Application of glutathione S-transferase gene ZmGST30 in drought resistance of corn

Abstract

The invention discloses application of glutathione S-transferase gene ZmGST30 in corn drought resistance, the ZmGST30 gene in corn is over-expressed to obtain an over-expressed plant, and drought phenotype observation and physiological index determination are performed on the ZmGST30 gene over-expressed plant to determine the drought resistance of corn. Results show that the relative water content of ZmGST30 overexpressed plant leaves is increased, the sensitivity of leaf stomata to the process of promoting stomata closing by abscisic acid (ABA) is increased, the water loss rate of in-vitro leaves is reduced, the drought resistance is remarkably enhanced, the ROS removal capacity of the ZmGST30 overexpressed plant is improved, and the H2O2 content is reduced and the ion permeability is reduced under drought stress. The gene positively regulates and controls drought stress, and the drought resistance of the corn can be remarkably improved by overexpression of the gene. According to the invention, ZmGST30 gene overexpression and ZmGST30 gene mutant strains are obtained by adopting a genetic engineering means, the effect of the ZmGST30 gene in regulating the drought resistance of the corn is clarified, a gene resource is provided for cultivating the drought-resistant corn, and a theoretical basis is provided for illuminating the effect of the ZmGST30 gene in coping with drought stress of plants.

Description

Technical Field

[0001] The invention relates to the technical field of biological gene engineering, and in particular to the application of glutathione S-transferase gene ZmGST30 in corn drought resistance. Background Art

[0002] Glutathione S-transferase (GST) is a multifunctional super-large family, which is widely present in microorganisms, animals and plants in the form of homologous or heterodimers. It participates in the detoxification of exogenous and endogenous compounds by catalyzing the toxic secondary metabolites produced in the organism and the electrophilic groups of foreign toxic substances to form complexes with the sulfhydryl groups of reduced glutathione and then excreted from the body. Excessive ROS such as hydrogen peroxide, superoxide and hydroxyl free radicals can cause programmed cell death by destroying the structure or function of lipids, proteins, carbohydrates and DNA, and have adverse effects on the growth and development of plants. Soybean nodules are rich in GSTs, which respond to ROS, regulate ROS homeostasis, and act as antioxidants to mark and remove oxidative degradation products.

[0003] GSTs not only play an important role in normal cell metabolism and detoxification of heterogeneous compounds, but can also be induced under different biological or abiotic stresses, thereby improving the ability of plants to tolerate adverse environmental conditions and resist damage. Overexpression of the rice OsGSTU4 gene in Arabidopsis can improve Arabidopsis' tolerance to salt stress and oxidative stress; overexpression of the VvGST13 gene in grapes increases the activity of SOD and POD in the plant, while reducing MDA content and electrolyte leakage, enhancing stress resistance and ROS scavenging ability or maintaining ROS homeostasis by increasing the activity of antioxidant enzymes.

[0004] At present, there have been some reports on the function of GST in corn stress, but most of them focus on the ability of GST to remove reactive oxygen in the body, and it is still unclear how it participates in drought stress response. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide an application of the glutathione S-transferase gene ZmGST30 in corn drought resistance. Using corn ND101 as a material and using transgenic technology, the ZmGST30 gene in corn is overexpressed to obtain overexpressed plants. The relative water content of leaves of ZmGST30 overexpressed plants is increased, the sensitivity of leaf stomata to the stomatal closure process promoted by abscisic acid (ABA) is increased, the water loss rate of detached leaves is reduced, and the drought resistance is significantly enhanced. In addition, the ROS scavenging ability of ZmGST30 overexpressed plants is improved, which is manifested by the reduction of H2O2 content and ion permeability under drought stress.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] Application of glutathione S-transferase gene ZmGST30 or its encoded protein or biological material containing its encoded gene in improving drought resistance of corn, wherein the nucleotide sequence of the glutathione S-transferase gene ZmGST30 is any one of the following:

[0008] A1) the nucleotide sequence shown in Seq ID No.1;

[0009] A2) a nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added as the nucleotide sequence shown in SEQ ID No. 1 and the nucleotide sequence expresses a protein with the same function;

[0010] A3) A nucleotide sequence that hybridizes to the sequence shown in Seq ID No. 1 under stringent conditions.

[0011] Application of glutathione S-transferase gene ZmGST30 in breeding new drought-resistant corn varieties,

[0012] The nucleotide sequence of the glutathione S-transferase gene ZmGST30 is any one of the following:

[0013] A1) the nucleotide sequence shown in Seq ID No.1;

[0014] A2) a nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added as the nucleotide sequence shown in SEQ ID No. 1 and the nucleotide sequence expresses a protein with the same function;

[0015] A3) A nucleotide sequence that hybridizes to the sequence shown in Seq ID No. 1 under stringent conditions.

[0016] Preferably,

[0017] The nucleotide sequence of the transcript of the glutathione S-transferase gene ZmGST30 is shown in Seq ID No.2, and the amino acid sequence of the encoded protein is shown in Seq ID No.3;

[0018] Or the nucleotide sequence of the transcript of the glutathione S-transferase gene ZmGST30 is shown in Seq ID No.4, and the amino acid sequence of the encoded protein is shown in Seq ID No.5.

[0019] Preferably,

[0020] The biological material is any one of the following B1) to B9):

[0021] B1) a nucleic acid molecule encoding the protein according to claim 1;

[0022] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0023] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0024] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0025] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0026] B6) transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0027] B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

[0028] The primer pair sequences for amplifying the glutathione S-transferase gene ZmGST30 are shown in Seq ID No.7 and Seq ID No.8.

[0029] Preferably,

[0030] The drought resistance of corn is improved by increasing the expression level of glutathione S-transferase gene ZmGST30.

[0031] Preferably,

[0032] The ZmGST30 gene in corn was overexpressed to obtain overexpression plants, which improved the drought resistance of corn, including: the relative water content of leaves of ZmGST30 overexpression plants increased; the sensitivity of leaf stomata to the stomatal closure process promoted by abscisic acid increased; the water loss rate of detached leaves decreased; and the ROS scavenging ability of ZmGST30 overexpression plants was improved, which was manifested by the decrease of H2O2 content and ion permeability under drought stress.

[0033] A method for improving drought resistance of corn,

[0034] By increasing the expression of glutathione S-transferase gene ZmGST30, the drought resistance of corn is improved;

[0035] The nucleotide sequence of the glutathione S-transferase gene ZmGST30 is any one of the following:

[0036] A1) the nucleotide sequence shown in Seq ID No.1;

[0037] A2) a nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added as the nucleotide sequence shown in SEQ ID No. 1 and the nucleotide sequence expresses a protein with the same function;

[0038] A3) A nucleotide sequence that hybridizes to the sequence shown in Seq ID No. 1 under stringent conditions.

[0039] The beneficial effects of the present invention are:

[0040] In the examples of the present invention, genetic engineering methods were used to obtain a ZmGST30 gene overexpression strain, clarifying the role of the ZmGST30 gene in regulating corn drought resistance, and improving corn tolerance to drought stress through genetic modification, providing genetic resources for obtaining corn materials with high drought resistance through genetic improvement methods, and also providing a theoretical basis for clarifying the role of the ZmGST30 gene in plant response to drought stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0042] Figure 1 Figure 2 shows the expression level detection results of ZmGST30 gene overexpression plants

[0043] Figure 2 Phenotypes of ZmGST30 gene overexpressing plants and wild type under drought treatment

[0044] Figure 3 The relative water content of leaves of ZmGST30 gene overexpressing plants and wild type plants

[0045] Figure 4 Figure 2 shows the stomatal aperture of ZmGST30 gene overexpressing plants and wild type plants.

[0046] Figure 5 The water loss rate of detached leaves of ZmGST30 gene overexpressing plants and wild type

[0047] Figure 6 H2O2 content in ZmGST30 gene overexpressing plants and wild type

[0048] Figure 7 Figure 2 shows the ion leakage rate of ZmGST30 gene overexpressing plants. DETAILED DESCRIPTION

[0049] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the following embodiments are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0050] The maize ZmGST30 gene involved in the present invention has a gene number of Zm00001eb021720 in Zeamays (B73_RefGen_v5), a gene number of Zm00001d029708 in Zeamays (B73_RefGen_v4), and a version encoding of GRMZM2G044383 in Zea mays (B73_RefGen_v3). The ZmGST30 gene Genomic DNA consists of 1,305 bases, with a total of 2 transcripts. The gene has 1 exon and 2 introns, and its nucleotide sequence is shown in Seq ID No.1.

[0051] The present invention provides an isolated nucleic acid molecule comprising a sequence selected from the group consisting of:

[0052] (1) The nucleotide sequence shown in Seq ID No. 1, the different transcripts generated by it and the amino acid sequence tables of the encoded proteins are shown in Seq ID No. 2-5, and the actual insertion sequence for constructing the overexpression material is shown in Seq ID No. 6.

[0053] (2) Different transcripts generated by the nucleotide sequence shown in Seq ID No.1 and the amino acid sequences of their encoded proteins.

[0054] (3) The nucleotide sequence shown in Seq ID No. 1 is replaced, deleted and / or added with one or more nucleotides to express the same or functionally deficient or mutated protein.

[0055] (4) A nucleotide sequence that hybridizes with the sequence shown in Seq ID No. 1 under stringent conditions.

[0056] Since the same DNA sequence in corn can produce different transcripts and translate into different proteins, the different transcripts produced by this sequence and the different proteins translated are all within the scope of protection of this patent.

[0057] According to the nucleotide sequence disclosed in the present invention, the ZmGST30 gene is overexpressed in corn through transgenic technology to obtain plants with improved drought resistance at the seedling stage, thereby providing gene resources for breeding new varieties of drought-resistant plants.

[0058] The maize recipient material inbred line used in the following examples is ND101; the Agrobacterium strain is GV3101; the cDNA of B73 as a PCR template and the pSuper1300:GFP as a vector were provided by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.

[0059] Example 1 Obtaining ZmGST30 gene overexpressing corn plants

[0060] 1. Construction and detection of ZmGST30 gene overexpression vector

[0061] Total RNA was extracted from B73 corn (Zea mays L.), and cDNA was obtained by reverse transcription. Using cDNA as a template, the sequences of the designed primers are shown in Seq ID No.7 and Seq ID No.8. The CDS region of the target gene ZmGST30 was amplified using upstream and downstream primers and high-fidelity enzymes. The PCR system was (50 μL): 5 μL 5×Phusion PCR Buffer, 4 μL 2.5 mM dNTPmix, 2.5 μL Primer 1 (10 μM), 2.5 μL Primer 2 (10 μM), 1 μL template DNA, 1.5 μL DMSO, 0.5 μL Phusion DNA Polymerase (2 U / μL). PCR program: 98℃ pre-denaturation for 3 minutes; 98℃ for 20 seconds, 60℃ for 20 seconds, 72℃ for 2 minutes and 30 seconds, 35 cycles; 72℃ extension for 5 seconds, and PCR products were stored at 10℃. The PCR product was purified using 1% agarose gel and gel recovery, and the purified fragment was connected to the constructed pBCXUN:GFP vector with Ubiquitin promoter by TA cloning, and the T4 ligase was used for ligation reaction at 16°C for 8 hours. The clone construction was completed by heat-stimulated transformation of Escherichia coli and screening of positive clones. The constructed plasmid pBCXUN:GFP-ZmGST30 was sequenced, and the sequencing results and target sequences were compared and analyzed, and the vector containing the complete and correctly sequenced target gene sequence was selected for subsequent experiments.

[0062] Primer-F: 5'-CGCCATAAACGAGTCCCA-3' (Seq ID No. 7)

[0063] Primer-R: 5'-GCACCTGAACAGCATCCC-3' (Seq ID No. 8)

[0064] 2. Identification of maize plants overexpressing the ZmGST30 gene

[0065] The constructed recombinant expression vector pBCXUN:GFP-ZmGST30 containing the target gene was transformed into Agrobacterium GV3101 strain. The transgenic operation was carried out by Agrobacterium-mediated transgenic operation into the immature embryos of the maize inbred line ND101, and the callus was formed after dedifferentiation, and then differentiated into tissue organs to obtain T0 generation transgenic plants.

[0066] The identified T0 generation plants are transplanted into a greenhouse to produce T1 generation transgenic plant offspring of the inbred line; the T1 generation transgenic plant offspring are self-pollinated to obtain T2 generation transgenic seeds; the T2 generation transgenic plants are self-pollinated again to obtain T3 generation transgenic plant seeds; in each generation, the PCR amplification method is used to identify positive transgenic plants, and strict self-pollination is performed to obtain the next generation of seeds.

[0067] In order to detect the expression efficiency of ZmGST30 gene in T3 transgenic lines, RNA was extracted from the leaves of the recipient ND101 and three transgenic lines OE1, OE2, and OE3 at the seedling stage, and reverse transcribed into corresponding cDNA. Fluorescence quantitative PCR experiment was performed to detect its expression level. The results are shown in Figure 2. Figure 1 As shown, the transcription levels of three independent overexpression lines OE1, OE2, and OE3 were all overexpressed. Subsequent experiments were performed using T3 generation transgenic homozygous seeds.

[0068] Example 2 Application of ZmGST30 gene overexpressing plants in drought stress

[0069] 1. Phenotypic detection of ZmGST30 gene overexpressing plants under soil drought treatment

[0070] Imported soil, peat soil, and vermiculite were mixed in equal proportions and placed in small pots, with 140 g of soil in each small pot. Seeds of wild-type and ZmGST30 gene overexpressing plants OE1, OE2, and OE3 were sown in the soil, 4 seeds per pot, evenly distributed. When the seedlings grow to the three-leaf and one-heart stage, thinning was performed, and 3 plants with relatively consistent growth size and state were retained in each pot. After a week, water was added to the tray, and after the water was fully absorbed, all small pots were transferred to another dry tray. The wild-type and overexpressing plants were divided into 2 groups and treated separately: the control group plants continued to be cultivated normally and watered on time, and the drought treatment group plants were no longer watered and subjected to drought stress treatment. Observe regularly every day, and take photos and record according to the drought conditions of the plants after about 11-12 days. Figure 2 The figure shows the phenotypic results of wild-type and overexpression plants under drought treatment conditions, with the left side showing normal watering and the right side showing drought treatment conditions. As can be seen from the figure, the ZmGST30 gene overexpression plants have improved drought resistance compared to the control and have a drought resistance phenotype.

[0071] 2. Detection of relative water content in leaves of plants overexpressing ZmGST30 gene

[0072] Observe the state of the seedlings in the pot. When the drought phenotype appears, take 9 leaves of the overexpression plants OE1, OE2, OE3 and the control plants under normal watering and drought conditions, weigh the weight of the leaves and record it as W1; then put the leaf segments into a 50mL centrifuge tube, add distilled water, let the leaves absorb water to saturation (about 30h), then wipe the water on the leaves with absorbent paper, weigh them again and record them as W2; after weighing, dry the leaves (80℃, more than 8h), measure the dry weight and record it as W3. Calculate the relative water content, relative water content = (W1-W3) / (W2-W3)×100%, the results are as follows Figure 3 As shown, under drought conditions, the relative water content of leaves of overexpressing plants OE1, OE2, and OE3 was significantly increased compared with that of the wild-type parent control material, indicating that their water retention capacity was significantly higher than that of the parent control material.

[0073] 3. Detection of the effect of exogenous ABA on stomatal aperture in ZmGST30 gene overexpressing plants

[0074] This operation used leaves of wild-type and ZmGST30 gene overexpressing plants as the operation objects, and set up a control group and an experimental group (10μM ABA treatment) respectively. Apply transparent nail polish on the back of the corn leaves, tear off the lower epidermis, take pictures under a microscope, and use Image J software to count the stomatal opening (expressed by the horizontal diameter of the stomatal opening). The results of the stomatal movement experiment are shown in Figure 4 As shown in the data, under control conditions, there was no difference in stomatal aperture among the plants, but under ABA treatment conditions, the stomatal aperture of the ZmGST30 gene overexpressing plants was smaller than that of the wild type and was sensitive to the effects of ABA, indicating that the ZmGST30 gene responds to the regulation of plant stomatal movement and plays an important role in plant response to drought stress.

[0075] 4. Detection of water loss rate of detached leaves of plants overexpressing ZmGST30 gene

[0076] The second fully expanded leaf of corn seedlings that have grown normally to the V2 stage was removed and the initial fresh weight was immediately weighed using an electronic balance. The in vitro treatment was continued under light, and the leaf weight was recorded every 1 hour. The water loss rate of the in vitro leaves was calculated by recording the values. Figure 5 As shown, the water loss rate of detached leaves of the overexpressing plants was slightly lower than that of the wild type, indicating that the drought resistance phenotype of the ZmGST30 gene overexpressing material was mainly caused by the lower water loss rate of the plants.

[0077] 5. Determination of H2O2 content in ZmGST30 gene overexpressing plants before and after drought stress

[0078] Corn plants were cultured with sufficient water until the 2-leaf stage and then divided into normal watering and drought treatment (no more watering after sufficient watering). When the wild-type strain showed a drought phenotype of leaf wilting, samples were taken to test the H2O2 content of the leaves. Figure 6 As shown in the figure, the experimental results showed that there was no significant difference in the H2O2 accumulated in the plants overexpressing the ZmGST30 gene and the wild-type plants before drought treatment. When the plants were subjected to drought stress, the H2O2 content in the body increased, and the ZmGST30 gene overexpression line could significantly reduce the H2O2 accumulated in the plants. This result shows that GST can reduce the accumulation of ROS in the plants when the plants are subjected to drought stress and protect the plant tissue cells from stress damage.

[0079] 6. Detection of ion leakage rate in ZmGST30 gene overexpressing plants

[0080] The second leaf of the ZmGST30 gene overexpressing material and the wild-type plant showing drought phenotype was cut off to detect leaf ion leakage. The experimental results are as follows Figure 7 As shown in the figure, under normal plant growth conditions, there is not much difference in ion leakage rate between the ZmGST30 gene overexpression material and the wild type, but when the plant shows a drought stress phenotype, the overexpression material has a lower ion leakage rate than the wild type, indicating that the degree of stress damage to the cells of the overexpression material is lower than that of the wild type. This shows that when plants suffer from drought stress, GST can maintain cell homeostasis and reduce the degree of stress damage to plants.

[0081] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

[0082] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of glutathione S-transferase gene ZmGST30 or its encoded protein or biological material containing its encoded gene in improving drought resistance of corn, wherein the nucleotide sequence of the glutathione S-transferase gene ZmGST30 is any one of the following: A1) the nucleotide sequence shown in Seq ID No.1; A2) a nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added as the nucleotide sequence shown in SEQ ID No. 1 and the nucleotide sequence expresses a protein with the same function; A3) A nucleotide sequence that hybridizes to the sequence shown in Seq ID No. 1 under stringent conditions.

2. Application of glutathione S-transferase gene ZmGST30 in breeding new drought-resistant corn varieties, The nucleotide sequence of the glutathione S-transferase gene ZmGST30 is any one of the following: A1) the nucleotide sequence shown in Seq ID No.1; A2) a nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added as the nucleotide sequence shown in SEQ ID No. 1 and the nucleotide sequence expresses a protein with the same function; A3) A nucleotide sequence that hybridizes to the sequence shown in Seq ID No. 1 under stringent conditions.

3. The use according to claim 1 or 2, characterized in that: The nucleotide sequence of the transcript of the glutathione S-transferase gene ZmGST30 is shown in Seq ID No.2, and the amino acid sequence of the encoded protein is shown in Seq ID No.3; Or the nucleotide sequence of the transcript of the glutathione S-transferase gene ZmGST30 is shown in Seq ID No.4, and the amino acid sequence of the encoded protein is shown in Seq ID No.

5.

4. The use according to claim 1 or 2, characterized in that: The biological material is any one of the following B1) to B9): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

5. The primer pair sequences for amplifying the glutathione S-transferase gene ZmGST30 are shown in Seq ID No.7 and Seq ID No.

8.

6. The use according to claim 1 or 2, characterized in that: The drought resistance of corn is improved by increasing the expression level of glutathione S-transferase gene ZmGST30.

7. The use according to claim 6, characterized in that: The ZmGST30 gene in corn was overexpressed to obtain overexpression plants, which improved the drought resistance of corn, including: the relative water content of leaves of ZmGST30 overexpression plants increased; the sensitivity of leaf stomata to the stomatal closure process promoted by abscisic acid increased; the water loss rate of detached leaves decreased; and the ROS scavenging ability of ZmGST30 overexpression plants was improved, which was manifested by the decrease of H2O2 content and ion permeability under drought stress.

8. A method for improving drought resistance of corn, characterized in that: By increasing the expression of glutathione S-transferase gene ZmGST30, the drought resistance of corn is improved; The nucleotide sequence of the glutathione S-transferase gene ZmGST30 is any one of the following: A1) the nucleotide sequence shown in Seq ID No.1; A2) a nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added as the nucleotide sequence shown in SEQ ID No. 1 and the nucleotide sequence expresses a protein with the same function; A3) A nucleotide sequence that hybridizes to the sequence shown in Seq ID No. 1 under stringent conditions.

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