Application of OsHIPP36 gene in regulating arsenic tolerance in rice

Abstract

The present invention provides an application of the OsHIPP36 gene in regulating arsenic tolerance in rice, belonging to the field of genetic engineering technology. The present invention provides an application of the OsHIPP36 gene in regulating arsenic tolerance in rice, wherein the amino acid sequence of the protein encoded by the OsHIPP36 gene is shown in SEQ ID NO.1. The present invention obtains OsHIPP36 gene mutant plants by constructing a transgenic vector. After external addition of As(III) treatment, it was found that the mutant plants had significantly improved tolerance to As(III), specifically, the root length of the OsHIPP36 gene mutant plants under As(III) treatment was significantly longer than that of the Nipponbare wild-type plants, indicating that OsHIPP36 plays an important role in regulating arsenic tolerance in rice.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to application of the OsHIPP36 gene in regulating arsenic tolerance in rice. Background Art

[0002] Arsenic is a toxic metal that is ubiquitous in the environment. Its main sources include mining and smelting, fossil fuel combustion, and agricultural activities. Widespread arsenic contamination in soil poses a greater threat to rice growth. Flooded rice soil is an anaerobic environment, making it easier for arsenic to be reduced from pentavalent to trivalent, making As(III) more soluble in water, and its mobility and root adsorption capacity also increase. As(III) is mainly absorbed by rice roots through the silicon uptake pathway. Therefore, as a silicon hyperaccumulator, rice is more susceptible to absorbing As(III) from the environment. In addition, As(V) can also be taken up by rice through the phosphorus transport pathway.

[0003] In organisms, the movement of metal ions is often accomplished through the help of metalloproteins. Heavy metal-associated isoprenylated plant proteins (HIPPs) are a large family of metalloproteins. Their specialized structures allow them to bind to metal ions and act as carriers for their movement within cells. Consequently, HIPPs have been shown to play a role in the accumulation and detoxification of heavy metal ions. Currently, there are few reports on whether rice HIPP genes affect arsenic tolerance in rice. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of the OsHIPP36 gene in regulating arsenic tolerance in rice, which has potential application value in improving arsenic tolerance in rice.

[0005] The present invention provides an application of the OsHIPP36 gene in regulating arsenic tolerance in rice. The amino acid sequence of the protein encoded by the OsHIPP36 gene is shown in SEQ ID NO.1.

[0006] Preferably, the nucleotide sequence of the CDS sequence of the OsHIPP36 gene is shown as SEQ ID NO.2.

[0007] Preferably, the regulating arsenic tolerance of rice comprises knocking out or knocking down the OsHIPP36 gene to improve the arsenic tolerance of rice.

[0008] The present invention also provides an sgRNA targeting the OsHIPP36 gene, the nucleotide sequence of the sgRNA is shown in SEQ ID NO.3.

[0009] Preferably, the sgRNA includes sgRNA-S having a nucleotide sequence as shown in SEQ ID NO.4 and sgRNA-A having a nucleotide sequence as shown in SEQ ID NO.5.

[0010] The present invention also provides a recombinant vector for editing the OsHIPP36 gene, wherein the recombinant vector comprises the sgRNA described in the above scheme.

[0011] The present invention also provides a method for improving arsenic tolerance in rice using the CRISPR / Cas9 system, comprising the following steps:

[0012] The recombinant vector described in the above scheme was transferred into the recipient rice to edit the OsHIPP36 gene.

[0013] The present invention also provides OsHIPP36 gene mutants associated with arsenic tolerance in rice, including OsHIPP36-1 mutant and / or OsHIPP36-3 mutant; the OsHIPP36-1 mutant has an A base deletion at the 205bp position of the OsHIPP36 gene fragment as shown in the nucleotide sequence of SEQ ID NO.2; the OsHIPP36-3 mutant has a G base inserted between 205bp and 206bp of the OsHIPP36 gene fragment as shown in the nucleotide sequence of SEQ ID NO.2.

[0014] The present invention also provides the use of the OsHIPP36 gene mutant described in the above scheme in regulating arsenic tolerance in rice.

[0015] The present invention also provides a primer set for identifying the OsHIPP36 gene mutant described in the above scheme, comprising an upstream primer having a nucleotide sequence as shown in SEQ ID NO.6 and a downstream primer as shown in SEQ ID NO.7.

[0016] The present invention provides the use of the OsHIPP36 gene in regulating arsenic tolerance in rice. The amino acid sequence of the protein encoded by the OsHIPP36 gene is shown in SEQ ID NO. 1. The present invention constructs a recombinant expression vector for a mutant OsHIPP36 gene using CRISPR / Cas9 technology, thereby obtaining a homozygous rice line for the OsHIPP36 gene mutation. After external treatment with As(III), it is found that the mutant plants have significantly improved As(III) tolerance. Specifically, under As(III) treatment, the root length of the OsHIPP36 gene mutant plants is significantly longer than that of the Nipponbare wild-type plants, indicating that the deletion of the OsHIPP36 gene significantly improves the tolerance of rice to arsenic, and OsHIPP36 plays an important role in regulating the arsenic tolerance of rice. Therefore, the OsHIPP36 gene has potential application value in improving the arsenic tolerance of rice. At the same time, the present invention also lays a theoretical and application foundation for the use of the OsHIPP36 gene to cultivate arsenic-resistant rice varieties. The present invention obtains OsHIPP36 gene mutant plants by constructing a transgenic vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0018] Figure 1 This is the identification result of mutant plants of OsHIPP36 gene obtained by CRISPR / Cas9 technology;

[0019] Figure 2 This is the phylogenetic tree analysis result of OsHIPP36 gene;

[0020] Figure 3 This is a diagram showing the phenotypic results of wild-type Nipponbare, hipp36-1, and hipp36-3 under arsenic treatment provided in the embodiments of the present invention;

[0021] Figure 4 This is a diagram showing the root growth results of wild-type Nipponbare, hipp36-1, and hipp36-3 under arsenic treatment provided in the examples of the present invention. DETAILED DESCRIPTION

[0022] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Reagents not specifically described in detail herein are conventional reagents and are commercially available; methods not specifically described in detail are conventional experimental methods and are known in the art.

[0024] The present invention provides the use of the OsHIPP36 gene in regulating arsenic tolerance in rice. The amino acid sequence of the protein encoded by the OsHIPP36 gene is shown in SEQ ID NO. 1, specifically:

[0025] MGDEKAAPKAGATADPVVLRMELHCAGCAQKVKKSIKHLAGVESVAADVATNTVVVAGTAEAAALKARIEAKTKKPVEVVSAGGGGAAAKKPAAEPKAVKDDGGEKKDAQAKEEKGKKQPPEEKKPKEETVLLRIRLHCDGCADRIRRRIYKIKGVKEVVMDGNAKDEVKVSGT MDVPAMLTYLTEKLNRAVEAVAPGSKKDEKKKDKGGDADGGEKKKDAAGGDKKDKGKSIEVAGPSTAAAAASMAPAPAEASTYHVSPYGHGYFAYPQQQGPPPSYYQYYGGGNGDGVGYANPNAGGAGGYYHPHPNDVPTYQPPPSYPPYPYQLDMSPAPQLFSDENPNACSVM.

[0026] In the specific implementation of the present invention, the nucleotide sequence of the CDS sequence of the OsHIPP36 gene is shown in SEQ ID NO. 2, specifically:

[0027]

[0028] In the present invention, the CDS sequence of the OsHIPP36 gene was amplified using the total cDNA of Nipponbare as a template. The OsHIPP36 gene contains four exons and three introns. The cDNA sequence of the OsHIPP36 gene consists of 1047 nucleotides and encodes a total of 348 amino acids. The OsHIPP36 gene belongs to the HIPP family and has two HMA domains.

[0029] In a specific implementation of the present invention, the regulation of rice arsenic tolerance includes knocking out or knocking down the OsHIPP36 gene to improve rice arsenic tolerance.

[0030] The present invention also provides an sgRNA targeting the OsHIPP36 gene, wherein the nucleotide sequence of the sgRNA is shown in SEQ ID NO. 3, specifically: 5'-CGCGCTCAAGGCGAGGATCG-3'.

[0031] In the specific implementation of the present invention, the sgRNA includes sgRNA-S as shown in SEQ ID NO.4 and sgRNA-A as shown in SEQ ID NO.5; the nucleotide sequence of sgRNA-S shown in SEQ ID NO.4 is specifically: 5'-ggcaCGCGCTCAAGGCGAGGATCG-3'; the nucleotide sequence of sgRNA-A shown in SEQ ID NO.5 is specifically: 5'-aaacCGATCCTCGCCTTGAGCGCG-3'.

[0032] In the specific implementation process of the present invention, the method of using the sgRNA-S and sgRNA-A includes: mixing sgRNA-S, sgRNA-A and ddH2O, reacting, cooling, and obtaining an oligo dimer; the volume ratio of the sgRNA-S, sgRNA-A and ddH2O is 5:5:15; the reaction procedure is: 95°C metal bath for 3 minutes; the cooling procedure is: natural cooling at room temperature for 10 minutes.

[0033] The present invention also provides a recombinant vector for editing the OsHIPP36 gene, wherein the recombinant vector comprises the sgRNA described in the above scheme.

[0034] In the specific implementation process of the present invention, the base vector of the recombinant vector includes SK-gRNA and pC1300-Cas9. In the specific implementation process of the present invention, the pC1300-Cas9 and SK-gRNA are deposited in the Hangzhou Rice Research Institute, China, and are disclosed in the document "Upgrading the genome of an elitejaponica rice variety Kongyu131 for lodging resistance improvement". The original link is: https: / / onlinelibrary.wiley.com / doi / 10.1111 / pbi.1396.

[0035] In a specific implementation of the present invention, the editing includes knockout.

[0036] The present invention has no particular limitation on the method for constructing the recombinant vector, and conventional methods in the art may be used.

[0037] The present invention also provides a method for improving arsenic tolerance in rice using the CRISPR / Cas9 system, comprising the following steps:

[0038] The recombinant vector described in the above scheme was transferred into the recipient rice to edit the OsHIPP36 gene.

[0039] The present invention also provides OsHIPP36 gene mutants associated with arsenic tolerance in rice, including OsHIPP36-1 mutant and / or OsHIPP36-3 mutant; the OsHIPP36-1 mutant has an A base deletion at the 205bp position of the OsHIPP36 gene fragment as shown in the nucleotide sequence of SEQ ID NO.2; the OsHIPP36-3 mutant has a G base inserted between 205bp and 206bp of the OsHIPP36 gene fragment as shown in the nucleotide sequence of SEQ ID NO.2.

[0040] The present invention also provides the use of the OsHIPP36 gene mutant described in the above scheme in regulating arsenic tolerance in rice.

[0041] In the specific implementation of the present invention, the use of the OsHIPP36 gene mutant in regulating rice arsenic tolerance includes the use of overexpressing the OsHIPP36 gene mutant in increasing rice arsenic sensitivity.

[0042] The present invention also provides a primer set for identifying the OsHIPP36 gene mutant described in the above scheme, comprising an upstream primer having a nucleotide sequence as shown in SEQ ID NO.6 and a downstream primer as shown in SEQ ID NO.7; the nucleotide sequence of the upstream primer shown in SEQ ID NO.6 is specifically: CTGCCGCCTCTCGTTTCTTTCT; the nucleotide sequence of the downstream primer shown in SEQ ID NO.7 is specifically: GCGTTGGTAGGGTTGCCTCTTG.

[0043] To further illustrate the present invention, the application of the OsHIPP36 gene provided by the present invention in regulating arsenic tolerance in rice is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Sequence analysis of the OsHIPP36 gene

[0046] To obtain the full-length CDS coding sequence of the OsHIPP36 gene, total RNA from the wild-type rice variety Nipponbare was extracted and reverse-transcribed into cDNA. Using the Nipponbare total cDNA as a template, the CDS coding sequence of the OsHIPP36 gene was amplified. The PCR product was sequenced and analyzed, yielding the CDS coding sequence shown in SEQ ID NO. 2.

[0047] The amplification primer sequences are as follows:

[0048] Upstream primer: 5'-ATGGGGGATGAGAAGGCTGC-3' (SEQ ID NO. 8)

[0049] Downstream primer: 5'-TCACATCACCGAGCAGGCGT-3' (SEQ ID NO. 9)

[0050] The PCR amplification system is shown in Table 1.

[0051] Table 1 PCR amplification system for amplifying the CDS sequence of the OsHIPP53 gene

[0052] Reagents 50μL system 2xPCR buffer for KODFX 25 μL 2mM dNTPs 10 μL Upstream primer 1.5 μL Downstream primer 1.5 μL KODFX enzyme 1 μL Template cDNA 2μL <![CDATA[ddH2O]]> 9μL

[0053] The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 68°C for 40 s, with 29 cycles; and complete extension at 68°C for 10 min.

[0054] Analysis of the coding region of the OsHIPP36 gene revealed that it contains four exons and three introns. The cDNA sequence of the OsHIPP36 gene consists of 1047 nucleotides encoding a total of 348 amino acids.

[0055] Phylogenetic analysis of the OsHIPP36 gene was performed using TB tools software. The results showed that the OsHIPP36 gene belongs to the HIPP family and has two HMA domains ( Figure 2 ).

[0056] Example 2

[0057] Obtaining OsHIPP36 gene mutant strains OsHIPP36-1 and OsHIPP36-3 and identifying homozygous mutant strains.

[0058] Based on the coding sequence of the OsHIPP36 gene and the target design principles of CRISPR / Cas9, the target sgRNA sequences for the mutant materials were designed and constructed as follows:

[0059] Target sgRNA: 5'-CGCGCTCAAGGCGAGGATCG-3' (SEQ ID NO.3)

[0060] After mixing at a volume ratio of sgRNA-S:sgRNA-A:ddH2O=5:5:15, the mixture was placed in a 95°C metal bath for 3 minutes and cooled naturally at room temperature for 10 minutes to obtain an oligo dimer for use; the SK-gRNA was digested with AarI restriction endonuclease at 50°C in a water bath for 1 hour; after the digestion was completed, the gel was recovered.

[0061] The oligo dimer and SK-gRNA digestion product obtained above were enzymatically linked using T4 enzyme, wherein the volume ratio of SK-gRNA digestion product, oligo dimer, T4 enzyme and T4 Buffer was 1:7:1:1. After mixing, the mixture was allowed to stand at room temperature for 4 hours. DH5α competent cells were transformed and positive monoclonal screening was performed using ampicillin-resistant culture medium. Positive monoclonal clones were detected by colony PCR, and the successfully constructed intermediate recombinant vector was obtained after sequencing.

[0062] The intermediate recombinant vector obtained above was digested with KpnI and BglII restriction endonucleases at 50°C in a water bath for 1 hour; the pC1300-Cas9 vector was digested with KpnI and BamHI restriction endonucleases at 50°C in a water bath for 1 hour; the vectors were connected using T4 enzyme at a volume ratio of SK-gRNA intermediate recombinant vector digestion product: pC1300-Cas9 vector digestion product: T4 enzyme: T4 Buffer = 4:4:1:1. After mixing, the mixture was allowed to stand at room temperature for 4 hours to obtain the recombinant vector.

[0063] The constructed recombinant vector was transformed into DH5α competent cells, and positive single clones were screened using kanamycin-resistant culture medium to obtain a positive clone solution. The recombinant plasmid was then extracted from the resulting positive clone solution. Subsequent transgenic work was then commissioned to Wuhan Boyuan Biotechnology Co., Ltd.

[0064] The resulting transgenic plants were identified. DNA was extracted from wild-type Nipponbare and the plant samples to be identified. PCR amplification was performed using the mutant identification primers described below, followed by first-generation sequencing. The resulting sequencing results were compared with the PCR amplification sequencing results of Nipponbare to determine whether the OsHIPP36 gene was successfully mutated.

[0065] The identification primer sequences are as follows:

[0066] OsHIPP36-F:CTGCCGCCTCTCGTTTCTTTCT(SEQ ID NO.6)

[0067] OsHIPP36-R: GCGTTGGTAGGGTTGCCTCTTG (SEQ ID NO.7)

[0068] The PCR amplification system is shown in Table 2.

[0069] Table 2 Identification primer PCR amplification system

[0070] Reagents 20μL system 2×RapidTaqMasterMix 10 μL OsHIPP53-F 1 μL OsHIPP53-R 1 μL Sample DNA 1 μL <![CDATA[ddH2O]]> 7μL

[0071] The PCR amplification reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s, repeated for 32 cycles; and complete extension at 72°C for 5 min.

[0072] Sequencing analysis of the transgenic plants yielded two homozygous mutant lines, designated OsHIPP36-1 and OsHIPP36-3. Sequencing results revealed that the OsHIPP36-1 mutant harbored a single-base deletion, an A at position 205 of the OsHIPP36 gene coding sequence; while the OsHIPP36-3 mutant harbored a single-base insertion, a G between 205 and 206 bp of the OsHIPP36 gene coding sequence.

[0073] The results of identification of mutation sites in OsHIPP36 gene transgenic plants are as follows Figure 1 shown.

[0074] Example 3

[0075] Verification of the tolerance of OsHIPP36 gene mutants to As(Ⅲ) treatment

[0076] To determine the regulatory role of the OsHIPP36 gene in arsenic tolerance in rice, wild-type Nipponbare, OsHIPP36-1, and OsHIPP36-3 were simultaneously treated with As(III) and their phenotypic changes were observed. Seven-day-old rice seedlings were cultured, and seedlings with consistent growth were selected and transferred to Kimura B rice nutrient solution containing or without 10 μM NaAsO2 for further culture. After seven days of culture, phenotypic data, including plant height, root length, and biomass, were collected and measured.

[0077] Under the treatment condition without arsenic, the growth of OsHIPP36-1, OsHIPP36-3 and wild-type Nipponbare was basically the same; under the treatment condition of 10μMAs(Ⅲ), the growth of OsHIPP36-1 and OsHIPP36-3 was significantly better than that of Nipponbare. Figure 3 The plant height and root length of wild-type Nipponbare and mutant plants were measured under normal conditions and arsenic treatment conditions. Under normal conditions, there was no difference in plant height and root length between the mutant and Nipponbare. However, under arsenic treatment, the root length of the mutant plant was significantly longer than that of Nipponbare. Figure 4 Based on the above results, it can be concluded that the mutants OsHIPP36-1 and OsHIPP36-3 have higher tolerance to As(Ⅲ), and the OsHIPP36 gene plays a regulatory role in arsenic tolerance in rice.

[0078] In summary, the present invention mutated the OsHIPP36 gene through genetic engineering techniques, resulting in screening of the OsHIPP36 mutant lines, OsHIPP36-1 and OsHIPP36-3. Experimental verification showed that the mutant lines exhibited superior phenotypic data compared to the wild-type Nipponbare under arsenic treatment, indicating that the mutant lines were more tolerant to arsenic. This experimental confirmation is the first to demonstrate that the OsHIPP36 gene sensitizes rice to arsenic. Given the application of the OsHIPP36 gene under arsenic stress, it is believed that this gene has potential application value in improving rice resistance to arsenic stress. This invention also lays a solid theoretical and practical foundation for the use of the OsHIPP36 gene in breeding arsenic-tolerant rice varieties.

[0079] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for improving arsenic tolerance in rice using the CRISPR / Cas9 system, characterized in that: The following steps are involved: The recombinant vector was transferred into the recipient rice. OsHIPP36 Gene editing; described OsHIPP36 The nucleotide sequence of the CDS sequence of the gene is shown in SEQ ID NO. 2; The editing is as shown in SEQ ID NO.2 OsHIPP36 The A base is deleted at the 205 bp position of the gene fragment; or, the nucleotide sequence is as shown in SEQ ID NO.2 OsHIPP36 A G base was inserted between 205 bp and 206 bp of the gene fragment; The recombinant vector includes sgRNA; The sgRNA includes sgRNA-S with a nucleotide sequence as shown in SEQ ID NO.4 and sgRNA-A with a nucleotide sequence as shown in SEQ ID NO.

5.

2. Use of the method according to claim 1 in improving arsenic tolerance in rice.

Images