Application of OsNCED3 gene overexpression to improvement of alkali resistance of rice

Abstract

The invention discloses application of OsNCED3 gene overexpression to improvement of alkali resistance of rice, belongs to the field of gene engineering, and particularly relates to application of OsNCED3 gene overexpression to improvement of saline-alkaline resistance of rice and increase of yield. The invention provides application of OsNCED3 gene overexpression to improvement of alkali resistance of rice in order to solve the serious problems that existing rice in Songnen plain soda saline-alkali soil suffers from an inhibition effect of high pH alkali stress, and is slow in growth and development and low in yield. The invention provides an OsNCED3 related to alkali resistance of rice and a coding gene thereof, and a method for improving the yield of rice in saline-alkali soil by overexpressing the target gene in the rice. The gene provided by the invention is of great significance to cultivation and improvement of saline-alkali tolerant rice varieties, improvement of saline-alkali tolerance and improvement of rice yield, and can improve saline-alkali tolerance and yield of soda saline-alkali soil rice and economic income of farmers.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and particularly relates to an application of OsNCED3 gene overexpression in improving the salt-alkali tolerance of rice and increasing the yield. Background Art

[0002] Soil salinization, as a global environmental and agricultural disaster, not only causes serious deterioration of the ecological environment, but also brings serious saline-alkali stress hazards to the growth and development of crops. It is a key limiting factor that seriously restricts agricultural production. The Songnen Plain is one of the main concentrated distribution areas of saline-alkali land in my country, accounting for 7.7% of the total area of ​​saline-alkali land in the country. The main components of its soil are Na2CO3 and NaHCO3, and crops are mainly subject to severe high pH alkaline stress. Rice is an important food crop. Developing and planting rice in soil salinization areas is one of the effective measures to efficiently utilize soda saline-alkali land, but its growth suffers from many damages caused by saline-alkali stress: including osmotic stress, ion toxicity and high pH alkaline stress. Compared with other types of stress, alkaline stress has a higher pH value, which will cause excessive accumulation of reactive oxygen species (ROS) in the root system, aggravate root damage and seedling death of rice, cause more serious damage to the growth and development of rice, and hinder rice growth and yield formation. It is considered to be the dominant obstacle factor inhibiting plant growth. Therefore, it is necessary to use genetic engineering technology to improve the alkaline resistance of rice while increasing rice production capacity. Summary of the invention

[0003] In order to solve the serious problems that rice in soda saline-alkali land in Songnen Plain suffers from the inhibitory effect of high pH alkali stress, slow growth and development and low yield, the present invention provides the application of OsNCED3 gene overexpression to improve the alkali resistance of rice.

[0004] The invention discloses an application of overexpressing the OsNCED3 gene to improve the alkali resistance of rice. The CDS sequence of the OsNCED3 gene is shown in the sequence table as SEQ ID NO.1.

[0005] Furthermore, the present invention improves the alkali resistance of rice by increasing the expression of the OsNCED3 gene.

[0006] Furthermore, the method for overexpressing the OsNCED3 gene in rice of the present invention is as follows:

[0007] First, RNAiso Reagent was used to extract total RNA from rice roots, stems, leaves, leaf sheaths or inflorescences, and the integrity and extraction concentration of RNA were detected by 1% agarose electrophoresis and a microconcentration meter; cDNA synthesis was performed using a reverse transcription kit; then primers for gene cloning were designed according to the found mRNA sequence of OsNCED3 (AY838899), F: 5'-GCTCGGTCTCACACACTC-3' and R: 5'-GCTCCCTCTGGTCACTTCC-3', and PCR amplification was performed; polymerase chain reaction conditions included four stages: pre-denaturation, denaturation, withdrawal and extension, and the reaction conditions were as follows: 94°C for 8 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 60 sec, for a total of 30 cycles; post-extension at 72°C for 8 min;

[0008] 2. Obtain the CDS sequence of the OsNCED3 gene by PCR amplification; after the PCR product is connected to the pMD18-T vector, it is transformed into Escherichia coli, and a single colony is picked for colony PCR and agarose gel electrophoresis, and the bacterial liquid plasmid with the correct electrophoresis band is extracted for sequencing comparison. The plasmid with the correct sequence is connected to the pCAMBIA-1301 expression vector and transformed into Escherichia coli by the freeze-thaw method. The plasmid with the correct PCR and sequencing is transformed into EHA105 Bacillus; the reaction system is:

[0009]

[0010] Furthermore, the amino acid sequence encoded by the OsNCED3 gene of the present invention is shown in the sequence listing SEQ ID NO.2.

[0011] The CDS sequence of the OsNCED3 gene of the present invention consists of 1827 bases, the reading frame of the gene is from the 1st to the 1827th bases at the 5' end, and the full length of mRNA is 2060 bp.

[0012] The amino acid residue sequence of OsNCED3 described in the present invention is a protein composed of 608 amino acid residues, and belongs to a typical PLN02258 superfamily gene in rice.

[0013] The present invention utilizes any vector that can guide the expression of foreign genes in plants, introduces the coding gene of OsNCED3 provided by the present invention into rice recipient cells and overexpresses it, and can obtain transgenic cell lines and transgenic plants with improved alkaline resistance. When using the gene of the present invention to construct a plant expression vector, any enhancing promoter or inducible promoter can be added before its transcription initiation nucleotide.

[0014] In order to facilitate the identification and screening of transgenic plant cells or plants, the vector used is processed and modified, such as adding plant selectable markers, such as GUS gene, BAR gene and GFP gene, etc. Or antibiotic markers with resistance, such as ampicillin, kanamycin, etc. The expression vector carrying OsNCED3 of the present invention can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues are cultivated into plants. The transformed host can be either a monocot or a dicot.

[0015] The present invention increases ABA content and significantly reduces the degree of damage to rice after alkali stress by overexpressing the OsNCED3 gene in rice. The OsNCED3 gene effectively regulates the salt-alkali tolerance of rice in saline-alkali soil, increases the number of effective panicles per unit area and the harvest index, and thus increases the yield of rice. The gene of the present invention is of great significance for cultivating and improving salt-alkali tolerant rice varieties, improving salt-alkali tolerance and rice yield. The present invention can improve the salt-alkali tolerance and yield of rice in soda saline-alkali land and the economic income of farmers, and plays a key role in improving and cultivating new rice germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the PCR amplification result of the OsNCED3 gene in Example 1.

[0017] Figure 2 This is the result of the specific expression of OsNCED3 gene in tissues in Example 2.

[0018] Figure 3 This is the root growth of wild-type and OsNCED3 transgenic rice lines under alkaline stress treatment in Example 4.

[0019] Figure 4 This is the root staining of the wild-type and OsNCED3 transgenic rice lines under alkaline stress treatment in Example 4.

[0020] Figure 5 The aboveground growth of wild-type and OsNCED3 transgenic rice lines under alkaline stress in Example 4.

[0021] Figure 6 This is the determination of antioxidant enzyme activity in the roots of wild-type and OsNCED3 transgenic rice under alkaline stress in Example 5.

[0022] Figure 7 These are the yield results of wild-type and OsNCED3 transgenic rice lines grown in saline-alkali greenhouse paddy fields in Example 6. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0025] Specific implementation method 1: In this implementation method, the overexpression of OsNCED3 gene is used to improve the alkali resistance of rice. The nucleotide sequence of the OsNCED3 gene is shown in the sequence table SEQ ID NO.1.

[0026] The OsNCED3 gene in this embodiment is named 9-cis-epoxycarotene dioxygenase gene.

[0027] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that the alkali tolerance of rice is improved by increasing the expression of the OsNCED3 gene. Other steps and parameters are the same as those of specific embodiment 1.

[0028] By using any vector that can guide the expression of foreign genes in plants, the coding gene of OsNCED3 provided in this embodiment is introduced into rice recipient cells to obtain transgenic cell lines and transgenic plants with improved alkaline resistance. When using the gene of the present invention to construct a plant expression vector, any enhancing promoter or inducible promoter can be added before its transcription initiation nucleotide.

[0029] Specific embodiment 3: This embodiment is different from specific embodiment 1 or 2 in that: Method for overexpressing OsNCED3 gene in rice:

[0030] First, RNAiso Reagent was used to extract total RNA from rice roots, stems, leaves, leaf sheaths or inflorescences, and the integrity and extraction concentration of RNA were detected by 1% agarose electrophoresis and a microconcentration meter; cDNA synthesis was performed using a reverse transcription kit; then primers for gene cloning were designed according to the found mRNA sequence of OsNCED3 (AY838899), F: 5'-GCTCGGTCTCACACACTC-3' and R: 5'-GCTCCCTCTGGTCACTTCC-3', and PCR amplification was performed; polymerase chain reaction conditions included four stages: pre-denaturation, denaturation, withdrawal and extension, and the reaction conditions were as follows: 94°C for 8 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 60 sec, for a total of 30 cycles; post-extension at 72°C for 8 min;

[0031] 2. Obtain the CDS sequence of the OsNCED3 gene by PCR amplification; after the PCR product is connected to the pMD18-T vector, it is transformed into Escherichia coli, and a single colony is picked for colony PCR and agarose gel electrophoresis, and the bacterial liquid plasmid with the correct electrophoresis band is extracted for sequencing comparison. The plasmid with the correct sequence is connected to the pCAMBIA-1301 expression vector and transformed into Escherichia coli by the freeze-thaw method. The plasmid with the correct PCR and sequencing is transformed into EHA105 Bacillus; the reaction system is:

[0032]

[0033] The other steps and parameters are the same as those in the first specific implementation.

[0034] In step 2 of this embodiment, the CDS sequence of the OsNCED3 gene obtained by PCR amplification consists of 1827 base pairs, and the reading frame is from the 1st to the 1827th base at the 5' end, encoding a protein consisting of 608 amino acid residues, which contains an RPE65 conserved domain.

[0035] Specific embodiment 4: This embodiment differs from specific embodiment 1 or 2 in that: the amino acid sequence encoded by the OsNCED3 gene is shown in the sequence table SEQ ID NO. 2. Other steps and parameters are the same as those of specific embodiment 1.

[0036] Example 1 Cloning of OsNCED3 gene and construction of overexpression vector

[0037] RNAiso Reagent was used to extract total RNA from roots, stems, leaves, leaf sheaths and inflorescences of Dongdao No. 4 (a product line developed by the Northeast Institute of Geography and Agricultural Ecology, Chinese Academy of Sciences). The integrity and extraction concentration of RNA were tested by 1% agarose electrophoresis and a microconcentration meter. The synthesis of cDNA was carried out according to the instructions of the Reverse Transcriptase M-MLV Kit of Takara Biotech. The primers for gene cloning were designed based on the mRNA sequence of OsNCED3 (AY838899), F: 5'-GCTCGGTCTCACACACTC-3' and R: 5'-GCTCCCTCTGGTCACTTCC-3'. PCR amplification (25 μl) of the target gene sequence was performed according to the following reaction system and conditions. The reaction system is shown in Table 1. The polymerase chain reaction conditions included four stages: pre-denaturation, denaturation, withdrawal and extension. The reaction conditions were as follows: 94°C for 8 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 60 sec, for a total of 30 cycles; post-extension at 72°C for 8 min. Figure 1 is the PCR amplification result of OsNCED3. Figure 1Middle M: ​​DL2000 DNAMarker; Lanes 1, 2, and 3: PCR results of target genes. OsNCED3 was obtained by PCR amplification and consisted of 1827 base pairs. The reading frame was from the 1st to the 1827th base at the 5' end, encoding a protein consisting of 608 amino acid residues, which contained an RPE65 conserved domain. After the PCR product was connected to the pMD18-T vector, Escherichia coli was transformed. Single colonies were picked for colony PCR and agarose gel electrophoresis. The bacterial liquid plasmid with the correct electrophoresis band was extracted and sent to Sangon Sequencing Company for sequencing comparison. The plasmid with the correct sequence was connected to the pCAMBIA-1301 expression vector and transformed into Escherichia coli by freeze-thaw method. The plasmid with the correct PCR and sequencing was transformed into EHA105 Bacillus, laying the foundation for subsequent rice genetic transformation and gene function research.

[0038] Table 1 PCR amplification reaction system

[0039]

[0040] Example 2 Tissue-specific expression of OsNCED3 gene

[0041] Total RNA from the root, stem, leaf, leaf sheath and inflorescence of the rice variety Dongdao No. 4 was extracted and reverse transcribed into cDNA, using the same method as in Example 1. The expression of the OsNCED3 gene in different rice tissues was detected using real-time fluorescence quantitative PCR. According to the instructions of SYBR Premix Ex TaqTMⅡ (purchased from Bao Biotechnology Co., Ltd.), the DNA amplification and fluorescent chemical detection experiments were completed using the StepOnePlus instrument. Among them, rice OsActin was used as the internal reference gene, and the primer sequences were F: 5'-TTCCAGCCTTCCTTCATA-3', R: 5'-AACGATGTTGCCATATAGAT-3'. The primer sequences of the OsNCED3 gene are F: 5'-GACTGCTTCTGCTTCCACCTCTGG-3'; R: 5'-AGGCGGTCGTCGGACTCGTT-3', and the specific reaction system is shown in Table 2.

[0042] Table 2 qPCR amplification reaction system

[0043]

[0044]

[0045] The specific reaction conditions were: 95°C for 30 sec; 95°C for 5 sec, 60°C for 30 sec, for a total of 40 cycles; post-extension at 72°C for 8 min; Figure 2 The expression specificity of OsNCED3 gene in different tissues.

[0046] After the reaction is completed, the amplification curve and melting curve of Real Time PCR are confirmed, and a standard curve is prepared for PCR quantification. The results showed that the OsNCED3 gene was expressed at the highest level in rice roots and inflorescences, and at the lowest level in leaves, showing the specificity of gene expression in different rice tissues.

[0047] Example 3 Overexpression of OsNCED3 gene in rice

[0048] The present invention uses the method of Agrobacterium transformation to carry out rice genetic transformation and introduce the target gene OsNCED3 into rice recipient cells. The specific method is as follows:

[0049] 3.1 Induction and culture of rice callus

[0050] Remove the husks from the newly harvested rice seeds, put them in a conical flask, add 70% ethanol to soak and disinfect for 1 minute, rinse with sterile distilled water for 3-5 times, then shake and disinfect with 50% sodium hypochlorite solution for 15 minutes, and shake and rinse with sterile distilled water again for 8-10 times. Put them on sterile absorbent filter paper to absorb the water, and then inoculate them into callus induction medium in a sterile clean bench. Cultivate rice callus tissue under 28°C light conditions for about 15-20 days to obtain rice embryonic callus tissue, which is transferred to N6D medium (macroelements, iron salts, trace elements, organic matter, L-proline, 2,4-D, sucrose, pH = 5.8) and pre-cultured under light for 5 days.

[0051] 3.2 Agrobacterium infection and co-cultivation

[0052] The rice callus with good appearance and growth vitality after pre-culture was transferred to the AAM resuspension transformation solution (macroelements, iron salts, trace elements, organic matter, amino acids, acetosyringone, sucrose, glucose, pH = 5.2) in the clean bench, and after shaking for 90 seconds, it was placed on sterile filter paper to absorb the excess Agrobacterium bacterial solution, and the rice callus was transferred to the co-cultivation medium and cultured in the dark for 3 days away from light.

[0053] Preparation method of AAM resuspended transformation liquid: In a sterile clean bench, inoculate Agrobacterium EHA105 containing the target gene vector plasmid into a solid culture medium containing 50 mg / L kanamycin YEP, seal and invert in a dark incubator at 28°C for 2-3 days, pick a single colony and transfer it to a liquid culture medium containing 50 mg / L kanamycin YEP, shake it on a shaker at 28°C and 130 rpm for 2 days. Collect the bacterial liquid precipitate by centrifugation, resuspend the Agrobacterium liquid with 100 mL AAM liquid culture medium with 20 mg / L acetosyringone, OD600 = 0.2, and obtain the AAM resuspended transformation liquid.

[0054] 3.3 Degerming and screening of rice callus

[0055] Transfer the co-cultivated callus to a glass bottle in a clean bench. Rinse with sterile water for 5 times, and place on a shaker to wash thoroughly to fully wash off the Agrobacterium inside the callus. Sterile distilled water containing carbenicillin can also be added to rinse again for 10-15 minutes to inhibit bacteria. Then place the callus on sterile filter paper to absorb the water until it is dry. Finally, inoculate the callus on the screening medium containing the screening agent, culture and screen in a 28℃ light incubator for about 3 weeks, and change the medium once a week. Transfer the well-growing resistant callus to the regeneration medium, subculture and change the medium regularly until the regenerated seedlings appear and then transfer to the rooting medium. After the roots grow out, open the breathable lid of the rooting bottle, add sterile water, and harden the seedlings for 3-5 days. Wash off the solid culture medium, transplant to a pot, and manage it in a greenhouse or artificial climate chamber as usual. The overexpression of the OsNCED3 gene in rice is completed, and the OsNCED3 transgenic rice strains with stable inheritance are obtained through generation-by-generation propagation and PCR positive identification.

[0056] Example 4 Growth of wild-type and OsNCED3 transgenic rice lines under alkaline stress

[0057] 4.1 Cultivation of rice seedlings and alkaline stress treatment

[0058] The rice seeds were soaked in 0.1% (W / V) HgCl2 solution for 10 minutes for disinfection, and then rinsed repeatedly with distilled water. Then, distilled water was added to the beaker to soak the seeds, and placed in a constant temperature incubator at 30°C for 48 hours. Then, the seeds were evenly spread on the bottom of the culture dish and germinated in a constant temperature incubator at 28°C for 24 hours. The rice seeds with the same germination were selected and sown on a 96-well black culture plate and placed in a black plastic culture box for distilled water culture for 5 days. Then, 1 / 2 Kimura B nutrient solution was used for culture in an artificial climate chamber, and the culture was continued to the three-leaf stage under the setting of 25°C (light) / 20°C (darkness) and a photoperiod of 12 hours. The rice seedlings were then transferred to 15mM Na2CO3 (pH: 10.82±0.032, EC: 2.80±0.002mS / cm) solution for culture to simulate alkaline stress conditions, laying the foundation for subsequent phenotypic observations and various physiological and biochemical index determinations.

[0059] 4.2 Observation of rice growth phenotype after alkaline stress treatment

[0060] The wild-type and transgenic rice seedlings were observed and photographed for 3 days after alkaline stress treatment, and the root and aboveground leaf growth status was observed and recorded. It was found that the roots of the wild-type rice seedlings were severely damaged after alkali treatment, the growth was slow, the leaves were withered and curled, and yellowing was obvious. The wilting was serious and could not grow normally. The transgenic rice seedlings that overexpressed the OsNCED3 gene were significantly weakened by the alkaline stress, grew better, and the survival rate was significantly higher than that of the wild-type control group. In order to further verify the damage of alkaline stress to the roots of rice, the present invention was identified by the method of Evans blue staining. Evans blue is an azo dye used to determine cell viability, and the blue color it presents can be used to evaluate the cell state. When plant cells are damaged by adverse stress, Evans blue can penetrate broken or unstable membranes and stain these damaged cells, and more stained cells can be observed. 2cm root tips of wild-type and transgenic rice seedlings were cut, 0.25% Evans blue solution was added to soak the leaves for 30 minutes, and then rinsed repeatedly with deionized water until the blue color disappeared. The stained root tips were placed under a stereo microscope for observation and photographed. The results showed that the wild-type was stained darker, the dye penetrated more into the damaged cells, and the roots were severely damaged. The roots of transgenic rice were stained lighter and the cells were less damaged, indicating that the overexpression of this gene plays a very important role in regulating rice to alleviate alkaline stress and improve rice alkaline tolerance.

[0061] Figure 3 The root growth of wild type (WT) and OsNCED3 transgenic rice lines (OE-1, OE-2, OE-3) under alkaline stress in this example; Figure 4 The root staining of wild type (WT) and OsNCED3 transgenic rice lines (OE-1, OE-2, OE-3) under alkaline stress in this example; Figure 5 The aboveground growth of wild-type (WT) and OsNCED3 transgenic rice lines (OE-1, OE-2, OE-3) under alkaline stress in this example.

[0062] Example 5 Determination of antioxidant enzyme activities in roots of wild-type and OsNCED3 transgenic rice under alkaline stress

[0063] Fresh root samples (0.1 g) of rice treated with alkaline stress for 3 days and untreated control were placed in 2 mL sterile centrifuge tubes, quickly frozen in liquid nitrogen, and then the activities of SOD, POD, CAT and APX were determined according to conventional methods. Superoxide dismutase (SOD) activity was determined by nitroblue tetrazolium photoreduction method; peroxidase (POD) activity was determined by guaiacol method; catalase (CAT) activity was determined by quartz colorimetry; ascorbate peroxidase (APX) activity was directly determined by APX activity based on ascorbic acid. Sampling and testing were repeated 3 times biologically and 3 times technically to ensure the reliability and accuracy of the test results and reduce errors.

[0064] Figure 6 Results of antioxidant enzyme activity determination in roots of wild type (WT) and OsNCED3 transgenic rice (OE-1, OE-2, OE-3) under alkaline stress. Figure 6 It can be seen that under normal conditions, there was no significant difference in the antioxidant enzyme activities in the roots of wild-type and OsNCED3 transgenic rice. After 3 days of alkaline stress treatment, the SOD, POD, CAT and APX activities in the roots of OsNCED3 transgenic rice were significantly increased compared with the wild-type (P<0.05).

[0065] Example 6 Planting and yield determination of wild-type and OsNCED3 transgenic rice in saline-alkali paddy field greenhouse

[0066] The wild-type and T2-generation OsNCED3 transgenic rice lines were planted in a transgenic experimental greenhouse (with saline-alkali paddy fields configured in proportion). After sowing, field management, sampling, investigating the growth status of rice at each stage (tillering, jointing, booting, heading, flowering, pollination, and filling), and conducting a unified survey of yield components at harvest, mainly including rice thousand-grain weight, fruit setting rate, number of effective panicles per square meter, and number of grains per panicle, the theoretical yield was calculated, and the data were analyzed by biostatistics.

[0067] Note: Theoretical yield = number of effective ears × number of grains per ear × fruit setting rate × thousand-grain weight

[0068] Figure 7 The results of single plant yield of wild type (WT) and OsNCED3 transgenic rice lines (OE-1, OE-2, OE-3) grown in saline-alkali greenhouse paddy fields are shown in Figure 2. Figure 7It can be seen that through the calculation of the theoretical yield formula, the theoretical yield of each plant of the three OsNCED3 transgenic rice lines is significantly higher than that of the wild type, indicating that in saline-alkali soil, overexpression of the OsNCED3 gene can improve the yield of rice by improving the alkali resistance of rice. While cultivating new alkali-resistant rice varieties, the present invention can quickly improve the rice production capacity, providing a theoretical scientific basis for exploring the potential of improving the rice production capacity in soda saline-alkali land in the Songnen Plain.

Claims

1. Application of OsNCED3 gene overexpression to improve rice alkali tolerance, characterized in that: The CDS sequence of the OsNCED3 gene is shown in the sequence listing SEQ ID NO.

1.

2. The use of OsNCED3 gene overexpression to improve rice alkali tolerance according to claim 1, characterized in that: Improving alkaline tolerance in rice by increasing the expression of the OsNCED3 gene.

3. The use of OsNCED3 gene overexpression to improve rice alkali tolerance according to claim 1 or 2, characterized in that: Methods for overexpressing the OsNCED3 gene in rice: First, RNAiso Reagent was used to extract total RNA from rice roots, stems, leaves, leaf sheaths or inflorescences, and the integrity and extraction concentration of RNA were detected by 1% agarose electrophoresis and a microconcentration meter; cDNA synthesis was performed using a reverse transcription kit; then primers for gene cloning were designed according to the found mRNA sequence of OsNCED3 (AY838899), F: 5'-GCTCGGTCTCACACACTC-3' and R: 5'-GCTCCCTCTGGTCACTTCC-3', and PCR amplification was performed; polymerase chain reaction conditions included four stages: pre-denaturation, denaturation, withdrawal and extension, and the reaction conditions were as follows: 94°C for 8 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 60 sec, for a total of 30 cycles; post-extension at 72°C for 8 min; 2. Obtain the CDS sequence of the OsNCED3 gene by PCR amplification; after the PCR product is connected to the pMD18-T vector, it is transformed into Escherichia coli, and a single colony is picked for colony PCR and agarose gel electrophoresis, and the bacterial liquid plasmid with the correct electrophoresis band is extracted for sequencing comparison. The plasmid with the correct sequence is connected to the pCAMBIA-1301 expression vector and transformed into Escherichia coli by the freeze-thaw method. The plasmid with the correct PCR and sequencing is transformed into EHA105 Bacillus; the reaction system is:

4. The use of OsNCED3 gene overexpression to improve rice alkali tolerance according to claim 1, characterized in that: The amino acid sequence encoded by the OsNCED3 gene is shown in the sequence listing SEQ ID NO.2.

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