Populus euphratica peareb1 gene and application thereof in improving drought resistance of plants

Abstract

The application 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. The nucleic acid sequence of the Populus euphratica PeAREB1 gene is shown as SEQ ID NO. 1, and the amino acid sequence coded by the gene is shown as SEQ ID NO. 2. The gene is constructed into a plant expression vector, and is transformed into plants, so that the drought resistance of the plants can be improved, and the application prospect in breeding of plants with stress resistance is good.

Description

Technical Field

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

[0002] Drought is one of the major environmental stresses affecting plant growth and survival, posing a serious threat to agricultural production and ecosystem security, particularly in the context of intensifying global climate change. Drought stress leads to water loss in plants, directly impacting photosynthesis, nutrient absorption, cellular turgor, and metabolic activity, thereby reducing plant growth rate, yield, and viability. In severe cases, it can even lead to plant death.

[0003] Understanding how plants cope with water shortages and improving crop drought tolerance are 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 regulating stomatal aperture, activating antioxidant systems, and accumulating osmotic regulating substances. In order to ensure the normal growth and development of plants and cope with complex climatic conditions, breeding stress-tolerant plant varieties has become one of the main goals of scientific research. Scientists have already attempted to improve plant photosynthesis and drought resistance by regulating plant stomatal aperture, and to cultivate stress-resistant plants with high light efficiency and high water use efficiency.

[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 present invention provides a drought-resistant Populus euphratica PeAREB1 gene, the nucleic acid sequence of which 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, comprising 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, the above recombinant vector, the expression cassette 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 regulate 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 be used to 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

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

[0023] Figure 2 The following are images of stomata on the leaves of transgenic Arabidopsis thaliana plants and wild-type Arabidopsis thaliana under a microscope;

[0024] Figure 3 Stomatal aperture was measured at 40x magnification in the epidermis of mature leaves of Col-0 and three T3 generations of Arabidopsis plants overexpressing PeAREB1. Data are the mean plus standard error of 30 replicates. Stomatal aperture in the PeAREB1-overexpressing lines was significantly smaller than that in the wild type under drought stress. Data were analyzed using a one-way ANOVA. Significant differences are indicated by different letters. DETAILED DESCRIPTION

[0025] The present invention provides a Populus euphratica gene PeAREB1, the nucleotide sequence of which is shown in SEQ ID NO. 1 and is 1368 bp in length, and the encoded protein sequence is shown in SEQ ID NO. 2 and consists of 455 amino acids. The nucleotide sequence contains a stop codon, which does not translate into 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 isolation and cloning of target fragments, and the transformation of Arabidopsis thaliana by Agrobacterium inflorescence infection, followed by phenotypic identification of the transformed plants. Overexpression of the PeAREB1 gene can enhance the drought tolerance of Arabidopsis thaliana and increase the survival rate of Arabidopsis thaliana. This gene can be well applied in ecological restoration, plant drought resistance and genetic improvement.

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

[0032] The PeAREB1 gene of the present invention can be linked 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 through conventional biotechnology methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation, ultimately obtaining plants with improved drought resistance.

[0033] The expression vector including the PeAREB1 gene of the present application 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 application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

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

[0036] The production process, experimental method or detection method involved in the embodiments of the present application are all conventional methods in the prior art without special instructions, and the name and / or abbreviation thereof all belong to the conventional name in the art and are very clear and explicit in the related application field. The skilled person in the art can understand the conventional process steps and apply the corresponding equipment according to the name, and implement it according to the 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 application do not have special restrictions on the source, and are conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional methods well known to those skilled in the art.

[0038] Example 1: Construction of expression vector

[0039] The nucleotide sequence shown as SEQ ID NO. 1 is obtained by biological method. It is used as a template to perform PCR amplification by 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 ligated 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 sequenced vector was transformed into Agrobacterium GV3101 by electroporation.

[0045] Example 2 Agrobacterium-mediated genetic transformation

[0046] Planting of Arabidopsis thaliana: Sterilized Arabidopsis thaliana seeds were evenly spread on 1 / 2 MS (Coolaber) medium for culture. Vernalization was carried out at 4°C in the dark for 3 days. Then, the seeds were transferred to a growth chamber or growth chamber 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 experimental manipulation as required. Culture conditions: 21°C, relative humidity 60%, light intensity 80 μmol m -2 s -1 , 16h light / 8h dark.

[0047] Genetic transformation of Arabidopsis thaliana: When Arabidopsis thaliana reaches the peak flowering stage, the floral dip method is used. Agrobacterium tumefaciens carrying the PeAREB1 gene is inoculated into LB medium supplemented with kanamycin and rifampicin. Culture the medium in a shaking incubator at 28°C until the OD value is approximately 0.8. After centrifugation, the cells are harvested and resuspended in 5% sucrose solution. 100 μl of Silwet-77 is added to 250 ml of the resuspended medium. Select Arabidopsis thaliana plants with healthy flowering and well-developed inflorescences. The inflorescences should not be fully open during the inflorescence dip. Dip the Arabidopsis inflorescence into the Agrobacterium suspension for approximately 1 minute. Ensure the inflorescence is completely soaked and gently shake the plant to ensure better access to the pollen mother cells. After inoculation, cover the plant with a transparent plastic bag or lid to maintain humidity and temperature, facilitating Agrobacterium infection. Keep the plant in the dark for 12 hours. After transformation, harvest the T0 generation seeds.

[0048] Screening of positive seedlings: 35S::PeAREB1-pBWA(V)HS-GFP was transformed into wild type Col-0 background to obtain overexpression lines and the seed of the line containing PeAREB1 gene was T0 generation. The screening of T1 generation positive seedlings was to select about 10 lines with normal root growth on 1 / 2MS medium containing hygromycin resistance, and then the seed of each line was collected by transplanting and single plant. The screening of T2 generation positive seedlings was to select lines with about 75% positive rate on 1 / 2MS medium containing hygromycin resistance, and then the positive seedlings were determined by combining the method of PCR amplification of PeAREB1 gene (primers SEQ ID NO. 3 and SEQ ID NO. 4), and the seed of each line was collected by single plant. The screening of T3 generation positive seedlings was to further select lines with more than 95% positive rate on hygromycin plate, and then 3 lines with relatively high expression were selected for subsequent phenotype observation by combining qRT-PCR detection of expression level.

[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 value to 5.8 with 1mol / L KOH, add 0.5g Phytagel to each 250mL medium, and sterilize at 121℃ for 20min. After sterilization, when the medium is cooled to about 55℃, add ampicillin (Amp) and fungal inhibitor (PPM) to a final concentration of 20mg / L, and then pour into culture dishes. When screening transgenic plants in the follow-up, add hygromycin to a final concentration of 25mg / L when the medium is cooled to about 55℃.

[0051] LB medium (1L): weigh 10g peptone, 5g yeast extract and 10g NaCl, dissolve in 950mL deionized water, mix well, and then add deionized water to 1L, sterilize at 121℃ for 20min.

[0052] LA medium (1L): weigh 10g peptone, 5g yeast extract and 10g NaCl, dissolve in 950mL deionized water, mix well, and then add deionized water to 1L. Add 3.75g agar powder to each 250mL medium, sterilize at 121℃ for 20min. Cool to about 55℃ before pouring into culture dishes. Then add kanamycin (Kan) and rifampicin (Rif) according to experimental requirements, shake the medium well, and then pour into culture dishes.

[0053] Antibiotic preparation: kanamycin (Kan): 50mg / mL, water soluble, filter sterilization; rifampicin (Rif): 50mg / mL, dissolved in DMSO, filter sterilization. Divide and store in sterile EP tubes at -20℃ for standby.

[0054] 5xTEDA (10 ml) preparation: 0.25 g PEG8000, 5 ml 1M Tris-HCl, 0.101 g MgCl2(MW: 203.3), 0.5 ml 1M DTT, 100 μl T5 exonuclease, -80°C storage.

[0055] Example 3 Gene expression analysis

[0056] T3 generation positive seedlings obtained by positive seedling screening, each strain was planted respectively. The leaves of these positive strains were cut and stored in liquid nitrogen. The total RNA was extracted by RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit. The cDNA was reverse transcribed under the action of reverse transcriptase according to the operation instruction of M5 Sprint qPCR RT kit with gDNA remover reverse transcription kit.

[0057] The actin gene of rice was used as an internal reference gene, and the cDNA of the positive transgenic strain was PCR amplified and then subjected to qRT-PCR reaction analysis.

[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] Bio-Rad CFX96 Real-Time System was used, and the reaction program was as follows: 95°C for 15 sec, 60°C for 15 sec, 72°C for 30 sec, 40 cycles. Finally, 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] 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 lines 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 those 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 Size 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 from the wild-type and three overexpressing strains at the same stage of development. Attach the lower epidermis of each leaf to transparent tape on a similarly labeled slide. Use a razor blade to scrape away the mesophyll cells, leaving only the epidermal cells. Be extremely careful when scraping to preserve the guard cell morphology. Add deionized water to the slide with the lower epidermal cells and cover 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 stomata 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 provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. An application of the Populus euphratica PeAREB1 gene in improving drought tolerance in Arabidopsis thaliana, characterized in that: The nucleic acid sequence of the Populus euphratica PeAREB1 gene is shown in SEQ ID NO.

1.

2. A method for improving drought tolerance of Arabidopsis thaliana, characterized in that: The method comprises constructing the Populus euphratica PeAREB1 gene into an expression vector pBWA(V)HS-GFP, and infecting Arabidopsis thaliana inflorescences with host cell Agrobacterium GV3101 to express the Populus euphratica PeAREB1 gene in the plant Arabidopsis thaliana; the nucleic acid sequence of the Populus euphratica PeAREB1 gene is shown in SEQ ID NO.

1.

3. Application of the Populus euphratica PeAREB1 gene in reducing stomatal aperture in Arabidopsis thaliana, characterized in that: The nucleic acid sequence of the Populus euphratica PeAREB1 gene is shown in SEQ ID NO.1.

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