Application of OsHIPP36 gene in regulation and control of arsenic tolerance of rice

Abstract

The invention provides application of an OsHIPP36 gene in regulation and control of arsenic tolerance of rice, and belongs to the technical field of gene engineering. The invention provides application of an OsHIPP36 gene in regulation and control of arsenic tolerance of rice. The amino acid sequence of protein coded by the OsHIPP36 gene is shown as SEQ ID NO. 1. According to the invention, an OsHIPP36 gene mutant plant is obtained by constructing a transgenic vector. After As (III) is externally added for treatment, the tolerance of the mutant plant to As (III) is obviously improved, specifically, under As (III) treatment, the root length of the OsHIPP36 gene mutant plant is obviously longer than that of a Nipponbare wild type plant, and it is indicated that the OsHIPP36 plays an important role in the process of regulating and controlling the arsenic tolerance of rice.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to application of OsHIPP36 gene in regulating arsenic tolerance of 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. Large-scale arsenic contamination in the soil will pose a greater threat to the growth of rice. Flooded rice soil is an anaerobic environment, and arsenic is more easily reduced from pentavalent to trivalent. As(III) is more soluble in water, and its mobility and root adsorption capacity are also increased. As(III) is mainly absorbed by rice roots through the silicon absorption pathway. Therefore, as a silicon hyperaccumulator, rice is more likely to absorb As(III) in 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 usually accomplished with the help of metal proteins. Heavy metal-associated isoprenyl plant proteins (HIPPs) are a large family of metal proteins. Their specific structure can bind to metal ions and can serve as carriers for the movement of metal ions in cells. Therefore, HIPPs have also been shown to play a role in the accumulation and detoxification of heavy metal ions. At present, there are few reports on whether the rice HIPP gene affects the arsenic tolerance of 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 of rice, which has potential application value in improving arsenic tolerance of rice.

[0005] The present invention provides application of OsHIPP36 gene in regulating arsenic tolerance of 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, and 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 of 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 an OsHIPP36 gene mutant associated with arsenic tolerance in rice, wherein the OsHIPP36 gene mutant includes an OsHIPP36-1 mutant and / or an 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 of 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 with 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 an application of the OsHIPP36 gene in regulating arsenic tolerance of rice, and 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 of the OsHIPP36 gene mutation by CRISPR / Cas9 technology, and then obtains a homozygous rice strain of the OsHIPP36 gene mutation. After external As(III) treatment, it is found that the mutant plant has significantly improved tolerance to As(III), which is specifically manifested in that the root length of the OsHIPP36 gene mutant plant is significantly longer than that of the Nipponbare wild-type plant under As(III) treatment, 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, and the present invention also lays a theoretical and application foundation for cultivating arsenic-resistant rice varieties using the OsHIPP36 gene. 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 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 labor.

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

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

[0020] Figure 3 The phenotypic result diagram of the 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 embodiments of the present invention. DETAILED DESCRIPTION

[0022] The principles and features of the present invention are described below in conjunction with 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 those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0024] 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, 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 specific implementation process of the present invention, the CDS sequence of the OsHIPP36 gene is amplified using the total cDNA of Nipponbare as a template. The OsHIPP36 gene contains 4 exons and 3 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 2 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, 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 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; 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 of the present invention, the basic vector of the recombinant vector includes SK-gRNA and pC1300-Cas9. In the specific implementation of the present invention, the pC1300-Cas9 and SK-gRNA are both deposited in the Hangzhou Rice Research Institute, China, and are disclosed in the document [Upgrading the genome of an elitejaponica rice variety Kongyu131for 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 of 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 an OsHIPP36 gene mutant associated with arsenic tolerance in rice, wherein the OsHIPP36 gene mutant includes an OsHIPP36-1 mutant and / or an 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 of 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 whose nucleotide sequence is shown in SEQ ID NO.6 and a downstream primer 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 OsHIPP36 gene

[0046] In order to obtain the full-length CDS coding sequence of the OsHIPP36 gene, total RNA of the wild-type rice variety Nipponbare was extracted and reverse transcribed into cDNA. The CDS sequence of the OsHIPP36 gene was amplified using the total cDNA of Nipponbare as a template, and the CDS coding sequence of the OsHIPP36 gene was obtained after sequencing and analysis of the PCR product, as 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 OsHIPP53 gene

[0052] Reagents 50μL system 2xPCR buffer for KODFX 25μL 2mM dNTPs 10μL Upstream primer 1.5μL Downstream primer 1.5μL KODFX enzyme 1μL 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] The coding region of OsHIPP36 gene was analyzed and it was determined that OsHIPP36 gene contained 4 exons and 3 introns. The cDNA sequence of OsHIPP36 gene consisted of 1047 nucleotides and encoded 348 amino acids.

[0055] Phylogenetic analysis of the OsHIPP36 gene was performed using TB tools software. The results showed that the OsHIPP36 gene belonged to the HIPP family and had 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] According to the coding sequence of the OsHIPP36 gene and the target design principle of CRISPR / Cas9, the target sgRNA sequence of the mutant material was 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, place in a 95℃ metal bath for 3 minutes, and cool naturally at room temperature for 10 minutes to obtain oligo dimer for use; use AarI restriction endonuclease to digest SK-gRNA at 50℃ in a water bath for 1 hour; after digestion, perform gel recovery.

[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, and the digestion conditions were 50°C and water bath for 1 hour; the pC1300-Cas9 vector was digested with KpnI and BamHI restriction endonucleases, and the digestion conditions were 50°C and water bath for 1 hour; the vectors were connected using T4 enzyme, and the volume ratio was 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 monoclonal screening was performed using kanamycin-resistant culture medium to obtain positive clone bacterial solution; the recombinant plasmid was extracted from the obtained positive clone bacterial solution. Wuhan Boyuan Biotechnology Co., Ltd. was then commissioned to complete the subsequent transgenic work.

[0064] The obtained transgenic plants were identified, and DNA was extracted from the wild-type Nipponbare and the plant samples to be identified, respectively. PCR amplification was performed using the mutant identification primers described below, followed by first-generation sequencing. The obtained sequencing results were compared with the Nipponbare PCR amplification sequencing results 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 10μL OsHIPP53-F 1μL OsHIPP53-R 1μL Sample DNA 1μL <![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, extension at 72°C for 15 s, repeated for 32 cycles; and complete extension at 72°C for 5 min.

[0072] After analyzing the sequencing results of the transgenic plants, two homozygous mutant lines were obtained, named OsHIPP36-1 and OsHIPP36-3. The sequencing results showed that the OsHIPP36-1 mutant had a single base deletion, that is, the A base was deleted at the 205bp position of the nucleotide sequence encoding the OsHIPP36 gene; the OsHIPP36-3 mutant had a single base insertion, with a G base inserted between the 205bp and 206bp of the nucleotide sequence encoding the OsHIPP36 gene.

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

[0074] Example 3

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

[0076] In order to determine the regulatory role of the OsHIPP36 gene on arsenic tolerance in rice, wild-type Nipponbare, OsHIPP36-1 and OsHIPP36-3 were used as experimental materials, and As(Ⅲ) treatment was carried out at the same time to observe the phenotypic changes of each. The rice seedlings were cultured to 7 days old, and the seedlings with the same growth were selected and transferred to Kimura B rice nutrient solution without and containing 10μM NaAsO2 for further cultivation. After 7 days of cultivation, the phenotypic data of the rice plants were collected and measured, including plant height, root length and biomass.

[0077] Under the treatment condition without arsenic, the growth of OsHIPP36-1 and OsHIPP36-3 was basically the same as that of wild-type Nipponbare. 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, but 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 considered that the mutants OsHIPP36-1 and OsHIPP36-3 have higher tolerance to As(Ⅲ), and the OsHIPP36 gene has a regulatory effect on arsenic tolerance in rice.

[0078] In summary, the present invention mutates the OsHIPP36 gene through genetic engineering technology, and obtains the mutant strains OsHIPP36-1 and OsHIPP36-3 of the OsHIPP36 gene through screening, and experimentally verifies that under arsenic treatment conditions, the phenotypic data of the mutant strains are better than those of the wild-type Nipponbare, indicating that the mutant strains are more tolerant to arsenic. The present invention experimentally confirms for the first time that the OsHIPP36 gene makes rice sensitive to arsenic, and in view of the application of the OsHIPP36 gene under arsenic stress, it can be considered that the gene has potential application value for improving the resistance of rice to arsenic stress, and the present invention also lays a good theoretical and application foundation for cultivating arsenic-resistant rice varieties using the OsHIPP36 gene.

[0079] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. 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.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the CDS sequence of the OsHIPP36 gene is shown in SEQ ID NO.

2.

3. The use according to claim 1 or 2, characterized in that: The regulation of rice arsenic tolerance includes knocking out or knocking down the OsHIPP36 gene to improve rice arsenic tolerance.

4. A sgRNA targeting the OsHIPP36 gene, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID NO.

3.

5. The sgRNA according to claim 4, characterized in that The sgRNA includes sgRNA-S whose nucleotide sequence is shown in SEQ ID NO.4 and sgRNA-A whose nucleotide sequence is shown in SEQ ID NO.

5.

6. A recombinant vector for editing the OsHIPP36 gene, characterized in that: The recombinant vector comprises the sgRNA according to claim 4 or 5.

7. A method for improving arsenic tolerance in rice using the CRISPR / Cas9 system, characterized in that: The following steps are involved: The recombinant vector of claim 6 is introduced into the recipient rice to edit the OsHIPP36 gene.

8. A mutant of the OsHIPP36 gene associated with arsenic tolerance in rice, characterized in that: The OsHIPP36 gene mutants include OsHIPP36-1 mutant and / or OsHIPP36-3 mutant; The OsHIPP36-1 mutant has a base A deletion at the 205 bp 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 205 bp and 206 bp of the OsHIPP36 gene fragment as shown in the nucleotide sequence of SEQ ID NO.

2.

9. Use of the OsHIPP36 gene mutant according to claim 8 in regulating arsenic tolerance in rice.

10. A primer set for identifying the OsHIPP36 gene mutant according to claim 8, characterized in that: It includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.6 and a downstream primer as shown in SEQ ID NO.7.

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