Populus euphratica PeAREB1 gene and application thereof in improving drought resistance of plants

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a populus euphratica PeAREB1 gene and application thereof in improving drought resistance of plants. According to the populus euphratica PeAREB1 gene, the nucleotide sequence of the gene is as shown in SEQ ID NO.1, and the amino acid sequence coded by the gene is as shown in SEQ ID NO.2. When the gene is used for constructing a plant expression vector and transforming a plant, the drought tolerance of the plant can be improved, and the gene has a relatively good application prospect in stress-resistant breeding of the plant.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and in particular relates to a Populus euphratica PeAREB1 gene and an application thereof in improving plant drought resistance. Background Art

[0002] Drought is one of the main environmental stresses that affects plant growth and survival, especially in the context of intensified global climate change, which poses a serious threat to agricultural production and ecosystem security. Drought stress causes plant water loss, directly affecting photosynthesis, nutrient absorption, cell turgor and metabolic activities, thereby reducing plant growth rate, yield and survival ability, and in severe cases can lead to plant death.

[0003] Understanding how plants cope with water shortages and improving crop drought tolerance is particularly important for ensuring food security, accelerating ecological restoration and desertification control, and responding to climate change. Studies have shown that under drought stress, plants will enhance their drought tolerance by adjusting the opening of stomata, activating antioxidant systems, accumulating osmotic regulating substances, and other measures. In order to ensure the normal growth and development of plants and cope with complex climatic conditions, breeding stress-resistant plant varieties has become one of the main goals of scientific research. At present, scientists have tried to improve plant photosynthesis and drought resistance by adjusting the opening of plant stomata, and cultivate stress-resistant plants with high light efficiency and high water utilization rate.

[0004] Therefore, discovering genes that can be used to improve plant drought resistance is of great significance to plant genetic engineering research. Summary of the invention

[0005] The purpose of the present invention is to provide a Populus euphratica PeAREB1 gene, which can regulate the stomatal aperture of plants and improve the drought resistance of plants.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides a drought-resistant Populus euphratica PeAREB1 gene, the nucleic acid sequence of the gene is shown as SEQ ID NO.1.

[0008] Preferably, the amino acid sequence encoded by the gene is shown as SEQ ID NO.2.

[0009] The present invention also provides application of the Populus euphratica PeAREB1 gene in improving plant stress resistance.

[0010] Preferably, the stress resistance is drought resistance.

[0011] The present invention also provides a recombinant vector or expression cassette for improving plant stress resistance, wherein the recombinant vector or expression cassette contains the coding region sequence of the Populus euphratica PeAREB1 gene, and the nucleic acid sequence of the Populus euphratica PeAREB1 gene is shown in SEQ ID NO.1.

[0012] The present invention also provides a recombinant bacterium for improving plant stress resistance, wherein the recombinant bacterium contains the Populus euphratica PeAREB1 gene; the nucleic acid sequence of the Populus euphratica PeAREB1 gene is shown in SEQ ID NO.1.

[0013] The present invention also provides a method for improving plant stress resistance, which comprises constructing the Populus euphratica PeAREB1 gene into a plant expression vector, transforming the plant, and expressing the gene in the plant; the nucleic acid sequence of the Populus euphratica PeAREB1 gene is shown in SEQ ID NO.1.

[0014] Preferably, the stress resistance is drought resistance.

[0015] Preferably, the plant is Arabidopsis thaliana.

[0016] The present invention also provides the use of the above gene or the above recombinant vector or the expression box or the above recombinant bacteria in regulating the stomatal aperture of plants.

[0017] Beneficial effects of the present invention:

[0018] (1) The PeAREB1 gene can improve plant drought resistance and has the function of regulating stomatal aperture. Plants with overexpression of the PeAREB1 gene can significantly regulate epidermal stomatal aperture.

[0019] (2) The PeAREB1 gene can be constructed into an expression vector and expressed in plants to exert drought resistance.

[0020] (3) Plants transformed with the expression vector of the PeAREB1 gene of the present invention can cultivate new varieties of drought-resistant and high-yielding plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] Figure 1Real-time quantitative PCR (RT-PCR) was used to detect the expression level of PeAREB1 gene in wild type Arabidopsis thaliana (Wild type, Col-0) and three independent T3 generation PeAREB1 gene overexpression lines with different expression levels (PeAREB1-OE-1, PeAREB1-OE-2 and PeAREB1-OE-3). Arabidopsis Actin gene was used as the internal reference gene. The data are the mean of three replicates plus the standard error.

[0023] Figure 2 The stomatal images of the leaves of Arabidopsis transgenic plants and wild-type Arabidopsis under a microscope;

[0024] Figure 3 The stomatal opening size of the epidermis of the middle part of mature leaves of Col-0 and three PeAREB1 overexpressing Arabidopsis thaliana T3 generations was measured under a microscope at 40 times. The data are the average of 30 replicates plus the standard error; the stomatal opening of the PeAREB1 gene overexpressing strain under drought stress is significantly smaller than that of the wild type. The data were analyzed by one-way ANOVA, and significant differences are indicated by different English letters. DETAILED DESCRIPTION

[0025] The present invention provides a Populus euphratica gene PeAREB1, the nucleotide sequence of the gene is shown in SEQ ID NO.1, the length is 1368 bp, the encoded protein sequence is shown in SEQ ID NO.2, and it consists of 455 amino acids. The nucleotide sequence contains a stop codon, and the stop codon does not translate the protein.

[0026] Nucleotide sequence of PeAREB1-1:

[0027]

[0028] Encoding protein sequence PeAREB1-1:

[0029] MGTNFNFKNFSNDPSDAVGGRPPGNSPLTRQSSIYSLTFDELQNTMGGSLGKDFGSMNMDELLKNIWSAEETQTIATATSTGVQEGGALQRQGSLTLPRTLSQRTVDEVWKDMS KEYVINGTSAGAANNVPQRQPTLGGMTLEEFLLRAGVAIEDIQVAPKVNTNGGLLGDLSRSANNSLAIGFQQNRGVGLDNDNTNQISLQSSNLPLNVNGVRSNQAQVQQQQQQQI FPKQPNMGYVTQPNNDSTNQISLQSSNLPLNVNGVRSNQAQVQQQQIFPKQPNLGYVTQMPLQSGPGIRGGMLGIGDQGMDSGLMQGGGMGVVGLGGIATGSPANQLSSDGIGKS NGDTSSVSPVPYVFRESVRGRRAGGAVEKVVERRQRRMIKNRESAARSRARKQAYTMELEAEVAKLKEENEELRKKQAEMMEIQKNQVAEMMNMQQGGKKRCLRRTQTGPW(SEQ ID NO.2)

[0030] The present invention includes the separation and cloning of target fragments, and the transformation of Arabidopsis by Agrobacterium inflorescence infection, and then phenotypic identification of the transformed plants. Overexpression of the PeAREB1 gene can enhance the drought tolerance of Arabidopsis and increase the survival rate of Arabidopsis. The gene can be well applied in ecological restoration, plant drought resistance and genetic improvement.

[0031] The gene PeAREB1 of the present invention is found to have a phenotype of reduced stomatal aperture and enhanced drought resistance in leaves of transgenic Arabidopsis plants under drought stress after heterologous overexpression of PeAREB1 in Arabidopsis, confirming the function of the gene in regulating stomatal development of Arabidopsis and its application in improving drought resistance and survival rate of Arabidopsis.

[0032] The PeAREB1 gene of the present invention can be connected to any vector that can normally express the gene. The overexpression vector carrying the coding region of the PeAREB1 gene of the present invention can be introduced into other plant cells by conventional biotechnology methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, etc., to finally obtain plants with improved drought resistance.

[0033] The expression vector comprising the PeAREB1 gene of the present invention can be used to transform hosts (various plants including Arabidopsis thaliana) to cultivate drought-resistant and high-yield plant varieties.

[0034] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit DP441 was purchased from Tiangen Biotech, Co., Ltd.; M5 Sprint qPCR RT kit with gDNA remover reverse transcription kit MF949 was purchased from PolyMei Company.

[0036] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0037] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0038] Example 1: Construction of expression vector

[0039] The nucleotide sequence shown in SEQ ID NO.1 was obtained by biological methods. It was used as a template and PCR amplification was performed using the following primers:

[0040] PeAREB1-F:

[0041] 5'-GGGGTACCATGGGGACGAATTTCAACTTCA-3'(SEQ ID NO.3)

[0042] PeAREB1-R:

[0043] 5'-CGGGATCCTCACCATGGACCTGTCTGA-3'(SEQ ID NO.4)

[0044] The amplified PeAREB1 gene fragment was connected to the overexpression vector pBWA(V)HS-GFP in 5×TEDA buffer to obtain the recombinant expression vector 35S::PeAREB1-pBWA(V)HS-GFP. The correctly sequenced vector was transformed into Agrobacterium GV3101 by electroporation.

[0045] Example 2 Agrobacterium-mediated genetic transformation

[0046] Planting of Arabidopsis materials: Sterilized Arabidopsis seeds were evenly spread on 1 / 2MS (Coolaber) medium for culture, vernalized at 4°C in dark for 3 days, and then moved to a growth box or growth room for culture. After the seeds germinated to the four-leaf stage, seedlings with consistent growth were selected and transplanted to new soil for further culture or related experimental operations were performed according to the experimental requirements. Culture conditions: 21°C, relative humidity of 60%, light intensity of 80 μmol m -2 s -1 , 16h light / 8h dark.

[0047] Genetic transformation of Arabidopsis: When Arabidopsis grows to the flowering stage, the floral dip method is used. The Agrobacterium strain containing the PeAREB1 gene is inoculated into LB medium, and kanamycin and rifampicin are added to culture in a shaking incubator at 28°C until the OD value is about 0.8. After centrifugation, the bacteria are resuspended in a 5% sucrose solution. Add 100ul of silwet-77 to 250ml of the resuspension solution, and select Arabidopsis plants with healthy flowering and well-developed inflorescences. The inflorescence should be in a state of not fully opening during the dip. Dip the inflorescence of Arabidopsis into the Agrobacterium suspension for generally 1 minute. Make sure the inflorescence is completely soaked and shake the plant gently so that the Agrobacterium can better contact the pollen mother cells. After the dip, the plant can be covered with a transparent plastic bag or lid to maintain humidity and temperature to help Agrobacterium infection. Dark treatment for 12h, after transformation, collect T0 generation seeds.

[0048] Screening of positive seedlings: 35S::PeAREB1-pBWA(V)HS-GFP was transformed into the wild-type Col-0 background to obtain overexpression strains and seeds of the strains, which were seeds of overexpression strains containing the PeAREB1 gene in the T0 generation. Screening of positive seedlings in the T1 generation was to select about 10 strains whose roots could grow normally on a 1 / 2MS culture medium plate containing hygromycin resistance, and transplant and harvest the seeds individually. Screening of positive seedlings in the T2 generation was to select strains with a positive rate of about 75% on a 1 / 2MS culture medium plate containing hygromycin resistance, and to determine positive seedlings by combining PCR amplification of the PeAREB1 gene (primers SEQ ID NO.3 and SEQ ID NO.4), and harvest seeds individually for each strain. Screening of positive seedlings in the T3 generation was to further select strains with a positive rate greater than 95% on a hygromycin plate, and to combine qRT-PCR detection of expression levels, and select three strains with relatively high expression levels for subsequent phenotypic observations.

[0049] Medium components and their formulations:

[0050] 1 / 2MS medium (1L): Weigh 2.156g MS and 10g sucrose, dissolve in 950mL deionized water, adjust the pH to 5.8 with 1mol / LKOH, add 0.5g Phytagel to every 250mL medium, and sterilize at 121℃ for 20min. After sterilization, add ampicillin (Amp) and fungal inhibitor (PPM) at a final concentration of 20mg / L when the medium is cooled to about 55℃, and pour into the culture dish. When the transgenic plants are screened later, add hygromycin at a final concentration of 25mg / L when the medium is cooled to about 55℃.

[0051] LB medium (1 L): Weigh 10 g of peptone, 5 g of yeast extract and 10 g of NaCl, dissolve in 950 mL of deionized water, mix well and make up to 1 L with deionized water, and sterilize at 121°C for 20 min.

[0052] LA medium (1L): Weigh 10g of peptone, 5g of yeast extract and 10g of NaCl, dissolve in 950mL of deionized water, mix well and dilute to 1L with deionized water. Add 3.75g of agar powder to every 250mL of medium and sterilize at 121℃ for 20min. Pour into the culture dish after cooling to about 55℃. Add kanamycin (Kan) and rifampicin (Rif) according to the experimental requirements, shake the medium thoroughly and pour into the culture dish.

[0053] Preparation of antibiotics: Kanamycin (Kan): 50 mg / mL, dissolved in water, sterilized by filtration; Rifampicin (Rif): 50 mg / mL, dissolved in DMSO, sterilized by filtration. Dispense equally into sterile EP tubes and store at -20°C for future use.

[0054] Preparation of 5×TEDA (10 ml): 0.25 g PEG8000, 5 ml 1 M Tris-HCl, 0.101 g MgCl2 (MV: 203.3), 0.5 ml 1 M DTT, 100 μl T5 exonuclease, stored at -80°C.

[0055] Example 3 Gene Expression Analysis

[0056] Each T3 positive seedling line obtained by positive seedling screening was planted separately. The leaves of these positive lines were cut and stored in liquid nitrogen. The total RNA was extracted using RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. The reverse transcriptase was used to reverse transcribe into cDNA according to the M5 Sprint qPCR RT kit with gDNA remover reverse transcription kit operating instructions.

[0057] The rice actin gene (Actin) was used as an internal reference gene, and the cDNA of the positive transgenic lines was amplified by PCR, and then qRT-PCR reaction analysis was performed.

[0058] The qRT-PCR reaction system was as follows: 2 μL cDNA (50 ng / μL), 5 μL Universal SYBR Green Supermix (Bio-Rad), 0.2 μL forward primer (FP, 10 μmol / L), 0.2 μL reverse primer (RP, 10 μmol / L), and ddH2O was added to 10 μL.

[0059] The reaction program was as follows using the Bio-Rad CFX96 Real-Time System: 95°C for 15 sec, 60°C for 15 sec, 72°C for 30 sec, 40 cycles. Finally, the comparative Ct method was used for data processing.

[0060] PeAREB1 expression detection primers (SEQ ID NO.5-6):

[0061] PeAREB1-qF(5'-CCATAGCAACAGCCACCTCT-3') and PeAREB1-qR(5'-TCAAGCACCGTTTCTTACCC-3');

[0062] Internal reference gene Actin detection primers (SEQ ID NO.7-8):

[0063] RT-Actin-F(5'-GCTAACCGTGAGAAGATGAC-3')

[0064] RT-Actin-R(5'-CTAGCATAAAGCGACAGGAC-3').

[0065] Using the expression level of PeAREB1 in wild-type Arabidopsis plants as a control, three independent strains with expression levels significantly higher than the control were screened. The test results are as follows: Figure 1 As shown, the expression levels of the PeAREB1 gene in the three Arabidopsis overexpression lines PeAREB1-OE-1, PeAREB1-OE-2 and PeAREB1-OE-3 were significantly higher than that in the wild type (Col-0), indicating that the PeAREB1 gene has been successfully heterologously transformed into Arabidopsis plants.

[0066] Example 3: Statistics of stomatal aperture in Arabidopsis epidermis

[0067] Use the scraping method: mark the experimental plants that are 2-3 weeks old to reduce errors caused by human factors during the experiment.

[0068] Take two leaves of the same period from the wild type and the three overexpression lines, stick the lower epidermis of the leaves to the transparent glue of the slides with the same label, scrape the mesophyll cells with a blade, leaving only the epidermal cells, and be as careful as possible when scraping to ensure the morphology of the guard cells. Add deionized water to the slide with the lower epidermal cells and cover it with a coverslip.

[0069] Observe the leaves under the objective lens of different magnifications of the optical microscope, such as Figure 2 As shown, the stomatal morphology in the middle of both sides of the main leaf veins was observed and photographed by adjusting the objective lens magnification according to experimental requirements and saved in TIF or JPG format.

[0070] Counting: Use the software Image J to count the length and width of the stomata in each photo.

[0071] like Figure 3 As shown in the figure, it was found that under drought stress, the epidermal stomatal aperture of the three PeAREB1 overexpression lines of Arabidopsis thaliana was significantly smaller than that of the wild type (Col-0).

[0072] 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. A drought-resistant Populus euphratica PeAREB1 gene, characterized in that: The nucleic acid sequence of the gene is shown in SEQ ID NO.

1.

2. The Populus euphratica PeAREB1 gene according to claim 1, characterized in that: The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. Use of the Populus euphratica PeAREB1 gene according to claim 1 in improving plant stress resistance.

4. The use according to claim 3, characterized in that: The stress resistance is drought tolerance.

5. A recombinant vector or expression cassette for improving plant stress resistance, characterized in that: The recombinant vector or expression cassette contains the coding region sequence of the Populus euphratica PeAREB1 gene, and the nucleic acid sequence of the Populus euphratica PeAREB1 gene is shown as SEQ ID NO.

1.

6. A recombinant bacterium for improving plant stress resistance, characterized in that: The recombinant bacteria contains the Populus euphratica PeAREB1 gene; the nucleic acid sequence of the Populus euphratica PeAREB1 gene is shown in SEQ ID NO.

1.

7. A method for improving plant stress resistance, characterized in that: The Populus euphratica PeAREB1 gene is constructed into a plant expression vector and transformed into plants to be expressed in the plants; the nucleic acid sequence of the Populus euphratica PeAREB1 gene is shown in SEQ ID NO.

1.

8. The method for improving plant stress resistance according to claim 7, characterized in that: The stress resistance is drought tolerance.

9. The method for improving plant stress resistance according to claim 7, characterized in that: The plant is Arabidopsis thaliana.

10. Use of the gene according to any one of claims 1 to 2, the recombinant vector or expression cassette according to claim 5, or the recombinant bacterium according to claim 6 in regulating plant stomatal aperture.

Images