Application of OsGTF5 gene in regulation and control of rice plant brittleness, plant height and heading period

Abstract

The invention discloses application of an OsGTF5 gene in regulation and control of rice plant brittleness, plant height and / or heading period, and belongs to the technical field of rice breeding. The coding sequence of the OsGTF5 gene is as shown in SEQ ID No.1 (sequence identifier number 1). During specific application, the OsGTF5 gene of wild rice is silenced in a mutation or knockout mode, and the mechanical strength of an obtained rice plant line and the rice plant is reduced, the plant height is reduced and / or the heading period is delayed; or transforming the OsGTF5 gene in the OsGTF5 gene silenced rice strain to obtain a genetic complementary rice strain, so that the mechanical strength, the plant height and / or the heading period time of the rice plant can be recovered. According to the invention, a gtf5 mutant is identified by a Mutmap method, and experimental research proves that the OsGTF5 gene is a gene for regulating and controlling the brittleness, the height and the heading period of a rice plant. Valuable resources are provided for cultivation of new brittle rice varieties and exploration of a heading period regulation mechanism, and the method has important significance in the aspects of renewable biomass resource utilization and the like.

Description

Technical Field

[0001] The invention relates to application of OsGTF5 gene in regulating rice plant fragility, plant height and heading period, and belongs to the technical field of rice breeding. Background Art

[0002] Rice is widely grown in East Asia, South Asia, the Middle East, the West Indies and Latin America, with more than 110,000 local varieties, providing more than one-fifth of the global human calorie consumption. As an important food crop, rice's yield-related agronomic traits such as heading date, stalk strength and efficient use of post-harvest straw have always attracted much attention.

[0003] Rice straw is an important renewable biomass resource with a wide range of applications, such as fermentation as feed, biomass fuel, culture soil, bio-oil and other raw materials. At present, the main straw treatment methods are burning, enzymatic hydrolysis and microbial degradation, which are highly polluting to the environment and have low utilization efficiency. The efficient utilization of rice straw is an important issue facing current rice production.

[0004] Heading period is a key trait for the transition from vegetative growth to reproductive growth, and is closely related to the photosensitivity and temperature sensitivity of rice. Currently, the cultivation of varieties with long basic vegetative growth period and weak photosensitivity is the main breakthrough point for unrestricted rice planting in different regions and seasons, and is an important goal of rice breeding. It is closely related to rice yield.

[0005] Plants have evolved tough cell walls to resist physical and chemical damage in nature, which is the main reason for the low straw recovery rate. The strength of plant stems depends on the composition and structural characteristics of the cell wall. The main components of plant cell walls are cellulose, hemicellulose, lignin and pectin. Cellulose molecules provide tensile strength, and the intermolecular hydrogen bonds between adjacent cellulose molecules make them strongly adhere to each other in overlapping parallel arrangements, forming highly ordered crystal aggregates (cellulose microfibrils). These crystal aggregates are further bonded by hemicellulose, pectin to regulate ion balance, adhere to cells and maintain cell wall integrity signal transmission, lignin and pectin fill the gaps to form a complex network, further enhancing its tensile strength and rigidity. The mature plant cell wall structure is divided into primary wall, secondary wall and intercellular layer. The secondary wall is the main determinant of mechanical support. Therefore, the secondary wall composition of brittle mutants may have changed, resulting in their inability to maintain the integrity of cell wall functions. Brittle rice mutant plants have the characteristics of reduced mechanical strength and easy crushing of straw, and are excellent new materials for breeding rice varieties. At the same time, the delay in the heading period of rice increases the duration of basic vegetative growth, which lays a rich material foundation for the later reproductive growth.

[0006] So far, the reported rice brittle mutants usually show that the loss of genes interferes with the normal biosynthesis of secondary walls, causing changes in the cellulose and lignin content in the cell walls, thereby reducing the mechanical strength of rice stems. For example, BC15 and BC19 genes are related to cellulose synthesis; the loss of BC14 gene causes changes in the arrangement of fiber bundles, etc. These studies have laid the foundation for exploring the regulatory network of rice plant brittleness, but the specific mechanism of action and its use in breeding remain to be studied. It is of great significance to explore genes that are insensitive to light and temperature and regulate the heading period of rice.

[0007] In summary, the rice brittle mutant provides a new perspective for the study of efficient straw utilization and a new direction for the study of heading period regulation, which is of great significance for the future breeding of new varieties and the development and utilization of germplasm resources. Summary of the invention

[0008] In view of the above-mentioned prior art, the present invention provides the use of the OsGTF5 gene (Xylan arabinosyltransferase) in regulating the fragility, plant height and / or heading period of rice plants.

[0009] The present invention is achieved through the following technical solutions:

[0010] The application of OsGTF5 gene in regulating rice plant fragility, plant height and / or heading period, the coding sequence of the OsGTF5 gene is shown in SEQ ID No.1, and the amino acid sequence of the OsGTF5 protein is shown in SEQ ID No.2.

[0011] Furthermore, in a specific application, the OsGTF5 gene of wild-type rice is silenced or the expression level of the OsGTF5 gene is reduced by mutation or knockout, and the resulting rice strain has lower mechanical strength, shorter plant height and / or delayed heading period compared with the wild-type rice.

[0012] Or: the coding region of the OsGTF5 gene or the complete OsGTF5 gene is transformed into an OsGTF5 gene-silenced rice line, or the expression level of the OsGTF5 gene is increased, and the resulting genetically complemented rice line has restored mechanical strength, plant height and / or heading period of the rice plant compared to the OsGTF5 gene-silenced rice line.

[0013] Furthermore, the mutation is selected from EMS mutagenesis; and the knockout is selected from CRISPR-Cas9 gene editing technology knockout.

[0014] Furthermore, the nucleotide sequence of the coding region of the mutated OsGTF5 gene is as shown in SEQ ID No.3, and the A at position 136 of the coding region of the OsGTF5 gene is mutated to T; or as shown in SEQ ID No.5, two bases are deleted at positions 90 and 91 of the coding region of the OsGTF5 gene; or as shown in SEQ ID No.6, three bases are deleted at positions 53, 90 and 91 of the coding region of the OsGTF5 gene.

[0015] Furthermore, the complete nucleotide sequence of the OsGTF5 gene is shown in SEQ ID No. 4, which includes a complete 5'UTR, exons, introns, and 3'UTR (2793 bp before ATG to 1225 bp after TAG).

[0016] Furthermore, the variety of rice is Nipponbare (Nip).

[0017] The use of OsGTF5 protein in regulating rice plant fragility, plant height and / or heading period, the amino acid sequence of the OsGTF5 protein is shown in SEQ ID No.2.

[0018] Furthermore, in a specific application, the OsGTF5 gene of wild-type rice is silenced or the expression level of the OsGTF5 gene is reduced by mutation or knockout, and the resulting rice strain has lower mechanical strength, shorter plant height and / or delayed heading period compared with the wild-type rice.

[0019] Or: the coding region of the OsGTF5 gene or the complete OsGTF5 gene is transformed into an OsGTF5 gene-silenced rice line, or the expression level of the OsGTF5 gene is increased, and the resulting genetically complemented rice line has restored mechanical strength, plant height and / or heading period of the rice plant compared to the OsGTF5 gene-silenced rice line.

[0020] The nucleotide sequence of the coding region of the mutated OsGTF5 gene is shown as SEQ ID No.3, or as SEQ ID No.5, or as SEQ ID No.6.

[0021] The application of a mutant OsGTF5 gene or its related biological materials in the preparation of transgenic rice. The transgenic rice has reduced mechanical strength, shorter plant height and / or delayed heading period. The related biological materials include a recombinant vector containing the mutant OsGTF5 gene and a transformant containing the mutant OsGTF5 gene.

[0022] A method for regulating rice plant brittleness, plant height and / or heading period: silencing the OsGTF5 gene of wild-type rice by mutation or knockout, or reducing the expression level of the OsGTF5 gene, so that the obtained rice strain has lower mechanical strength, shorter plant height and / or delayed heading period compared with the wild type;

[0023] Or: the coding region of the OsGTF5 gene or the complete OsGTF5 gene is transformed into an OsGTF5 gene-silenced rice line, or the expression level of the OsGTF5 gene is increased, and the resulting genetically complemented rice line has restored mechanical strength, plant height and / or heading period of the rice plant compared to the OsGTF5 gene-silenced rice line.

[0024] Furthermore, the mutation is selected from EMS mutagenesis; and the knockout is selected from CRISPR-Cas9 gene editing technology knockout.

[0025] The OsGTF5 gene, NCBI accession number is LOC4342012, and its coding sequence is shown in SEQ ID No. 1. The OsGTF5 gene contains 2 exons and 1 intron, and has only one transcript, encoding 460 amino acids, the amino acid sequence is shown in SEQ ID No. 2, and the encoded protein is located in the Golgi apparatus in the cytoplasm. After conservative domain prediction and analysis, the OsGTF5 protein may be a xylan arabinose transferase, which contains a hidden Markov model PF04577 and belongs to the GT61 family (Glycosyltransferase family 61, GT61) in glycosyltransferase.

[0026] The present invention identified a gtf5 mutant through the Mutmap technology. In the mutant, the 136th A in the coding region of the OsGTF5 gene (on the second exon) mutated to T, resulting in premature termination of translation, and the corresponding coding sequence is shown in SEQ ID No.3. The mutant plant exhibits phenotypes such as shorter plant height, reduced mechanical strength, easy breakage of stems and leaves, and delayed heading period. In agricultural production, the straw of the mutant is easier to crush than that of the wild type, which is of great significance in improving the utilization rate of renewable biomass, and has great application value in biomass conversion, pulp and papermaking industry; in breeding, the mutant has a longer vegetative growth period, which provides new materials for breeding new varieties in the future.

[0027] The present invention constructs a complementary vector pCAMBIA1300-OsGTF5 for the OsGTF5 gene, and genetically transforms the gtf5 mutant to obtain positive strains Com1 and Com2, whose plant height, leaf and stem strength, and heading period are all restored. The present invention also constructs a knockout vector of the gene mutation through the CRISPR-Cas9 system, and genetically transforms the wild-type Nipponbare to obtain positive strains KO1 and KO2, and observes that they show a phenotype similar to that of the mutant, that is, the plant height becomes shorter, the stem and leaves are easy to break, and the heading period is delayed. These experiments further prove that the loss of the OsGTF5 gene is the main reason for the gtf5 mutant plant to show fragility, shorter plant height and delayed heading period. The OsGTF5 gene is a gene that regulates the fragility and heading period of rice plants, and can be used to prepare transgenic plants. The present invention is of great significance to crop improvement and improving lodging resistance, and has broad application prospects in future agricultural product processing. The present invention provides valuable resources for breeding new brittle rice varieties and exploring the mechanism of heading period regulation, and is of great significance in the utilization of renewable biomass resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Schematic diagram of the comparison of plant height and internode length between the wild type (Nip) and the gtf5 mutant, wherein, A: plants in the jointing stage, scale bar = 10 cm; B: plants in the filling stage, scale bar = 10 cm; C: plants in the heading stage, scale bar = 10 cm; D: comparison of the length of each internode, scale bar = 1 cm, 5 cm.

[0029] Figure 2 : Schematic diagram comparing the plant heights of the wild type (Nip), gtf5 mutant, complementation strain (Com1, Com2), and OsGTF5 gene knockout strain (KO1, KO2), wherein A: plants of the wild type, gtf5 mutant, and complementation strain, scale bar = 10 cm; B: plants of the wild type, gtf5 mutant, and OsGTF5 gene knockout strain, scale bar = 10 cm.

[0030] Figure 3 : Schematic diagram of the sequencing verification results of the complementary strain, the mutant base position shows a double peak.

[0031] Figure 4 : Schematic diagram of sequencing verification results of gene knockout strains, where the mutation position is represented by base deletion.

[0032] Figure 5 : Schematic diagram of subcellular localization of OsGTF5 protein, where A is OsGTF5-GFP fusion protein, B is Golgi marker, and C is the merged image of A and B.

[0033] Figure 6: Schematic diagram of plant height comparison analysis of the wild type (Nip), gtf5 mutant, complementation strain (Com1, Com2) and OsGTF5 gene knockout strain (KO1, KO2), where * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0034] Figure 7 : Schematic diagram of comparative analysis of internode lengths of the wild type (Nip), gtf5 mutant, complementation strain and OsGTF5 gene knockout strain, where * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001; A: first internode length; B: second internode length; C, third internode length; D, fourth internode length.

[0035] Figure 8 : Schematic diagram of comparative analysis of leaf and stem strength of wild type (Nip), gtf5 mutant, complementation line and OsGTF5 gene knockout line, where * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001; A: strength determination and analysis of the first internode; B: strength determination and analysis of the second internode; C, strength determination and analysis of the third internode; D, strength determination and analysis of the fourth internode; E: strength determination and analysis of the top leaf; F: strength determination and analysis of the top two leaves; G, top leaf strength phenotype of wild type (Nip), gtf5 mutant, complementation line and OsGTF5 gene knockout line; H, third internode strength phenotype of wild type (Nip), gtf5 mutant, complementation line and OsGTF5 gene knockout line.

[0036] Fig. 9 : Schematic diagram of comparative analysis of heading date among wild type (Nip), gtf5 mutant, complementation lines (Com1, Com2) and OsGTF5 gene knockout lines (KO1, KO2), where * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0038] Unless otherwise specified, the instruments, reagents, and materials involved in the following examples are conventional instruments, reagents, and materials already available in the prior art and can be obtained through regular commercial channels. The experimental methods involved in the following examples, if no specific experimental conditions are specified, are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer.

[0039] Example 1: Obtaining rice gtf5 mutants

[0040] The rice gtf5 mutant was derived from the EMS mutant pool of japonica rice Nipponbare. After five generations of continuous self-pollination, the mutant's traits were stably inherited, showing: plant fragility (low mechanical strength, easy breakage of stems and leaves), shorter plant height, and delayed heading period. The comparison of plant height and internode length between the rice gft5 mutant and the wild-type Nipponbare is shown in the figure. Figure 1 As shown by Figure 1 It can be seen that the plant height and internode length of the rice gtf5 mutant at the jointing stage, filling stage and heading stage are lower than those of the wild-type Nipponbare.

[0041] Further genetic analysis of the gtf5 mutant was performed using a hybrid combination of japonica rice Nipponbare. The F1 plant phenotype did not segregate, but the F2 population contained 155 plants with normal plant height and heading period, and 46 plants with brittleness, dwarfing, and delayed heading period. The chi-square test showed that the segregation ratio was 3:1 (χ 2 =0.48), indicating that the brittleness, dwarfing and delayed heading date of gtf5 mutant plants are controlled by a pair of recessive nuclear genes.

[0042] Example 2: Identification of gtf5 mutants using the Mutmap method

[0043] In order to identify the controlling genes that cause plant brittleness, dwarfing and delayed heading period, the gtf5 mutant was backcrossed with the wild type, and the plants showing brittleness in the backcross F2 segregating population were used for Mutmap identification. According to the candidate genes, referring to the LOC_Os06g49320 site information in the rice genome database (http: / / rice.uga.edu / cgi-bin / sequence_display.cgi?orf=LOC_Os06g49320.1), a pair of amplification primers OsGTF5-seq-F and OsGTF5-seq-R (see Table 1) were designed on both sides of the OsGTF5 sequence for PCR amplification to obtain the sequence containing the complete coding region of OsGTF5, which was compared with the wild type sequence.

[0044] The sequence difference between the mutant and Nipponbare was obtained by resequencing, and the InDel marker was designed to accurately locate and shorten the physical interval. Finally, the 136th A mutation in the coding region (second exon) of the LOC_Os06g49320 gene was obtained, resulting in premature termination of translation. The coding sequence of the mutant is shown in SEQ m No.3.

[0045] Table 1 Primer sequences used in the experiment

[0046]

[0047] Example 3: Identification of OsGTF5 gene function

[0048] To confirm that the phenotype of the gtf5 mutant is caused by the loss of OsGTF5 gene function, genetic complementation and OsGTF5 gene knockout experiments were performed on the gtf5 mutant and wild type, respectively, as described below.

[0049] (1) Using Nipponbare genomic DNA as a template, PCR amplification was performed with primers OsGTF5-EcoRI-F and OsGTF5-EcoRI-R (see Table 1) to obtain the full-length sequence of the OsGTF5 gene, which was then homologously recombined with the pCAMBIA1305 vector digested with EcoRI and transformed into Escherichia coli. The plaques were sequenced and verified. The plasmids that were verified to be correct by sequencing were transformed into Agrobacterium tumefaciens EHA105 to verify the transgenic complementation of the gtf5 mutant. The plant genetic transformation was completed by Wuhan Boyuan Biotechnology Co., Ltd., and finally the positive plants with gene complementation were obtained, which showed a phenotype similar to the wild type, that is, the plant height, stem and leaf strength, and heading period were restored. Among them, the comparison of plant height was as follows: Figure 2 As shown in A.

[0050] (2) Construction of OsGTF5 gene knockout strain using CRISPR-Cas9 system. The vector used to create the knockout strain using CRISPR-Cas9 was pCAMBIA1300-SK-5G, which was purified and recycled after AaRI digestion. A target site was designed on the first exon of the rice OsGTF5 gene to knock out the OsGTF5 gene. The sequence of the sgRNA designed according to the target sequence was: 5'-GATGTAGGTGAGGACGCAGA-3', and the oligo DNA primers designed for the target site were OsGTF5-AarI-F and OsGTF5-AarI-R (see Table 1). The primers were reacted at 100°C for 5 minutes and cooled at room temperature. The vector and the fragment were connected using T4 ligase, and Escherichia coli was transformed. The plaques were sequenced and verified. The plasmids verified to be correct by sequence sequencing were transformed into Agrobacterium tumefaciens EHA105. The plant genetic transformation was completed by Wuhan Boyuan Biotechnology Co., Ltd., and Nipponbare was used as the transformation receptor. Finally, OsGTF5 gene-edited positive plants were obtained, which showed a phenotype similar to that of the gtf5 mutant, that is, the plant height became shorter, the stem and leaf strength was reduced, and the heading period was delayed. Among them, the comparison of plant height was as follows Figure 2 As shown in B.

[0051] The complementary strains and OsGTF5 gene knockout strains constructed above were sequenced and verified. Figure 3 , Figure 4As shown, the mutant base position of the complementation strain showed a double peak, and the mutant position of the OsGTF5 gene knockout strain showed a 2-base deletion and a 3-base deletion.

[0052] Example 4: Subcellular localization of OsGTF5 protein

[0053] According to NCBI conserved domain prediction (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi), OsGTF5 contains the DUF563 conserved domain, and the prediction of the LocTree 3 (https: / / www.rostlab.org / services / loctree3 / ) website shows that its subcellular localization is in the Golgi apparatus. A subcellular localization experiment was performed on it. In this example, a subcellular localization vector was constructed. The vector construction process is as follows:

[0054] The full-length cDNA of the OsGTF5 gene (i.e., the sequence shown in SEQ ID NO.1) was amplified using primers OsGTF5-GFP-EcoRI-F and OsGTF5-GFP-EcoRI-R (see Table 1), and then the fragment was recombined into the EcoRI site of the pYBA1132 vector (pYBA1132 vector was kindly donated by Dr. Yao Lei of Beijing Academy of Agricultural and Forestry Sciences) by homologous recombination, and finally the OsGTF5-GFP recombinant vector was formed. The OsGTF5-GFP fusion protein was transiently expressed in tobacco cells, and the fluorescence signal was detected by confocal microscopy. The experimental results showed that the OsGTF5-GFP fusion protein was localized in the Golgi apparatus, such as Figure 5 shown.

[0055] Example 5: Plant height determination of wild type, gtf5 mutant, complementation strain and knockout strain

[0056] Plant height is a manifestation of cell morphology. The plant heights of wild-type Nipponbare (31 strains), gtf5 mutant (28 strains), complementary strains (Com1: 51 strains; Com2: 48 strains) and knockout strains (KO1: 41 strains; KO2: 30 strains) were measured, and the plant height data were compared and analyzed for significant differences using T-test. * indicates t-test Pvalue < 0.05; ** indicates t-test Pvalue < 0.01; *** indicates t-test Pvalue < 0.001. The statistical results are as follows Figure 6As shown in Table 2, the average plant heights of the wild type, gtf5 mutant, complementary strains (Com1, Com2) and knockout strains (KO1, KO2) were 79.7 cm, 72.6 cm, 80.8 cm, 82.7 cm, 70.0 cm and 68.2 cm, respectively. Compared with the wild type, the plant heights of the gtf5 mutant and knockout strains were significantly reduced by 7.1 cm, 9.7 cm and 11.5 cm, respectively, while the plant height of the complementary strains did not change significantly.

[0057] Table 2 Trait data statistics

[0058] Characteristics Nip gtf5 Com1 Com2 KO1 KO2 Plant height / cm 79.7±2.7 72.6±4.0 80.9±3.8 82.7±3.7 70.0±2.7 68.2±4.3 Length of the first section / cm 2.2±0.8 1.8±0.7 2.1±0.8 2.6±1.1 1.5±0.7 1.6±0.9 Length of the second section / cm 7.1±1.3 6.0±1.3 7.2±1.3 7.7±1.5 4.5±1.4 5.0±1.1 Length of the third section / cm 13.3±1.4 12.2±1.1 14.3±2.3 14.0±1.3 10.9±1.3 11.7±1.1 Length of the fourth section / cm 32.2±1.6 28.6±2.2 33.8±3.8 33.8±1.6 30.3±2.2 30.4±2.2 First section crisp / N 78.2±27.4 15.9±4.7 57.39±27.1 63.5±34.4 12.0±5.4 17.5±8.1 The second section is crisp / N 115.4±10.9 24.3±11.3 106.1±19.5 118.8±9.0 22.9±11.8 30.3±13.8 The third section is crisp / N 110.5±11.7 25.9±10.7 94.5±20.4 104.2±19.0 27.0±15.2 29.3±11.3 The fourth quarter is crisp / N 66.9±20.1 14.4±6.6 57.1±14.2 54.2±15.8 15.8±5.9 16.7±6.0 Daoyiyecui / N 13.2±3.4 3.1±1.5 14.3±3.5 14.8±3.1 ＜1 ＜1 Two leaves crisp / N 12.3±2.9 1.6±0.6 11.6±3.5 12.4±2.6 ＜1 ＜1 Heading period / d 64.1±1.2 69.4±1.4 65.5±1.9 64.5±1.2 74.5±3.1 70.7±1.4

[0059] The length of each internode is statistically Figure 7 , as shown in Table 2, Figure 7 As shown in Table 2, compared with the wild type, the number of internodes of the gtf5 mutant remained unchanged, and the length of each internode was significantly shortened. Compared with the gtf5 mutant, the length of each internode of the complementary strain was restored. Among them, the average length of the first node of Nip, gtf5, Com1, Com2, KO1, and KO2 was 2.2cm, 1.8cm, 2.1cm, 2.6cm, 1.5cm, and 1.6cm, respectively, the average length of the second node was 7.1cm, 6.0cm, 7.2cm, 7.7cm, 4.5cm, and 5.0cm, the average length of the third node was 13.3cm, 12.2cm, 14.3cm, 14.0cm, 10.9cm, and 11.7cm, and the average length of the fourth node was 32.2cm, 28.6cm, 33.8cm, 33.8cm, 30.3cm, and 30.4cm, respectively.

[0060] Example 6: Measurement of mechanical strength of stems and leaves of wild type, gtf5 mutant, complemented strain and knockout strain

[0061] Mechanical strength reflects the lodging resistance of plants. The mechanical strength of leaves and stems of wild-type Nipponbare, gtf5 mutant, complementation strain and knockout strain was measured, and T-test was used to compare and analyze the significant differences. The statistical results are as follows: Figure 8As shown in Table 2, the mechanical strength of the first section of 27 Nip strains, 21 gtf5 strains, 16 Com1 strains, 36 Com2 strains, 18 KO1 strains and 24 KO2 strains was measured; the mechanical strength of the second section of 43 Nip strains, 32 gtf5 strains, 27 Com1 strains, 44 Com2 strains, 42 KO1 strains and 36 KO2 strains was measured; the mechanical strength of the third section of 40 Nip strains, 33 gtf5 strains, 24 Com1 strains, 44 Com2 strains, 40 KO1 strains and 37 KO2 strains was measured; the mechanical strength of the fourth section of 40 Nip strains, 33 gtf5 strains, 24 Com1 strains, 38 Com2 strains, 47 KO1 strains and 35 KO2 strains was measured. The results showed that compared with the wild type, the mechanical strength of the gtf5 mutant and knockout strains was significantly reduced, and compared with the gtf5 mutant, the mechanical strength of the complemented strain was significantly restored. Similarly, the mechanical strength of the top leaf of 37 Nip strains, 38 gtf5 strains, 27 Com1 strains and 39 Com2 strains was measured; the mechanical strength of the top leaf of 52 Nip strains, 38 gtf5 strains, 27 Com1 strains and 39 Com2 strains was measured. The results showed that compared with Nip and complementary strains, the gtf5 leaf mutant showed significantly reduced brittleness, and the mechanical strength of the leaves of the knockout strains was less than 1N, which was difficult to detect.

[0062] Example 7: Heading period statistics of wild type, gtf5 mutant, complementation line and knockout line

[0063] The heading period of wild-type Nipponbare (37 strains), gtf5 mutant (31 strains), complementary strains (23 strains Com1; 23 strains Com2) and knockout strains (30 strains KO1; 30 strains KO2) was counted (Fuyang, Hangzhou), and the heading period data were compared and analyzed with T-test for significant differences. The statistical results are shown in the figure below. Fig. 9 As shown in Table 2, the heading period of Nip, gtf5 mutant, Com1, Com2, KO1 and KO2 were 64.1 days, 69.4 days, 65.5 days, 64.5 days, 74.5 days and 70.7 days, respectively. Compared with the wild type, the heading period of mutants and knockout lines was significantly delayed by 5.3 days, 10.4 days and 6.3 days, respectively.

[0064] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. Application of the OsGTF5 gene in regulating rice plant fragility, plant height and / or heading period, the coding sequence of the OsGTF5 gene is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: In a specific application, the OsGTF5 gene of wild-type rice is silenced or the expression level of the OsGTF5 gene is reduced by mutation or knockout, and the resulting rice strain has lower mechanical strength, shorter plant height and / or delayed heading period compared with the wild-type rice. Or: the coding region of the OsGTF5 gene or the complete OsGTF5 gene is transformed into an OsGTF5 gene-silenced rice line, or the expression level of the OsGTF5 gene is increased, and the resulting genetically complemented rice line has restored mechanical strength, plant height and / or heading period of the rice plant compared to the OsGTF5 gene-silenced rice line.

3. The use according to claim 2, characterized in that: The mutation is selected from EMS mutagenesis; the knockout is selected from CRISPR-Cas9 gene editing technology knockout.

4. The use according to claim 2, characterized in that: The nucleotide sequence of the coding region of the mutated OsGTF5 gene is as shown in SEQ ID No.3, where the A at position 136 of the coding region of the OsGTF5 gene is mutated to T; or as shown in SEQ ID No.5, where two bases are deleted at positions 90 and 91 of the coding region of the OsGTF5 gene; or as shown in SEQ ID No.6, where three bases are deleted at positions 53, 90 and 91 of the coding region of the OsGTF5 gene; The complete nucleotide sequence of the OsGTF5 gene is shown in SEQ ID No. 4, including a 5'UTR region and a 3'UTR region.

5. The use according to any one of claims 1 to 4, characterized in that: The variety of rice is Nipponbare.

6. Application of OsGTF5 protein in regulating rice plant fragility, plant height and / or heading period, wherein the amino acid sequence of the OsGTF5 protein is shown in SEQ ID No.

2.

7. The use according to claim 6, characterized in that: In a specific application, the OsGTF5 gene of the wild-type rice is silenced or the expression level of the OsGTF5 gene is reduced by mutation or knockout, so as to obtain a rice strain that cannot normally express the OsGTF5 protein or has a reduced expression level of the OsGTF5 protein. Compared with the wild-type, the mechanical strength of the rice plant is reduced, the plant height is shortened, and / or the heading period is delayed; or: transforming the coding region of the OsGTF5 gene or the complete OsGTF5 gene into an OsGTF5 gene-silenced rice line, or increasing the expression level of the OsGTF5 gene, to obtain a genetically complemented rice line, in which the mechanical strength, plant height and / or heading time of the rice plant are restored compared with the OsGTF5 gene-silenced rice line; The coding sequence of the OsGTF5 gene is shown in SEQ ID No.1; the complete nucleotide sequence of the OsGTF5 gene is shown in SEQ ID No.

4.

8. A mutant OsGTF5 gene, characterized in that: The nucleotide sequence of its coding region is shown as SEQ ID No.3, or as SEQ ID No.5, or as SEQ ID No.

6.

9. Use of the mutated OsGTF5 gene or its related biological materials according to claim 8 in preparing transgenic rice; the related biological materials include: A recombinant vector containing a mutated OsGTF5 gene, and a transformant containing a mutated OsGTF5 gene.

10. A method for regulating rice plant fragility, plant height and / or heading period, characterized in that: The OsGTF5 gene of wild-type rice is silenced by mutation or knockout, or the expression level of the OsGTF5 gene is reduced, so that the obtained rice strain has lower mechanical strength, shorter plant height and / or delayed heading date compared with the wild-type rice; or: transforming the coding region of the OsGTF5 gene or the complete OsGTF5 gene into an OsGTF5 gene-silenced rice line, or increasing the expression level of the OsGTF5 gene, to obtain a genetically complemented rice line, in which the mechanical strength, plant height and / or heading time of the rice plant are restored compared with the OsGTF5 gene-silenced rice line; The coding sequence of the OsGTF5 gene is shown in SEQ ID No.1; the complete nucleotide sequence of the OsGTF5 gene is shown in SEQ ID No.4.

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