Application of corn glutathione S-transferase gene ZmGST24 in plant drought resistance

Abstract

The invention relates to an application of a corn glutathione S-transferase gene ZmGST24 in drought resistance of plants. The ZmGST24 gene is over-expressed in a plant, so that the relative water content of 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, and the drought resistance is remarkably enhanced; in addition, the ROS removal capability of the ZmGST24 overexpressed plant is improved, and the H2O2 content is reduced under drought stress. The ZmGST24 gene can be used for breeding new drought-resistant varieties.

Description

Technical Field

[0001] The invention relates to the field of gene breeding, in particular to the application of a corn glutathione S-transferase gene ZmGST24 in plant drought resistance. Background Art

[0002] Glutathione S-transferase (GST) is a detoxification enzyme commonly found in various organisms. It protects organisms from various biological and abiotic stresses by catalyzing the electrophilic groups of toxic secondary metabolites and exogenous toxic substances produced in the organism to form a complex with the sulfhydryl group of reduced glutathione. Since GST was first discovered in corn in the 1970s, GST has been discovered in many plants, such as wheat, dwarf pine, tobacco, Arabidopsis, soybean, potato, sugarcane, barley, chickpea, dianthus, sorghum, etc.

[0003] GST plays an important role in repairing oxidative damage caused by biotic and abiotic stresses and is closely related to plant growth and development and the defense mechanism against biotic and abiotic stresses. The Arabidopsis gene ThGSTZ1 can strongly respond to abiotic stress treatments. Under NaCl and mannitol stress, the GST, GPX activities and reactive oxygen scavenging abilities of Arabidopsis overexpressing ThGSTZ1 and Arabidopsis seedlings were higher than those of control plants. In addition, under abiotic stress, GST can also regulate redox homeostasis by changing glutathione content and redox state, making it tolerant to a variety of abiotic stresses. Transgenic Arabidopsis plants overexpressing tomato LeGSTU2 enhance their resistance to drought stress by increasing the activity of antioxidant enzymes that scavenge excess ROS; the GST gene of cotton shows tissue-specific expression and is induced or inhibited by salt stress.

[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 little is known about how it participates in drought stress response. Summary of the invention

[0005] In view of the defects in the prior art, the purpose of the present invention is to provide an application of a maize glutathione S-transferase gene ZmGST24 (GRMZM2G032856_Genomic DNA) in plant drought resistance. The present invention improves the drought resistance of the plant by overexpressing the ZmGST24 gene in the plant, and the ZmGST24 gene can be used in the breeding of new drought-resistant plant varieties.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] The application of maize glutathione S-transferase gene ZmGST24 in plant drought resistance is characterized in that the application is:

[0008] By overexpressing the ZmGST24 gene in plants, the drought resistance of plants can be improved and new drought-resistant varieties can be bred;

[0009] The nucleotide sequence of the ZmGST24 gene is:

[0010] (1) As shown in Seq ID No.1, the nucleotide sequence transcribed from the nucleotide sequence is shown in Seq ID No.2, the amino acid sequence of the protein encoded by the nucleotide sequence of Seq ID No.2 is shown in Seq ID No.3; the actual insertion sequence for constructing the overexpression material is shown in Seq ID No.4.

[0011] (2) Different transcripts generated by the nucleotide sequence shown in Seq ID No. 1 and the amino acid sequences of their encoded proteins;

[0012] (3) A nucleotide sequence expressing the same or functionally deficient or mutated protein by replacing, deleting and / or adding one or more nucleotides from the nucleotide sequence shown in Seq ID No. 1.

[0013] (4) A nucleotide sequence that hybridizes with the nucleotide sequence shown in Seq ID No. 1 under stringent conditions.

[0014] The primers used for amplifying ZmGST24 gene are characterized in that: the primer sequences are shown in Seq ID No.5-6.

[0015] The application of the maize glutathione S-transferase gene ZmGST24 in plant drought resistance has the following beneficial effects:

[0016] Overexpression of the ZmGST24 gene in plants increased the relative water content of plant leaves, increased the sensitivity of leaf stomata to the process of stomatal closure promoted by abscisic acid (ABA), reduced the water loss rate of detached leaves, and significantly enhanced drought resistance. In addition, the ROS scavenging ability of ZmGST24-overexpressing plants was improved, which was manifested by the reduction of H2O2 content under drought stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention has the following accompanying drawings:

[0018] Figure 1 The expression level detection results of ZmGST24 transgenic overexpression plants;

[0019] Figure 2 Phenotypes of ZmGST24 overexpressing plants and wild type under drought treatment;

[0020] Figure 3 Relative water content of leaves of ZmGST24 overexpressing plants and wild type;

[0021] Figure 4 Stomatal aperture of ZmGST24 overexpressing plants and wild type;

[0022] Figure 5 Water loss rate of detached leaves of ZmGST24-overexpressing plants and wild type;

[0023] Figure 6 H2O2 content in ZmGST24 overexpressing plants and wild type. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0025] 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.

[0026] Example 1, Obtaining ZmGST24 Overexpressing Corn Plants

[0027] 1. Construction and detection of ZmGST24 gene overexpression vector

[0028] Total RNA was extracted from B73 corn (Zea mays L.), and cDNA was obtained by reverse transcription. Primers (primers are as follows) were designed using cDNA as a template, and the CDS region of the target gene ZmGST24 was amplified using upstream and downstream primers and high-fidelity enzymes. The PCR product was purified using 1% agarose gel and gel recovery methods, and the purified fragment was connected to the constructed pSuper1300:GFP vector with Ubiquitin promoter by TA cloning. Escherichia coli was transformed and positive clones were screened to complete the cloning construction. The constructed plasmid was sequenced, and the sequencing results and the target sequence were compared and analyzed, and the vector containing the complete and correctly sequenced target gene sequence was selected for subsequent experiments.

[0029] Primer-F: 5'-TCACGCACAACCCAGTCC-3'; (Seq ID No. 5)

[0030] Primer-R: 5'-CGATGAAGTCAGCCCAGAA-3'. (Seq ID No.6)

[0031] 2. Identification of Overexpressing Transgenic Maize Plants

[0032] The constructed pSuper1300:GFP recombinant expression vector 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 through tissue culture.

[0033] 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.

[0034] In order to detect the expression efficiency of ZmGST24 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 experiments were performed to detect its expression level ( Figure 1 ). T3 generation transgenic homozygous seeds were used for subsequent experiments.

[0035] Example 2, Application of ZmGST24 overexpressing plants in drought stress

[0036] 1. Phenotypic detection of ZmGST24 overexpressing plants under soil drought treatment

[0037] Imported soil: peat soil: vermiculite were mixed in equal proportions and put into small pots, with 140g of soil in each small pot. Seeds of wild-type and ZmGST24 overexpressing plants 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 ZmGST24 gene overexpression plants have improved drought resistance compared to the control and have a drought resistance phenotype.

[0038] 2. Detection of relative water content in leaves of ZmGST24 overexpressing plants

[0039] Observe the state of the seedlings in the pot. When the drought phenotype appears, take 9 leaves from the overexpression plants and the control plants under normal watering and drought conditions, weigh the leaves and record the weight as W1; then put the leaf segments into a 50mL centrifuge tube, add distilled water, let the leaves absorb water to saturation (about 24h), 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% ( Figure 3 ).

[0040] 3. Detection of the effect of exogenous ABA on stomatal aperture in ZmGST24 overexpressing plants

[0041] This operation used leaves of wild-type and ZmGST24 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 figure, under control conditions, there was no difference in stomatal aperture among the plants, but under ABA treatment conditions, the stomatal aperture of the ZmGST24 overexpressing plants was smaller than that of the wild type and was sensitive to the effects of ABA, indicating that ZmGST24 responds to the regulation of plant stomatal movement and plays an important role in plant response to drought stress.

[0042] 4. Detection of water loss rate in detached leaves of ZmGST24 overexpressing plants

[0043] 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 ZmGST24 overexpressing material was mainly caused by the lower water loss rate of the plants.

[0044] 5. Determination of H2O2 content in ZmGST24 overexpressing plants before and after drought stress

[0045] Experimental results ( Figure 6 ) showed that there was no significant difference in the amount of H2O2 accumulated in ZmGST24-overexpressing plants and wild-type plants before drought treatment. When the plants were subjected to drought stress, the amount of H2O2 in the body increased, and the ZmGST24-overexpressing strain could significantly reduce the amount of H2O2 accumulated in the plants. This result indicates that GST can reduce the accumulation of ROS in plants when they are subjected to drought stress, protecting plant tissue cells from stress damage.

[0046] The present invention has been described in detail above with general description and specific embodiments, and some modifications or improvements may be made to it on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

[0047] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

Claims

1. Application of maize glutathione S-transferase gene ZmGST24 in plant drought resistance, characterized in that: The application is: By overexpressing the ZmGST24 gene in plants, the drought resistance of plants can be improved and new drought-resistant varieties can be bred; The nucleotide sequence of the ZmGST24 gene is: (1) As shown in Seq ID No.1, the nucleotide sequence transcribed from the nucleotide sequence is shown in Seq ID No.2, and the amino acid sequence of the protein encoded by the nucleotide sequence of Seq ID No.2 is shown in Seq ID No.3; the actual insertion sequence for constructing the overexpression strain is shown in Seq ID No.4; (2) Different transcripts generated by the nucleotide sequence shown in Seq ID No. 1 and the amino acid sequences of their encoded proteins; (3) A nucleotide sequence expressing the same or functionally deficient or mutated protein by replacing, deleting and / or adding one or more nucleotides from the nucleotide sequence shown in Seq ID No.

1. (4) A nucleotide sequence that hybridizes with the nucleotide sequence shown in Seq ID No.

1.

2. Primers for amplifying ZmGST24 gene, characterized in that: The primer sequences are shown in Seq ID No.5-6.

Images