Application of HSP70-5 and coding gene thereof in regulation and control of saline-alkaline tolerance of plants
By regulating the expression and activity of the HSP70-5 protein in plants, the problem of regulating plant salt-alkali stress was solved, and plants with different salt-alkali tolerances were cultivated, thus achieving adaptive regulation of saline-alkali environments.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHINA AGRI UNIV
- Filing Date
- 2024-10-22
- Publication Date
- 2026-04-24
AI Technical Summary
Existing technologies are insufficient to effectively regulate plant responses to salt and alkali stress, leading to salinized soils that restrict crop growth and impair cell function.
By introducing or regulating the expression and activity of HSP70-5 protein, recombinant vectors and nucleic acid molecules can be used to overexpress or downregulate HSP70-5 protein in plants, thereby enhancing or weakening its expression level and activity in plants, and thus regulating the salt and alkali tolerance of plants.
It has enabled the regulation of plant salt and alkali tolerance, cultivating plants with high or low salt and alkali tolerance, and improving or reducing their tolerance to salt and alkali, which is manifested by salt-sensitive phenotype or changes in leaf color and plant height.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to the application of HSP70-5 and its encoding gene in regulating plant salt and alkali tolerance. Background Technology
[0002] Soil salinization is a growing trend worldwide, severely limiting crop growth and becoming a major factor restricting agricultural production. The harm caused by salt-alkali stress to plants lies in the high concentration of sodium (Na) in saline-alkali soils. + Excessively high pH levels can lead to the accumulation of salt ions within plant cells, causing ion toxicity and affecting normal cell function in multiple ways. In alkaline soil environments, most metal elements, except for alkali metals, will form insoluble salts, such as Na. + As the most abundant alkali metal element in nature, it will accumulate in alkaline soil, causing soil salinization.
[0003] With the rapid development of molecular biology, genomics, genetics, biochemistry and gene editing technology, the study of the molecular mechanisms of plant resistance to salt and alkali stress has been continuously deepened, and many new genes or proteins have been involved in regulating the salt and alkali stress response process. Summary of the Invention
[0004] The technical problem solved by this invention is how to regulate the salt and alkali tolerance of plants.
[0005] To address the aforementioned problems, the present invention provides the application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins.
[0006] The use of the protein or gene expression regulator or substance regulating the activity or content of said protein provided by this invention in any of the following: 1) Application in regulating plant salt and alkali tolerance; 2) Application in the preparation of products that regulate plant salt and alkali tolerance; 3) Application in cultivating plants with altered salt and alkali tolerance; 4) Application in the preparation of products that cultivate plants with altered salt and alkali tolerance; 5) Applications in plant breeding; The protein is any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No. 2; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No. 2, by substitution and / or deletion and / or addition of one or more amino acid residues; Proteins that have more than 80% identity and the same function of any of the amino acid sequences defined in (a3), (a1), and (a2); The fusion protein is obtained by attaching a tag to the end of any of the proteins defined in (a4), (a1), ..., (a3). In the above-mentioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.
[0007] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.
[0008] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.
[0009] In the above applications, the protein is derived from corn ( Zea mays L. ).
[0010] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein HSP70-5.
[0011] In this invention, the regulation can be increased, enhanced, or improved; the regulation can also be decreased, weakened, or reduced.
[0012] In this article, the enhancement, increase, or upregulation of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase, or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.
[0013] In this article, regulating the expression of the gene encoding the protein may mean enhancing, increasing, or upregulating the expression of the gene encoding the protein.
[0014] In the above applications, the substance regulating gene expression or the substance regulating protein activity or content can be a biological material related to the protein described above, and the biological material can be any of the following: c1) The nucleic acid molecule that encodes the protein described above; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2).
[0015] In the above applications, c1) the nucleic acid molecule can be any of the following DNA molecules: d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3; d2) The coding region sequence is the DNA molecule shown in SEQ ID No. 1 of the sequence listing; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above; d4) A DNA molecule that hybridizes under strict conditions with a nucleotide sequence defined by d1) or d2) and encodes the protein described above.
[0016] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.
[0017] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specific vectors: PCBC-MT1T2, pBUE411.
[0018] Existing plant expression vectors can be used to construct structures containing... HSP70-5 Recombinant gene expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).
[0019] The present invention also provides a method for altering the salt tolerance of plants, wherein the method involves enhancing, increasing or upregulating the activity and / or content of the proteins described above in the target plant, or / and enhancing, increasing or upregulating the expression level of the encoding genes of the proteins described above, in order to reduce the salt tolerance of plants.
[0020] The present invention also provides a method for cultivating plants with low salt tolerance, comprising enhancing, increasing or upregulating the expression level of the gene encoding the protein described above in the target plant, and / or, the activity and / or content of the protein to obtain a plant with low salt tolerance, wherein the plant with low salt tolerance has a weaker salt tolerance than the target plant.
[0021] In the above method, enhancing, improving or upregulating the expression of the gene encoding the protein described above in the plant includes introducing the nucleic acid molecule described above (c1), the expression cassette described above (c2), or the recombinant vector described above (c3) into the target plant to obtain a plant with low salt and alkali tolerance.
[0022] In one specific embodiment, enhancing, improving, or upregulating the expression of the gene encoding the protein described above in the plant includes introducing the nucleic acid molecule, expression cassette, or recombinant vector described above into the target plant to obtain a plant with low salt and alkali tolerance.
[0023] The present invention also provides a method for cultivating highly salt-tolerant plants, comprising downregulating or weakening or reducing the expression level of the encoding gene of the protein described above in the target plant, and / or, the activity and / or content of the protein to obtain a highly salt-tolerant plant, wherein the salt-tolerant plant has a stronger salt-tolerant ability than the target plant.
[0024] In this article, the purpose of breeding includes cultivating plants with high salt and alkali tolerance; the purpose of breeding also includes cultivating plants with low salt and alkali tolerance.
[0025] In this article, the low salt and alkali tolerance is specifically manifested as a stronger salt-sensitive phenotype, with more purple leaves and a lower plant height.
[0026] In this article, the corn mentioned may be corn B73-329.
[0027] In this article, the recombinant microorganism may be Agrobacterium EHA105.
[0028] The present invention also provides the proteins and / or the biological materials described above.
[0029] In the above applications or methods, the plant may be any of the following: N1) Monocotyledons; N2) Plants of the order Poales; N3) Gramineae plants; N4) Plants of the genus *Zea*; N5) Corn.
[0030] This study demonstrates that, compared with the wild-type B73-329, the HSP70-5 overexpressing lines exhibit a significantly more sensitive phenotype to salt and alkali stress, indicating that the function of the HSP70-5 protein is to reduce the plant's tolerance to high salt and alkali conditions. Attached Figure Description
[0031] Figure 1 Phenotypic results of wild-type maize strains B73-329 and HSP70-5 overexpression lines in soil treated with NaHCO3. Detailed Implementation
[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0034] Unless otherwise specified, the quantitative experiments in the following examples are all repeated 5 times, and the results are averaged.
[0035] The maize B73-329 in the following examples is described in: Liu M, Zhang S, Li W, Zhao X, Wang XQ. Identifying yield-related genes in maize based on ear trait plasticity. Genome Biol. 2023;24(1):94. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention and may not be used for any other purpose.
[0036] Example 1 HSP70-5 Application of genes in regulating plant salt and alkali tolerance HSP70-5 The cDNA sequence of the gene is SEQ ID No. 1, the genome sequence is SEQ ID No. 3, and the amino acid sequence of the protein HSP70-5 encoded by the gene is SEQ ID No. 2.
[0037] Maize B73-329 and HSP70-5 overexpression lines were obtained from the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University. The construction method of the HSP70-5 overexpression lines is as follows: 1. Culture medium preparation LB medium (1000mL): 10g tryptone, 5g yeast extract, 10g NaCl, 8g agarose (LB liquid medium does not contain agarose).
[0038] 2. Amplified fragments, gel recovery, enzyme digestion and ligation The upstream and downstream primer sequences were designed based on the sequence of the HSP70-5 gene, with F: 5'-aaattagtagaaaaataaaaactgactcggatacCTGGCCACCATTTCTTTCCCCTCTCC-3' and R: 5'-tcgagggagatttttcaaatcagtgcgcaATGTTAACCATATAAATAATTCCTATTAT-3'. After amplification, the target fragment was approximately 3059 bp (containing the CDS sequence of HSP70-5 with nucleotide sequence SEQ ID No. 1). The target fragment was then ligated into the starting vector P1300super-MT1T2 (the nucleotide sequence of the starting vector is SEQ ID No. 4) to obtain the recombinant vector P1300-MT1T2-HSP70-5.
[0039] The recombinant vector P1300-MT1T2-HSP70-5 is described as follows: It is a recombinant vector obtained by replacing the BciVI and FSPI recognition site sequences of the starting vector P1300super-MT1T2 with the coding gene sequence of Hsp70 (SEQ ID No. 1), while keeping other sequences of the starting vector P1300super-MT1T2 unchanged.
[0040] 3. Escherichia coli transformation culture Thaw 50 μL of competent E. coli cells on ice, add recombinant vector P1300-MT1T2-HSP70-5, mix gently, and incubate on ice for 30 min. Heat shock at 42°C for 30 s, then quickly transfer the centrifuge tube to ice for 2 min. Add 700 μL of sterile LB medium (antibiotic-free) to each centrifuge tube, mix well, and incubate at 37°C, 200 rpm for 1 h to resuscitate the strain. Centrifuge at 5000-6000 rpm for 5 min, aspirate a portion of the supernatant, and vortex. Plate the cells (containing 50 μg / mL of KANA antibiotic) at 37°C for 12-16 h. Pick single colonies based on colony growth. Perform colony PCR using the Gel Extraction Kit (Omega, catalog number QYM10016).
[0041] The PCR products were verified by electrophoresis to obtain positive clones. These clones were then selected for culture, plasmids were extracted using a kit, and sequencing was performed, successfully yielding recombinant cells. 4. Agrobacterium-mediated transformation (2-3 days) 1) Thaw 50 μL of EHA105 Agrobacterium competent cells on ice, add 1-2 μL of plasmid DNA, mix gently, and incubate on ice for 30 min.
[0042] 2) Freeze in liquid nitrogen for 1 minute, heat shock in a 37°C water bath for 5 minutes, and then quickly transfer the centrifuge tube to an ice bath for 2 minutes.
[0043] 3) Add 1 ml of sterile LB medium (without antibiotics) to each centrifuge tube, mix well, and incubate at 28°C and 180 rpm for 3 h to revive the strain.
[0044] 4) Centrifuge at 5000-6000 rpm for 5 minutes, then aspirate a portion of the supernatant and shake.
[0045] 5) Plate coating. (Contains 50 ug / ml kanamycin + 40 ug / ml rifampicin) Incubate in the dark at 28℃ for 2-3 days.
[0046] The HSP70-5 overexpression line was finally obtained.
[0047] Example 2 HSP70-5 Application of genes in plant salt and alkali tolerance Test samples: maize seeds of B73-329 and HSP70-5 overexpression lines, with B73-329 as the control.
[0048] Planted in pure vermiculite without potting soil, the corn was irrigated with a 1 / 2 corn nutrient solution (potassium sulfate (0.75 mM), magnesium sulfate (0.65 mM), calcium nitrate (2 mM), potassium chloride (0.1 mM), potassium dihydrogen phosphate (0.25 mM), zinc sulfate (0.001 mM), manganese sulfate (0.001 mM), copper sulfate (0.0001 mM), ferrous sulfate (0.001 mM), boric acid (0.001 mM)). Approximately 7-10 days after planting (at the three-leaf stage), the corn was irrigated with a 1 / 2 corn nutrient solution containing 100 mM NaHCO3 (for salt-alkali stress), repeating this every 7-10 days at a rate of 2 liters per tray. The growth rate and leaf yellowing phenotype were observed after approximately 30-40 days.
[0049] The results are as follows Figure 1 As shown, under the condition of 100 mM NaHCO3 treatment, the HSP70-5 overexpression line showed a stronger salt-sensitive phenotype than the wild-type B73-329, with more purple leaves and lower plant height.
[0050] The above results indicate that HSP70-5 can negatively regulate plant salt and alkali tolerance.
[0051] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. The use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in any of the following; 1) Application in regulating plant salt and alkali tolerance; 2) Application in the preparation of products that regulate plant salt and alkali tolerance; 3) Application in cultivating plants with altered salt and alkali tolerance; 4) Application in the preparation of products that cultivate plants with altered salt and alkali tolerance; 5) Applications in plant breeding; The protein is any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No. 2; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No. 2, by substitution and / or deletion and / or addition of one or more amino acid residues; a3) Proteins that share more than 80% identity with the amino acid sequence defined by a1) or a2) and have the same function; a4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in a1)-a3).
2. The application according to claim 1, characterized in that, The protein is derived from corn.
3. The application according to claim 1 or 2, characterized in that, The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application of claim 1 or 2, and the biological material is any one of the following: c1) The nucleic acid molecule encoding the protein; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2).
4. The application according to claim 3, characterized in that: c1) The nucleic acid molecule is any of the following DNA molecules. d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3; d2) The coding region sequence is the DNA molecule shown in SEQ ID No. 1 of the sequence listing; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein of claim 1; d4) Hybridizes under stringent conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein of claim 1.
5. A method for altering the salt and alkali tolerance of plants, characterized in that: The method is to enhance, increase, or upregulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase, or upregulate the expression level of the gene encoding the protein described in claim 1 or 2, in order to reduce the plant's salt tolerance.
6. A method for cultivating plants with low salt and alkali tolerance, characterized in that, This includes enhancing, increasing, or upregulating the expression level of the gene encoding the protein described in claim 1 or 2 in the target plant, and / or, the activity and / or content of the protein, to obtain a low salt-alkali tolerant plant, wherein the low salt-alkali tolerant plant has weaker salt-alkali tolerance than the target plant.
7. The method according to claim 6, characterized in that, The enhancement, improvement, or upregulation of the expression of the gene encoding the protein of claim 1 or 2 in the plant comprises introducing the nucleic acid molecule of claim 4c1), the expression cassette of claim 4c2), or the recombinant vector of claim 4c3) into the target plant to obtain a plant with low salt tolerance.
8. The protein as described in claim 1 or 2 and / or the biomaterial as described in claim 3 or 4.
9. The method according to any one of claims 5-7, characterized in that, The plant is any one of the following: N1) Monocotyledons; N2) Plants of the order Poales; N3) Gramineae plants; N4) Plants of the genus *Zea*; N5) Corn.