Application of rice OsPIP2;2 gene in improvement of seedling resistance to bacterial blight

Abstract

The application discloses application of a rice OsPIP2;2 gene in improvement of seedling stage resistance to bakanae disease, a nucleotide sequence of the OsPIP2;2 gene is shown as SEQ ID NO. 1, and the OsPIP2;2 gene has a positive regulation effect on the resistance of rice to the bakanae disease. The application constructs mutants Ospip2;2-1 and Ospip2;2-2 with 93-11 material as a genetic background by using a gene editing technology, and phenotype identification is carried out seven days after inoculation of bakanae disease bacteria, and the result shows that the relative seedling length of the Ospip2;2-1 and Ospip2;2-2 mutants is significantly higher than that of a wild type; OsPIP2;2 overexpression lines OE-1 and OE-2 with Nipponbare as a genetic background are obtained by using an agrobacterium-mediated genetic transformation, and phenotype identification is carried out seven days after inoculation of bakanae disease bacteria, and the result shows that the relative seedling length of the OsPIP2;2 overexpression lines OE-1 and OE-2 is significantly lower than that of the wild type. In summary, the overexpression line of the rice OsPIP2;2 gene has better resistance to the bakanae disease than the mutant of the rice OsPIP2;2 gene, that is, the OsPIP2;2 gene has a positive regulation effect on the resistance of rice to the bakanae disease.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding and genetic engineering, and particularly relates to application of rice OsPIP2;2 gene in improving resistance to bakanae disease at the seedling stage. Background Art

[0002] Rice bakanae disease, also known as leggy growth disease, occurs in all rice-growing areas of China. Affected grains often fail to germinate or emerge after sowing. During the seedling stage, the disease manifests as tall, thin seedlings with elongated leaves and sheaths, pale yellow leaves, and poor root development. Some affected seedlings may die before transplanting, severely impacting rice yield and quality.

[0003] Current control measures for rice bakanae disease primarily include establishing disease-free seed-keeping fields, soaking rice seeds, and using pesticides. Reports indicate that rice OsPIP2;2 is a channel protein that facilitates water transport and plays a role in maintaining cell membrane integrity, effectively protecting rice cells from electrolyte leakage caused by physiological drought stress. Overexpression of OsPIP2;2 significantly enhances H2O transport and drought resistance, and also increases proline and polyamine concentrations. However, the role of OsPIP2;2 in regulating seedling bakanae disease resistance has not been reported. Summary of the Invention

[0004] In view of the above problems, the present invention aims to provide an application of the rice OsPIP2;2 gene in improving resistance to bakanae disease at the seedling stage.

[0005] The application of rice OsPIP2;2 gene in improving seedling resistance to bakanae disease. The nucleotide sequence of OsPIP2;2 gene is shown in SEQ ID NO.1. The OsPIP2;2 gene has a positive regulatory effect on rice resistance to bakanae disease.

[0006] Furthermore, the amino acid sequence of the protein encoded by the OsPIP2;2 gene is shown in SEQ ID NO.2.

[0007] Furthermore, the relative seedling length of the mutant of the OsPIP2;2 gene is significantly longer than that of the wild type.

[0008] Furthermore, the relative seedling length of the OsPIP2;2 gene overexpression strain was significantly lower than that of the wild type.

[0009] A PCR amplification primer for amplifying rice OsPIP2;2 gene, wherein the sequence of the upstream primer of the PCR amplification primer is shown as SEQ ID NO.3, and the sequence of the downstream primer of the PCR amplification primer is shown as SEQ ID NO.4.

[0010] A method for preparing an overexpression strain that overexpresses the rice OsPIP2;2 gene comprises:

[0011] Design target sites and PCR amplification primers;

[0012] The PCR amplification primers are acted on the target site, PCR amplification is performed using rice cDNA as a template, and the PCR product is purified and recovered to obtain the OsPIP2;2 gene CDS fragment;

[0013] The OsPIP2;2 gene CDS fragment is amplified by PCR using primers with homologous recombination adapters, and the PCR product is purified and recovered to obtain a target fragment containing the OsPIP2;2 gene;

[0014] The target fragment carrying the OsPIP2;2 gene was constructed into the pCAMBIA1300 vector to obtain a recombinant plasmid;

[0015] The recombinant plasmid is used to transform Agrobacterium, and the transformed Agrobacterium is used to transform rice to obtain a rice OsPIP2;2 gene overexpression material.

[0016] Furthermore, after obtaining the rice OsPIP2;2 gene overexpression material, the method further comprises:

[0017] Screening and identifying the rice OsPIP2;2 gene overexpression material;

[0018] The resistance phenotype of the rice OsPIP2;2 gene overexpressing material was identified at the seedling stage.

[0019] Furthermore, the upstream primer sequence used for screening rice OsPIP2;2 gene overexpression materials is shown in SEQ ID NO.13, and the downstream primer sequence is shown in SEQ ID NO.14.

[0020] A method for identifying resistance to seedling bakanae disease at the seedling stage, based on rice OsPIP2;2 gene overexpression material, comprises:

[0021] Select healthy and plump seeds.

[0022] Sterilize the seed surface with 0.1% mercuric chloride solution for 5 minutes;

[0023] Rinse the surface of the sterilized seeds, place the rinsed seeds in a seedling box, add an appropriate amount of distilled water, set the ambient temperature to 30°C, and place the seedling box under light and dark conditions for 12 hours respectively;

[0024] Phenotypic identification was performed seven days after the rice seedlings were inoculated with Bakanae pathogen.

[0025] A method for preparing a mutant lacking the rice OsPIP2;2 gene comprises:

[0026] Design target sites and PCR amplification primers;

[0027] Using pCBC-MT1T2 as a template, PCR amplification was performed using the PCR amplification primers, and the PCR product was purified and recovered to obtain an MT1T2-PCR intermediate vector containing the OsPIP2 and 2gRNA target sequences;

[0028] The MT1T2-PCR intermediate vector was constructed into the pHUE411 vector to obtain a plasmid carrying the OsPIP2;2 gene target fragment;

[0029] The plasmid carrying the OsPIP2;2 gene target fragment is used to transform Agrobacterium, and the transformed Agrobacterium is used to transform rice to obtain a rice OsPIP2;2 gene mutant.

[0030] The beneficial effects of the present invention are:

[0031] (1) The present invention uses gene editing technology to construct mutants Ospip2;2-1 and Ospip2;2-2 with 93-11 material as the genetic background. Phenotypic identification was performed seven days after inoculation with the baklaying pathogen. The results showed that the relative seedling lengths of the Ospip2;2-1 and Ospip2;2-2 mutants were significantly longer than those of the wild type. Agrobacterium-mediated genetic transformation was used to obtain OsPIP2;2 overexpression strains OE-1 and OE-2 with Nipponbare as the genetic background. Phenotypic identification was performed seven days after inoculation with the baklaying pathogen. The results showed that the relative seedling lengths of the OsPIP2;2 overexpression strains OE-1 and OE-2 were significantly shorter than those of the wild type. In summary, the rice OsPIP2;2 gene overexpression strains have better resistance to baklaying disease than the rice OsPIP2;2 gene mutants, that is, the OsPIP2;2 gene has a positive regulatory effect on rice baklaying disease resistance.

[0032] (2) The present invention isolated and cloned the OsPIP2;2 gene from rice, and demonstrated for the first time through mutants and overexpression transgenic materials that the gene is involved in the regulation of rice seedling blanching disease.

[0033] (3) The present invention provides a basis for screening rice varieties resistant to bakanae disease and also provides important gene resources for improving and enhancing rice resistance to bakanae disease, which is of great significance to production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the experimental result of the resistance of rice OsPIP2;2 gene mutant material to seedling disease at the seedling stage;

[0035] Figure 2 This is the experimental result of the resistance of rice OsPIP2;2 gene overexpression material to seedling blanching disease at the seedling stage. DETAILED DESCRIPTION

[0036] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Any modifications or substitutions to the methods, steps, or conditions of the present invention, without departing from the spirit and substance of the present invention, fall within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, and instruments used in the examples herein are commercially available; unless otherwise specified, all technical means in the examples herein are conventional means well known to those skilled in the art.

[0037] The primers and sequencing used in the present invention were completed by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd., and the various restriction endonucleases used in the experiment were purchased from Kangrun Jingxing (Suzhou) Biotechnology Co., Ltd. The genome extraction kit was purchased from Zhengzhou Suling Biotechnology Co., Ltd., the reverse transcription kit was purchased from Novozymes Biotechnology Co., Ltd., and the plasmid extraction kit and gel recovery kit were purchased from Meiji Biotechnology Co., Ltd. The methods were all carried out according to the instructions.

[0038] Example 1: Gene cloning and mutant construction

[0039] The rice MSU7.0 database (http: / / rice.uga.edu / index.shtml) was used to obtain the nucleotide sequence of the rice OsPIP2;2 gene shown in SEQ ID NO.1 and the amino acid sequence of the protein encoded by the rice OsPIP2;2 gene shown in SEQ ID NO.2.

[0040] Log in to the website http: / / skl.scau.edu.cn / targetdesign / to screen targets. The target sequences of the OsPIP2;2 gene are shown in SEQ ID NOs. 5 and 6. Primers were designed based on these target sequences. The primers for the OsPIP2;2 target 1 sequence are shown in SEQ ID NOs. 7 and 8, and the primers for the OsPIP2;2 target 2 sequence are shown in SEQ ID NOs. 9 and 10. PCR amplification was performed using pCBC-MT1T2 as a template using the four primers shown in SEQ ID NOs. 7, 8, 9, and 10. The amplification system was: 1 μL of pCBC-MT1T2, 1 μL of each primer, 15 μL of the mix, and 10 μL of ddH2O. The amplification program was as follows: 98°C for 30 s, 98°C for 10 s, 58°C for 5 s, and 72°C for 5 s, for 32 cycles, and 72°C for 1 min. The PCR product was purified and recovered to obtain the MT1T2-PCR target sequence containing the gRNA for the OsPIP2;2 gene. The pHUE411 vector was digested with BsaI, and the MT1T2-PCR gel-recovered product was constructed into the pHUE411 vector by homologous recombination to obtain the pHUE411+MT1T2-PCR vector. The resulting pHUE411+MT1T2-PCR vector was transformed into Agrobacterium tumefaciens, which was then transformed into the wild-type indica rice variety 93-11. The PCR amplified products were sequenced and compared with the wild-type, revealing the identification of OsPIP2;2 mutants in the 93-11 background. The upstream and downstream primers for screening homozygous CRISPR / Cas9 mutants of the OsPIP2;2 gene are shown in SEQ ID NOs. 11 and 12.

[0041] Example 2: Construction of overexpression transgenic material

[0042] The rice cDNA was used as a template for PCR amplification, and the PCR product was purified and recovered to obtain the OsPIP2;2 gene CDS fragment. The sequence of the upstream primer of the PCR is shown in SEQ ID NO. 3, and the sequence of the downstream primer is shown in SEQ ID NO. 4. The obtained OsPIP2;2 gene CDS fragment was used as a template, and a primer with a homologous recombination linker was used for PCR amplification. The PCR product was purified and recovered to obtain a CDS fragment with a homologous recombination linker. The sequence of the upstream primer of the PCR is shown in SEQ ID NO. 13, and the sequence of the downstream primer is shown in SEQ ID NO. 14. The target fragment with the OsPIP2;2 gene was constructed into a pCAMBIA1300 vector by a homologous recombination method to obtain a recombinant plasmid. The recombinant plasmid was transformed into Agrobacterium, and the Agrobacterium with the transformed plasmid was transformed into a wild-type japonica rice variety Nipponbare. A hygromycin resistance tag was used for PCR amplification to screen positive overexpression materials. The sequence of the upstream primer is shown in SEQ ID NO. 15, and the sequence of the downstream primer is shown in SEQ ID NO. 16.

[0043] Example 3: Phenotype analysis of gene mutant and overexpression transgenic materials

[0044] Using the constructed OsPIP2;2 CRISPR / Cas9 mutants Ospip2;2-1, Ospip2;2-2 and wild-type 93-11, overexpression OE-1, OE-2 and wild-type Nipponbare rice varieties, seedling stage bakanae disease resistance identification was carried out. The specific method is as follows: 100 healthy and full seeds were selected each time, surface sterilized with 0.1% mercuric chloride solution for 5 min, washed with distilled water for 3 times, the surface of the seeds was wiped dry, placed in a seedling box, added with appropriate amount of distilled water, placed in a light / dark condition of 12 h each at 30°C, the humidity of the air had no requirement, and the seedlings were grown in sterile water before and after inoculation to ensure the seedlings were moist. The experiment was completed in the seedling stage, and the nutritional conditions were not high. Phenotype identification was carried out seven days after inoculation of bakanae disease bacteria. The results showed that the relative seedling length of the Ospip2;2-1 and Ospip2;2-2 mutants was significantly higher than that of the wild type (93-11) (P<0.05); the relative seedling length of the OsPIP2;2 overexpression strains OE-1 and OE-2 was significantly lower than that of the wild type (P<0.05). It can be seen that the OsPIP2;2 gene has a positive regulation on the bakanae disease resistance of rice. Figure 1 Figure 2

[0045] ​​It is understandable that the above embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, the above technical features can be freely combined, and several deformations and improvements can be made, all of which fall within the scope of protection of the present invention. Therefore, all equivalent changes and modifications made to the scope of the claims of the present invention should fall within the scope of coverage of the claims of the present invention.

Claims

1. Application of rice OsPIP2;2 gene in improving resistance to Bakanae disease at the seedling stage, characterized in that: The nucleotide sequence of the OsPIP2;2 gene is shown in SEQ ID NO.

1. The OsPIP2;2 gene has a positive regulatory effect on the resistance to bakanae disease in rice.

2. The use of the rice OsPIP2;2 gene in improving resistance to Bakanae disease at the seedling stage according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the OsPIP2;2 gene is shown in SEQ ID NO.

2.

3. The use of the rice OsPIP2;2 gene in improving resistance to Bakanae disease at the seedling stage according to claim 1, characterized in that: The relative seedling length of the mutant of the OsPIP2;2 gene was significantly longer than that of the wild type.

4. The use of the rice OsPIP2;2 gene in improving resistance to Bakanae disease at the seedling stage according to claim 1, characterized in that: The relative seedling length of the OsPIP2;2 gene overexpression strain was significantly lower than that of the wild type.

Images