Application of a gene AT2G28200 in improving plant drought resistance

Abstract

The present invention belongs to the field of plant genetic engineering, and specifically relates to the application of AT2G28200 gene in improving plant drought resistance. The present invention uses a genetic transformation method to overexpress the AT2G28200 gene in Arabidopsis thaliana. After biological function verification, the drought resistance of the transgenic plant is significantly improved, the drought resistance of the plant can be effectively improved, the water consumption of the plant can be effectively reduced in production, and a new gene resource is provided for plant drought resistance molecular breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to the application of a segment of AT2G28200 gene in improving the drought resistance of plants. Background Art

[0002] Climate change and water shortage caused by human activities have posed challenges to global agricultural production. This situation may become more serious in the next 30 years. It is estimated that by 2050, more than 50% of the earth's areas will experience water shortages and droughts. Abiotic stresses such as drought can seriously affect the growth and development of plants, resulting in significant losses in global agriculture. Therefore, improving the drought resistance / tolerance of crops themselves and breeding drought-resistant / tolerant crop varieties have become the key to reducing agricultural losses and an important way to achieve sustainable agricultural development. However, since the molecular and genetic mechanisms of plant drought resistance / tolerance have not been fully elucidated, drought-resistant / tolerant crop varieties are scarce, which limits the cultivation and application of drought-resistant / tolerant varieties. Therefore, in-depth exploration of drought resistance / tolerance-related genes has important theoretical significance and practical value for the cultivation and breeding of drought-resistant crop varieties.

[0003] During the evolution process, plants have formed a complex regulatory network to cope with drought, which mainly includes drought avoidance, drought resistance, drought tolerance and drought recovery after stress. Among them, drought resistance and drought tolerance are the main mechanisms to improve plant drought resistance. "Drought avoidance" refers to plants avoiding adversity by adjusting their growth cycle and completing their growth and reproduction in a relatively adaptive environment. "Drought resistance" refers to plants coping with drought by adjusting morphological and physiological characteristics such as root growth, leaf structure, stomatal movement, photosynthesis, etc., optimizing water absorption and reducing water loss. "Drought tolerance" means that plants maintain cell expansion and remove harmful substances through osmotic regulation and antioxidant defense systems, thereby enduring moderate and severe drought. In the drought stress response, many drought response genes are activated and participate in a complex transcriptional regulatory network.

[0004] Transcription factors (TFs) such as DREB, ERF, MYB, WRKY and zinc finger transcription factors play an important role in plant growth regulation by regulating the expression of downstream genes and responding to changes in the external environment. Among them, zinc finger protein transcription factors are very abundant in plants. Literature reports show that plants such as Arabidopsis, petunia, wheat, cotton, soybean and rice all contain zinc finger protein transcription factors. Arabidopsis contains 176 C2H2-type zinc finger proteins, which are divided into three subfamilies: A, B and C. They play an important role in plant growth and development. Since multiple factors synergistically affect plant growth and development, the role of specific zinc finger proteins in plant growth and development and plant stress and drought resistance has not yet been fully elucidated.

[0005] Patent 103361359A "A gene of C2H2 zinc finger transcription factor of Alpine mustard, amino acid sequence of the encoded product" discloses "a nucleotide sequence of C2H2 zinc finger transcription factor FZF gene of Alpine mustard", which can improve the frost resistance of plants. However, there are few reports on genes that can improve the drought resistance of plants. Therefore, it is necessary to provide a gene for plant drought resistance to improve the drought resistance of plants. Summary of the invention

[0006] In order to solve the above technical problems, the inventors discovered and determined the drought resistance effect of the AT2G28200 gene, and applied the AT2G28200 gene to transgenic plants to improve the drought resistance of plants.

[0007] After long-term screening, the inventors found a AT2G28200 gene in Arabidopsis. The gene was cloned and in-depth research showed that after Arabidopsis overexpressed the AT2G28200 gene, its tolerance to drought stress was significantly enhanced; and the tolerance of plants with AT2G28200 gene mutation to drought stress was significantly reduced, indicating that the AT2G28200 gene of the present invention has the function of improving plant drought tolerance, can provide new gene resources for molecular breeding of stress-resistant crops, and can be used to cultivate new stress-resistant varieties and new materials, showing important value in the field of plant genetic engineering.

[0008] The AT2G28200 gene is derived from Arabidopsis thaliana and has the function of drought stress resistance.

[0009] To achieve the above object, the present invention is specifically implemented by the following technical solutions. Application of AT2G28200 gene in improving plant drought resistance, the nucleotide sequence of the AT2G28200 gene is shown in SEQ ID NO.1. The inventor cloned the coding region sequence of the AT2G28200 gene shown in SEQ ID NO.1, and proved through experiments that it has the ability to improve plant drought resistance.

[0010] SEQ ID NO.1:

[0011] ATGATGATGGGTCAAGATGAGGTTGGGAGTGATCAGACGCAAATCATAAAAGGGAAACGTACGAAGCGACAAAGATCGTCTTCGACGTTTGTGGTGACGGCGGCGACAACAGTGACTTCAACAAGTTCATCGGCCGGTGGAAGTGGAGGAGAAAGAGCTGTTTCAGATGAATACAACTCGGCGGTTTCGTCTCCGGTGACTACTGATTGTACGCAAGAAGAAGAAGACATGGCGATTTGTCTCATCATGTTAGCTCGTGGGACAGTTCTTCCATCGCCGGATCTCAAGAACTCGAGAAAAATTCATCAGAAGATTTCGTCGGAGAATTCTAGTTTCTATGTGTACGAGTGTAAAACGTGTAACCGGACGTTTTCGTCGTTCCAAGCACTTGGTGGACACAGAGCGAGCCACAAGAAGCCGAGGACGTCGACTGAGGAAAAGACTAGACTACCCCTGACGCAACCCAAGTCTAGTGCATCAGAAGAAGGGCAAAACAGTCATTTCAAAGTTTCCGGCTCAGCCCTAGCTTCACAGGCAAGTAACATCATCAACAAGGCAAACAAAGTACACGAGTGTTCCATCTGCGGTTCTGAGTTCACTTCCGGGCAAGCTCTCGGTGGTCACATGAGGCGGCACAGGACAGCCGTAACCACGATTAGCCCCGTTGCAGCCACCGCAGAAGTAAGCAGAAACAGTACAGAGGAAGAGATTGAGATCAATATAGGCCGTTCGATGGAACAGCAGAGGAAATATCTACCGTTGGATCTTAATCTACCAGCACCAGAAGATGATCTAAGAGAGTCAAAGTTTCAAGGGATAGTATTCTCAGCAACACCAGCGTTAATAGATTGTCATTACTAG

[0012] Preferably, the amino acid sequence of the protein encoded by the AT2G28200 gene is shown in SEQ ID NO.2.

[0013] SEQ ID NO.2:

[0014] MMMGQDEVGSDQTQIIKGKRTKRQRSSSTFVVTAATTVTSTSSSAGGSGGERAVSDEYNSAVSSPVTTDCTQEEEDMAICLIMLARGTVLPSPDLKNSRKIHQKISSENSSFYVYECKTCNRT FSSFQALGGHRASHKKPRTSTEEKTRLPLTQPKSSASEEGQNSHFKVSGSALASQASNIINKANKVHECSICGSEFTSGQALGGHMRRHRTAVTTISPVAATAEVSRNSTEEEIEINIGRSMEQ QRKYLPLDLNLPAPEDDLRESKFQGIVFSATPALIDCHY

[0015] A method for improving plant drought resistance, comprising constructing a transgenic plant using the AT2G28200 gene of claim 1. The AT2G28200 gene is expressed or overexpressed in the plant by transgenic means to increase the expression level and / or activity of the AT2G28200 gene, thereby obtaining a transgenic plant; the transgenic plant has higher drought resistance than the recipient plant.

[0016] Preferably, a specific primer pair is used to amplify the AT2G28200 gene, and the sequences of the specific primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0017] SEQ ID NO.3:

[0018] Upstream primer: ATGATGATGGGTCAAGATGAGGTTG

[0019] SEQ ID NO.4:

[0020] Downstream primer: GTAATGACAATCTATTAACGCTGGTGTT

[0021] Preferably, the AT2G28200 gene is overexpressed using a recombinant vector, wherein the recombinant vector contains the nucleotide sequence shown in SEQ ID NO.1.

[0022] Preferably, the basic vector of the recombinant vector includes pCAMBIA1391 vector, and the AT2G28200 gene is inserted between the restriction sites of BamH I of the expression vector.

[0023] Preferably, the recombinant vector is introduced into cells to form transformants.

[0024] Preferably, the transformant is Agrobacterium tumefaciens and / or a plant cell, and the organism is transgenic Arabidopsis thaliana.

[0025] Preferably, the recipient plant is Arabidopsis, petunia, wheat, cotton, soybean or rice.

[0026] A primer pair for amplifying the above AT2G28200 gene, wherein the specific primer pair sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, and are used for amplifying the gene shown in SEQ ID NO.1.

[0027] A recombinant vector for improving the drought resistance of plants, the recombinant vector contains the nucleotide sequence shown as SEQ ID NO.1.

[0028] A transformant for improving the drought resistance of plants, the transformant contains the above recombinant vector.

[0029] Through the above technical scheme, the beneficial effects achieved by the present invention are:

[0030] The present invention provides a drought-resistant AT2G28200 gene, which can be overexpressed in Arabidopsis thaliana by genetic transformation, significantly improving the drought resistance of plants, effectively improving the drought resistance of plants, effectively reducing plant water consumption in production, and providing new gene resources for plant drought-resistant molecular breeding. Genetic engineering methods can be used to increase the expression of the gene in other crops, thereby improving the drought resistance of other crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The AT2G28200 gene cloning result provided by the present invention;

[0032] Figure 2 The AT2G28200 gene overexpression vector map provided by the present invention;

[0033] Figure 3 The positive seedling identification result of Arabidopsis thaliana T1 generation plants provided by the present invention;

[0034] Figure 4 The quantitative results of the AT2G28200 positive plants provided by the present invention;

[0035] Figure 5 The phenotypic results of the AT2G28200 positive transgenic plants provided by the present invention under simulated drought stress treatment;

[0036] Figure 6 The root length statistics of the AT2G28200 positive transgenic plants provided by the present invention under simulated drought stress treatment;

[0037] Figure 7 This is the drought-resistant phenotype result of the AT2G28200-positive transgenic plants provided by the present invention. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with specific embodiments. The given embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention in any way.

[0039] The experimental methods in the following examples are all conventional experimental methods unless otherwise specified. The reagents and consumables used in the following examples are all commercially available products unless otherwise specified.

[0040] Example 1: Acquisition of AT2G28200 gene

[0041] Take young leaves of Arabidopsis thaliana, use a plant genomic RNA extraction kit to extract total RNA, detect it by agarose gel electrophoresis and detect the concentration, then use a reverse transcription kit to reverse transcribe the RNA into cDNA, and store it at -20°C for later use. Using the obtained Arabidopsis thaliana genomic DNA as a template, use the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4, the upstream primer is: ATGATGATGGGTCAAGATGAGGTTG; the downstream primer is: GTAATGACAATCTATTAACGCTGGTGTT, and the AT2G28200 gene is obtained by PCR amplification. The PCR amplification reaction conditions are: 94°C pre-denaturation for 3min, 94°C denaturation for 15min, 56°C annealing for 15s, 72°C extension for 1min, a total of 30 cycles, and store at 4°C. The PCR product is detected by electrophoresis ( Figure 1 ) and sent to BGI for verification. After sequencing comparison, we cloned the AT2G28200 gene as shown in SEQ ID NO.1.

[0042] Example 2: Construction and genetic transformation of plant overexpression vector

[0043] 2.1 Construction of overexpression vector

[0044] The AT2G28200 gene obtained in Example 1 was inserted into the plant binary expression vector pCAMBIA1391 by homologous recombination to construct the pCAMBIA1391-ZAT5 recombinant expression vector ( Figure 2 ). The specific process is as follows:

[0045] ① Use BamH I (Takara) to digest the expression vector and purify it with a gel recovery kit to obtain a linearized expression vector. Mix the target gene and the linearized vector at a molar ratio of 3:1, place at 37°C for about 30 minutes for recombination reaction, and then proceed to the next transformation experiment.

[0046] ② Take 10μL of the reaction solution and add it to 50μL of DH5α competent cells. Mix gently and incubate on ice for 30min. Heat shock at 42℃ for 45 seconds and then quickly cool on ice for 2min. Then add 800μL of SOD liquid medium and incubate at 37℃ for 1h. Centrifuge at 4500rpm for 2min, collect the cells, evenly spread them on LB solid medium, and culture them in a 37℃ incubator overnight.

[0047] ③ Pick several clones on the transformation plate and identify positive clones by colony PCR, then pick the corresponding single clone in LB liquid medium (containing Kana antibiotics), culture overnight at 200rpm and 37℃ for plasmid extraction. Further identify the accuracy of the vector by plasmid PCR and sequencing.

[0048] 2.2 Genetic transformation

[0049] The pCAMBIA1391-ZAT5 recombinant expression vector obtained above was introduced into Agrobacterium GV3101, and then transformed into Arabidopsis plants. The specific process is as follows:

[0050] ① Take about 1μg of the recombinant expression vector and add it to 100μl of Agrobacterium competent cells, mix well and place in an ice bath for 30 minutes. Then freeze in liquid nitrogen for 1 minute, transfer to a 37℃ water bath for 5 minutes, and quickly place in an ice bath for 2 minutes. Add 800μl of YEP liquid culture medium, shake and culture at 220rpm and 28℃ for 4 to 6 hours. Centrifuge at 4500rpm for 1min, take an appropriate amount of bacterial liquid and spread it on the YEP solid culture medium containing the corresponding antibiotics (50μg / ml of Kana and 50mg / l of rifampicin), and culture it upside down at 28℃ for about 2 to 3 days.

[0051] ② Pick several positive clones for colony PCR verification, then pick the corresponding single colony in YEP liquid medium, culture in the dark at 28℃ and 220rpm, and then expand the culture until OD600 is 0.8. Use 5% sucrose solution (containing 0.05% Silwet L-77) to prepare the resuspension solution, resuspend the bacterial solution and mix well for later use.

[0052] ③Use Arabidopsis seeds in advance, disinfect with sodium hypochlorite, sow on seedling medium, and transfer to soil one week later. After flowering, use the inflorescence infection method to infect with the above-mentioned resuspended Agrobacterium transferred with the pCAMBIA1391-ZAT5 expression vector. After infection, the plants are cultured in the dark in a tissue culture incubator (24°C, 16h / 8h day / night cycle) for one day, and then transferred to normal conditions for growth.

[0053] 2.3 Screening of T3 transgenic positive plants

[0054] After harvesting the seeds of the transgenic infected plants, they were first disinfected with sodium hypochlorite and then screened for resistance on a medium containing 15 mg / L hygromycin. Plants that turned yellow and gradually died were non-transgenic plants, and seedlings that grew green true leaves and took roots were selected for planting. Figure 3 The plants with numbers are positive plants, and the wild type (WT) plants have no vectors, so there are no bands), and false positive plants are further eliminated. Continuous planting and identification are carried out to finally obtain T3 generation positive transgenic plants.

[0055] 2.4 Quantitative expression verification of T3 transgenic positive plants

[0056] To further verify the expression of AT2G28200 in the positive seedlings of the genome, three plants were selected from the positive seedlings identified above, and the wild type (WT) was used as the control. The total RNA of the leaves was extracted using an RNA extraction kit, and then the cDNA was obtained using a reverse transcription kit (Quanshijin Biotechnology Co., Ltd.). The cDNA was used as a template and Arabidopsis ACTIN2 was used as an internal reference for qRT-PCR expression verification. The qRT-PCR primer pair for the Arabidopsis ACTIN2 internal reference was: actin-F: 5'-ACATTGTGCTCAGTGGTGGA-3'; actin-R: 5'-CTTGGTGCAAGTGCTGTGAT-3'; the quantitative qRT-PCR primer pair for the target gene ZAT5 was: ZAT5-F: 5'-GCTCAGCCCTAGCTTCACAG-3'; ZAT5-R: 5'-TGAACTCAGAACCGCAGATG-3'. The expression of the AT2G28200 gene in the transgenic plants was detected using the non-transgenic wild type Arabidopsis as the control. The results are as follows Figure 4 Among the three Arabidopsis transgenic lines tested, the expression of the AT2G28200 gene in Line 1 plants was about 17 times that of the control plants, and the expression of the AT2G28200 gene in Line 2 and Line 3 plants was about 15 times that of the control plants, indicating that the expression level of the AT2G28200 gene in the overexpression transgenic plants was significantly increased.

[0057] Example 3: Drought tolerance test

[0058] 3.1 Drought tolerance testing of ZAT5-overexpressing transgenic plants

[0059] The drought stress experiment was carried out using the T3 generation positive transgenic plants overexpressing the AT2G28200 gene in Example 3. T-DNA was purchased from the NASC website and inserted into the AT2G28200 gene to obtain an Arabidopsis loss-of-function mutant, which was used as a negative control (labeled as ZAT5); Arabidopsis Columbia Col-0 plants (labeled as WT) were used as normal controls.

[0060] We used high-salt medium and mannitol hypertonic medium to simulate drought treatment, and the seeds of wild type (marked as WT), mutant (marked as ZAT5) and overexpression strain (marked as ZAT5-OX) were surface-sterilized and sown on 1 / 2MS medium containing 250mM NaCl or 250mM mannitol, and placed in a plant culture room at 23±1℃ with a photoperiod of 16 hours light and 8 hours dark for 2 to 3 weeks for drought tolerance testing. Figure 5 Under NaCl or mannitol stress, plant growth was inhibited to varying degrees, but the root length of ZAT5 overexpressing transgenic plants was inhibited less than that of the zat5 mutant and was slightly longer than the control, indicating that the AT2G28200 gene has a certain drought resistance function.

[0061] 3.2 Root length detection of ZAT5 overexpressing transgenic plants under drought stress

[0062] The root length of plants under salt stress was statistically analyzed. Compared with WT and ZAT5 transgenic plants grown on normal medium, the root length of WT was 85% of the normal state under 250mM NaCl stress and 43% of the normal state under 250mM mannitol stress; while the root length of AT2G28200 gene mutant strains under 250mM NaCl and 250mM mannitol stress was only 52% and 19% of the normal strain, respectively. The root length of ZAT5 overexpression strains was comparable to that of WT, thus reflecting a certain degree of drought tolerance ( Figure 6 ).

[0063] 3.3 Drought resistance testing of ZAT5-overexpressing transgenic plants

[0064] The T3 generation positive transgenic plants with overexpression of AT2G28200 gene in Example 3 were used for drought stress experiment. T-DNA was purchased from NASC website and inserted into AT2G28200 gene to obtain Arabidopsis functional loss mutant, which was used as negative control; Arabidopsis Columbia Col-0 plant (WT) was used as normal control. The wild type (marked as WT), mutant (marked as ZAT5) and overexpression strain (marked as ZAT5-OX) were sown, and after normal growth for 3 weeks, water control treatment was carried out for 14 days and then re-watered to observe the growth of transgenic plants.

[0065] The results show that if Figure 7 As shown in the figure, the transgenic plants overexpressing the AT2G28200 gene had no significant difference in growth compared with the control plants under normal conditions. After 14 days of drought stress and rehydration, the growth of the mutant plants did not improve, and the leaves of the wild-type control plants and the ZAT5 overexpressing transgenic lines began to turn green. The survival rates of the control plants were statistically analyzed. After the stress, the control plants were severely wilted and damaged. After 2 weeks of drought stress and rehydration, no mutant plants survived, while the survival rate of the wild-type control plants was only 10%, and the AT2G28200 gene overexpression line was as high as 41%. The results showed that the drought resistance of the AT2G28200 transgenic Arabidopsis lines was significantly higher than that of the wild-type Arabidopsis control, and overexpression of the AT2G28200 gene could improve the drought tolerance of plants.

[0066] The AT2G28200 gene can also be overexpressed in other crops through genetic engineering and transgenic methods, which can improve the drought resistance of crops such as petunia, wheat, cotton, soybeans or rice.

[0067] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for improving drought resistance of Arabidopsis thaliana, characterized in that: A transgenic plant is constructed using the AT2G28200 gene with a nucleotide sequence as shown in SEQ ID NO.1; the AT2G28200 gene is amplified using a specific primer pair, and the DNA sequence of the specific primer pair is shown in SEQ ID NO.3 and SEQ ID NO.4; the amplified product is connected into a plant recombinant vector by homologous recombination; the recombinant vector comprises the nucleotide sequence as shown in SEQ ID NO.1, the recombinant vector comprises a basic vector, the basic vector is a pCAMBIA1391 vector, and the AT2G28200 gene is inserted between the restriction sites of BamH I of the recombinant vector.

2. Use of a gene segment AT2G28200 in improving drought tolerance in Arabidopsis thaliana, characterized in that: The nucleotide sequence of the AT2G28200 gene is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that: The amino acid sequence of the protein encoded by the AT2G28200 gene is shown in SEQ ID NO.2.

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