Application of the rice OsCLE48 gene in improving plant salt tolerance

Abstract

The present invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of rice OsCLE48 gene in improving plant salt-alkali tolerance. The nucleotide sequence of the OsCLE48 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded secreted small peptide is shown in SEQ ID NO.2. The OsCLE48 gene was knocked out using CRISPR gene editing technology, and a rice mutant with functional loss was successfully obtained. Studies have shown that knocking out the gene significantly enhances the tolerance of rice to salt-alkali stress, and the survival rate and biomass of the mutant under saline-alkali conditions are significantly better than those of wild-type rice. In addition, through the same gene editing technology, the homologous genes of the OsCLE48 gene can be knocked out in crops such as corn and barley to improve the salt-alkali tolerance of these plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of rice OsCLE48 gene in improving the salt-alkali tolerance of plants. Background Art

[0002] Salt-alkali stress refers to excessive salt and alkaline substances in the soil, which deteriorates the growth environment of rice and affects its growth and development. The metabolic disorder caused by salt-alkali stress will weaken the rice root system's ability to absorb water and nutrients, inhibit photosynthesis, and then lead to short plants, yellowing leaves, and reduced fruiting rate. In severe cases, it can cause large-scale yield reduction or even total crop failure. In addition, salt-alkali stress can also destroy the soil structure, causing compaction and preventing the root system from growing normally.

[0003] At present, the countermeasures for salinity stress include: improving rice resistance by breeding salt-tolerant varieties; improving soil, such as irrigation and salt removal, applying organic fertilizers and gypsum, etc., to reduce soil salinity; and reasonable irrigation and fertilization to enhance rice resistance. At the same time, genetic engineering technology is also actively used to explore ways to improve rice resistance to salt and alkali through genetic improvement.

[0004] The CLE (CLAVATA3 / EMBRYO SURROUNDING REGION-RELAT ED) family is an important type of signal peptide in plants, which is widely involved in the regulation of plant growth, development and stress response. Studies have shown that CLE peptides enhance the adaptability of plants under drought and salt stress by regulating the differentiation and development of root cap cells. CLE peptides can also activate specific signaling pathways and regulate the ion balance of plant cells, thereby reducing the inhibitory effect of salt on plant growth. In addition, the CLE family also plays an important role in enhancing plant resistance to diseases.

[0005] Currently, research on the role of CLE peptides in rice salt-alkali tolerance is still in its early stages. Summary of the invention

[0006] In order to solve the above problems, the present invention provides the use of OsCLE48 gene in improving the salt-alkali tolerance of plants.

[0007] Furthermore, the nucleotide sequence of the OsCLE48 gene is as shown in SEQ ID NO.1 or has 95% or more consistency or identity with the nucleotide sequence shown in SEQ ID NO.1 and expresses the same functional protein.

[0008] Furthermore, the amino acid sequence of the OsCLE48 protein is as shown in SEQ ID NO.2 or a fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.2.

[0009] The invention also provides application of OsCLE48 protein in improving salt-alkali tolerance of plants.

[0010] The present invention also provides the application of OsCLE48 gene and OsCLE48 protein in salt-alkali tolerant plant breeding.

[0011] Furthermore, the plants include rice, corn and barley.

[0012] The present invention also provides a method for improving the salt-alkali tolerance of plants, which comprises knocking out the OsCLE48 gene in the plant or reducing the expression of the OsCLE48 protein.

[0013] The present invention also provides a salt-tolerant plant breeding method, characterized in that it comprises the above method for improving the salt-alkali tolerance of plants.

[0014] The present invention has the following beneficial effects:

[0015] 1. This paper cloned and analyzed for the first time the CLE family gene OsCLE48 that responds to saline-alkali stress in rice, and published its nucleotide sequence, amino acid sequence and promoter sequence, which is of great significance for clarifying the molecular mechanism of rice salt-alkali tolerance regulation and breeding.

[0016] 2. The present invention confirms that the OsCLE48 gene is involved in the salt-alkali tolerance of rice by means of transgenic means, wherein knocking out the gene significantly improves the salt-alkali tolerance of rice, indicating that OsCLE48 is a negative regulatory factor.

[0017] 3. The OsCLE48 gene has homologous genes in barley and corn. Knocking out the homologous genes in these crops can improve their salt-alkali tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 .Schematic diagram of OsCLE48 gene editing, where: (A) is the gene structure of OsCLE48 and its gene editing type, (B) is the protein structure diagram of OsCLE48 and the amino acid sequence of the conserved peptide segment;

[0020] Figure 2.Expression pattern diagram of OsCLE48 gene, wherein: (A) is the expression pattern diagram of OsCLE48 gene in different tissues of rice, (B) is the expression pattern diagram of OsCLE48 gene in rice root under saline-alkali stress (20mM Na2CO3 (pH=9)) at different treatment times;

[0021] Figure 3 .The phenotypic differences of mutant materials of OsCLE48 gene under saline-alkali stress, where: (A) is a plant phenotype photo, showing the difference in salt-alkali tolerance between mutant materials and wild type after treatment with 20mM Na2CO3 (pH=9) for 10 days at the age of 14 days, (B) the survival rate of wild type and mutant materials after control and treatment, (C) the comparison of root and aboveground fresh weight of wild type and mutant materials after control and treatment.

[0022] Figure 4 .Subcellular localization map of OsCLE48 protein.

[0023] Figure 5 .Amino acid sequence alignment of rice OsCLE48 homologous genes in barley and corn, where the red boxes are functional peptide sequences. DETAILED DESCRIPTION

[0024] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] If you need to obtain the OsCLE48 gene knockout mutant strain of the present invention, you can contact Professor Wu Dezhi of the College of Agriculture of Hunan Agricultural University, address: 409, South of Life Science Building, Hunan Agricultural University, Furong District, Changsha City, Hunan Province; Postal Code: 410128.

[0030] The primer sequences involved in the following examples are shown in the table below:

[0031]

[0032] Example 1: Identification of Zhonghua11 and its mutant (cle48) and cloning of the OsCLE48 gene

[0033] To further study the function of OsCLE48 gene in rice salt-alkali tolerance, the present inventors constructed a loss-of-function mutant of OsCLE48 gene by CRISPR / Cas9 gene editing technology in the rice variety Zhonghua 11.

[0034] The specific steps are as follows:

[0035] 1) Seed treatment and cultivation

[0036] The mutant seeds were surface sterilized, treated with 3% (v / v) hydrogen peroxide solution for 30 minutes, and then washed with distilled water for 5 times. The sterilized seeds were sown in germination boxes and cultured in an incubator for 10 days to obtain rice seedlings.

[0037] 2) DNA extraction and PCR amplification

[0038] Young and tender rice leaves were cut and rice genomic DNA was extracted using the CTAB method. The extracted DNA was used as a template and primers OsCLE48-CDS-F (SEQ ID NO.4) and OsCLE48-CDS-R (SEQ ID NO.5) were used for PCR amplification to amplify the coding region of the target gene. The DNA of wild-type Zhonghua 11 was used as a control for amplification, and the obtained PCR product was confirmed by sequencing, and the results showed that the nucleotide sequence of the fragment was consistent with SEQ ID NO.1.

[0039] 3) Amplification of the OsCLE48 promoter region

[0040] Using wild-type Zhonghua 11 DNA as a template, primers OsCLE48-promoter-F (SEQ ID NO.8) and OsCLE48-promoter-R (SEQ ID NO.9) were used for PCR amplification to obtain a promoter region of about 2 kb. Sequencing results showed that the nucleotide sequence of the promoter was shown in SEQ ID NO.3.

[0041] 4) Mutant identification

[0042] Among the constructed mutants, the present invention selected two homozygous mutant strains: cle48-1 and cle48-5. Through sequencing analysis, cle48-1 inserted one A base into the OsCLE48 gene, while cle48-5 inserted two A bases ( Figure 1 A), Both mutations resulted in frameshift mutations, indicating that both strains are functional knockout mutants.

[0043] 5) Gene translation and amino acid sequence analysis

[0044] The CDS sequence of the cloned OsCLE48 gene was translated using SnapGene software to obtain its encoded amino acid sequence (SEQ ID NO.2), as shown in the schematic diagram. Figure 1 As shown in B.

[0045] The PCR reaction program involved above is: 95°C for 5 min, 95°C for 30 s, 60°C for 30 s, 72°C for 20 s, 72°C for 5 min, 35 cycles.

[0046] The PCR reaction system is as follows:

[0047] PrimerSTAR (Takara) System μL 2×Mix 10 Primer F / R 1 / 1 <![CDATA[ddH2O]]> 7 DNA template 1

[0048] Example 2: Analysis of the expression pattern of the OsCLE48 gene

[0049] In order to explore the expression pattern of OsCLE48 gene in different tissues and under saline-alkali stress, the present invention analyzed the expression level of OsCLE48 gene in different tissues of rice Zhonghua 11 and after saline-alkali stress treatment by fluorescence quantitative q-PCR. The results showed that the gene was mainly expressed in the rice root system and was significantly upregulated by saline-alkali stress ( Figure 2 ).

[0050] The experimental steps are as follows:

[0051] 1) Plant material preparation

[0052] According to the method of Example 1, rice midflowers were cultured to 11 to 2 weeks old. The root, basal stem, leaf blade, petiole and other tissues of rice were collected respectively, and 3 biological replicates were set for each tissue. The collected tissue samples were quickly frozen with liquid nitrogen for subsequent RNA extraction.

[0053] 2) RNA extraction and reverse transcription

[0054] Total RNA of rice was extracted using the RC411 kit provided by Novagen. After extraction, the concentration of RNA was measured using Nanodrop and quantified to 500 ng for subsequent reverse transcription reaction. RNA was reverse transcribed into cDNA using a reverse transcription kit (R323, Novagen).

[0055] 3) qPCR reaction

[0056] Using the specific primers OsCLE48-CDS-F (SEQ ID NO.4) and OsCLE48-CDS-R (SEQ ID NO.5) of the OsCLE48 gene and the Actin gene as an internal reference, the expression level of the OsCLE48 gene in different tissues and at different time points under salinity-alkali stress was detected.

[0057] 4) qPCR reagents and reaction conditions

[0058] The experiment used TSINGKE TSE401 ArtiCanCEO SYBR qPCR Mix kit and performed qPCR reaction according to the kit instructions. Three technical replicates were performed for each sample. The qPCR reaction system is shown in the following table:

[0059] SYBRgreen system μL 2×Mix 10 Primer F / R 0.5 / 0.5 <![CDATA[ddH2O]]> 7 cDNA template 2

[0060] Example 3: Identification of salt-alkali tolerance of Zhonghua11 and mutant (cle48) plants

[0061] This example further verifies the effect of OsCLE48 gene knockout on rice salt-alkali tolerance through salt-alkali stress tolerance experiments. The specific steps are as follows:

[0062] 1) Cultivation of rice seedlings

[0063] After surface disinfection, the rice Zhonghua11, OsCLE48-1, and OsCLE48-5 mutant seeds were placed in Kimura B nutrient solution for hydroponics and cultured until the rice seedlings were two weeks old.

[0064] 2) Salt and alkali treatment

[0065] Two weeks later, the seedlings were transferred to Kimura B nutrient solution containing 20 mM Na2CO3 (pH=9) for 10 days as the salt-alkali stress treatment group. A normal nutrient solution treatment group was set up as the control during the same period.

[0066] 3) Phenotypic observation and survival rate recording

[0067] After the treatment, the growth of rice in each group was recorded. Figure 3 As shown in A and 3B, after saline-alkali stress, the growth of OsCLE48 mutant plants was significantly better than that of wild-type Zhonghua11, as shown by better plant status and higher survival rate.

[0068] 4) Fresh weight determination

[0069] After the stress treatment, the aboveground parts and roots of rice in each group were collected and their fresh weights were measured. Figure 3 As shown in C, the aboveground fresh weight of mutants OsCLE48-1 and OsCLE48-5 was significantly higher than that of wild-type Zhonghua11 after saline-alkali stress, indicating that knockout of OsCLE48 helps to enhance the salt-alkali tolerance of rice.

[0070] Example 4: Subcellular localization of OsCLE48

[0071] In order to clarify the subcellular localization of the OsCLE48 gene, the inventors constructed a subcellular localization vector of OsCLE48 and transformed it into Nicotiana benthamiana for expression. The results showed that OsCLE48 was localized in the extracellular region of the cell ( Figure 4 ), further confirming that the gene encodes a secretory small peptide. The experimental steps are as follows:

[0072] 1) Construction of subcellular localization vector of OsCLE48

[0073] The cloned OsCLE48 coding region was used as a template, and primers OsCLE48-ZL035-F (SEQ ID NO.6) and OsCLE48-ZL035-R (SEQ ID NO.7) were used for PCR amplification, and the amplified product was recovered. Subsequently, the subcellular localization vector ZL035 was digested with SalI enzyme (37°C for 30 minutes), and the digested product was recovered. OsCLE48 was cloned into the ZL035 vector by homologous recombination, and after successful sequencing verification, it was transformed into Agrobacterium strain EHA105.

[0074] 2) Agrobacterium culture and tobacco injection

[0075] The transformed Agrobacterium monoclone was cultured overnight in 5 ml LB medium until OD600 reached 1.0. After collecting the cells, resuspend them in tobacco transformation buffer to OD600 = 0.8 and let stand at room temperature for 3 hours. Mix with an equal volume of tobacco cell membrane localization marker (AtPIP2A-RFP) Agrobacterium strain and inject into Nicotiana benthamiana leaves. The formula of tobacco transformation buffer is as follows:

[0076] Tobacco transformation buffer formula (pH = 5.6) concentration <![CDATA[MgCl2 (Magnesium Chloride)]]> 10mM MES (sodium methyl ester sulfonate) 10mM AS(Acetosyringone) 150μM <![CDATA[ddH2O (double distilled water)]]> -

[0077] 3) Fluorescence microscopy

[0078] After injection, tobacco was cultured in an incubator for 48 hours, and fluorescence signals were captured using a laser confocal microscope (Zeiss LSM 980). The results showed that OsCLE48 was localized in the cell apoplast region, with no overlapping signals with the cell membrane localization marker, indicating that the gene played a role in the cell apoplast.

[0079] Example 5: Analysis of species conservation of OsCLE48 gene

[0080] In order to explore the conservation of OsCLE48 gene in other plants, the inventors used the BlAST method to search for homologous sequences of this gene in corn and barley in the EnsemblPlants database. The OsCLE48 homologous genes retrieved in corn and barley showed a high degree of conservation with the rice OsCLE48 gene. The comparison results showed that these homologous genes were almost identical in amino acid sequence, with only one amino acid difference found in the functional peptide sequence (see Figure 5 ). The analysis results showed that the OsCLE48 gene was significantly conserved among different plant species, suggesting that it may play an important role in regulating salt-alkali tolerance.

[0081] In summary, the OsCLE48 gene negatively regulates the salt-alkali tolerance of rice. Knocking out the gene or reducing its expression can enhance the salt-alkali tolerance of rice. The conservation of this gene in other crops (such as corn and barley) further supports its potential application value in regulating plant salt-alkali tolerance. This provides a theoretical basis and practical basis for using the OsCLE48 gene to improve salt-alkali tolerance in other crops in the future.

Claims

1. OsCLE48 The application of the gene in improving the salt-alkali tolerance of plants is characterized in that: Knockout in plants OsCLE48 Genes to improve the salt and alkaline tolerance of plants; Said OsCLE48 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The plant is rice.

2. OsCLE48 The application of the gene in salt-alkali tolerant rice breeding is characterized in that: Knockout in rice OsCLE48 Genes to improve rice's salt-alkali tolerance; Said OsCLE48 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A method for improving the salt-alkali tolerance of rice, characterized in that: Knockout in rice OsCLE48 Gene, improves the salt-alkali tolerance of rice; OsCLE48 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. A salt-alkali tolerant rice breeding method, characterized in that: The invention comprises the method for improving the salt-alkali tolerance of rice as described in claim 3.

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