grains appear in the lox6 mutant, and the scale bar is 1 cm).

[0041] Figure 3is the preliminary positioning result of the LOX6 gene; among them, A is the distribution map of Δ(SNP-index) on the whole rice genome (the point indicated by the arrow in the figure is located on chromosome 4, which is the gene LOX6); B is a schematic diagram of the gene structure of the candidate gene LOX6 (in the figure, black squares represent exons; black lines represent introns; white squares represent UTRs; red triangles indicate that the lipoxygenase gene LOX6 has a missense mutation here (C to T), and the amino acid changes from proline Pro to leucine Leu (the three underlined bases represent codons, and the three-letter amino acid represents the corresponding encoded amino acid)); C is the sequencing peak map of the wild type WT and mutant lox6 mutation sites.

[0042] Figure 4 This is the predicted structure of the protein encoded by the lipoxygenase gene LOX6 (in the figure, LH2: Lipoxygenase homology, lipoxygenase homology domain; LOX: Lipoxygenases, lipoxygenase domain; the red triangle indicates the position of the mutated amino acid).

[0043] Figure 5 Figure 1 is a spikelet phenotype of a plant with LOX6 knockout gene; A is a knockout of LOX6 gene based on CRISPR / Cas9 technology (in the figure, red vertical lines represent two target sites T1 and T2; "-" represents base deletion; red bases represent inserted bases; blue bases represent: protospacer adjacent motif (PAM)); B is a primary branch phenotype of the knockout plant (scale is 3 cm); C is a spikelet phenotype of the knockout plant (scale is 1 cm). DETAILED DESCRIPTION

[0044] The present invention will be described in further detail below in conjunction with the examples, but embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods in the following examples where specific experimental conditions are not specified are generally based on conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0045] The wild indica rice variety "Ruanhua B" (abbreviated as RHB) involved in the embodiments of the present invention is a conventional rice variety in this field and has been published in the National Rice Database (https: / / www.ricedata.cn / variety / var is / 617292.htm); the rice variety Zhonghua No. 11 (hereinafter referred to as ZH11) involved in the embodiments is a conventional rice variety in this field and can be purchased through conventional commercial channels.

[0046] Example 1 Isolation and genetic analysis of the rice spikelet development abnormality mutant lox6

[0047] The rice spikelet development abnormality mutant lox6 was obtained by inducing the wild-type indica rice variety RHB with ethyl methanesulfonate (EMS). Specifically, RHB seeds were soaked in a 0.5% mass concentration of EMS solution for 12 hours. The soaked seeds were planted until they were grain-filled and fruit-bearing, and individual seeds were harvested to obtain a mutant pool. Seeds from the mutant pool were then planted, and the rice spikelet development abnormality mutant lox6 was screened through phenotypic identification. Compared with the wild-type WT, the spikelet development abnormality of the lox6 mutant ( Figure 1 ), the grains will have double grains after maturity ( Figure 2 Different types of spikelets appeared simultaneously on the same spike. Based on the lemma morphology of the mutants, the spikelets of the mutants can be divided into four types: normal type (no difference between the mutant and the wild type), degenerate type (the palea is degenerate or missing to varying degrees), inclosed type (the tips of the two lemmas are hooked and cannot close), and multiple lemmas (compared with the wild type, there is an additional lemma-like structure). The number of stamens in the degenerate and inclosed spikelets changed. Compared with the wild type, the number of stamens in the degenerate spikelets was ≤6, and the number of stamens in the inclosed spikelets was ≥6. The pistils of the inclosed spikelets were also abnormal, and multiple ovary grains appeared in the mutants.

[0048] Seeds were harvested from mutant heterozygous plants and planted as segregating populations. Among the segregating populations, 165 plants showed normal spikelets and 65 plants showed abnormal spikelet development, for a total of 230 plants. The segregation ratio of normal spikelet plants to plants with abnormal spikelet development was 3:1 (χ 2 0.05=1.3<3.84, p=0.25>0.05), indicating that the mutant trait is controlled by a single recessive nuclear gene.

[0049] Example 2 Localization and Transgenic Verification of Rice Spikelet Development Control Gene LOX6

[0050] The present invention uses the MutMap+ strategy (MutMap+: genetic mapping and mutant identification without crossing in rice. [J]. PloS one, 2013, 8(7): e68529. DOI: 10.1371 / journal.pone.0068529.) to locate the gene of the rice spikelet development abnormality mutant lox6. The specific steps are as follows:

[0051] (1) At the heading stage, 65 normal plants and 65 plants with spikelet development abnormalities were collected from the segregating population of rice (indica rice variety RHB), and leaves of each were mixed in equal amounts to construct a wild-type DNA pool with normal phenotype and a mutant DNA pool. These two DNA pools were subjected to whole-genome sequencing analysis. Only chromosome 4 had a significant peak in the rice genome ( Figure 3 ).

[0052] (2) Chromosome 4 was analyzed, and Δ(SNP-index) ≥ 0.4 was used as the screening criterion to screen out 4 candidate SNP sites, of which 2 SNPs were located in the intergenic region, 1 SNP was located in the gene intron, and only 1 SNP was located in the seventh exon of gene LOC9267158 (NCBI accession number (Gene ID): 9267158; https: / / www.ncbi.nlm.nih.gov / gene / ?term=LOC9267158). The base C at position 735 of the LOX6 gene mutated to T, causing the amino acid it encodes to mutate from proline to leucine ( Figure 3 The site was further sequenced and verified. Using genomic DNA as a template, primers LOX6-F and LOX6-R were used for amplification. The obtained PCR product was sequenced and found that the mutant had a T base at this site, while the wild type had a C base. Therefore, the candidate gene was preliminarily determined to be the lipoxygenase gene LOX6 ( Figure 3 ). The gene was predicted to be LOX6 in the lipoxygenase family of genes ( Figure 4 ).

[0053] The primer sequences for sequencing verification of the LOX6 mutation site are:

[0054] LOX6-F: 5'-GACCACGACGTTCATGACC-3' (SEQ ID NO. 1);

[0055] LOX6-R: 5'-CTCAATTTTCCCACTTACCTC-3' (SEQ ID NO. 2).

[0056] Example 3 Knockout of Rice Spikelet Development Control Gene LOX6

[0057] 1. Construction of gene knockout vectors: The vectors used were pYLCRISPR / Cas9Pubi-H, pYLgRNA-OsU6a, and pYLgRNA-OsU6b, which have been published in the literature (Ma X, Liu YG. CRISPR / Cas9-Based Multiplex Genome Editing in Monocot and Dicot Plants [J]. 2016). The CRISPR / Cas9 knockout vector was constructed using a strategy of expressing two guide RNAs (sgRNAs) per vector. The two sgRNAs were designed to target positions 1 to 23 of the first exon of the LOX6 gene (SEQ ID NO. 3: 5'-ATGGCGTCGTTGGAGTTGCT GGG -3') and the seventh exon 720 to 741 (SEQ ID NO.4: 5'- CCT GCCGGGGCGTGCCAAACAG-3'), the underline is the PAM sequence that conforms to the NGG sequence. The first sgRNA target sequence of LOX6 was connected to pYLgRNA-OsU6a, and the second sgRNA target sequence was connected to pYLgRNA-OsU6b. After constructing a complete expression cassette by target linker ligation amplification method, the expression cassette was cloned into the pYLCRISPR / Cas9Pubi-H vector containing the Cas9 enzyme expression frame to obtain a complete recombinant vector. The specific construction method was carried out according to the method described in the reference (Zeng Dongchang, Ma Xingliang, Xie Xianrong, et al. Operation method for construction and mutation analysis of plant CRISPR / Cas9 multi-gene editing vector [J]. Chinese Science, 2018, 048(007): P.783-794.). The constructed knockout vector was transferred into ZH11 by Agrobacterium-mediated genetic transformation, and the genetic transformation was entrusted to Wuhan Boyuan Biological Co., Ltd.

[0058] 2. Genotypic identification and phenotypic investigation of the obtained transgenic materials. DNA was extracted from leaves of the transgenic materials. The sequence near the first target site was amplified by PCR using primers LOX6-T1-F (SEQ ID NO. 5: 5'-TCGGGCTACTCGTCTTCT CGT-3') and LOX6-T1-R (SEQ ID NO. 6: 5'-GTCCCCTTCCCTGCAAAACCC-3'). The sequence near the second target site was amplified by PCR using primers LOX6-F (SEQ ID NO. 1) and primer LOX6-R (SEQ ID NO. 2). The genotype was then identified by sequencing the PCR products. Sequencing was provided by Qingke Biotechnology (Guangzhou). According to the division of rice panicle development stages proposed by Ding Ying (Ding Ying, Li Naiming, Xu Xuebin, et al. Observation on the development of rice panicles and grain filling process [J]. Acta Agricultural Sinica, 1959), the spikelet phenotypes of ZH11 and rice LOX6 knockout mutants were observed at the eighth stage of panicle development.

[0059] The results are as follows Figure 5 As shown: base insertions and deletions occurred in the target genes of the knockout materials. The knockout plants were named KO-1 (a base T was inserted between the 11th and 12th positions of the first exon of the lipoxygenase gene LOX6), KO-2 (a base G was inserted between the 729th and 730th positions of the seventh exon of the lipoxygenase gene LOX6), and KO-3 (a four-base deletion of AGTT from the 14th to 17th positions of the first exon of the lipoxygenase gene LOX6). Compared with the wild type ZH11, the knockout plants all showed abnormal spikelet development, that is, the mutation of the lipoxygenase gene LOX6 regulates the development of rice spikelets.

[0060] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Lipoxygenase gene LOX6 The application of the invention in regulating the development of rice spikelets is characterized by: The lipoxygenase gene LOX6 The gene number on NCBI is LOC 9267158; The regulation of rice spikelet development is through the lipoxygenase gene LOX6 The mutation causes abnormal development of spikelets in rice plants; The abnormal development of spikelets includes changes in the shape of the glumes and the number of stamens and pistils; The changing of the husk morphology is changing from a normal type to a degenerate type, an open type or a multi-lemma type; The degenerate type is characterized by varying degrees of palea degradation or absence; the unclosed type is characterized by the two palea tips being hooked and unable to close; the multi-lemma type is characterized by an additional lemma-like structure compared to wild-type rice; The change in stamen number is manifested in that the number of stamens in the degenerate spikelets is reduced, while the number of stamens in the open spikelets is increased compared to wild-type rice. The change in pistil number is manifested in that the pistils of the non-closed spikelets have more ovary grains compared to wild-type rice; The lipoxygenase gene LOX6 Mutation can be achieved by any of the following methods: (a) Lipoxygenase gene LOX6 The 735th base C of the seventh exon was replaced by a T base; (b) Lipoxygenase gene LOX6 A T base is inserted between positions 11 and 12 of the first exon; (c) Lipoxygenase gene LOX6 A G base was inserted between positions 729 and 730 of the seventh exon; (d) Lipoxygenase gene LOX6 There is a 4-base deletion of AGTT from position 14 to position 17 of the first exon.

2. A lipoxygenase gene LOX6 A mutant characterized in that The nucleotide sequence is any of the following: (a) Lipoxygenase gene LOX6 The 735th base C of the seventh exon was replaced by a T base; (b) Lipoxygenase gene LOX6 A T base is inserted between positions 11 and 12 of the first exon; (c) Lipoxygenase gene LOX6 A G base was inserted between positions 729 and 730 of the seventh exon; (d) Lipoxygenase gene LOX6 The 4 bases AGTT are deleted from position 14 to 17 of the first exon; The lipoxygenase gene LOX6 The gene number on NCBI is LOC 9267158.

3. Containing the lipoxygenase gene according to claim 2 LOX6 expression vectors or recombinant microorganisms of mutants.

4. The lipoxygenase gene according to claim 2 LOX6 The use of a mutant of in regulating rice spikelet development is characterized by: The regulation of rice spikelet development is through the lipoxygenase gene LOX6 The mutation causes abnormal development of spikelets in rice plants; The abnormal development of spikelets includes changes in the shape of the glumes and the number of stamens and pistils; The changing of the husk morphology is changing from a normal type to a degenerate type, an open type or a multi-lemma type; The degenerate type is characterized by varying degrees of palea degradation or absence; the unclosed type is characterized by the two palea tips being hooked and unable to close; the multi-lemma type is characterized by an additional lemma-like structure compared to wild-type rice; The change in stamen number is manifested in that the number of stamens in the degenerate spikelets is reduced, while the number of stamens in the open spikelets is increased compared to wild-type rice. The change in pistil number is manifested in that the pistils of the non-closed spikelets have more ovary grains compared with wild-type rice.

5. The lipoxygenase gene according to claim 2 LOX6 Application of mutants in the preparation of transgenic rice.

6. A method for editing the lipoxygenase gene of claim 1 LOX6 The application of a gRNA target sequence in regulating rice spikelet development is characterized in that, The lipoxygenase gene LOX6 The gRNA target sequences are as follows: (1) Located in the lipoxygenase gene LOX6 The nucleotide sequence of positions 1 to 23 of the first exon is shown in SEQ ID NO. 3; and (2) Located in the lipoxygenase gene LOX6 The nucleotide sequence of positions 720 to 741 of the seventh exon is shown in SEQ ID NO. 4; The regulation of rice spikelet development is through the lipoxygenase gene LOX6 The mutation causes abnormal development of spikelets in rice plants; The abnormal development of spikelets includes changes in the shape of the glumes and the number of stamens and pistils; The changing of the husk morphology is changing from a normal type to a degenerate type, an open type or a multi-lemma type; The degenerate type is characterized by varying degrees of palea degradation or absence; the unclosed type is characterized by the two palea tips being hooked and unable to close; the multi-lemma type is characterized by an additional lemma-like structure compared to wild-type rice; The change in stamen number is manifested in that the number of stamens in the degenerate spikelets is reduced, while the number of stamens in the open spikelets is increased compared to wild-type rice. The change in pistil number is manifested in that the pistils of the non-closed spikelets have more ovary grains compared with wild-type rice.

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