Method for improving rice salt tolerance by oszfnf9 gene and application thereof

Abstract

This invention discloses a method and application for improving salt tolerance in rice using the OsZNF9 gene. The method includes reducing the expression of the OsZNF9 gene in rice or knocking out the OsZNF9 gene in rice. The nucleotide sequence of the OsZNF9 gene is shown in SEQ ID NO.3. This invention, through differential expression of the OsZNF9 gene in rice, found that knocking out OsZNF9 significantly improved the salt tolerance of rice, with a survival rate significantly higher than that of YueTai B (YB); overexpression of OsZNF9 significantly decreased the salt tolerance of rice, with a survival rate lower than that of YB. This indicates that OsZNF9 is closely related to rice salt tolerance, and reducing the expression level of OsZNF9 can improve rice salt tolerance. Therefore, the OsZNF9 gene provides a powerful means and tool for using molecular marker-assisted breeding and genetic engineering to cultivate new salt-tolerant rice varieties, and has great application potential.

Description

Technical Field

[0001] This invention relates to the field of crop genetic engineering technology, and particularly to... OsZNF9 Methods and applications of gene-based methods to enhance salt tolerance in rice. Background Technology

[0002] Rice is a major crop worldwide, widely cultivated and feeding more than half the world's population. However, with increasing population, severe environmental degradation, and frequent extreme weather events, food security remains a prominent issue. Therefore, increasing rice yield is crucial for maintaining global food security, especially in my country. my country is a country with over 500 million mu (approximately 33 million hectares) of saline-alkali land; improving the utilization of this land is essential for increasing rice yield. Therefore, identifying and cloning genes involved in salt tolerance regulatory networks, and discovering novel salt-tolerant genes through genetic mutation and genetic engineering methods, are crucial for cultivating new salt-tolerant rice varieties.

[0003] The ZNF (zinc finger) transcription factor family is widely distributed in plants, especially rice and Arabidopsis thaliana. Previous studies have shown that this gene family participates in seed development, flowering regulation, and biotic and abiotic stress, possessing a considerable number of biological functions. OsZNF9 This gene is a novel C3HC4 type zinc finger gene that we have identified. Studies have shown that this gene is involved in the regulation of salt tolerance in rice and can be widely used in the breeding and utilization of salt-tolerant rice varieties. Summary of the Invention

[0004] Based on the rice discovered in this invention OsZNF9 The relationship between genes and rice salt tolerance; the purpose of this invention is to provide a method for improving rice salt tolerance, and thereby providing... OsZNF9 The application of genes in breeding.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect of the present invention, a method for improving salt tolerance in rice is provided, the method comprising the steps of: reducing... OsZNF9 Gene expression in rice may... OsZNF9 Gene knockout in rice, or reduction OsZNF9 The activity of proteins in rice. Among them, the... OsZNF9 The nucleotide sequence of the gene is shown in SEQ ID NO.3. (Decrease) OsZNF9 Gene expression in rice may... OsZNF9 Gene knockout in rice is preferably achieved through RNAi technology or CRISPR / Cas9 technology.

[0007] Furthermore, the aforementioned OsZNF9 Gene knockout in rice involves the following steps: construction OsZNF9 Gene knockout vectors are used to transform the gene into rice tissues or cells.

[0008] Furthermore, the aforementioned OsZNF9 The basic vectors for gene knockout vectors include crop modification vectors, which include one of binary Agrobacterium vectors and vectors that can be used for crop micro-bombardment. The binary Agrobacterium vectors include pYLCRISPR / Cas9Pubi-B, pYLCRISPR / Cas9P35S-H, pYLCRISPR / Cas9P35S-N or other editing technology-related vectors, such as TALENs, ZFNs, etc.

[0009] Furthermore, the transformation method includes microinjection, genetic transformation of Agrobacterium street, or transformation via one of Ti plasmids, Ri plasmids, or viral vectors.

[0010] Furthermore, the aforementioned OsZNF9 Gene knockout in rice involves the following steps:

[0011] Construction of knockout vectors: OsZNF9 The CDS region sequence on the gene was used as the target to design a knockout site. The gene was edited using the CRISPR / Cas9 system, and a gRNA expression cassette was constructed using overlapping PCR. The expression cassette was then cloned into the pYLCRISPR / Cas9 vector using the Golden Gate ligation method, thus obtaining a gRNA containing the knockout site. OsZNF9 -KO knockout vector;

[0012] Knockout vector OsZNF9 -KO was transferred into EHA105 Agrobacterium, and positive Agrobacterium strains that could be used to infect rice tissues were obtained by screening using the knockout vector and the characteristics of Agrobacterium itself.

[0013] The positive Agrobacterium strain was used to infect rice callus tissue, which was then cultured in the dark on hygromycin selection medium to obtain positive transgenic callus tissue.

[0014] The positive callus tissue was differentiated, rooted, and transplanted to obtain T0 generation transgenic plants;

[0015] T1 generation rice plants with improved salt tolerance were obtained through conventional molecular marker detection and rice cultivation methods.

[0016] In a second aspect of the invention, a method for regulating salt tolerance in rice is provided. OsZNF9 Genes, the ones mentioned OsZNF9The gene has a nucleotide sequence as shown in SEQ ID NO.3.

[0017] In a third aspect of the invention, a OsZNF9 Protein, the OsZNF9 The amino acid sequence of the protein is shown in SEQ ID NO.4.

[0018] In a fourth aspect of the invention, a method comprising the above is provided. OsZNF9 Knockout vectors for gene knockout sites.

[0019] In a fifth aspect of the invention, a transformant or transgenic line comprising the knockout vector is provided.

[0020] In a sixth aspect of the invention, the aforementioned OsZNF9 Genes, as described OsZNF9 The application of the protein, the knockout vector or the transformant, and the transgenic line in improving the salt tolerance of rice or cultivating salt-tolerant rice varieties, wherein the application is achieved by reducing... OsZNF9 Gene expression in rice, OsZNF9 Gene knockout or reduction in rice OsZNF9 The activity of the protein in rice was realized. Furthermore, the application of the OsZNF9 homolog in monocotyledonous grasses in improving salt tolerance in these crops was demonstrated.

[0021] In a sixth aspect of the invention, it is provided that OsZNF9 Application of genes as molecular markers in rice breeding.

[0022] This invention has at least the following technical effects or advantages: This invention provides a method and application for improving the salt tolerance of rice, by reducing... OsZNF9 The expression level of this gene can be used to improve the salt tolerance of rice, indicating that this gene can be used as a marker to improve salt tolerance in the breeding of salt-tolerant rice crops. Therefore... OsZNF9 Genes provide powerful means and tools for using molecular marker-assisted breeding and genetic engineering to cultivate salt-tolerant rice varieties, and have enormous application potential. Attached Figure Description

[0023] Figure 1 yes OsZNF9 Gene structure diagram, containing a C3HC4 type RING finger domain;

[0024] Figure 2 yes OsZNF9 Model structures of genes in overexpressed rice materials;

[0025] Figure 3 yes OsZNF9 Overexpression of rice materials before and after salt treatment OsZNF9 Expression level identification;

[0026] Figure 4 yes OsZNF9 Base editing in knockout rice materials;

[0027] Figure 5 yes OsZNF9 The phenotypic performance of overexpressed and knockout rice materials under salt treatment conditions. Detailed Implementation

[0028] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0029] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0030] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0031] The rice in the following examples was cultivated according to normal management methods: First, fresh rice seeds need to be soaked and germinated. After the seeds show white sprouts, they can be sown in a prepared hydroponic box. When the seedlings reach the four-leaf-one-heart stage, they can be treated.

[0032] The technical solution of this application embodiment is to solve the above-mentioned technical problems, and the general idea is as follows:

[0033] In our previous research on the salt tolerance mechanism of rice, we identified a novel C3HC4 type zinc finge gene. OsZNF9 ( Figure 1 Studies have shown that this gene is involved in the regulation of salt tolerance in rice.

[0034] This invention utilizes differential expression in rice. OsZNF9 Gene discovery: Knockout in rice variety YB OsZNF9 Afterwards, the salt tolerance of rice significantly improved, and its survival rate was significantly higher than that of YB, indicating that... OsZNF9 Closely related to rice salt tolerance, reducing OsZNF9 The expression level of this substance can improve the salt tolerance of rice.

[0035] OsZNF9The functional domains of this gene are highly conserved in other important food crops (wheat, maize, etc.), indicating that this gene has a similar molecular mechanism in the regulation of salt tolerance in other crops. Therefore OsZNF9 Homologous genes found in other important food crops can also be applied to improve the salt tolerance of other food crops.

[0036] Therefore, according to a typical embodiment of the present invention, a method for participating in rice salt regulation is provided. OsZNF9 Genes, the ones mentioned OsZNF9 The gene has a nucleotide sequence as shown in SEQ ID NO.3.

[0037] The OsZNF9 Genes have the following characteristics:

[0038] (1) Its nucleotide sequence is the genomic base sequence shown in SEQ ID NO.1.

[0039] (2) Its nucleotide sequence is the transcribed sequence shown in SEQ ID NO.2;

[0040] (3) Its nucleotide sequence is the cDNA sequence shown in SEQ ID NO.3;

[0041] The nucleotide sequence shown in SEQ ID NO.1 consists of 3636 bases, including exons, introns and a 3'UTR.

[0042] The nucleotide sequence shown in SEQ ID NO.2 is a transcription sequence.

[0043] The nucleotide sequence shown in SEQ ID NO.3 is a cDNA coding sequence.

[0044] According to another typical embodiment of the present invention, a method is provided. OsZNF9 Proteins involved in regulating salt tolerance in rice, in order to... OsZNF9 Proteins are easy to study and utilize; tags as shown in Table 1 can be attached to the amino or carboxyl ends of the protein sequence. OsZNF9 The amino acid sequence of the protein is shown in SEQ ID NO.4.

[0045] Table 1 - Tags and their amino acid sequences

[0046]

[0047] OsZNF9 Genes that can regulate salt tolerance in rice are of great significance in the breeding of salt-tolerant rice varieties.

[0048] According to another typical embodiment of the present invention, a method for improving salt tolerance in rice is provided, the method comprising the following steps: reducing... OsZNF9 Gene expression in rice may... OsZNF9 Gene knockout in rice, or reduction OsZNF9 The activity of proteins in rice. Among them, the... OsZNF9 The nucleotide sequence of the gene is shown in SEQ ID NO.3. (Decrease) OsZNF9 Gene expression in rice may... OsZNF9 Gene knockout in rice can be achieved using RNAi technology or CRISPR / Cas9 technology.

[0049] In the above technical solution, the term "will" OsZNF9 Gene knockout in rice involves the following steps: construction OsZNF9 Gene knockout vectors are used to transform the gene into rice tissues or cells.

[0050] The OsZNF9 The basic vector for gene knockout vectors includes crop modification vectors, which include one of binary Agrobacterium vectors and vectors that can be used for crop micro-bombardment. The binary Agrobacterium vectors include pYLCRISPR / Cas9Pubi-B, pYLCRISPR / Cas9P35S-H, pYLCRISPR / Cas9P35S-N, or other editing technology-related vectors, such as TALENs, ZFNs, etc. The transformation methods include microinjection, genetic transformation via Agrobacterium streets, or through one of Ti plasmids, Ri plasmids, or viral vectors.

[0051] As one specific implementation method, the described method is as follows: OsZNF9 Gene knockout in rice involves the following steps:

[0052] On the CRISPR-GE website (http: / / skl.scau.edu.cn / ), enter the target design subroutine and design a knockout site using the CDS region sequence on the OsZNF9 gene as the target. Generally, select a target site with a GC content of 45%~70%, an off-target estimate of less than 0.6, and a suitable location. Edit the gene using the CRISPR / Cas9 system, construct a gRNA expression cassette using overlapping PCR, and clone the expression cassette into the pYLCRISPR / Cas9 vector using the Golden Gate ligation method to obtain the OsZNF9-KO knockout vector containing the knockout site.

[0053] carrier OsZNF9 -KO was transferred into EHA105 Agrobacterium, and positive Agrobacterium strains that could be used to infect rice tissues were obtained by screening using the knockout vector and the characteristics of Agrobacterium itself.

[0054] The positive Agrobacterium strain was used to infect rice callus tissue, which was then cultured in the dark on hygromycin selection medium to obtain positive transgenic callus tissue.

[0055] The positive callus tissue was differentiated, rooted, and transplanted to obtain T0 generation transgenic plants;

[0056] T1 generation rice plants with improved salt tolerance were obtained through conventional molecular marker detection and rice cultivation methods.

[0057] The aforementioned OsZNF9 Genes, as described OsZNF9 The protein, the knockout vector, the transformant, and the transgenic line can all be used to improve the salt tolerance of rice or to cultivate salt-tolerant rice varieties. These applications work by reducing... OsZNF9 Gene expression in rice, OsZNF9 Gene knockout or reduction in rice OsZNF9 The activity of proteins is realized in rice.

[0058] The following will provide a detailed description of a method for improving salt tolerance in rice, along with its application, using examples and experimental data.

[0059] Example 1

[0060] 1. OsZNF9 Obtaining the full-length gene fragment

[0061] Primer pairs were designed using rice Yuetai B (YB) cDNA as a template. OsZNF9- F / R and corresponding recombinant sequences were added to the 5' end of each primer. The primer sequences are shown in Table 2. PCR amplification was performed, and the products were sequenced for analysis. The nucleotide sequence of the amplified gene fragment is shown in SEQ ID NO.3. In other embodiments, the gene fragment with the nucleotide sequence shown in SEQ ID NO.3 can be directly synthesized.

[0062] Table 2 - Primer Sequences

[0063]

[0064] 2. OsZNF9 Construction of gene overexpression vectors

[0065] The product obtained by amplification using primer pair OsZNF9-F / R was inserted into the expression vector pCAMBIA1301-35SN (available from Miaoling Plasmid Platform, catalog number P0380) containing a strong promoter via a recombination reaction. Positive clones were screened using the marker gene on the vector to obtain the recombinant expression vector. OsZNF9- OE.

[0066] 3. OsZNF9Obtaining transgenic plants with overexpressed genes

[0067] well-built OsZNF9- OE vectors can be transferred into Agrobacterium tumefaciens EHA105 via electroporation or heat shock. Agrobacterium tumefaciens In this study, positive Agrobacterium strains that can be used to infect rice tissues were screened using vectors and the characteristics of Agrobacterium itself.

[0068] Use containing recombinant plasmids OsZNF9- Recombinant Agrobacterium tumefaciens strain OE infected rice YB callus tissue and cultured in the dark on selection medium containing 50 mg / L hygromycin to obtain positive transgenic callus tissue. The positive callus tissue was differentiated, rooted, and transplanted to obtain T0 generation transgenic plants. T1 generation plants were obtained through conventional molecular marker detection and rice cultivation methods. OsZNF9 Model structures of genes in overexpressed rice materials, such as Figure 2 As shown.

[0069] 4. OsZNF9 Yield detection of gene overexpression plants

[0070] (1) Detection by qRT-PCR OsZNF9 Gene expression levels:

[0071] The 35S promoter, as a strong promoter in plants, can enhance the expression level of target genes in plants. It is obtained using conventional RNA extraction methods. OsZNF9 Total RNA from overexpressing plants and wild-type plants was collected, and the corresponding cDNA was obtained using a reverse transcription kit (Invitrogen). Primers were used... OsZNF9- qRT-PCR detection using RT-F / R primer pairs OsZNF9 The expression level was determined; the PCR product of primer Actin-RT-F / R was used as an internal control; the primer sequences are shown in Table 3.

[0072] Table 3 - Primer Sequences

[0073]

[0074] OsZNF9 Overexpression materials OsZNF9 The expression level identification results are as follows Figure 3 As shown. The results show that in the overexpressed materials OsZNF9 The expression level was significantly increased, especially after salt treatment.

[0075] (2) Salt tolerance test of transgenic plants:

[0076] Overexpression materials and YB seeds were soaked and germinated using standard methods. They were then transplanted into hydroponic boxes and placed in a 28°C incubator. When the seedlings reached the four-leaf stage (one bud), they were cultured in Yoshida nutrient solution containing 200 mM NaCl for 3 days, followed by rehydration in normal nutrient solution for 5-10 days. The survival rate of the materials was then statistically analyzed.

[0077] OsZNF9 The phenotypic performance of overexpression materials under salt treatment conditions is as follows: Figure 5 As shown. Overexpression in rice OsZNF9 The salt tolerance of rice decreased significantly, and its survival rate was lower than that of YB, indicating that... OsZNF9 Negative regulation of salt tolerance in rice.

[0078] Example 2

[0079] 1. OsZNF9 Selection of gene knockout sites

[0080] by OsZNF9 The CDS region sequence on the gene was used as a target to design a knockout site. The gene was then edited using the CRISPR / Cas9 system to achieve the desired alteration. OsZNF9 The purpose is to influence biological function through structure. Primer sequences involved are shown in Table 4.

[0081] Table 4. Primer sequences

[0082]

[0083] 2. OsZNF9 Construction of gene knockout vector

[0084] gRNA expression cassettes were constructed using overlapping PCR with primers U6aT#, gRT#+U6a, UF, gR-R, Pgs-L, and Pps-R. These cassettes were then cloned into the pYLCRISPR / Cas9 vector using the Golden Gate ligation method. This yielded a pYLCRISPR / Cas9 vector containing a knockout site. OsZNF9 Knockout vectors are used for gene transformation.

[0085] 3. OsZNF9 Obtaining gene knockout materials

[0086] Successfully built OsZNF9 The knockout vector was transferred into EHA105 Agrobacterium via electroporation or heat shock. Agrobacterium tumefaciens In the strain, rifampicin and kanamycin were used to screen for positive Agrobacterium strains containing the knockout vector.

[0087] YB callus was infected with a positive Agrobacterium strain containing a knockout vector and cultured in the dark on a selection medium containing 50 mg / L hygromycin to obtain positive transgenic callus. The positive callus was differentiated, rooted, and transplanted to obtain T0 generation plants. T1 generation plants were obtained through routine molecular detection and rice cultivation methods.

[0088] 4. OsZNF9 Detection of gene knockout materials

[0089] (1) Detection by PCR sequencing OsZNF9 Editing status:

[0090] Products containing knockout sites in the rice genome were amplified using CRISPR-F / R primers, and sequencing was used to determine the editing status of the knockout sites. Since the knockout sites are located in the coding region of the gene, the CRISPR / Cas9 system can efficiently alter the gene's function by editing these sites. OsZNF9 Base editing in knockout rice materials is as follows: Figure 4 As shown.

[0091] (2) Salt resistance test of the knockout material:

[0092] The knockout materials and YB seeds were soaked and germinated using standard methods. They were then transplanted into hydroponic boxes and placed in a 28°C incubator. When the seedlings reached the four-leaf stage (one bud), they were cultured in Yoshida nutrient solution containing 200 mM NaCl for 3 days, followed by rehydration in normal Yoshida nutrient solution for 5-10 days. The survival rate of the materials was then statistically analyzed.

[0093] OsZNF9 The phenotypic behavior of the knockout material under salt treatment conditions, such as Figure 5 As shown. Knockout in rice YB cells. OsZNF9 Afterwards, the salt tolerance of rice was significantly improved, and its survival rate was significantly higher than that of YB, indicating that knocking out or reducing salt tolerance was the key factor. OsZNF9 The expression level significantly improved the salt tolerance of rice.

[0094] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0095] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0096] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for improving salt tolerance in rice, characterized in that, The method includes: reducing OsZNF9 Gene expression in rice may... OsZNF9 Gene knockout in rice; wherein, the aforementioned OsZNF9 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. The method for improving salt tolerance in rice according to claim 1, characterized in that, The OsZNF9 Gene knockout in rice includes: constructing OsZNF9 Gene knockout vectors are used to transform the gene into rice tissues or cells.

3. The method for improving salt tolerance in rice according to claim 2, characterized in that, The OsZNF9 The basic vectors for gene knockout vectors include crop modification vectors, and the transformation methods include microinjection or Agrobacterium-mediated genetic transformation.

4. The method for improving salt tolerance in rice according to claim 3, characterized in that, The crop modification vector includes one of a binary Agrobacterium vector and a vector for crop micro-bombardment.

5. The method for improving salt tolerance in rice according to claim 4, characterized in that, The binary Agrobacterium vector includes pYLCRISPR / Cas9Pubi-B, pYLCRISPR / Cas9P35S-H, or pYLCRISPR / Cas9P35S-N.

6. A kind OsZNF9 The application of genes in improving rice salt tolerance or breeding salt-tolerant rice varieties is characterized by: reduce OsZNF9 Gene expression in rice may... OsZNF9 Gene knockout in rice; wherein, the aforementioned OsZNF9 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

Images