Rice SDT6 gene, its encoded protein and its application

Abstract

The present invention relates to a rice SDT6 gene, a protein encoded therein and an application thereof. The present invention provides an SDT6 gene for regulating rice plant type, and the nucleotide sequence of the SDT6 gene is shown in SEQ ID NO.2 or SEQ ID NO.3. The amino acid sequence of the protein encoded by the SDT6 gene is shown in SEQ ID NO.1. The present invention screened a mutant with semi-dwarf stem and increased tillering and successfully cloned the target gene for regulating the phenotype. After the gene was knocked out in the japonica rice ZH11 material by CRISPR / Cas9 technology, the rice plant height was significantly reduced and the tillering was significantly increased. The gene function was further confirmed by gene function complementation verification, and the gene SDT6 was preliminarily verified to regulate the plant height and tillering of rice, and it was found that it was related to the auxin regulation mechanism in rice. The present application is of great significance in breeding rice, indica or japonica rice with excellent yield and / or lodging resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of gene SDT6 in regulating the plant type of rice. Background Art

[0002] The plant type of rice refers to the morphological characteristics of the plant, mainly including indicators such as plant height and panicle type. Rice with a good plant type often has a moderate plant height and a relatively compact plant type. A moderate plant height is conducive to the efficient utilization of light energy and the absorption of nutrients. Excessively tall rice plants are prone to insufficient light energy in the lower leaves, affecting photosynthesis, and are easily affected by the environment and lodge, thereby affecting the yield. A moderate plant height can ensure sufficient light energy for the upper leaves of the plant, improve the photosynthesis efficiency, and is conducive to the mutual support between individual rice plants. This plant type helps to give full play to photosynthesis and can maximize the absorption of light energy. At the same time, rice with a good panicle type often can increase the number of tillers on the panicle, improve the panicle grain capacity and the yield per panicle, thereby increasing the total yield. Therefore, the plant type is the key to achieving high yields. The plant type of rice determines the spatial distribution, photosynthesis efficiency, lodging resistance, and stress resistance of rice, and plays a crucial role in the exploration of rice yield.

[0003] The history of dwarfing breeding in rice has a long development. Since the late 1950s and 1960s, work related to dwarfing breeding for plant type improvement has been carried out. Related research shows that dwarfing breeding based on the dwarf gene semi-dwarf1 (sd1) has greatly improved the yield potential of rice, and this breeding behavior is called the "Green Revolution". The semi-dwarf breeding of the "Green Revolution" is achieved by regulating the biosynthesis and signal transduction of the plant hormone gibberellin (GA). The semi-dwarf gene SD1 of rice encodes the GA synthase GA-20ox2, and the mutation of this gene leads to the inhibition of GA synthesis, the accumulation of the plant growth inhibitor DELLA protein, and the inhibition of rice growth, thereby producing a semi-dwarf phenotype. Dwarfing breeding improves the lodging resistance and economic coefficient of rice by reducing the plant height of rice, increasing the rice yield by 20% - 30% compared with the original local varieties, and achieving the first leap in rice yield. Since then, the dwarfing breeding of rice has been widely concerned.

[0004] Recently, a research team from the Chinese Academy of Agricultural Sciences used genome-wide association study (GWAS) technology to identify the Arabidopsis auxin transporter ABCB1 homologous gene SD8 related to plant height in rice, and created SD8 gene-edited mutants sd8-1 and sd8-2 under the Nipponbare (NIP) background using CRISPR-Cas9 technology. The plant heights of sd8-1 and sd8-2 were significantly reduced, the flag leaf angles became smaller, the plants were more upright, and the yield per plant was not affected. The results of field trials showed that the sd8-1 knockout line had a significant yield increase effect (about 10%) under dense planting conditions, while there was no significant difference in the yield of sd8-1 compared with the conventional rice NIP under normal planting density conditions. The research of this team on improving the rice plant type by editing the auxin transport gene SD8 in rice without affecting the yield traits provides a new strategy for increasing the yield of rice under dense planting conditions using a single gene.

[0005] Although a large number of rice dwarf mutants have been identified, most of them are accompanied by adverse traits such as rolled leaves, floral organ variations, and shriveled seeds, and cannot be applied to breeding, resulting in sd1 being almost the only dwarf germplasm resource. The widespread application of this single gene may pose a potential risk of genetic diversity loss. In addition, sd1 materials usually also show disadvantages such as poor resistance and low nitrogen use efficiency, which to a certain extent restricts the breeding of new rice varieties. Therefore, exploring new dwarf gene resources is very important for further improving rice high-yield breeding.

[0006] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, although the applicant studied a large number of literatures and patents when making this invention, all details and contents are not listed in detail due to space limitations. However, this does not mean that this invention does not possess the features of these prior arts. On the contrary, this invention already possesses all the features of the prior arts, and the applicant reserves the right to add relevant prior arts in the background art. Summary of the Invention

[0007] On the one hand, the present application provides an auxin regulatory protein. The sequence of the auxin regulatory protein includes the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence of the auxin regulatory protein includes an amino acid sequence or derivative generated by adding, substituting, inserting, or deleting one or more amino acids or homologous sequences of other species in the amino acid sequence shown in SEQ ID NO.1.

[0008] On the other hand, the present application provides an auxin regulatory protein obtained by chemical modification or genetic engineering means and having the function of regulating the rice plant type. The amino acid sequence of the auxin regulatory protein includes an amino acid sequence or derivative generated by adding, substituting, inserting, or deleting one or more amino acids or a homologous sequence of other species in the amino acid sequence shown in SEQ ID NO.1.

[0009] On the other hand, the present application provides an auxin regulatory gene. The auxin regulatory gene encodes an auxin regulatory protein. The auxin regulatory gene is named SDT6 (LOC_Os06g44500). The nucleotide sequence of the auxin regulatory gene includes a mutant sequence, allele, or derivative generated by adding, substituting, inserting, or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3.

[0010] The gene SDT6 is annotated as an F-box protein (FBK) containing a Kelch repeat domain. Related studies have shown that F-box proteins usually function as a subunit of the E3 ubiquitin ligase SCF complex.

[0011] On the other hand, the present application provides the use of a protein containing the amino acid sequence shown in SEQ ID NO.1 in regulating the rice plant type.

[0012] According to a preferred embodiment, the rice plant type includes plant height, tiller number, root number, and root length.

[0013] On the other hand, the present application provides the use of a gene encoding an auxin regulatory protein in regulating the rice plant type, wherein the gene sequence encoding the auxin regulatory protein includes the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3.

[0014] On the other hand, the present application provides a rice seed for reducing the plant height or improving the lodging resistance of a plant. The rice seed has been genetically modified to reduce the expression level of a protein whose sequence includes the amino acid sequence shown in SEQ ID NO.1, or to reduce the expression level of a gene whose sequence includes the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3.

[0015] On the other hand, the present application provides a rice plant for reducing the plant height or improving the lodging resistance of a plant. The plant has been genetically modified to reduce the expression level of a protein whose sequence includes the amino acid sequence shown in SEQ ID NO.1, or to reduce the expression level of a gene whose sequence includes the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3.

[0016] Genetic modification involves the act of altering the genetic information of a plant or seed using gene editing techniques or homologous hybridization techniques.

[0017] On the other hand, the present application provides an agricultural composition. The agricultural composition comprises a plant line that has been genetically modified to reduce the expression level of a protein whose sequence contains the amino acid sequence shown in SEQ ID NO.1, or to reduce the expression level of a gene whose sequence contains the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3. The plant line is, for example, a gramineous crop comprising a rice line.

[0018] On the other hand, the present application provides a genetic engineering toolkit. The genetic engineering toolkit includes at least one active molecule for downregulating the expression level of a gene encoding a protein with the amino acid sequence shown in SEQ ID NO.1, or for downregulating the expression level of a protein with the amino acid sequence shown in SEQ ID NO.1, or for reducing the expression level of a gene with the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3. The active molecule is, for example, a cell, a recombinant vector, a CTP / CPP nanocarrier for large-scale delivery of polypeptide biomolecular complexes in plants (for example, a nanoparticle-based directed biomolecular delivery tool launched by Mai'anna, namely foliar spray technology).

[0019] According to the relevant experimental results recorded in the examples, after knocking out or inactivating the gene SDT6 by the CRISPR / Cas9 editing technique, the plant height of rice decreased and tillering increased. The present invention has successfully cloned the gene SDT6 for the first time at the molecular level, providing a new gene resource for in-depth understanding of the mechanism of plant architecture regulation and molecular improvement breeding of rice plant architecture. Based on this, rice improvement aimed at maintaining crop yield stability, increasing crop yield, understanding the auxin regulation mechanism in crops, regulating auxin in crops, or increasing the landscape value of crops can adopt the method of regulating the gene SDT6 or the protein encoded by the gene SDT6.

[0020] On the other hand, the present application provides a method for reducing the plant height of a plant or improving the lodging resistance of a plant. The method comprises the following steps:

[0021] Downregulating the expression level of a gene encoding a protein with the amino acid sequence shown in SEQ ID NO.1 in rice,

[0022] or downregulating the expression level of a protein with the amino acid sequence shown in SEQ ID NO.1 in rice,

[0023] or reducing the activity of a protein with the amino acid sequence shown in SEQ ID NO.1 in rice.

[0024] According to a preferred embodiment, a method for downregulating the expression level of a gene encoding a protein with the amino acid sequence shown in SEQ ID NO.1 in rice, or downregulating the expression level of a protein with the amino acid sequence shown in SEQ ID NO.1 in rice, or reducing the activity of a protein with the amino acid sequence shown in SEQ ID NO.1 in rice comprises:

[0025] Knocking out the base G at position 296 of the gene;

[0026] Or adding a base G at position 297 of the gene;

[0027] Or adding a base T at position 295 of the gene.

[0028] On the other hand, the present application provides a method for screening auxin-deficient phenotype rice. The method comprises the following steps:

[0029] Detecting the expression level of a protein with the amino acid sequence shown in SEQ ID NO.1,

[0030] Or detecting the activity of a protein with the amino acid sequence shown in SEQ ID NO.1,

[0031] Or detecting the expression level of a gene encoding a protein with the amino acid sequence shown in SEQ ID NO.1.

[0032] On the other hand, the present application provides a recombinant vector. The recombinant vector comprises a functional gene operably linked to a vector, wherein the nucleotide sequence of the functional gene is shown in SEQ ID NO.2 or SEQ ID NO.3. Preferably, the basic vector of the recombinant vector includes a prokaryotic expression vector and / or a eukaryotic expression vector. More preferably, the recombinant vector includes a binary Agrobacterium vector or a vector suitable for plant gene gun bombardment, such as pCAMBIA3301, pCAMBIA2300, pCAMBIA2301, pCAMBIA1300, pCAMBIA1301, pWM101, pGreen0029, pBI121, pBin19, pCAMBIA1301-UbiN, etc. or other derived plant expression vectors. More preferably, the recombinant vector can use enhancers, including translation enhancers or transcription enhancers, and these enhancer regions can be the ATG start codon (within the reading frame of the coding sequence) or the adjacent region start codon (within the reading frame of the coding sequence), etc.

[0033] The recombinant vector can use a gene encoding an enzyme or a luminescent compound that can produce a color change, an antibiotic marker with resistance, or an anti-chemical reagent marker gene expressed in plants to screen for positive plants. For example: GUS reporter gene.

[0034] The recombinant vector involved in this application is not only applicable to transferring into rice to regulate the expression level of gene SDT6 or the protein encoded thereby, but also capable of being transferred into other gramineous plants, tobacco or Arabidopsis thaliana for scientific research exploration.

[0035] On the other hand, the present invention provides a plant cell transformation vector containing the gene SDT6 that regulates the plant type of rice.

[0036] On the other hand, the present invention provides a recombinant cell containing the gene SDT6 that regulates the plant type of rice.

[0037] On the other hand, the present invention provides a recombinant cell containing the protein encoded by the gene SDT6 that regulates the plant type of rice. The specific construction method of the recombinant cell can be the Agrobacterium-mediated method, the gene gun method, the electroporation method, the pollen tube introduction method, the liposome fusion method, and any other method that can introduce the plasmid into the transformed plant cell or tissue.

[0038] On the other hand, the present invention also provides the application of the gene SDT6, the protein encoded by the gene SDT6, the recombinant vector containing the gene SDT6, and the recombinant cell containing the gene SDT6 in regulating the plant type of indica rice or japonica rice. Preferably, the regulation is achieved by inhibiting or reducing the expression of the gene SDT6 in rice.

[0039] On the other hand, the present invention also provides the application of the gene SDT6, the protein encoded by the gene SDT6, the recombinant vector containing the gene SDT6, and the recombinant cell containing the gene SDT6 in regulating the plant height of indica rice or japonica rice. Preferably, the regulation is achieved by inhibiting or reducing the expression of the gene SDT6 in rice.

[0040] On the other hand, the present invention also provides the application of the gene SDT6, the protein encoded by the gene SDT6, the recombinant vector containing the gene SDT6, and the recombinant cell containing the gene SDT6 in regulating the tillering phenotype of indica rice or japonica rice. Preferably, the regulation is achieved by inhibiting or reducing the expression of the gene SDT6 in rice. Preferably, the tillering phenotype includes the number of tillers.

[0041] On the other hand, the present invention also provides the application of the gene SDT6, the protein encoded by the gene SDT6, the recombinant vector containing the gene SDT6, and the recombinant cell containing the gene SDT6 in regulating the stem phenotype of indica rice or japonica rice. Preferably, the regulation is achieved by inhibiting or reducing the expression of the gene SDT6 in rice.

[0042] On the other hand, the present invention also provides the application of gene SDT6, the protein encoded by gene SDT6, the recombinant vector containing gene SDT6, and the recombinant cell containing gene SDT6 in regulating the content of auxin IAA in indica rice or japonica rice. Preferably, the regulation is achieved by inhibiting or reducing the expression of gene SDT6 in rice. Preferably, by inhibiting or reducing the expression of gene SDT6, the content of active auxin is reduced and the content of amino - acidified auxin is increased.

[0043] On the other hand, the present invention also provides the application of gene SDT6, the protein encoded by gene SDT6, the recombinant vector containing gene SDT6, and the recombinant cell containing gene SDT6 in regulating the root phenotype of indica rice or japonica rice. Preferably, the regulation is achieved by inhibiting or reducing the expression of gene SDT6 in rice. Preferably, the root phenotype includes the number of root hairs and the degree of bending of seminal roots and stems.

[0044] On the other hand, the present invention also provides a method for improving the stress resistance of rice. This method uses gene editing technology to inhibit the expression of gene SDT6 or reduce the expression level of gene SDT6.

[0045] On the other hand, the present invention also provides a method for improving the stress resistance of rice. This method uses gene knockout, RNA interference or natural variants to inhibit the expression of gene SDT6 or reduce the expression level of gene SDT6. Preferably, this method reduces the plant height of rice by inhibiting the expression of gene SDT6 or reducing the expression level of gene SDT6, thereby increasing the lodging - resistance of rice. Preferably, this method reduces the internode length of the rice stem by inhibiting the expression of gene SDT6 or reducing the expression level of gene SDT6, thereby increasing the lodging - resistance of rice.

[0046] On the other hand, the present invention also provides a method for regulating the tiller number of rice, which increases the tiller number of rice by inhibiting the expression of gene SDT6 or reducing the expression level of gene SDT6.

[0047] On the other hand, the present invention also provides the application of gene SDT6 for regulating rice plant type or the protein encoded by gene SDT6 for regulating rice plant type in breeding indica rice or japonica rice with excellent yield and / or lodging - resistance. Brief Description of the Drawings

[0048] Figure 1It is the phenotypic analysis diagram of mutant sdt6. Among them, A is the comparison diagram of the plant types of wild type and sdt6 mutant at the seedling stage; B is the comparison diagram of the plant types of wild type and sdt6 mutant at the mature stage; C is the statistical chart of plant heights of wild type and sdt6 mutant at the mature stage; D is the statistical chart of tillers of wild type and sdt6 mutant at the mature stage; E is the comparison diagram of internode lengths of wild type and sdt6 mutant at the mature stage; F is the statistical chart of internode lengths of wild type and sdt6 mutant at the mature stage; G is the comparison diagram of leaf lengths of wild type and sdt6 mutant; H is the comparison diagram of panicle types of wild type and sdt6 mutant at the mature stage; I is the statistical chart of leaf lengths and panicle lengths of wild type and sdt6 mutant;

[0049] Figure 2 It is the gene mapping of sdt6 mutant. Among them, A is the distribution diagram of SNPs in the sdt6 mutant bulk pool on the chromosome; B is the schematic diagram of the SDT6 protein structure; C is the schematic diagram of the SDT6 protein mutation site;

[0050] Figure 3 It is the phenotypic diagram of the knockout lines of SDT6 in japonica rice ZH11 material. Among them, A is the comparison diagram of the plant types of wild type and CR-1, CR-2, CR-3 at the mature stage; B is the statistical chart of plant heights of wild type and CR-1, CR-2, CR-3 at the mature stage; C is the statistical chart of tillers of wild type and CR-1, CR-2, CR-3 at the mature stage;

[0051] Figure 4 It is the phenotype of the complementary positive plants of sdt6 mutant. Among them, A is the comparison diagram of the plant types of wild type and sdt6, COM-1, COM-2 at the mature stage; B is the statistical chart of plant heights of wild type and sdt6, COM-1, COM-2 at the mature stage; C is the statistical chart of tillers of wild type and sdt6, COM-1, COM-2 at the mature stage;

[0052] Figure 5 It is the determination diagram of the contents of different types of auxin in the leaves of wild type and sdt6 mutant. Among them, A is the statistical chart of IAA content in the leaves of wild type and sdt6; B is the statistical chart of IAA-Glc content in the leaves of wild type and sdt6; C is the statistical chart of IAA-Asp content in the leaves of wild type and sdt6; D is the statistical chart of IAA-Glu content in the leaves of wild type and sdt6; E is the statistical chart of OxIAA content in the leaves of wild type and sdt6;

[0053] Figure 6 It is the comparison diagram of the root response of wild type and sdt6 mutant seedlings to exogenous IAA treatment. Among them, A is the comparison diagram of the root growth of wild type and sdt6 mutant seedlings under exogenous IAA treatment; B is the statistical chart of the root growth of wild type and sdt6 mutant seedlings under exogenous IAA treatment;

[0054] Figure 7It is the phenotype diagram of wild type, sdt6 mutant and SDT6 transgenic material related to auxin, among which A is the wild type and sdt6 geotropism response diagram; B is the wild type and SDT6-CR, SDT6-OE geotropism response diagram; C is the wild type and sdt6 root bending statistics diagram; D is the wild type and sdt6 shoot bending statistics diagram; E is the wild type and SDT6-CR2, SDT6-OE1 root bending statistics diagram; F is the wild type and SDT6-CR2, SDT6-OE1 shoot bending statistics diagram; G is the wild type root hair growth; H is the SDT6-CR root hair growth; I is the wild type root hair growth enlarged diagram in G; J is the SDT6-CR root hair growth enlarged diagram in H. Specific implementation method

[0055] The following is a detailed description with reference to the accompanying drawings.

[0056] Plant type is the three-dimensional arrangement of plant organs, which is affected to some extent by environmental factors such as light, temperature, plant density and humidity. However, due to the involvement of multiple molecular regulatory mechanisms and hormone signaling pathways, the composition of plant structure is inherently complex. So far, although many genes involved in the auxin signaling pathway and affecting rice grain type and grain weight have been cloned, the functional relationship between these genes is very complex, and their genetic diversity information in rice populations is also unclear. Therefore, like most cloned rice genes, genes known to be involved in the auxin signaling pathway are difficult to apply to the genetic improvement of rice, especially the improvement of yield traits.

[0057] The plant type model that was mainly established around high yield in the past may not be suitable for the new goal of taking into account high yield, high quality, high efficiency and greenness. In the future, we need to make full use of the latest theoretical and technological achievements of modern biology, especially bioinformatics, phenotype groups, gene editing, molecular design breeding, whole genome selection, etc., to analyze the external and internal relationships between various organs, and build and optimize the precise plant type model of rice. On this basis, we will clarify the molecular mechanism and regulatory network and put it into practice in plant type breeding to achieve a higher level of comprehensive "ideal rice plant type".

[0058] This invention identified a mutant sdt6 with reduced plant height and increased tillering from the mutant library of indica rice Shuhui 600 (R600) induced by ethyl methanesulfonate (EMS). The gene was located using MutMap technology combined with co-segregation verification, and the SDT6 gene was preliminarily explored for its role in regulating rice plant type through gene cloning, gene editing, phenotypic analysis, etc., proving that inactivation of the SDT6 gene would shorten rice plant height and increase tillering.

[0059] Example

[0060] In this invention, mutants with semi-dwarf and increased tillering were screened, and the target gene regulating this phenotype was successfully cloned. After knocking out this gene in japonica rice ZH11 material by CRISPR / Cas9 technology, the plant height of rice decreased significantly and tillering increased significantly. Gene function complementation verification further confirmed the gene function, and it was preliminarily verified that the regulation of rice plant height and tillering by gene SDT6 may be related to auxin. This example includes the following research processes and research results.

[0061] 1. Creation of mutant materials

[0062] A large number of mutants were obtained by EMS mutagenesis of indica rice variety R600 (wild type, WT). A mutant material sdt6 with reduced plant height and increased tillering was screened from this mutant library. After multiple generations of self-crossing, the mutant sdt6 with a stable genetic phenotype was finally screened. All rice materials were planted in the experimental base of Sichuan Agricultural University in Wenjiang District, Chengdu City, Sichuan Province and the southern breeding base in Lingshui Li Autonomous County, Hainan, and all were under conventional management.

[0063] 2. Analysis of semi-dwarf phenotype of mutants

[0064] Through field agronomic trait investigation, it was found that there was no obvious change in the plant height of the mutant at the seedling stage compared with the wild type ( Figure 1 A), and at the mature stage, the plant height of the mutant decreased by about 20 cm compared with the wild type, showing semi-dwarfism ( Figure 1 B, Figure 1 C, Figure 1 D). The lengths of each internode of the mutant at the mature stage were measured, and the results showed that each internode of the mutant was significantly shorter than that of the wild type ( Figure 1 E, Figure 1 F). In addition, the leaf length and panicle length of the mutant were both smaller than those of the wild type ( Figure 1 G, Figure 1 H, Figure 1 I).

[0065] 3. Mapping and cloning of candidate genes

[0066] sdt6 was crossed with wild type R600 to obtain the BC1F1 generation. The phenotype of BC1F1 plants was consistent with that of the wild type, indicating that sdt6 is a recessive mutation. The phenotypes of wild type and mutant plants in the BC1F2 population were statistically analyzed in the field. After chi-square test, it was found that the ratio of the number of plants with normal plant type and mutant plant type conforms to 3:1. This shows that the phenotype of mutant sdt6 is controlled by a pair of recessive single genes.

[0067] In this example, 30 individual plants with mutant phenotypes were selected from the F2 population, and pooled DNA sequencing was performed on them. Using the genome of R600 as the reference sequence, analysis was carried out through the MutMap pipeline. The linked region was identified by SNP-index. Finally, in this example, the candidate gene of the mutant was mapped to chromosome 6( Figure 2 A), the CDS sequence of this gene is shown in SEQ ID NO.2, and the amino acid sequence encoded by this gene is shown in SEQ ID NO.1, as shown in Table 1. By screening the linked region of chromosome 6, it was found that the sdt6 mutant had a single-base C-T mutation in the coding region of the candidate gene, resulting in the amino acid encoded by it changing from Ser to Phe( Figure 2 C).

[0068] To further verify this candidate gene, in this example, 40 plants with mutant phenotypes were reselected for cosegregation verification, and it was found that the gene variation at this locus cosegregated with the mutant phenotype. Bioinformatics analysis showed that this candidate gene encodes a protein of the FBKs family. This type of protein usually serves as a subunit of the SCF complex and has E3 ubiquitin ligase activity. Its N-terminus contains a conserved F-box domain and its C-terminus contains a Kelch repeat domain( Figure 2 B). Based on the above results, in this example, it is considered that this gene is the candidate gene.

[0069] Table 1

[0070]

[0071]

[0072]

[0073] 4. Verification of SDT6 gene knockout

[0074] To verify whether the SDT6 gene is the target gene, in this example, the CRISPR / Cas9 technology was used to edit the SDT6 gene under the background of ZH11. Through sequencing identification, in this example, a total of 3 knockout types were obtained, namely CR-1 (deletion of one base G), CR-2 (insertion of one base G), and CR-3 (insertion of one base T). The above mutations all caused premature termination of protein translation. Further investigation of the agronomic traits of the knockout plants showed that compared with ZH11, the plant height of the CR plants was significantly reduced and the tillering was significantly increased( Figure 3 A、 Figure 3 B、 Figure 3 C). As Figure 3As shown in Figure B, the height of wild-type ZH11 is 103.5 cm, the height of CR-1 is 79.87 cm, the height of CR-2 is 76.67 cm, and the height of CR-3 is 75.6 cm. As Figure 3 As shown in Figure C, the tiller number of wild-type ZH11 is 7.8, the tiller number of CR-1 is 12.4, the tiller number of CR-2 is 14, and the tiller number of CR-3 is 14.5.

[0075] 5. Complementary verification of the function of the SDT6 gene

[0076] To further verify whether the mutant phenotype is caused by the variation of the SDT6 gene, this example conducted a complementary verification of gene function. The gDNA containing the wild-type SDT6 gene was transferred into the mutant sdt6 to obtain sdt6 complementary transgenic plants COM-1 and COM-2. The plant height and tiller number of the positive complementary plants were restored to the wild-type level ( Figure 4 A, Figure 4 B, Figure 4 C). This indicates that the SDT6 gene is the target gene regulating the sdt6 mutant phenotype. As Figure 4 Shown in Figure B, the height of wild-type R600 is 130.5 cm, the height of the sdt6 mutant is 109.6 cm, the height of COM-1 is 130.3 cm, and the height of COM-2 is 131.8 cm. As Figure 4 Shown in Figure C, the tiller number of wild-type R600 is 5.3, the tiller number of the sdt6 mutant is 9.6, the tiller number of COM-1 is 5.7, and the tiller number of COM-2 is 5.6.

[0077] 6. SDT6 is involved in regulating the rice plant type through the auxin-related pathway

[0078] Research has shown that auxin is an important phytohormone regulating plant height. Therefore, in this example, the contents of different types of auxin in the leaves of wild-type R600 and sdt6 were measured. Compared with the wild-type, the content of active IAA in sdt6 was significantly reduced ( Figure 5 A and Figure 5 B, where IAA-Glc is a substance enhancing the biological activity of auxin), and the contents of IAA storage forms IAA-Asp ( Figure 5 C), IAA-Glu ( Figure 5 D) and the inactivated form oxIAA ( Figure 5 E) were significantly increased. This indicates that the content of active IAA in sdt6 decreased while the content of amino-grouped IAA increased. As Figure 5 Shown in Figure A, the content of active IAA in wild-type (WT) R600 is 12.1 ng / g, and the active IAA in sdt6 is 8.4 ng / g. As Figure 5As shown in C, the content of aminated IAA (IAA-Asp) in wild-type (WT) ZH11 is 26.3 ng / g, and that in sdt6 is 40.9 ng / g.

[0079] In this example, wild-type R600 and sdt6 rice seedlings grown in a hydroponic box were externally treated with IAA for 7 days, and then the growth of their roots was investigated and statistically analyzed. As Figure 6 shown in B, when the applied IAA content was 1 μM, the root length of wild-type ZH11 (WT) was 10.86 cm, while that of sdt6 was 12.34 cm. When the applied IAA content was 5 μM, the root length of wild-type ZH11 (WT) was 8.67 cm, while that of sdt6 was 10.74 cm. When the applied IAA content was 10 μM, the root length of wild-type ZH11 (WT) was 7.53 cm, while that of sdt6 was 8.46 cm. The results showed that the growth of the roots of wild-type and mutant seedlings was inhibited after IAA treatment, but the inhibitory effect of IAA on the root growth of sdt6 was significantly weakened compared with that of the wild type, and sdt6 was insensitive to IAA treatment ( Figure 6 A, Figure 6 B).

[0080] Research has shown that auxin can promote plant growth and development and root hair formation. Therefore, in this example, after the seeds were disinfected with 70% ethanol, they were placed on a 1 / 2MS solid medium containing 0.6% agar. After 24 hours, by observing the root hair phenotypes of wild-type ZH11 and SDT6-CR, it was found that the root hairs of SDT6-CR were significantly fewer than those of the wild type, showing an auxin deficiency phenotype ( Figure 7 G, Figure 7 H, Figure 7 I, Figure 7 J). Therefore, it can be reasonably confirmed that the gene SDT6 is involved in the auxin pathway and affects the root phenotype of rice.

[0081] Due to the effect of gravity, the auxin concentration on the proximal side is higher than that on the distal side. The stem is insensitive to auxin. With a high auxin concentration on the proximal side, the growth is fast, and the stem bends upward; the root is sensitive to auxin. With a high auxin concentration on the proximal side, the growth is inhibited, and the growth is slow, and the root bends downward. Therefore, in this example, after the disinfected seeds were placed vertically for 2 days, the seeds were then placed horizontally on a 1 / 2MS medium containing 0.6% agar.

[0082] As Figure 7 shown in C, the root curvature of wild-type ZH11 (WT) is 62.66°, while that of sdt6 is 49.75°. As Figure 7As shown in D, the bud curvature of wild-type ZH11 (WT) was 61.99°, while that of sdt6 was 46.62°. As Figure 7 shown in E, the root curvature of wild-type ZH11 (WT) was 59.98°, while that of CR-2 was 51.25° and that of OE-1 was 81.2°. As Figure 7 shown in F, the bud curvature of wild-type ZH11 (WT) was 54°, while that of CR-2 was 42.6° and that of OE-1 was 71.47°. OE-1 is an overexpression line of the gene SDT6.

[0083] By observing the bending degree of seminal roots and shoots, it was found that compared with the wild type, the gravitropic responses of sdt6 and SDT6-CR were weakened, and auxin deficiency phenotypes appeared, as Figure 7 shown in C, Figure 7 shown in D, Figure 7 shown in E, Figure 7 shown in F. While the gravitropic response of SDT6-OE was enhanced ( Figure 7 shown in E, Figure 7 shown in F), further confirming that SDT6 is involved in the auxin-related pathway in rice.

[0084] It should be noted that the above specific embodiments are exemplary. Those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the description and drawings of the present invention are illustrative and do not constitute a limitation to the claims. The protection scope of the present invention is defined by the claims and their equivalents. The description of the present invention contains multiple inventive concepts. For example, "preferably" and "according to a preferred embodiment" indicate that the corresponding paragraphs disclose an independent inventive concept. The applicant reserves the right to file divisional applications based on each inventive concept. Throughout the text, the features guided by "preferably" are only optional ways and should not be understood as must be set. Therefore, the applicant reserves the right to abandon or delete relevant preferred features at any time.

Claims

1. Use of a protein having an amino acid sequence as shown in SEQ ID NO.1 in regulating rice plant height or rice tiller number, characterized in that: The application is to reduce the plant height of rice or increase the tiller number of rice by knocking out or inhibiting the protein shown in SEQ ID NO.

1.

2. Use of a gene having a nucleotide sequence as shown in SEQ ID NO.2 or SEQ ID NO.3 in regulating rice plant type, characterized in that: The use refers to reducing the height of rice plants or increasing the number of rice tillers by knocking out or inhibiting the gene shown in SEQ ID NO.2 or SEQ ID NO.

3.

3. A method for reducing the plant height of rice, characterized in that: The following steps are involved: Down-regulating the expression level of the gene encoding the protein with the amino acid sequence as shown in SEQ ID NO.1 in rice, or down-regulating the expression level of the protein with the amino acid sequence as shown in SEQ ID NO.1 in rice, Or reduce the activity of the protein with the amino acid sequence as shown in SEQ ID NO.1 in rice.

4. A method for screening auxin-deficient rice phenotype, characterized in that: The following steps are involved: Detecting the expression level of a protein whose amino acid sequence is shown in SEQ ID NO.1 in rice, if the expression level of the protein whose amino acid sequence is shown in SEQ ID NO.1 in the rice is downregulated relative to that of the wild type, then the rice has a defective phenotype of reduced root hairs, reduced root curvature and reduced shoot curvature; or detecting the activity of a protein whose amino acid sequence is shown in SEQ ID NO.1 in rice; if the activity of the protein whose amino acid sequence is shown in SEQ ID NO.1 in the rice is downregulated relative to that in the wild type, the rice has a defective phenotype of reduced root hairs, reduced root curvature and reduced shoot curvature; Or detecting the expression level of a gene encoding a protein having an amino acid sequence as shown in SEQ ID NO.1 in rice; if the expression level of the gene encoding a protein having an amino acid sequence as shown in SEQ ID NO.1 in the rice is downregulated relative to that of the wild type, then the rice has a defective phenotype of reduced root hairs, and reduced root curvature and shoot curvature.

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