CN114656536A - ZmpHt1, protein 10 and application of coding gene thereof in regulation and control of saline-alkali tolerance of plants - Google Patents
ZmpHt1, protein 10 and application of coding gene thereof in regulation and control of saline-alkali tolerance of plants Download PDFInfo
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Abstract
The invention discloses ZmPut 1;10 protein and the application of the coding gene thereof in regulating and controlling the saline-alkali tolerance of plants. The protein provided by the invention is named ZmpHt1;10 protein, obtained from maize (Zea mays L.), is a protein represented by sequence 1 of the sequence listing. Encodes zmpth 1;10 protein gene, named ZmpHt1;10 gene, also belongs to the protection scope of the invention. The invention also protects ZmPut 1;10 protein in regulating and controlling the saline-alkali stress tolerance of plants. The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: mixing ZmPut 1;10 gene is introduced into a receptor plant to obtain a transgenic plant with increased saline-alkali stress tolerance. The invention has great application value for cultivating saline-alkali tolerant plants.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to ZmpHt1;10 protein and the application of the coding gene thereof in regulating and controlling the saline-alkali tolerance of plants.
Background
The tendency of soil salinization is continuously expanded in the world, the growth of crops is severely limited, and the tendency becomes an important factor for restricting agricultural production. The harm to plants caused by saline-alkali stress is high concentration of Na in saline-alkali soil+And has already been mixedA high pH. Along with the accumulation of salt ions in plant cells, the salt ions generate ion toxic effect on the cells, thereby influencing the cells to play normal functions in multiple aspects. In alkaline earth environments, most metal elements except alkali metal elements form insoluble salts, Na+As the alkali metal element with the highest content in nature, the alkali metal element is enriched in alkaline earth to cause salinization of soil.
With the rapid development of molecular biology, genomics, genetics, biochemistry and gene editing technology, the research on the molecular mechanism of plants for resisting saline-alkali stress is continuously and deeply carried out, and a plurality of new genes or proteins participate in the response process of regulating the saline-alkali stress.
Disclosure of Invention
It is an object of the present invention to provide ZmPht1;10 protein and the application of the coding gene thereof in regulating and controlling the saline-alkali tolerance of plants.
The protein provided by the invention is named ZmpHt1;10 protein, obtained from maize (Zea mays L.), is (a1) or (a2) as follows:
(a1) protein shown in a sequence 1 in a sequence table;
(a2) and (b) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1).
The labels are specifically shown in table 1.
TABLE 1 sequences of tags
| Label (R) | Residue of | Sequence of |
| Poly-Arg | 5-6 (generally 5) | RRRRR |
| Poly-His | 2-10 (generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
| HA | 9 | YPYDVPDYA |
Encodes zmpth 1;10 protein gene, named ZmpHt1;10 gene, which also belongs to the protection scope of the invention.
Specifically, zmpth 1; the 10 gene is a DNA molecule of (b1) or (b2) as follows:
(b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;
(b2) a DNA molecule shown in a sequence 3 of a sequence table.
Contains ZmpHt1;10 gene recombinant vector, expression cassette or recombinant bacteria are all in the protection scope of the invention.
The existing plant expression vector can be used for constructing ZmpHt1;10 gene.
When constructing a recombinant vector, any one of an enhanced, constitutive, tissue-specific or inducible promoter may be added in front of its transcription initiation nucleotide, and they may be used alone or in combination with other plant promoters. In addition, enhancers, including translational or transcriptional enhancers, may be used in the construction of recombinant vectors, and these enhancer regions may be ATG initiation codons or initiation codons in adjacent regions, but are necessarily in frame with the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plants, the recombinant vector used may be processed, for example, by adding a gene expressing an enzyme or a luminescent compound which produces a color change in a plant, an antibiotic marker having resistance, or a chemical-resistant marker gene, etc. From the viewpoint of transgene safety, the transformed plants can be directly screened for phenotypes without adding any selectable marker gene.
The plant expression vector can be a pBCXUN vector.
The recombinant vector can be specifically a recombinant plasmid pBCXUN-ZmpHt1 obtained by inserting a DNA molecule shown in a sequence 2 of a sequence table into a pBCXUN vector; 10.
the invention also protects ZmPut 1;10 protein in regulating and controlling the saline-alkali stress tolerance of plants. Specifically, the regulation is positive regulation, i.e., zmpth 1;10 protein is increased, and the salt alkali stress tolerance of the plant is increased. The regulation and control of the plant saline-alkali stress tolerance is to improve the plant saline-alkali stress tolerance.
The invention also protects the ZmPut 1;10 gene or the recombinant vector or the expression cassette or the recombinant bacterium in cultivating transgenic plants with increased salt and alkali stress tolerance.
The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: mixing ZmpHt1;10 gene is introduced into a receptor plant to obtain a transgenic plant with increased saline-alkali stress tolerance. Zmpth 1; the 10 gene is specifically introduced into a recipient plant by the recombinant vector.
The invention also provides a plant breeding method, which comprises the following steps: increasing ZmPut 1 in the plant of interest; 10 protein content and/or activity, thereby increasing the salt-alkali stress tolerance of the plant.
The plant is a monocotyledon or a dicotyledon.
The plant is a gramineous plant.
The plant is a plant of the genus zea.
The plant is corn.
The plant is corn B73-329.
The inventors of the present invention found that zmpth 1;10 participate in the saline-alkali stress response process to regulate and control the saline-alkali stress tolerance of plants. ZmPht1, compared to corn B73-329; 10 overexpression lines showed significantly increased salt-base stress tolerance. Thus, it is demonstrated that ZmPht1; the function of the 10 protein is to improve the stress tolerance of plants to high saline and alkaline. The invention has great application value for cultivating saline-alkali tolerant plants.
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FIG. 1 is a photograph of a plant of example 2.
FIG. 2 is a photograph of the root system of the plant of example 2.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Corn (Zea mays L.) B73-329 is described in: wangfang, Zi Peng Juan, Huang Yun, Wang Shi Wen, Wang Hai Feng, Chen Yifang, molecular mechanism of absorption and redistribution of phosphorus from corn [ A ],2018 national plant biology congress of discourse [ C ], 2018. The pBCXUN vector is an expression vector obtained by replacing the HYG gene (hptII, hygromycin resistance gene) of the pCXUN vector (GenBank: FJ905215.1, 06-JUL-2009) with the Bar gene (encoding phosphinothricin acetyltransferase) shown in sequence 4 of the sequence table and keeping the other nucleotides of pCXUN unchanged.
Example 1, zmpth 1;10 gene and discovery of protein encoded by same
A new protein is found from corn, and is shown as a sequence 1 in a sequence table and named as ZmpHt1;10 protein.
ZmpHt1 in maize genomic DNA; the 10 gene is shown as a sequence 3 in a sequence table.
In maize cDNA, zmptht 1; the open reading frame of the 10 genes is shown as a sequence 2 in a sequence table.
Example 2 acquisition of transgenic plants and characterization of salt and alkali stress tolerance
Construction of recombinant plasmid
Inserting a DNA molecule shown in a sequence 2 of a sequence table into a pBCXUN vector to obtain a recombinant plasmid pBCXUN-ZmpHt 1;10, sequencing verification has been performed.
Recombinant plasmid pBCXUN-ZmpHt 1;10, the transcription of the DNA molecule shown in the sequence 2 of the sequence table is started by a Ubi promoter and is stopped by an Nos terminator, so that ZmpHt1 is expressed; 10 protein.
II, ZmpHt1;10 Gene overexpression plant
1. Recombinant plasmid pBCXUN-ZmpHt 1;10, introducing the agrobacterium into the EHA105 to obtain the recombinant agrobacterium.
2. Adopting the recombinant agrobacterium prepared in the step 1 to infect embryonic callus of corn B73-329, then sequentially carrying out cocultivation and resistance screening (the resistance screening adopts herbicide glufosinate), and then sequentially carrying out pre-differentiation, differentiation and rooting to obtain T0Regenerating plants.
3、T0Carrying out PCR identification on the generation regeneration plant, and screening to obtain a transgenic plant; will T0Selfing the transgenic plant to obtain the seed T1Seed generation, T1The plant grown by the seed generation is T1Plant generation; will T1Selfing the plant to obtain the seed T2Seed generation, T2The plant grown by the seed generation is T2Plant generation; will T2Selfing the plant to obtain the seed T3Seed generation, T3The plant grown by the seed generation isT3And (5) plant generation.
4. Will T1Plant generation and sampled T2And carrying out PCR identification on the generation plants. For a certain T1Generation of plants, if the plants and T obtained by selfing the plants2All the generation plants are transgenic plants, and the selfing progeny of the plants are homozygous transgenic lines.
The PCR identification method in step 3 and step 4 is as follows: extracting genome DNA of plant leaves, carrying out PCR amplification by adopting a primer pair consisting of Ubi P-seq (corresponding to a Ubi promoter) and NosR-seq (corresponding to a Nos terminator), and if a specific amplification product is obtained, the plant is a transgenic plant.
Obtaining two homozygous transgenic strains which are ZmpHt1 respectively; strain 10-OX1 and ZmpHt1; strain 10-OX 2.
Thirdly, ZmpHt1; identification of salt alkali stress tolerance of 10 gene over-expressed plant
Test seeds: zmpth 1; t of strain 10-OX13Seed generation, zmpth 1; t of strain 10-OX23Seed generation, corn seeds of B73-329.
The test seeds were planted in vermiculite, irrigated with 1/2 Hoagland's nutrient solution, and cultured normally to the 3-leaf stage. Then the reaction was started with a solution containing 200mM NaHCO31/2 Hoagland's nutrient solution (pouring once every 7-10 days) and culturing for 28 days normally. The photographs were then observed and the survival was counted.
Five replicates were performed, with at least 5 biological replicates per sample per replicate set.
The photograph of the plant is shown in FIG. 1.
The photograph of the roots of the plants is shown in FIG. 2.
Zmpth 1 compared to maize B73-329 plants; the 10 gene over-expression plants show stronger salt-alkali stress tolerance, and concretely show that the leaves are greener, the plant height is higher, and the root system development is better.
The survival rate of the corn B73-329 plant is 0. Zmpth 1; the survival rate of the 10-OX1 strain plants is 70% -80%. Zmpth 1; the survival rate of the 10-OX2 strain plants is 50% -60%.
The above results indicate that zmpth 1; the 10 protein positively regulates the salt-base stress tolerance of plants.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> university of agriculture in China
<120> ZmpHt1, 10 protein and application of coding gene thereof in regulation and control of saline-alkali tolerance of plants
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Cys Ser Leu Ala Ser Gly Leu Ser Phe Ser Arg Lys Pro Lys Asp Ala
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Ile Ala Thr Leu Cys Phe Phe Arg Phe Trp Leu Gly Val Gly Ile Gly
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Gly Asp Tyr Pro Leu Ser Ala Thr Ile Met Ser Glu Tyr Ala Asn Lys
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Arg Thr Arg Gly Ala Phe Ile Ala Ala Val Phe Ala Met Gln Gly Leu
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Gly Asn Leu Ala Ala Gly Thr Val Val Leu Ala Thr Cys Ala Arg Phe
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cgatggcctc ctaaacggcc gttggatata acattatatc cgactgttgg acaccaaaat 2400
gagcggacgg tccggcccat gggcccggac ggtccgcgtg tcccgagatt agattaactc 2460
ggatgtttat ccttatctcg tgcgtgatta tccatctaat catgtgggag tttgttggct 2520
atctcttagg aaaaggtcca gacctcctcc cctataaata taaaggggta cggtcgattg 2580
agaacccccg aacacattcc aatcgaacca attaccttat ttactttttc tgccctagga 2640
gtagatgtag catagttcta gttgtagtct tccacatatc cacctccatc cctattcgac 2700
tctacgtcgt ctagatccgt cttggttggc ctgccgatcc caaaacaacc ctaggatctc 2760
acccctcccg ggggg 2775
<210> 4
<211> 552
<212> DNA
<213> Zea mays L.
<400> 4
atgagcccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga catgccggcg 60
gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg taccgagccg 120
caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta tccctggctc 180
gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc 240
aacgcctacg actggacggc cgagtcgacc gtgtacgtct ccccccgcca ccagcggacg 300
ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca gggcttcaag 360
agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc 420
ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa ctggcatgac 480
gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt cctgcccgtc 540
accgagattt ga 552
Claims (10)
1. A protein which is (a1) or (a2) as follows:
(a1) protein shown as a sequence 1 in a sequence table;
(a2) and (b) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1).
2. A gene encoding the protein of claim 1.
3. The gene of claim 2, wherein: the gene is a DNA molecule of (b1) or (b 2):
(b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;
(b2) a DNA molecule shown in a sequence 3 of a sequence table.
4. A recombinant vector, expression cassette or recombinant bacterium comprising the gene of claim 2 or 3.
5. Use of the protein of claim 1 for modulating salt-base stress tolerance in plants.
6. The use of the gene of claim 2 or 3 or the recombinant vector of claim 4 or the expression cassette of claim 4 for the production of transgenic plants with increased saline-alkali stress tolerance.
7. Use according to claim 5 or 6, characterized in that: the plant is a monocot.
8. A method of breeding a transgenic plant comprising the steps of: a transgenic plant having increased salt/alkali tolerance, which is obtained by introducing the gene according to claim 2 or 3 into a recipient plant.
9. A method of plant breeding comprising the steps of: increasing the content and/or activity of the protein of claim 1 in the plant of interest, thereby increasing the salt-base stress tolerance of the plant.
10. The method of claim 8 or 9, wherein: the plant is a monocot.
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| WO1999058657A2 (en) * | 1998-05-13 | 1999-11-18 | Pioneer Hi-Bred International, Inc. | Zea mays phosphate transporter genes and uses thereof |
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| CN106674337A (en) * | 2015-11-06 | 2017-05-17 | 中国农业大学 | Plant phosphorus transport protein ZmPHT1;7, and encoding gene and application thereof |
| WO2018146481A1 (en) * | 2017-02-09 | 2018-08-16 | Fujian Agriculture And Forestry University | Expression of a phosphate transporter for improving plant yield |
| CN110129337A (en) * | 2019-06-10 | 2019-08-16 | 山东大学 | The high affine phosphorus transporter body ZmPHT1 of corn;The deletion mutant of 5 gene promoters and its application |
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2020
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| WO1999058657A2 (en) * | 1998-05-13 | 1999-11-18 | Pioneer Hi-Bred International, Inc. | Zea mays phosphate transporter genes and uses thereof |
| CN104974235A (en) * | 2015-07-22 | 2015-10-14 | 中国农业大学 | Application of phosphorus absorption-related protein ZmPht1;5 in regulation of plant phosphorus absorption |
| CN106674337A (en) * | 2015-11-06 | 2017-05-17 | 中国农业大学 | Plant phosphorus transport protein ZmPHT1;7, and encoding gene and application thereof |
| WO2018146481A1 (en) * | 2017-02-09 | 2018-08-16 | Fujian Agriculture And Forestry University | Expression of a phosphate transporter for improving plant yield |
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| CN114656536B (en) | 2023-03-21 |
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