Application of dehydrin gene in plant drought resistance

Abstract

The invention belongs to the technical field of gene engineering, and discloses application of a dehydrin gene in plant drought resistance. The dehydrin gene is separated and extracted from perennial ryegrass and is specifically an LpDHN1 gene, and the nucleotide sequence of the dehydrin gene is shown as SEQ ID NO: 1. The dehydrin gene positively regulates the drought resistance of the plant, and the drought resistance of the plant can be improved by overexpressing the dehydrin gene. After the LpDHN1 gene is heterologously expressed in arabidopsis thaliana, the survival rate of a transgenic plant under drought stress is remarkably improved, and the ion permeability and malondialdehyde content of leaf cells of the transgenic plant under the drought stress condition are remarkably reduced. The invention provides a novel germplasm material for cultivation of drought-resistant plant varieties and improvement of drought-resistant characters of plants.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and relates to the application of dehydrin genes in plant drought resistance. Background Art

[0002] Perennial ryegrass (Lolium perenne) is a perennial sparse clump herbaceous plant of the genus Lolium. It can be used as a lawn grass and is also a cold-season livestock forage grass, widely planted throughout the country due to its advantages such as many tillers, resistance to trampling, beautiful plant shape, many tender leaves, fast lawn formation, strong regeneration ability, and high nutritional value. However, perennial ryegrass is sensitive to drought. Drought stress reduces the amount of water available to the plant, dehydrates the plant leaves, affects their growth and development, and causes leaf aging or even death. Most areas in northern my country are suffering from drought and water shortage. Due to the intensification of climate change, the usually humid south is also often hit by drought disasters. Therefore, it is of great significance to cultivate drought-resistant perennial ryegrass varieties.

[0003] Compared with conventional breeding techniques, molecular breeding techniques can accurately and quickly improve the quality of perennial ryegrass. Therefore, exploring the key genes that regulate the drought resistance of perennial ryegrass is of great significance for breeding drought-resistant ryegrass varieties. Dehydrin (DHN), also known as dehydrin, belongs to the second family of late embryogenesis abundant protein (LEA), and generally contains three conserved sequences: K, Y and S fragments. Among them, the K fragment is rich in lysine and can form an amphipathic α-helix by binding to phospholipid vesicles, which has the function of maintaining the stability of the cell membrane and preventing the denaturation of proteins on the cytoplasmic membrane. When plants are subjected to adverse stresses such as low temperature, drought and high salt, dehydrin will accumulate in large quantities to improve the tolerance of plants to adverse stress. At present, the mechanism of DHN protein involved in the regulation of perennial ryegrass stress resistance is still unclear, and there are few studies on perennial ryegrass drought resistance genes. Therefore, exploring a gene related to the regulation of perennial ryegrass drought resistance is of great significance for perennial ryegrass drought resistance breeding. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the application of dehydrin gene in plant drought resistance. The dehydrin gene is isolated and extracted from perennial ryegrass, specifically LpDHN1 gene, and its nucleotide sequence is shown in SEQ ID NO:1. Dehydrin gene positively regulates plant drought resistance, and overexpression of dehydrin gene can improve plant drought resistance. The present invention provides a new germplasm material for the cultivation of drought-resistant plant varieties and the improvement of plant drought resistance traits.

[0005] To achieve the technical purpose of the present invention, on the one hand, the present invention provides the use of a dehydrin gene in plant drought resistance, wherein the dehydrin gene is the LpDHN1 gene, and the nucleotide sequence of the LpDHN1 gene is shown in SEQ ID NO:1.

[0006] Furthermore, the LpDHN1 gene is induced to express under drought stress.

[0007] Specifically, the present invention analyzed the expression pattern of the LpDHN1 gene under drought stress and found that the relative expression level of the LpDHN1 gene was significantly increased after drought treatment of perennial ryegrass, indicating that the LpDHN1 gene was induced to express by drought stress and was related to the drought resistance of perennial ryegrass.

[0008] Furthermore, the LpDHN1 gene positively regulates the drought resistance of plants, and overexpression of the LpDHN1 gene improves the drought resistance of plants. Improving the drought resistance of plants includes increasing the survival rate of plants under drought stress, and also includes reducing the ion permeability and malondialdehyde content of leaf cells under drought stress.

[0009] Specifically, the present invention heterologously expresses the LpDHN1 gene in Arabidopsis plants, enhances the survival rate of transgenic plants under drought stress, and reduces the ion permeability and malondialdehyde content of leaf cells under drought stress.

[0010] On the other hand, the present invention claims a method for breeding drought-resistant plant varieties, overexpressing a dehydrin gene in the plant, wherein the dehydrin gene is the LpDHN1 gene, and the nucleotide sequence of the LpDHN1 gene is shown in SEQ ID NO:1.

[0011] Furthermore, the method for breeding drought-resistant varieties of the present invention comprises constructing a recombinant vector, transforming the recombinant vector into Agrobacterium, and infecting plants with the Agrobacterium carrying the recombinant vector to obtain the drought-resistant variety.

[0012] Furthermore, in the method for breeding drought-resistant varieties of the present invention, the recombinant vector comprises the LpDHN1 gene and an empty vector, and the empty vector is a Super1300-GFP vector. The Agrobacterium is GV3301, and the plant includes Arabidopsis thaliana.

[0013] Those skilled in the art can easily mutate the LpDHN1 gene of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the LpDHN1 gene isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode dehydrins and have the same function.

[0014] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the LpDHN1 gene of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0015] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0016] Those skilled in the art can use existing expression vectors to construct a recombinant vector containing the LpDHN1 gene. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector can also include the 3' end non-translated region of the foreign gene, that is, a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant gene (such as soybean storage protein gene) 3' end transcription non-translated region all have similar functions. When using the gene construction plant expression vector of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive and can be natural or synthetic. The translation initiation region can come from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and transformed plants can be directly screened by adversity.

[0017] The above-mentioned vector or expression vector can be a plasmid, cosmid, phage or virus vector.

[0018] The microorganisms may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0019] The above transgenic plant cell lines do not include propagation materials.

[0020] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0021] (1) The present invention clones a dehydrin family gene LpDHN1 induced by drought stress from perennial ryegrass, and its encoded protein is mainly located in the cell membrane. Through identification, it is found that the LpDHN1 gene is related to plant drought resistance. After drought treatment, the relative expression level of the LpDHN1 gene in perennial ryegrass plants is significantly increased. The present invention provides a new type of germplasm material for the cultivation of drought-resistant plant varieties and the improvement of plant drought resistance traits.

[0022] (2) The LpDHN1 gene positively regulates plant drought resistance. Heterologous expression of the LpDHN1 gene in Arabidopsis plants enhanced the survival rate of transgenic plants under drought stress and reduced the ion permeability and malondialdehyde content of leaf cells under drought stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the relative expression level of the LpDHN1 gene in perennial ryegrass under drought stress.

[0024] Figure 2 The following is the result of subcellular localization analysis of LpDHN1 protein. LpDHN1-GFP is the result of GFP signal observation; CBL-mcherry is the result of mcherry signal observation; Bridge is the result of bright field observation; Merged is the result of superposition of GFP signal, mcherry signal and bright field.

[0025] Figure 3 Phenotypic diagrams of wild-type Arabidopsis plants and LpDHN1 gene overexpressing Arabidopsis plants under normal conditions and drought stress. WT is a wild-type Arabidopsis plant; OE1 and OE3 are two transgenic plants overexpressing LpDHN1 gene.

[0026] Figure 4 The figure shows the statistical results of the survival rate of wild-type Arabidopsis plants and Arabidopsis plants overexpressing the LpDHN1 gene under normal conditions and drought stress.

[0027] Figure 5 The figure shows the statistical results of malondialdehyde content in wild-type Arabidopsis plants and Arabidopsis plants overexpressing the LpDHN1 gene under normal conditions and after drought stress.

[0028] Figure 6 The graph shows the statistical results of ion permeability of wild-type Arabidopsis plants and LpDHN1 gene overexpressing Arabidopsis plants under normal conditions and after drought stress. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is now described in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0030] Example 1

[0031] This example provides the acquisition of the LpDHN1 gene and the expression pattern analysis of the LpDHN1 gene under drought stress.

[0032] 1. Acquisition of LpDHN1 gene

[0033] Using the genome of perennial ryegrass as a template, specific primers (DHN1-F: 5'-ATGGAGTACCAGGGGCAGCAC-3'; DHN1-R: 5'-TCA GTGCGTCCCGCCGCCGGG-3') were designed for PCR amplification. The PCR product was purified, and the purified product was connected to the pEASY cloning vector and transferred into Escherichia coli DH5α to obtain positive clones. The positive clones were sent to Xi'an Qingke Biological Co., Ltd. for sequencing, and the nucleotide sequence of the LpDHN1 gene was obtained as shown in SEQ ID NO: 1.

[0034] 2. Analysis of the expression pattern of LpDHN1 gene under drought stress

[0035] Watering was stopped for perennial ryegrass seedlings growing normally in culture dishes, and leaves that had been watered for 24h and 48h were taken as drought treatment samples. mRNA was extracted and reverse transcribed into cDNA to detect the expression of LpDHN1 gene under drought stress ( Figure 1 ).Depend on Figure 1 It can be seen that the relative expression level of the LpDHN1 gene in perennial ryegrass increased significantly after drought treatment, indicating that the LpDHN1 gene was induced to express under drought stress and was related to the drought resistance of perennial ryegrass.

[0036] Example 2

[0037] This example provides subcellular localization analysis of LpDHN1 protein.

[0038] Using homologous recombination technology, 35S:LpDHN1-GFP vector was constructed and transferred into Agrobacterium competent GV3301. Agrobacterium transferred with 35S:LpDHN1-GFP vector and Agrobacterium containing 35S:CBL-mcherry vector (gift from Professor Fu Ying's laboratory at China Agricultural University) were used to infect tobacco leaves together and cultured in the dark for 2-3 days. 2The leaves were observed using a laser confocal microscope to observe GFP signals (green fluorescence), mCherry signals (red fluorescence) and bright field. Figure 2 shown.

[0039] Depend on Figure 2 It can be seen that the GFP signal of LpDHN1-GFP fusion protein overlaps with the CBL-mcherry signal localized in the cytoplasmic membrane, indicating that LpDHN1 can be localized in the cytoplasmic membrane.

[0040] Example 3

[0041] This example provides the effect of heterologous overexpression of the LPDHN1 gene on plant drought resistance.

[0042] 1. Construction of LpDHN1 transgenic Arabidopsis plants

[0043] The LpDHN1 gene was constructed into the Super1300-GFP vector using homologous recombination technology to obtain a recombinant vector, which was then transferred into the Agrobacterium competent GV3301. The Arabidopsis inflorescence was then infected with Agrobacterium. After the seeds were harvested, hygromycin was used to select homozygous to obtain a transgenic strain overexpressing the LpDHN1 gene.

[0044] 2. Analysis of drought resistance phenotype of Arabidopsis plants overexpressing LpDHN1 gene

[0045] Plant survival rate statistics: After about four weeks of drought treatment of wild-type (WT) and LpDHN1 gene overexpressing Arabidopsis plants grown under normal conditions, the phenotypes of the plants are shown in the figure below. Figure 3 As shown, the survival rates of different strains were statistically analyzed, and the statistical results are as follows Figure 4 As shown. Figure 3 It can be seen that after drought treatment, the leaves of wild-type Arabidopsis plants in the control group turned yellow and became smaller, and the plants wilted. The leaves of Arabidopsis plants with overexpression of the LpDHN1 gene showed significantly less yellowing, and the wilting of the plants was alleviated. Figure 4 It can be seen that the survival rate of Arabidopsis plants overexpressing the LpDHN1 gene after drought treatment is significantly improved, indicating that overexpression of the LpDHN1 gene can significantly improve the plant's drought resistance.

[0046] Determination of malondialdehyde (MDA) content: Take the leaves of the plant, grind them into a homogenate in a mortar, centrifuge at 12000rpm for 10min, and use the thiobarbituric acid colorimetric method to determine the malondialdehyde content. The results are as follows: Figure 5 As shown. Under drought conditions, plant cells undergo membrane lipid peroxidation and produce MDA. The accumulation of MDA is negatively correlated with the drought resistance of plants, which can reflect the drought resistance of plants. Figure 5It can be seen that there was no significant difference in the malondialdehyde content in the leaves of wild-type plants and transgenic plants grown under normal conditions. After drought treatment, the malondialdehyde content in the leaves of wild-type Arabidopsis plants was significantly higher than that of Arabidopsis plants overexpressing the LpDHN1 gene, indicating that overexpressing the LpDHN1 gene can significantly improve the drought resistance of plants.

[0047] Ion permeability (EL) determination: Take an appropriate amount of plant leaves and put them into a test tube containing 10 mL of deionized water. After 12 hours, use a conductivity meter to measure the conductivity R0. Then put the test tube into a boiling water bath and boil it for 15 minutes. After cooling, measure the conductivity R1. Calculate the ion permeability using the formula EL = R0 / R1 × 100%. The calculation results are as follows: Figure 6 As shown. Figure 6 It can be seen that there was no significant difference in the ion permeability of leaves between wild-type plants and transgenic plants grown under normal conditions. After drought treatment, the ion permeability of leaves of wild-type Arabidopsis plants was significantly higher than that of Arabidopsis plants overexpressing the LpDHN1 gene, indicating that the LpDHN1 gene positively regulates plant drought resistance.

[0048] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred embodiments of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. Application of dehydrin gene in plant drought resistance, characterized in that: The dehydrin gene is the LpDHN1 gene, and the nucleotide sequence of the LpDHN1 gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The LpDHN1 gene is induced to express under drought stress.

3. The use according to claim 1, characterized in that: The LpDHN1 gene positively regulates the drought resistance of plants.

4. The use according to claim 1, characterized in that: Overexpression of the LpDHN1 gene improves the drought resistance of plants.

5. The use according to claim 4, characterized in that: The improving the drought resistance of plants includes improving the survival rate of plants under drought stress.

6. The use according to claim 4, characterized in that: The method of improving the drought resistance of plants also includes reducing the ion permeability of leaf cells and the malondialdehyde content of leaf cells under drought stress of plants.

7. A method for breeding drought-resistant plant varieties, characterized in that: A dehydrin gene is overexpressed in the plant, wherein the dehydrin gene is the LpDHN1 gene, and the nucleotide sequence of the LpDHN1 gene is shown in SEQ ID NO:

1.

8. The cultivation method according to claim 7, characterized in that: The method comprises constructing a recombinant vector, transforming the recombinant vector into Agrobacterium, and infecting plants with the Agrobacterium carrying the recombinant vector to obtain the drought-resistant variety.

9. The cultivation method according to claim 8, characterized in that: The recombinant vector comprises the LpDHN1 gene and an empty vector; The empty vector is Super1300-GFP vector.

10. The cultivation method according to claim 8, characterized in that: The Agrobacterium is GV3301; The plants include Arabidopsis thaliana.

Images