Application of negative regulation of OsABA8ox1 in improvement of alkali resistance of rice and / or transfer level of rice stem sheath assimilate

Abstract

The invention belongs to the technical field of plant genetic engineering and plant genetic improvement, and particularly relates to application of negative regulation of OsABA8ox1 in improvement of alkali resistance of rice and / or transfer level of rice stem sheath assimilates. The OsABA8ox1 gene is a newly found key gene for promoting NSC transport of the stem sheaths of the rice and is also a main gene for improving alkali resistance of the rice, the NSC transport efficiency of the stem sheaths can be improved through genetic improvement, the maturing rate can be increased, the harvest index can be increased, meanwhile, the stress resistance of the rice is enhanced, the alkali resistance of the rice is remarkably improved, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and plant genetic improvement technology, specifically involving the application of negative regulation of OsABA8ox1 in improving the alkali resistance of rice and / or the level of assimilate transport in rice stems and sheaths. Background Technology

[0002] As the world's largest producer and consumer of rice, China relies on the stable growth of rice production as a core pillar for ensuring national food security. The supply of carbohydrates during the rice grain-filling stage directly determines the grain setting rate and final yield. These carbohydrates primarily originate from the synthesis of photosynthetic products in the top three leaves and the translocation of non-structural carbohydrates (NSCs) stored in the stem sheath before heading. Studies have shown that approximately 68% of the carbohydrates stored in the plant before heading are translocated to the panicle, contributing up to 30% to yield. Therefore, promoting the efficient translocation of NSCs from the stem sheath to the grain is a key approach to unlocking the yield potential of rice. Currently, it is known that moderate drought after flowering or exogenous application of abscisic acid (ABA) can regulate the plant senescence process, thereby promoting the translocation of NSCs from the stem sheath to the grain in rice and wheat. However, the relevant regulatory mechanisms are not yet fully understood, limiting the precise application of this pathway in high-yield breeding.

[0003] Globally, approximately one-third of the land is threatened by salinity stress. In my country, the area of ​​saline-alkali land exceeds 100 million hectares, of which the area of ​​soda-alkali land (mainly composed of NaHCO3 and Na2CO3) in Northeast China reaches 110 million mu (approximately 7.3 million hectares), and is expanding at a rate of 1.4% annually, severely restricting the planting area and yield stability of rice. ABA, as an important plant hormone, plays a central role in regulating plant stress responses. Studies have confirmed that applying ABA can effectively improve the alkali tolerance of rice, and patents (such as CN202411428195.X) disclose the ABA synthesis gene. OsNCED3 Overexpression significantly enhances alkali tolerance in rice. ABA homeostasis in plants is maintained by both synthesis and degradation processes, including the ABA 8'-carboxylase gene family. OsABA8ox These are key enzymes that mediate ABA breakdown. Currently, research on this family of genes mainly focuses on ABA metabolic regulation, and their function in rice stem and sheath NSC transport and alkali tolerance regulation has not yet been reported.

[0004] In the current field of rice genetic improvement, yield enhancement and stress resistance are mostly independent research directions: research on yield mainly focuses on the regulation of "source-sink" relationships such as photosynthetic efficiency and NSC translocation, while research on alkali tolerance focuses on stress response mechanisms such as ABA signaling pathways and ion balance. However, in actual production, rice often faces the dual pressure of yield enhancement and saline-alkali stress. Existing technologies lack gene targets that can simultaneously regulate stem-sheath NSC translocation efficiency and alkali tolerance, making it impossible to achieve synergistic improvement of high-yield traits and stress resistance traits. This has become a key technical bottleneck restricting the release of the yield potential of rice in saline-alkali land. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide an application of negative regulation of OsABA8ox1 in improving alkali tolerance in rice and / or the level of assimilate transport in rice stems and sheaths.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: Firstly, this invention provides a negative regulation OsABA8ox1 Application of gene expression in improving alkali tolerance and / or the level of assimilate transport in rice stems and sheaths. OsABA8ox1 The nucleotide sequence of a gene includes any of the following: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A nucleotide sequence as shown in SEQ ID NO.1 that, after substitution, deletion or insertion of one or more nucleotides, still has the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem and sheath; (3) A nucleotide sequence that is complementary to the nucleotide sequence described in (1) or (2), and can hybridize with it under strict conditions, and has the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem sheath.

[0007] Preferably, in the above applications, negative regulation OsABA8ox1 Gene expression or negative regulation of OsABA8ox1 protein levels has the following effects: (i) It has the effect of increasing the level of NSC translocation in rice stems and sheaths; (ii) It has the effect of increasing the seed setting rate of rice; (iii) It has the effect of increasing the rice harvest index; (iv) It has the effect of improving nitrogen utilization efficiency; (v) It has the effect of increasing the abscisic acid content in seedlings.

[0008] Preferably, in the above applications, by knocking out or knocking down the concentration of certain substances in rice... OsABA8ox1 The gene aims to improve rice's alkali resistance and / or the level of assimilate transport in rice stems and sheaths.

[0009] More preferably, in the above applications, knocking out or knocking down the concentration of certain substances in rice is used. OsABA8ox1 The gene involves cloning the sgRNA sequence into a CRISPR / Cas9 expression vector and transforming it into rice using Agrobacterium-mediated transformation to achieve knockout or knockdown of the gene in rice. OsABA8ox1 The purpose of the gene; the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3.

[0010] Secondly, the present invention provides an application of negative regulation of OsABA8ox1 protein levels in improving rice alkali tolerance and / or rice stem-sheath assimilate transport levels, wherein the amino acid sequence of the OsABA8ox1 protein includes any of the following: (1) The amino acid sequence as shown in SEQ ID NO.2; (2) An amino acid sequence as shown in SEQ ID NO.2, obtained by substitution, insertion or deletion of one or more amino acids, and still having the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem and sheath.

[0011] Preferably, in the above applications, negative regulation OsABA8ox1 Gene expression or negative regulation of OsABA8ox1 protein levels has the following effects: (i) It has the effect of increasing the level of NSC translocation in rice stems and sheaths; (ii) It has the effect of increasing the seed setting rate of rice; (iii) It has the effect of increasing the rice harvest index; (iv) It has the effect of improving nitrogen utilization efficiency; (v) It has the effect of increasing the abscisic acid content in seedlings.

[0012] Preferably, in the above applications, by knocking out or knocking down the concentration of certain substances in rice... OsABA8ox1 The gene aims to improve rice's alkali resistance and / or the level of assimilate transport in rice stems and sheaths.

[0013] More preferably, in the above applications, knocking out or knocking down the concentration of certain substances in rice is used. OsABA8ox1 The gene involves cloning the sgRNA sequence into a CRISPR / Cas9 expression vector and transforming it into rice using Agrobacterium-mediated transformation to achieve knockout or knockdown of the gene in rice. OsABA8ox1 The purpose of the gene; the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3.

[0014] Thirdly, embodiments of the present invention provide a method for constructing a rice mutant strain, wherein the rice mutant strain is constructed by knocking out or knocking down the... OsABA8ox1 Gene acquisition; OsABA8ox1 The nucleotide sequence of a gene includes any of the following: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A nucleotide sequence as shown in SEQ ID NO.1 obtained by substitution, deletion or insertion of one or more nucleotides, and still having the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem and sheath; (3) A nucleotide sequence that is complementary to the nucleotide sequence described in (1) or (2), and can hybridize with it under strict conditions, and has the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem and sheath; The construction method includes: cloning the sgRNA sequence into a CRISPR / Cas9 expression vector, transforming it into rice embryogenic callus tissue via Agrobacterium-mediated transformation, and obtaining transgenic plants; the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3.

[0015] Fourthly, the present invention provides a breeding method for rice, which includes: reducing the content and / or activity of OsABA8ox1 protein in conventional rice or hybrid rice parents, and obtaining offspring through hybridization technology; The amino acid sequence of the OsABA8ox1 protein includes any of the following: (1) The amino acid sequence as shown in SEQ ID NO.2; (2) An amino acid sequence as shown in SEQ ID NO.2, obtained by substitution, insertion or deletion of one or more amino acids, and still having the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem and sheath.

[0016] Preferably, in the above-mentioned rice breeding method, reducing the content and / or activity of OsABA8ox1 protein is achieved by knocking out or reducing the content of OsABA8ox1 protein in rice. OsABA8ox1 Genes achieve their purpose; knock out or knock down the gene in rice. OsABA8ox1 Genetic methods include: cloning the sgRNA sequence into a CRISPR / Cas9 expression vector, and then transforming it into rice using Agrobacterium-mediated transformation to achieve knockout or knockdown of the gene in rice. OsABA8ox1 The purpose of the gene; the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3.

[0017] The beneficial effects of this invention include: (1) This invention achieves its effect by knocking out OsABA8ox1 The gene prevents the normal expression of the OsABA8ox1 protein, hindering ABA degradation in the plant. This leads to an increase in ABA content in the stem and sheath during the grain-filling stage of rice, increased NSC translocation in the stem and sheath, and improved seed setting rate and yield.

[0018] (2) This invention achieves this by knocking out OsABA8ox1 The gene prevents the normal expression of the OsABA8ox1 protein, hindering the degradation of ABA in the plant and leading to an increase in the ABA content in rice seedlings. This makes the seedlings more alkali-resistant and improves their survival rate when exposed to alkali stress during the seedling stage.

[0019] (3) OsABA8ox1The gene is a newly discovered key gene that promotes the translocation of NSCs in rice stems and sheaths. It is also a major gene for improving rice alkali tolerance. Through genetic improvement, the translocation efficiency of NSCs in stems and sheaths can be improved, the seed setting rate can be increased, the harvest index can be increased, and the stress resistance and alkali tolerance of rice can be enhanced. It has broad application prospects. Attached Figure Description

[0020] Figure 1 for OsABA8ox1 Information on the target and mutation sites of the mutant; Figure 2 For the aboveground parts of ZH11 after alkali treatment OsABA8ox1 Gene expression levels; Figure 3 for OsABA8ox1 Overexpression materials OsABA8ox Expression level identification results; Figure 4 for OsABA8ox1 Phenotypic diagram of rice seedling alkali tolerance test regulated by genes; Figure 5 for OsABA8ox1 Gene regulation of rice seedling survival rate in alkali tolerance test; Figure 6 for OsABA8ox1 Fresh weight of rice seedlings in alkali tolerance test regulated by genes; Figure 7 for OsABA8ox1 The content of ABA (abscisic acid) in the gene-regulated alkali tolerance test of rice seedlings; Figure 8 for OsABA8ox1 NSC content in rice stem and sheath experiments regulated by genes; Figure 9 for OsABA8ox1 Phenotypic diagram of senescence in rice stem and sheath experiments regulated by genes; Figure 10 for OsABA8ox1 NSC transport concentration in rice stem-sheath gene regulation experiment; Figure 11 for OsABA8ox1 Plant height in a gene-regulated rice grain filling experiment; Figure 12 for OsABA8ox1 Seed setting rate in a gene-regulated rice grain filling experiment; Figure 13 for OsABA8ox1 Yield per plant in a gene-regulated rice grain filling experiment; Figure 14 for OsABA8ox1 Harvest index in a gene-regulated rice grain filling experiment; Figure 15 for OsABA8ox1 Nitrogen fertilizer productivity in a gene-regulated rice grain-filling experiment; Figure 16 for OsABA8ox1 Nitrogen agronomical use efficiency in a gene-regulated rice grain filling experiment; in, Figure 3 , Figures 6 to 8 , Figures 10 to 16 In this study, the data were analyzed using LSD (Least Significant Difference) multiple comparisons, where different letters indicated significant differences between different groups. Detailed Implementation

[0021] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0022] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0023] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0024] Example 1 In the following examples, the primers for preparing Oligo dimers and identifying mutant single plants are shown in Table 3.

[0025] Table 1 Primers required for Example 1

[0026] This invention provides a process for constructing and identifying OsABA8ox1 gene knockout mutants. Details are as follows: (1) In order to construct the CRISPR / Cas9 mutant of the OsABA8ox1 (LOC_Os02g47470) gene, single-suide RNAs (sgRNAs) were constructed using the Biogle CRISPR / Cas kit (BGK03 http: / / www.biogle.cn / ). (2) Oligo sequences (Oligonucleotides) were generated using an online website (BGK03). The process for preparing Oligo dimers is as follows: 1) Oligo dimer full length was amplified using 2x Phanta Mix high-fidelity polymerase. The PCR reaction system was prepared according to Table 2. The PCR reaction conditions were: pre-denaturation 95℃, 3 min, denaturation 95℃, 15 s, annealing 60℃, 15 s, extension 72℃, 2 min, 34 cycles, and final extension 72℃, 5 min. Table 2 Composition of the reaction system

[0027] 2) The DNA of the mutant plant was amplified using 2x Tap Mix polymerase. The PCR reaction system was prepared according to Table 2. PCR reaction conditions: pre-denaturation 95℃, 3 min, denaturation 95℃, 15 s, annealing 60℃, 15 s, extension 72℃, 30 s, 34 cycles, final extension 72℃, 5 min. Table 3 Composition of the reaction system

[0028] (3) Recovery and purification of PCR products: Add 10x Loading buffer to the PCR product, use DM 2000 DNA-Marker as a reference, electrophoresis on a 1% agarose gel, cut the agarose gel containing the target gene band under UV transmission, place it in a 1.5 mL centrifuge tube, and purify it according to the DNA gel extraction kit (Corning Axygen Co., Ltd.); send the purified Oligo dimer to a biological sequencing company (Qingke Biotechnology) for sequencing, and compare the sequencing results.

[0029] (3) Finally, the CRISPR / Cas9 vector PBGK03-OsABA8ox1 was transformed into Agrobacterium EHA105 and then into ZH11 (Zhonghua 11 rice). This genetic transformation was completed by Hangzhou Baige Biotechnology Co., Ltd. The target sequence is located at 193-212 bp of the full-length CDS sequence of OsABA8ox1 (nucleotide sequence as shown in SEQ ID NO:1, amino acid sequence as shown in SEQ ID NO:2), and the target sequence is: 5'-TTCAACAAGAAGCGGAACAA-3' (SEQ ID NO.3).

[0030] Two homozygous mutants, CR7 (OsABA80x1-CR7) and CR15 (OsABA80x1-CR15), were ultimately obtained. In CR7, a T was inserted at 202 bp of the full-length CDS sequence, resulting in a frameshift mutation and forming a stop codon at 203 bp. In CR15, the five bases CAAGA were deleted between 197 bp and 202 bp, failing to form a stop codon, and the amino acid sequence was altered. The mutated amino acid sequences are shown in SEQ ID NO:4 and SEQ ID NO:5, resulting in the OsABA8ox1 gene not being expressed normally (see...). Figure 1 ).

[0031] Example 2 The required primers for the following examples are shown in Table 4. Table 4 Primers used in Example 2

[0032] In the following example, the construction process of the overexpression material (ligation and transformation of OsABA8ox1-CDS (SEQ ID NO:1) with the Pubi-1300 vector) is as follows: 1) The Pubi-1300 vector was double-digested with KpnⅠ and HindⅢ. The digestion reaction system is shown in Table 5 below. Table 5 Enzyme digestion reaction system

[0033] 2) After mixing the above system, digest it at 37℃ for about 30 min. The digestion product is then recovered by gel electrophoresis to obtain the target fragment. The vector is then ligated using the ClonExpress Multis One Step Cloning Kit (Vazyme, Nanjing).

[0034] 3) Connect the system according to the system in Table 6 below: 37 React in a constant temperature water bath for 30 minutes, and then immediately place it on ice to cool the reaction solution; Table 6 Composition of the Connecting Reaction System

[0035] 4) Take 10 μL of recombinant product and add it to 100 μL of dissolved competent cells TOP10 (Weidi Bio, Shanghai) for genetic transformation; after overnight culture at 37 degrees Celsius and 200 rpm, select single colonies for positive identification. 5) For positive monoclonal bacteria, plasmids were extracted using the SanPrep column-based plasmid DNA mini-extraction kit (Sangon Biotech, Shanghai), and then sent for sequencing identification.

[0036] 6) The constructed transgenic vector was sent to Hangzhou Baige Biotechnology Co., Ltd. for genetic transformation. The obtained transgenic material was screened by hygromycin and identified by PCR. 7) RNA extraction from rice overexpression lines: Rice RNA was extracted using the Vazyme RNA extraction kit to obtain high-concentration, high-quality RNA, which was then reverse transcribed. 8) Reverse transcription was performed using the HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper). (Vazyme, Nanjing) cDNA obtained after reverse transcription was identified by real-time fluorescence PCR. 9) Expression level identification: Quantitative real-time PCR; qRT-PCR was performed using the Hieff™ qPCR SYBR Green Master Mix (Yeasen Biotech, Shanghai) kit, and the reaction was carried out in Step One Plus. TM The procedure was performed on an instrument from Applied Biosystems, USA.

[0037] 10) Gene-specific primers were designed using the Primer Blast online software on the NCBI website for real-time quantitative PCR amplification. Actin1 The gene was used as an internal reference gene. The reaction system was configured according to Table 7, and the instrument parameters were set according to the reaction conditions in Table 8. Finally, overexpressing plants OE3 and OE11 with high expression of the OsABA8ox1 gene were screened.

[0038] Table 7 Composition of the reaction system

[0039] Table 8 Reaction conditions

[0040] This invention provides a process for regulating seedling alkali tolerance using the OsABA8ox1 protein. The details are as follows: To investigate the role of OsABA8ox1 protein in seedling alkali resistance, OsABA8ox1 Mutants (CR7 and CR15, OsABA8ox1 Expression situation as follows Figure 2 (as shown) and OsABA8ox1 overexpression materials (OE3 and OE11, OsABA8ox1 Gene expression status, such as Figure 3 After soaking and germinating both wild-type (as shown) and wild-type ZH11, they were placed in incubators at 28℃, 70% relative humidity, and alternating light and dark cycles of 14h / 10h for normal hydroponics, and then their alkali tolerance was assessed. The method for assessing alkali tolerance is as follows: At the two- to three-leaf stage (BBCH12-13), wild-type and OsABA8ox1 transgenic rice seedlings (including OsABA8ox1 mutants and OsABA8ox1 overexpression materials) were subjected to hydroponic stress treatment with 15mmol / L Na2CO3 solution (pH 10.87) for 3 days, followed by 7 days of recovery culture. The survival rate, aboveground fresh weight, and malondialdehyde (MDA) content were used as indicators for evaluating alkali tolerance.

[0041] The actual growth conditions of different types of rice are as follows: Figure 4 As shown, OsABA8ox1 The mutants CR7 and CR15 exhibit greater tolerance to alkaline stress, while OsABA8ox1 Overexpression materials OE3 and OE11 are more sensitive to alkaline stress.

[0042] In addition, for OsABA8ox1 Repeated experiments on mutants revealed that the survival rates of CR7 and CR15 mutants were significantly higher than those of ZH11 (see...). Figure 5 The freshness also increased significantly. Figure 6 ).and, OsABA8ox1 The mutants CR7 and CR15 can increase ABA content in seedlings under normal (CK) and alkali stress (AS) conditions (see...). Figure 7 This enhances the activity of antioxidant enzymes and reduces the accumulation of reactive oxygen species.

[0043] The above data can illustrate that knockout OsABA8ox1 Genes can improve the alkali resistance of rice seedlings.

[0044] Example 3 In the following examples, the determination method for non-structural carbohydrates (NSC) is as follows: Stem and sheath samples were dried at 80°C to constant weight, pulverized, and then collected through a 100mm fine sieve for analysis. Soluble sugars were extracted using the hot ethanol method, repeated three times, and finally mixed and diluted to a fixed volume for soluble sugar content determination. Starch was extracted using the residue remaining after soluble sugar extraction, dried at 80°C, gelatinized in a boiling water bath, and extracted using a perchloric acid ice bath, repeated twice. The supernatants were combined, diluted to a fixed volume, and allowed to stand before determining the starch content. Both extracted soluble sugars and starch samples were determined using the anthrone colorimetric method. 100μL of sample was mixed with 1mL of anthrone reagent, heated in a boiling water bath for 10min, cooled to room temperature under running water, and 200μL of the reaction solution was transferred to a 96-well plate. The absorbance of the sample was measured at 625nm using a microplate reader. Based on the glucose standard curve, calculate the soluble sugar content and starch content (mg / gDW). The starch content is the concentration calculated from the glucose standard curve multiplied by 0.9. The NSC content is the sum of the soluble sugar content and the starch content.

[0045] Provided in the embodiments of the present invention OsABA8ox1 The statistical process and results of stem-sheath translocation and yield traits in transgenic materials are as follows: Normal planting was carried out in Changsha, Hunan Province during the spring and autumn seasons of 2023. OsABA8ox1 Mutants (CR7 and CR15) serve as reverse proof. OsABA8ox1 The role of genes in stem-sheath transport and grain filling, excluding mutants, was discussed in this study. OsABA8ox1 Overexpression materials (OE3 and OE11).

[0046] At the initial flowering stage of rice, uniformly growing panicles that flowered on the same day were tagged, with 100 panicles tagged for each material. At the heading stage (1 day after flowering), 12 days and 18 days after flowering, and at maturity (36 days after flowering), the top node of the main panicle was collected. For each variety, three stem sheaths were collected from each replicate at each time point, for a total of three replicates. At maturity, 10 individual plants of each variety were harvested for plant height and yield traits. NSC translocation = NSC concentration at grain-filling stage - NSC concentration at maturity; Harvest index = Yield per plant / Aboveground biomass 100%; Nitrogen fertilizer partial productivity = yield per plant / amount of nitrogen applied; Agricultural nitrogen use efficiency = (yield with nitrogen applied - yield without nitrogen applied) / amount of nitrogen applied.

[0047] The NSC content (total soluble sugars and starch, non-structural carbohydrates) in the stems and sheaths of rice was measured at the flowering and maturity stages, respectively. The results are as follows: Figure 8 As shown, the results indicated that during the flowering stage, the overexpressing lines accumulated less NSC, mainly due to a decrease in soluble sugar content. Furthermore, even at maturity, a significant amount of starch and soluble sugar remained accumulated in the stem sheaths, failing to be fully utilized. Moreover, the stem sheaths exhibited a senescent phenotype at maturity. OsABA8ox1 The material is expressed as late-blooming. OsABA8ox1 The mutant has a higher degree of stem and sheath senescence maturity. Figure 9 ).and OsABA8ox1 The NSC content of the mutant was not significantly different from that of ZH11 at the flowering stage, but at the maturity stage, the starch and soluble sugar content in the stem sheaths were significantly reduced, while the NSC translocation in the stem sheaths was significantly increased (see...). Figure 10 ). OsABA8ox1 The mutants CR7 and CR15 significantly improved the translocation efficiency of NSC in the stem sheath during the grain-filling stage.

[0048] in addition, OsABA8ox1 The mutants CR7 and CR15 were shorter in height, while OsABA8ox1 The overexpression materials OE3 and OE11 showed increased plant height (see...) Figure 11 This is because the ABA content in the plant changed, thus affecting the plant height of rice. OsABA8ox1 Overexpression materials OE3 and OE11 showed poor grain filling, decreased seed setting rate, reduced thousand-grain weight, and consequently, a significant decrease in yield per plant. OsABA8ox1 Although the mutants CR7 and CR15 result in shorter plants, OsABA8ox1 The seed setting rate of mutants CR7 and CR15 was significantly increased by 10% (see [link to relevant documentation]). Figure 12 The yield per plant increased by 2.4g and 3.5g compared to the WT (wt). Figure 13 ).

[0049] The above data can illustrate that knockout OsABA8ox1 Genes can improve the efficiency of NSC translocation in rice stems and sheaths during the grain-filling stage, promote grain filling, and increase the rice harvest index. Figure 14 Nitrogen fertilizer has a partial productivity effect ( Figure 15 ) and nitrogen fertilizer agronomic utilization ( Figure 16 ).

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Negative regulation OsABA8ox1 Application of gene expression in improving alkali tolerance and / or the level of assimilate transport in rice stems and sheaths. OsABA8ox1 The nucleotide sequence of a gene includes any of the following: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A nucleotide sequence as shown in SEQ ID NO.1 obtained by substitution, deletion or insertion of one or more nucleotides, and still having the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem and sheath; (3) A nucleotide sequence that is complementary to the nucleotide sequence described in (1) or (2), and can hybridize with it under strict conditions, and has the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem sheath.

2. Application of negative regulation of OsABA8ox1 protein levels in improving rice alkali tolerance and / or rice stem-sheath assimilate transport levels, wherein the amino acid sequence of OsABA8ox1 protein includes any of the following: (1) The amino acid sequence as shown in SEQ ID NO.2; (2) An amino acid sequence as shown in SEQ ID NO.2, obtained by substitution, insertion or deletion of one or more amino acids, and still having the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem and sheath.

3. The application according to claim 1 or 2, characterized in that, negative regulation OsABA8ox1 Gene expression or negative regulation of OsABA8ox1 protein levels has the following effects: (i) It has the effect of increasing the level of NSC translocation in rice stems and sheaths; (ii) It has the effect of improving the seed setting rate of rice; (iii) It has the effect of increasing the rice harvest index; (iv) It has the effect of improving nitrogen utilization efficiency; (v) It has the effect of increasing the abscisic acid content in seedlings.

4. The application according to claim 1 or 2, characterized in that, By knocking out or reducing the concentration of [certain substances] in rice OsABA8ox1 The gene aims to improve rice's alkali resistance and / or the level of assimilate transport in rice stems and sheaths.

5. The application according to claim 4, characterized in that, Knock out or knock down rice OsABA8ox1 The gene involves cloning the sgRNA sequence into a CRISPR / Cas9 expression vector and transforming it into rice using Agrobacterium-mediated transformation to achieve knockout or knockdown of the gene in rice. OsABA8ox1 The purpose of the gene; the nucleotide sequence of the sgRNA is shown in SEQ ID NO:

3.

6. A method for constructing a rice mutant, characterized in that, Rice mutants are produced by knocking out or knocking down the levels of certain nutrients in rice. OsABA8ox1 Gene acquisition; OsABA8ox1 The nucleotide sequence of a gene includes any of the following: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A nucleotide sequence as shown in SEQ ID NO.1 obtained by substitution, deletion or insertion of one or more nucleotides, and still having the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem and sheath; (3) A nucleotide sequence that is complementary to the nucleotide sequence described in (1) or (2), and can hybridize with it under strict conditions, and has the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem and sheath; The construction method includes: cloning the sgRNA sequence into a CRISPR / Cas9 expression vector, transforming it into rice embryogenic callus tissue via Agrobacterium-mediated transformation, and obtaining transgenic plants; The nucleotide sequence of sgRNA is shown in SEQ ID NO:

3.

7. A method for rice breeding, characterized in that, Rice breeding methods include: reducing the content and / or activity of OsABA8ox1 protein in conventional or hybrid rice parents, and obtaining offspring through hybridization techniques; The amino acid sequence of the OsABA8ox1 protein includes any of the following: (1) The amino acid sequence as shown in SEQ ID NO.2; (2) An amino acid sequence as shown in SEQ ID NO.2, obtained by substitution, insertion or deletion of one or more amino acids, and still having the function of improving the alkali resistance of rice and / or the level of assimilate transport in rice stem and sheath.

8. The rice breeding method according to claim 7, characterized in that, Reducing the content and / or activity of OsABA8ox1 protein by knocking out or reducing its activity in rice. OsABA8ox1 The genes achieved their purpose; Knock out or knock down rice OsABA8ox1 Genetic methods include: cloning the sgRNA sequence into a CRISPR / Cas9 expression vector, and then transforming it into rice using Agrobacterium-mediated transformation to achieve knockout or knockdown of the gene in rice. OsABA8ox1 The purpose of the gene; the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3.

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