Method for enhancing lodging resistance of rice and application thereof

Abstract

The present invention belongs to the technical fields of crop science and agricultural molecular biology, specifically relating to a method for enhancing rice lodging resistance and its application. By knocking out the OsCDPK14 gene, rice plant height is reduced and internode length is shortened without changing the number of rice nodes, thereby enhancing rice lodging resistance. The present invention reveals the function and role of the OsCDPK14 gene in the regulation of lodging resistance, providing a new technical solution for breeding highly lodging-resistant rice varieties, and has important breeding significance in agriculture.

Description

Technical Field

[0001] The invention belongs to the technical field of crop science and agricultural molecular biology, and particularly relates to a method for enhancing the lodging resistance of rice and an application thereof. Background Art

[0002] Increased nitrogen levels, overpopulation, soil density, disease, natural disasters (such as storm damage), sowing date, and seed type are all major factors contributing to cereal crop lodging. Lodging, defined as stem breakage and bending (stem lodging) or root lodging, is a major concern for farmers worldwide. Lodging severely impacts grain production for major cereal crops, particularly wheat and rice, with other indirect knock-on effects such as slower harvests, reduced grain quality, and increased drying costs. Crop lodging is generally categorized into three types. The first is bending lodging, where the internode bends midway without breaking the stalk. The second is breaking lodging, characterized by internodes breaking primarily below the third internode. Breaking lodging is more severe than bending lodging because the bent stalk can still transport photosynthetic assimilates from the leaves to the ear, which is essential for grain filling. The third type of lodging is rolling lodging, primarily due to a reduced root system penetrating the soil. In transplanted crops like rice, this lodging is less severe because their well-developed root systems typically reach deep into the soil. It's well known that rice lodging is the result of a combination of environmental factors. While external environmental factors like wind, snow, and rain are direct causes, the underlying and fundamental reason lies in the differences in the crop's genotype.

[0003] There are many characteristics of stem morphology, mainly including plant height, center of gravity height, internode length, stem diameter, stem wall thickness, plant fresh weight, plant dry weight and other indicators. Among them, plant height has a very significant effect on the lodging resistance of rice. Rice plants that are too short will cause reduced rice yields, and plants that are too tall will cause lodging. Rice plant height is mainly affected by genetic factors and external environmental factors. The internode length, number of internodes and panicle length jointly affect the rice plant height and center of gravity height, which is determined by the genetic factors of rice. External factors such as fertilization methods, different types of fertilizers, external temperature, and planting density also control the rice plant height and center of gravity height.

[0004] Currently, numerous technologies are available to cultivate highly lodging-resistant rice varieties through methods such as planting methods, fertilizers, and fertilizers. Many also utilize gene editing techniques to manipulate specific genes, such as DAXX1, PME28, REM, and LR2, to control lodging resistance. The rice genome reportedly contains 31 members of the CDPK gene family. While existing technologies have revealed the mechanisms by which rice CDPK genes contribute to disease resistance, male sterility, and seed set, the impact of CDPK genes on lodging resistance remains unknown. Therefore, exploring the functional role of rice kinase CDPK genes in the regulation of lodging resistance could lay the foundation for the development of highly lodging-resistant rice varieties and hold important breeding significance in agriculture. Summary of the Invention

[0005] The object of the present invention is to provide a method for enhancing lodging resistance and its application, specifically by knocking out the OsCDPK14 gene, reducing the plant height of rice, shortening the internode length without changing the number of rice nodes, thereby enhancing the lodging resistance of rice.

[0006] The present invention provides a method for enhancing the lodging resistance of rice, which comprises knocking out the OsCDPK14 gene by adopting gene editing technology.

[0007] The gene editing technology described in the present invention is CRISPR / Cas9 gene editing technology.

[0008] The method of knocking out the OsCDPK14 gene using gene editing technology in the present invention is to insert and / or delete bases in the OsCDPK14 gene.

[0009] The method of knocking out the OsCDPK14 gene using gene editing technology in the present invention comprises the following steps:

[0010] 1) Design the target sequence of the OsCDPK14 gene and construct the CRISPR / Cas9 gene editing vector for the OsCDPK14 gene using CRISPR / Cas9 gene editing technology;

[0011] 2) Construction of genetically engineered bacteria containing CRISPR / Cas9 gene editing vectors;

[0012] 3) Transform genetically engineered bacteria into rice to knock out the OsCDPK14 gene and enhance the rice's resistance to lodging.

[0013] The invention relates to the application of the method in cultivating transgenic rice.

[0014] The above applications include any of the following applications:

[0015] 1) Cultivate, screen, and identify transgenic rice with high lodging resistance;

[0016] 2) Study the expression regulation of lodging resistance traits in transgenic rice;

[0017] 3) Detection and prediction of plant height, internode length, and rice node number of transgenic rice;

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention reveals the function and role of the OsCDPK14 gene in the regulation of lodging resistance. By knocking out the OsCDPK14 gene, rice plant height is reduced, internode length is shortened, and the number of rice nodes remains unchanged, thereby enhancing rice lodging resistance. This invention provides a new technical solution for cultivating highly lodging-resistant rice varieties, facilitating the selection of lodging-resistant varieties, increasing the number of lines for grain production, and further addressing the current problem of rice lodging in production. This invention has important breeding significance and broad application prospects. It also has positive implications for the development of agricultural production and food security. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a map of the CRISPR / Cas 9 vector used to construct the OsCDPK14 knockout material in the present invention; A is a map of the pYLgRNA-OsU6a~c vector used to construct the OsCDPK14 knockout material, and B is a map of the pYLCRISPR / Cas9-MH vector used to construct the OsCDPK14 knockout material.

[0021] Figure 2 Flowchart of the operation for constructing the expression vector pYLCRISPR / Cas9-MH of the present invention.

[0022] Figure 3 Schematic diagram of the OsCDPK14 gene structure and specific knockout sites of the wild-type material and OsCDPK14 mutant material of the present invention, and a schematic diagram of the amino acid sequence of their protein; A is a schematic diagram of the OsCDPK14 gene structure, the sequence and position of each spcer, and specific knockout sites of the wild-type material and OsCDPK14 mutant material, wherein the white boxes on both sides of the gene structure schematic diagram are UTR sequences, the green boxes are exons, and the blue lines are introns; B is a schematic diagram of the amino acid sequence of the OsCDPK14 protein of the OsCDPK14 mutant and wild-type material.

[0023] Figure 4The present invention discloses field phenotypes of the Nipponbare wild type and the OsCDPK14 mutant at the flowering and maturity stages and their measurement results; A is a field phenotype diagram of the Nipponbare wild type and the OsCDPK14 mutant at the flowering stage under N2 treatment, B is a field phenotype diagram of the Nipponbare wild type and the OsCDPK14 mutant at the flowering stage under N4 treatment, C is a bar graph of the plant height measurement results of the Nipponbare wild type and the OsCDPK14 mutant at the flowering stage under N2 and N4 treatments, and D is a bar graph of the plant height measurement results of the Nipponbare wild type and the OsCDPK14 mutant at the flowering stage under N Figure 2 is a bar graph showing the tiller number measurement results under N2 and N4 treatments, E is the field phenotype of the Nipponbare wild type and the OsCDPK14 mutant at the mature stage under N2 treatment, F is the field phenotype of the Nipponbare wild type and the OsCDPK14 mutant at the mature stage under N4 treatment, G is a bar graph showing the plant height measurement results of the Nipponbare wild type and the OsCDPK14 mutant at the mature stage under N2 and N4 treatments, H is a bar graph showing the effective ear number measurement results of the Nipponbare wild type and the OsCDPK14 mutant at the mature stage under N2 and N4 treatments.

[0024] Figure 5 The internode length phenotypes of the mature Nipponbare wild type and the OsCDPK14 mutant of the present invention under different conditions; A is the internode length phenotype of the mature Nipponbare wild type and the OsCDPK14 mutant under N2 treatment, and B is the internode length phenotype of the mature Nipponbare wild type and the OsCDPK14 mutant under N4 treatment.

[0025] Figure 6 This is a statistical diagram of the internode length of the Nipponbare wild type and the OsCDPK14 mutant at the mature stage of the present invention.

[0026] Figure 7 Figures 2 and 3 are the expression patterns of the OsCDPK14 gene of the present invention in different tissue locations; A is the expression pattern of the OsCDPK14 gene in various parts of the plant (leaf, leaf sheath, root, stem, inflorescence, pistil, lemma, palea, ovary, embryo, and endosperm); and B is the expression pattern of the OsCDPK14 gene in various parts of the early heading stage (first leaf, second leaf, third leaf, first leaf sheath, second leaf sheath, third leaf sheath, internode I, internode II, internode III, node I, node II, node III, and ear).

[0027] Figure 8 This is the expression pattern of the OsCDPK14 gene in roots under different nutrient deficiency conditions of the present invention.

[0028] Figure 9 The wild type (right) and mutant (left) of the present invention fell over after a typhoon.

[0029] Figure 10This is a bar graph showing the lodging rates of the wild type and mutants after a typhoon in the field of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further illustrated below through specific embodiments.

[0031] Example 1 Construction of Crispr-Cas9 knockout mutant material

[0032] Using the rice genome-wide spacer database provided in the appendix of Miao et al. (2013), we entered the OsCDPK14 gene accession numbers, LOC_Os11g07040 and Os11g0171500, into the database and selected specific spacers. The nucleotide sequence of the OsCDPK14 gene is shown in SEQ ID NO:1. BsaI restriction sites were added to the 5' ends of the two complementary strands of the final spacer. Four spacers were selected and primers were designed. The primer sequences are shown in Table 1, SEQ ID NOs:2-9. The two complementary single-stranded primers were annealed in a PCR instrument using a gradient annealing process to synthesize double-stranded DNA. The reaction conditions were: 95°C, 2 min; 72°C, 2 min; 55°C, 2 min; 45°C, 2 min; 37°C, 2 min; 25°C, 2 min; and 15°C, hold. The resulting double-stranded DNA was diluted 10-fold for use. The CRISPR / Cas9 vector map used to construct OsCDPK14 knockout material is as follows Figure 1 As shown in (A and B), the operation flow chart of constructing the expression vector pYLCRISPR / Cas9-MH of the present invention is as follows Figure 2 shown.

[0033] Mutant materials can be constructed by transforming rice with CRISPR / Cas9 gene editing vectors and genetically engineered bacteria, obtaining T0 generation transgenic materials, extracting DNA, designing primers, and using PCR amplification technology to amplify and sequence the OsCDPK14 genome-specific sequence including the selected Spacer, to obtain three homozygous mutant strains (mu1, mu2, and mu3). Mu1 mutated to insert AA into SP3 and A into SP4; mu2 mutated to delete TC from SP3 and 95bp near SP4; and mu3 mutated to delete 51bp near SP1 and C into SP2. A schematic diagram of the OsCDPK14 gene structure and specific knockout sites of the wild-type material and OsCDPK14 mutant material of the present invention, as well as a schematic diagram of the amino acid sequence of their proteins, is shown in the figure below. Figure 3The nucleotide sequence of the SP1 site is shown in SEQ ID NO: 10, the nucleotide sequence of the SP2 site is shown in SEQ ID NO: 11, the nucleotide sequence of the SP3 site is shown in SEQ ID NO: 12, and the nucleotide sequence of the SP4 site is shown in SEQ ID NO: 13.

[0034] Table 1 Design of OsCDPK14-specific spacers

[0035]

[0036] Example 2 Field plot test

[0037] The experimental materials were planted at the Baima Experimental Base of Nanjing Agricultural University in Lishui District, Nanjing, Jiangsu Province. Two nitrogen fertilizer treatments were applied, calculated as pure nitrogen, at 75 kg / ha (N2) and 350 kg / ha (N4). Plant spacing was 20 cm, and row spacing was 20 cm. Three OsCDPK14 mutant lines (mu1, mu2, and mu3) were used. Plant height, tillering, internode length, and panicle length of the wild-type and mutant plants were measured and photographed at flowering and maturity.

[0038] The experimental results showed that at the flowering stage, the plant height of the mutant was significantly reduced compared with the wild type, with a decrease of 29.8% and 34.7% under N2 and N4 treatments respectively; there was no significant difference in tillering. The field phenotypes and measurement results of the Nipponbare wild type and OsCDPK14 mutant at the flowering stage were as follows: Figure 4 (A, B, C, and D). At maturity, the plant height of the mutant decreased by 35.4% and 32.1% under N2 and N4 treatments, respectively, compared with the wild type, and the number of effective ears increased significantly. The field phenotypes and measurement results of the Nipponbare wild type and OsCDPK14 mutant at maturity are shown in Figure 2. Figure 4 (E, F, G and H).

[0039] To further elucidate the reasons for the change in plant height, the panicle length and internode length of the wild type and mutant at maturity were measured and statistically analyzed. The results showed that, compared with the wild type, except for the fifth internode (IN V), the length of the remaining internodes was significantly shortened. The internode length phenotypes and statistical data of the Nipponbare wild type and OsCDPK14 mutant at maturity are shown in the figure. Figure 5 (A and B) and Figure 6 shown.

[0040] Example 3 Analysis of the expression pattern of the OsCDPK14 gene

[0041] (1) Analysis of the expression pattern of OsCDPK14 gene in different sites

[0042] Root and rhizome junction samples of Nipponbare wild-type rice were collected in the Baima field at the jointing stage, as well as from various plant parts during the early heading stage. These samples included the first leaf (D1), second leaf (D2), third leaf (D3), first leaf sheath (D1S), second leaf sheath (D2S), third leaf sheath (D3S), internode I, internode II, internode III, node I, node II, node III, and panicle. The samples were immediately frozen in liquid nitrogen and stored in a -80°C freezer until use. The samples were ground in liquid nitrogen and added to 1 mL of trizoL solution. Total RNA was then extracted, reverse transcribed, and cDNA synthesized. qRT-PCR was then performed to determine relative gene expression. The qRT-PCR primers for rice OsCDPK14 and ACTIN are shown in Table 2 (SEQ ID NOs: 14-21).

[0043] Table 2 qRT-PCR primers for rice OsCDPK14 and ACTIN

[0044]

[0045] The experimental results showed that the OsCDPK14 gene was expressed in all parts of rice, especially in the root-stem junction, node III, internode II and internode III, which were closely related to lodging resistance. Figure 7 (A and B).

[0046] Analysis of expression patterns of OsCDPK14 gene under different element deficiency conditions

[0047] Uniformly sized wild-type seeds of the Japanese cultivar Nipponbare were selected, shelled, and sterilized with 75% alcohol for one minute and 30% sodium hypochlorite solution for 30 minutes. The seeds were then rinsed with sterile water and, after the sodium hypochlorite had cleared away, sown in pre-sterilized 1 / 2 MS medium. After three days of incubation in the dark, they were cultured in the light until they had one leaf and one heart. Seeds with consistent growth were transplanted into 1 / 8 IRRI rice nutrient solution containing 1.25 mM (NH₄)₂SO₄ as the nitrogen source, pH 5.5. When they had three leaves and one heart in the full nutrient solution, they were treated with nitrogen, phosphorus, potassium, magnesium, manganese, and iron deficiency for five days. Five biological replicates were set for each strain within each treatment. Roots were dried, immediately frozen in liquid nitrogen, and stored in an ultra-low temperature freezer until needed. The samples were ground in liquid nitrogen and 1 mL of trizoL solution was added. Total plant RNA was then extracted, reverse transcribed, and cDNA synthesized. qRT-PCR was then performed to detect the relative expression levels of genes. The primers for the qRT-PCR reaction are shown in Table 2.

[0048] The expression patterns of OsCDPK14 gene in roots under different nutrient deficiency conditions are as follows Figure 8 The results showed that the expression of OsCDPK14 gene was significantly down-regulated by nitrogen deficiency, phosphorus deficiency and iron deficiency, and significantly up-regulated by potassium deficiency and manganese deficiency.

[0049] (3) Lodging resistance of OsCDPK14 mutant materials in practical applications

[0050] The experimental results after typhoon weather showed that the number of rice lodging was significantly reduced. After statistics, the lodging rate of OsCDPK14 mutant materials was significantly lower than that of wild-type materials. The lodging resistance of wild-type materials and OsCDPK14 mutant materials after typhoon weather was as follows: Figure 9 As shown, the bar graphs of the lodging resistance of the two materials are as follows Figure 10 shown.

[0051] After the OsCDPK14 gene was knocked out in the OsCDPK14 mutant material, its expression was restricted. The plant height of the OsCDPK14 mutant material was reduced, the internode length was shortened, and the number of rice nodes was not changed. These traits significantly improved the lodging resistance of rice compared with the original traits of the wild-type material.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for enhancing the lodging resistance of rice, characterized in that: The method is to use gene editing technology to knock out the OsCDPK14 gene, and the nucleotide sequence of the OsCDPK14 gene is shown in SEQ ID NO:

1.

2. The method for enhancing the lodging resistance of rice according to claim 1, characterized in that: The gene editing technology is CRISPR / Cas9 gene editing technology.

3. The method for enhancing the lodging resistance of rice according to claim 1, characterized in that: The method of knocking out the OsCDPK14 gene using gene editing technology is to insert and / or delete bases in the OsCDPK14 gene.

4. The method for enhancing the lodging resistance of rice according to claim 1, characterized in that: The method of knocking out the OsCDPK14 gene using gene editing technology comprises the following steps: 1) Design the target sequence of the OsCDPK14 gene and construct the CRISPR / Cas9 gene editing vector for the OsCDPK14 gene using CRISPR / Cas9 gene editing technology; 2) Construction of genetically engineered bacteria containing CRISPR / Cas9 gene editing vectors; 3) Transform genetically engineered bacteria into rice to knock out the OsCDPK14 gene and enhance the rice's resistance to lodging.

5. Use of the method according to claim 1 in cultivating transgenic rice with high lodging resistance.

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