CN117512002A - Eggplant protein for improving cold resistance of plants and application of coding gene - Google Patents
Eggplant protein for improving cold resistance of plants and application of coding gene Download PDFInfo
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- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
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- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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Abstract
The invention relates to an application of a protein derived from eggplant and capable of improving plant cold resistance and a coding gene, belonging to the technical field of bioengineering. The SmWRKY26 protein coding gene is transferred into the eggplant, so that the content of the SmWRKY26 protein in the eggplant is improved, photosynthesis of the eggplant after cold is enhanced, damage of cold injury to cell membranes is reduced, and the SmWRKY26 protein is proved to improve cold resistance of the eggplant.
Description
Technical Field
The invention relates to the technical field of biology, in particular to eggplant protein for improving cold resistance of plants and a coding gene thereof, and simultaneously relates to the improvement of the cold resistance of plants by utilizing the gene.
Background
Eggplant is one of main solanaceous vegetables in China, originates from the tropical region in southeast Asian, and belongs to a warm-loving crop. Eggplants are more susceptible to cold damage than other solanaceous crops. Cold damage is one of the important abiotic stresses of the cultivation of eggplants in the winter stubble and early spring stubble facilities in China. Cold damage can have adverse effects on the germination of eggplant seeds, the growth of seedlings, the flowering and fruiting of eggplants and other growth processes, so that the yield and the quality of eggplants are seriously threatened, and the sustainable development of the production of eggplants in winter and spring is restricted. Therefore, research on cold-resistant mechanism is developed, modern biotechnology is utilized to excavate cold-resistant genes, and cultivation of cold-resistant varieties suitable for facility cultivation is one of main targets of current eggplant production stress-resistant breeding.
Disclosure of Invention
The invention aims to provide the application of any one of the following substances in improving the cold resistance of plants:
(1) SmWRKY26 protein;
(2) A gene encoding a SmWRKY26 protein;
(3) Recombinant vectors, expression cassettes, recombinant bacteria or transgenic plant cell lines or tissues or organs containing genes encoding the SmWRKY26 protein.
The SmWRKY26 protein is any one of the following (1), (2) or (3):
(1) Consists of SEQ ID NO:1, and a protein consisting of an amino acid sequence shown in the formula 1;
(2) Setting SEQ ID NO:1 through substitution and/or deletion and/or addition of one or more amino acid residues and has the same function.
(3) And (3) connecting protein tags at the N end or/and the C end of the (1) or (2) to obtain the fusion protein.
In the above protein, the sequence SEQ ID NO:1 consists of 534 amino acid residues.
The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.
The gene for encoding the SmWRKY26 protein is any one of the following:
(1) The coding sequence is SEQ ID NO:2 from nucleotide 1 to nucleotide 1605;
(2) DNA molecule which hybridizes with the DNA molecule defined in (1) under stringent conditions and encodes a protein having the same function
(3) A DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in (1) and encoding a protein having the same function.
The stringent conditions are hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; alternatively, hybridization and washing of the membrane were performed at 65℃in a solution of 0.1 XSSPE (or 0.1 XSSC) and 0.1% SDS.
In the above application, the plant is a dicotyledonous plant or a monocotyledonous plant;
it is another object of the present invention to provide a method for breeding cold tolerant transgenic plants.
The method provided by the invention comprises the following steps: providing the expression level and/or activity of a DNA molecule encoding the protein SmWRKY26 in a plant of interest, to obtain a transgenic plant having a cold tolerance higher than that of said plant of interest;
the protein SmWRKY26 is any one of the following (1), (2) or (3):
(1) Consists of SEQ ID NO:1, and a protein consisting of an amino acid sequence shown in the formula 1;
(2) Setting SEQ ID NO:1 through substitution and/or deletion and/or addition of one or more amino acid residues and has the same function.
(3) And (3) connecting protein tags at the N end or/and the C end of the (1) or (2) to obtain the fusion protein.
The above transgenic plants have a cold tolerance higher than that of the plant of interest and a photosynthesis and cell permeability of the transgenic plants under the present cold stress higher and lower than that of the plant of interest, respectively.
In the above method, the improvement of the expression level and/or activity of the DNA molecule encoding the protein SmWRKY26 in the plant of interest can be achieved by introducing the DNA molecule encoding the protein SmWRKY26 into the plant of interest.
In the above method, the plant is a dicotyledonous plant or a monocotyledonous plant.
The invention will contain SEQ ID NO:2 into 'March eggplant' to obtain a transgenic plant which over-expresses SmWRKY 26; transgenic plants overexpressing SmWRKY26 have increased tolerance to cold injury compared to untransformed 'marshmania' plants. The SmWRKY26 is used for regulating plant cold tolerance, and the protein can be used for improving the cold tolerance of eggplants and has important application value for cultivating cold-tolerant eggplants.
Drawings
FIG. 1 shows the detection result of the relative expression amount of SmWRKY26 gene in a transgenic plant over-expressing SmWRKY 26;
FIG. 2 is a plant phenotype of transgenic plants overexpressing SmWRKY26 treated at 4℃with control plants;
FIG. 3 is a graph showing the determination of photosynthesis parameters after treatment of transgenic plants overexpressing SmWRKY26 at 4℃with control plants;
FIG. 4 shows the relative conductivity measurements of transgenic plants overexpressing SmWRKY26 after treatment with control plants at 4deg.C
Detailed Description
The present invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are to be considered in an illustrative sense only and are not intended to limit the invention.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
pFGC1008-HA vector is described in the following literature: wang Y et al, tomo HsfA 1a plays a critical role in plant drought tolerance by activating ATG genes and inducing autophagy.Autophagy,2015, 11:11, 2033-2047.
Example 1, protein SmWRKY26 and application of coding gene in regulating and controlling plant cold tolerance
The amino acid sequence of the eggplant protein SmWRKY26 is SEQ ID NO:1, the nucleotide sequence of the coding chain of the gene encoding the protein SmWRKY26 is SEQ ID NO:2, the gene for the protein SmWRKY26 was cloned from 'marshmania'.
1. Obtaining of eggplant protein SmWRKY26 gene and construction of recombinant expression vector
Extracting total RNA of leaves of the eggplant (Solanum melongena L.) variety 'March eggplant' in the four-leaf-one-heart stage, reversely transcribing the total RNA into cDNA, and then taking the cDNA as a template and utilizing an upstream primer F: TTGGCGCGCCATGGCTGCTTCAAGTTTCTC and downstream primer R: AGCGTCGACGCAAAGCAATGACTCCATAAAC PCR amplification was performed, and after the completion of the reaction, the PCR amplification product was subjected to 1% agarose gel electrophoresis, and a DNA fragment (1602 bp) was recovered and purified.
The vector pFGC1008-HA was digested with AscI and SaI to recover the vector backbone fragment. And (3) connecting the vector skeleton fragment with the recovered DNA fragment by using T4 ligase to obtain the recombinant vector. Sequencing confirmed SEQ ID NO:2 HAs been inserted into the pFGC1008-HA and kept the other nucleotide sequence of pFGC1008-HA from the recombinant vector pFGC1008-SmWRKY26-HA.
2. Acquisition of Agrobacterium tumefaciens containing recombinant vector
And (3) performing freeze thawing method on the obtained recombinant vector pFGC1008-SmWRKY26-HA to transform agrobacterium tumefaciens EHA105, and performing PCR identification to obtain positive recombinant bacteria, and performing genetic transformation on eggplants.
3. Genetic transformation of' March eggplant
1. Explant preparation
On an ultra-clean workbench, after the eggplant seeds are soaked in 75% ethanol for 30 seconds, the eggplant seeds are sterilized with 10% NaClO for 20 minutes, and the seeds are washed with sterile water for 5 times, so that residual sterilizing liquid is ensured to be washed off. Inoculating eggplant seeds into a 1/2 MS culture medium, culturing until the eggplant seeds germinate in the dark at 28 ℃, and transferring into illumination culture. 10 days after germination of the seeds, the cotyledons were fully expanded, cut into 4mm×4mm explant pieces with a blade, and the cotyledons were placed right side up in a co-culture medium for preculture for 1d.
2. Preparation of the liquor
Agrobacteria containing recombinant vector are streaked on YEP culture medium containing chloramphenicol (50 mg/L) and rifampicin (25 mg/L), single colony is selected and inoculated in YEP liquid culture medium containing corresponding antibiotics, and cultured at 28 ℃ for 200r/min overnight to OD 600 1.0. Centrifuging at 4000r/min10min, decanting the supernatant, adding 200 μM acetosyringone-containing liquid medium to resuspend to OD 600 0.2-0.3 for standby.
3. Infestation of the human body
The explants were immersed in the suspension and the dish was gently shaken and infected for 5min under darkness. And (3) sucking up the residual bacterial liquid on the surface of the explant, and placing the back of the explant upwards in a co-culture medium for co-culture for 2d in a dark place. And transferring the explant to a callus induction medium to induce callus. After 2 weeks, the callus was transferred to a bud induction medium for bud induction until the buds elongated to 1cm, and the buds were cut out and placed in an RMS medium for rooting. Subculturing was performed every 2 weeks during the induction of buds.
4. Identification of Positive plants
For the strain normally grown in hygromycin-containing medium, its genomic DNA was extracted by CTAB method, and pFGC1008-F: GATGTGACATCTCCACTGACGT and WRKY26R: AGCGTCGACGCAAAGCAATGACTCCATAAAC as a primer, PCR amplification was performed to determine whether T-DNA was inserted into 'Marsdenia' and 18 PCR positive plants were obtained as a result. And extracting total RNA of positive plant leaves by using a total RNA extraction kit, reversely transcribing the total RNA into cDNA, and homogenizing the concentration of the cDNA of the sample by using the constitutively expressed SmActin gene as an internal reference. The cDNA was then used as a template, WRKY26-qF: GCCTTCCGTGACATCTCATC and WRKY26-qR: AAACATGTCATCCCGAGGCT is primer to perform real-time fluorescence quantitative detection on SmWRKY26 gene expression level. As a result of the gene expression level in the untransformed wild type plant being 1, the expression level of SmWRKY26 in the multi-plant transgenic plant was significantly increased as compared with that in the wild type plant, as shown in FIG. 1.
4. Cold tolerance identification of transgenic eggplants
Cold stress treatment at 4 ℃ was performed on the transgenic eggplant OE overexpressing SmWRKY 26. The specific treatment method comprises the following steps: selecting full seeds of wild OE, sowing in a matrix, culturing at normal temperature under illumination, and transferring control plants and over-expression plants with the same size and growth vigor into a growth box at 4 ℃ for cold stress treatment for 5 days when the plants grow to four leaves and one core period. And then recovering at 25-28 ℃ for 3 days, carrying out character investigation, and carrying out chlorophyll fluorescence and relative conductivity detection and autophagosome observation on each plant. At the same time, 1 control group was set for parallel experiments (no cold stress treatment), and the experiments were repeated 3 times.
Chlorophyll fluorescence assays were detected using Imaging-PAM chlorophyll fluorescence Imaging system (Walz, germany).
The relative conductivities were determined as follows: 0.3g of the leaf blade is taken, washed and dried, sheared into fragments, placed into a 50ml centrifuge tube containing 25ml of ddH2O, shaken at 200rpm at room temperature for 2 hours, and then the conductivity E1 is measured, and then the water bath is carried out for 15 minutes, and the water bath is cooled to room temperature to measure the conductivity E2. Calculation is performed according to the relative conductivity formula: rec=e1/e2×100.
The experimental results of the cold stress treatment are shown in fig. 2, and the wild type and the over-expression plants under the normal growth condition have no obvious difference. After cold treatment, the leaves of the wild plants seriously wilt and lack green, and the over-expression plants only show slight water loss symptoms.
Under normal growth conditions, chlorophyll fluorescence parameter values of each plant were not different. Fv/Fm was reduced in plants under cold stress compared to normal temperature. Fv/Fm was significantly higher in the SmWRKY 26-overexpressing plants than in the wild-type (FIG. 3 a). Similarly, under normal growth conditions, the rate of CO2 assimilation at saturated light intensity was not substantially different in each strain, but Asat was drastically reduced for each strain after cold treatment, but Asat was significantly higher for the over-expressed plants than for the wild type (fig. 3 b).
Under normal growth conditions, the conductivity of wild type and overexpressing plants remained at a relatively low level with no difference. And after being stressed by cold injury, the permeability of each plant is enhanced, and the conductivity is increased. But the conductivity of the over-expressed plants was significantly lower than that of the wild type (fig. 4).
The experimental result shows that the transgenic strain with the over-expression SmWRKY26 can maintain higher photosynthesis and lower cell membrane permeability in the cold stress process, and shows stronger cold tolerance, and the over-expression of the SmWRKY26 gene can improve the cold tolerance of eggplant plants.
Claims (4)
1. Use of any of the following in improving cold tolerance of a plant:
(1) SmWRKY26 protein;
(2) A gene encoding a SmWRKY26 protein;
(3) Recombinant vectors, expression cassettes, recombinant bacteria or transgenic plant cell lines or tissues or organs containing genes encoding the SmWRKY26 protein.
The SmWRKY26 protein has an amino acid sequence shown in SEQ ID NO:1 is shown in the figure
The coding gene sequence is shown as SEQ ID NO: 2.
2. Use of any of the substances according to claim 1 for cultivating cold tolerant plants.
3. A method of growing a cold tolerant transgenic plant comprising the steps of: the expression quantity and/or the protein activity of a gene encoding a protein SmWRKY26 in a target plant are improved to obtain a transgenic plant, wherein the cold tolerance of the transgenic plant is higher than that of the target plant;
the protein SmWRKY26 is formed by SEQ ID NO:1, and a protein consisting of an amino acid sequence shown in the formula 1;
the plant is a monocotyledonous or dicotyledonous plant.
4. A method according to claim 3, characterized in that: the improvement of the expression quantity and/or the protein activity of the gene encoding the protein SmWRKY26 in the target plant is to introduce the gene encoding the protein SmWRKY26 into the target plant.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310900258.6A CN117512002A (en) | 2023-07-19 | 2023-07-19 | Eggplant protein for improving cold resistance of plants and application of coding gene |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310900258.6A CN117512002A (en) | 2023-07-19 | 2023-07-19 | Eggplant protein for improving cold resistance of plants and application of coding gene |
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