A protein and its encoding gene are applied to regulating drought tolerance of corn

Abstract

The application belongs to the technical field of plant molecular biology, and provides a protein and application of a coding gene thereof in regulating drought tolerance of corn, wherein the application takes a drought-sensitive corn inbred line B73 and a ZmWIP3 knockout (KO) transgenic line as test materials, verifies the role of the ZmWIP3 protein and the coding gene thereof in regulating drought tolerance of corn, and the results show that: after the ZmWIP3 gene is knocked out, the expression of the ZmWIP3 protein is inhibited, the antioxidant capacity of the corn is reduced under drought stress, the water loss rate of the corn leaves is accelerated, the growth of the corn seedlings is poor, the plant height and the leaf area are lower than those of the wild type, and the stem diameter is significantly lower than that of the wild type, which indicates that the ZmWIP3 protein and the coding gene thereof have a positive regulation on the drought tolerance of the corn.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to the application of ZmWIP3 protein and its encoding gene in regulating drought resistance in maize. Background Technology

[0002] Drought is the leading abiotic stress factor constraining the stability of global agricultural production. As the most widely cultivated cereal crop globally, maize's yield is highly susceptible to drought. In maize's drought resistance mechanisms, the jasmonic acid (JA) signaling pathway and aquaporins (AQPs) play crucial roles. JA is an important stress hormone, playing a central regulatory role in plant responses to drought, damage, and other stresses. Key components of the JA signaling pathway include JAZ family repressor proteins and transcription factors such as MYC. When JA levels rise, the SCF-COI1 complex mediates the degradation of JAZ proteins, relieving their inhibition of downstream transcription factors and thus initiating the expression of stress-responsive genes.

[0003] Aquaporins are a class of highly efficient and selective water-transporting channel proteins located on biological membranes, mainly divided into subfamilies such as plasma membrane intrinsic proteins (PIPs) and vacuolar membrane intrinsic proteins (TIPs). They dynamically regulate the hydroconductivity of cells and tissues, directly affecting the balance of water absorption, transport, and loss in plants. Studies have shown that the expression of many AQP genes (such as PIP2, TIP1, and TIP4 subfamily members) is drought-induced, and their loss of function or overexpression is directly associated with the drought-resistant phenotype of plants. Although it is known that both JA and AQPs are involved in drought resistance, how JA signaling precisely regulates the expression and activity of specific AQPs, and the direct molecular connections and signal transduction modules between them, remain unclear. Traditional breeding or single gene overexpression strategies often have limited effects or are accompanied by growth loss, lacking fine-grained regulation of upstream signaling pathways. Therefore, elucidating the core mechanism of JA regulation of the AQP regulatory axis and developing synergistic genetic improvement strategies based on this is both a frontier and a challenge in current crop drought resistance research. Current research has clearly established that coronatine (COR) regulates maize drought resistance by directly binding to the E-loop of the intrinsic plasma membrane protein ZmPIP2;5. Under drought stress, JA can also significantly increase maize leaf water potential, root hydraulic conductance, and ZmPIP2;5 gene expression, thereby improving maize drought resistance. Therefore, it is speculated that JA may have an indirect regulatory relationship with the upstream transcription factor ZmWIP3 of ZmPIP2;5, enhancing the water uptake capacity of maize roots. In summary, this invention uses the ZmWIP3 protein as the core of drought resistance, providing theoretical support for coping with drought stress in maize production. Summary of the Invention

[0004] The purpose of this invention is to provide an application of a protein and its encoding gene in regulating drought resistance in maize. By applying the ZmWIP3 protein and its encoding gene to positively regulate drought resistance in maize, a new approach and means can be provided to improve the drought resistance of maize.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] This invention provides the application of ZmWIP3 protein mimics and / or their encoding gene activators in the positive regulation of drought resistance in maize.

[0007] Preferably, the ZmWIP3 protein mimic and its encoded gene activator are used to enhance the antioxidant capacity of maize under drought stress.

[0008] Preferably, the ZmWIP3 protein mimic and its encoded gene activator are used to inhibit the rate of water loss from maize leaves.

[0009] Preferably, the ZmWIP3 protein mimic and its encoded gene activator are used to enhance maize plant height and leaf area, and inhibit maize seedling growth deterioration.

[0010] The present invention also provides a formulation for enhancing the drought resistance of maize, the formulation comprising the above-mentioned ZmWIP3 protein mimic and its encoding gene activator.

[0011] Preferably, the formulation includes various acceptable dosage forms, including aqueous solutions, emulsions, soluble liquids, water-in-oil emulsions, microemulsions, or suspensions.

[0012] The beneficial effects of this invention are:

[0013] The maize drought resistance protein ZmWIP3 provided by this invention has a significant impact on maize drought resistance. Knocking out the ZmWIP3 gene inhibits the expression of the ZmWIP3 protein, which reduces the antioxidant capacity of maize under drought stress, accelerates the rate of water loss from maize leaves, and results in poorer growth of maize seedlings, with reduced plant height and leaf area compared to the wild type, and a significantly reduced stem diameter. This indicates that the ZmWIP3 protein and its encoding gene have a positive regulatory effect on maize drought resistance, and that this protein and its encoding gene can be widely applied to drought resistance in other crops. Attached Figure Description

[0014] Figure 1 This is a diagram of the gene editing sites of the maize zmwip3 knockout lines in this invention;

[0015] Figure 2The phenotypes of the B73 wild-type and zmwip3 knockout lines in this invention are shown in different treatments (NW+W is normal water supply plus spraying, NW+M is normal water supply plus MeJA, DS+W is drought stress plus spraying, and DS+M is drought stress plus MeJA).

[0016] Figure 3 This is a morphological statistical result diagram of the zmwip3 knockout line in this invention among different treatments (A is plant height; B is stem diameter; C is leaf area; NW+W is normal water supply plus spraying, NW+M is normal water supply plus MeJA, DS+W is drought stress plus spraying, DS+M is drought stress plus MeJA; different lowercase letters represent significant differences between different treatments).

[0017] Figure 4 This invention describes the effect of exogenous methyl jasmonate on leaf physiology in zmwip3 knockout lines under drought stress (A represents relative chlorophyll content; B represents relative leaf water content; C represents detached leaf water loss rate; NW+W represents normal water supply plus spraying, NW+M represents normal water supply plus MeJA, DS+W represents drought stress plus spraying, and DS+M represents drought stress plus MeJA; different lowercase letters indicate significant differences between different treatments).

[0018] Figure 5 This is a graph showing the effect of exogenous methyl jasmonate on the overall antioxidant capacity of the zmwip3 knockout strain under drought stress in this invention (A is hydrogen peroxide content; B is peroxidase activity; C is ascorbate peroxidase activity; NW+W is normal water supply plus spraying, NW+M is normal water supply plus MeJA, DS+W is drought stress plus spraying, DS+M is drought stress plus MeJA; different lowercase letters represent significant differences between different treatments). Detailed Implementation

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] Example

[0023] The drought-sensitive maize inbred line B73 and the ZmWIP3 knockout (KO) transgenic line were used as experimental materials. B73 inbred lines were grown in pots with a substrate:vermiculite ratio of 3:1 (V:V). Four treatments were included: normal water supply + 5 mL deionized water (NW+W), normal water supply + 5 mL 100 μmol / L MeJA (NW+MeJA), drought stress + 5 mL deionized water (DS+W), and drought stress + 5 mL 100 μmol / L MeJA (DS+MeJA). Each pot contained 3 plants, and each treatment was replicated 3 times. After maize sprouted, sufficient water was applied. Water was withheld from the start of the drought stress treatment until the three-leaf stage, when severe drought was reached.

[0024] 1. Experimental Methods

[0025] 1.1 Leaf water potential measurement

[0026] Mature leaves from the same part of each plant were measured using a pressure chamber apparatus. Each line and treatment was replicated at least three times.

[0027] 1.2 Determination of relative water content in leaves

[0028] After rinsing and drying the collected leaves, their fresh weight was measured. The leaves were then soaked in water for 24 hours to fully absorb water and reach saturation. After drying, the saturated fresh weight was measured again. The leaves were then placed in a 105℃ oven for 20 minutes to kill the green, and then dried at 80℃ until constant weight was achieved. The dry weight was measured. The relative moisture content of the leaves was calculated using the following formula: RWC / % = [(fresh weight - dry weight) / (saturated fresh weight - dry weight)] × 100%.

[0029] 1.3 Assay of antioxidant enzyme activity

[0030] (1) Peroxidase (POD) can catalyze the oxidation of hydrogen, phenols, and amines by peroxidation. POD catalyzes the oxidation of specific substrates by H2O2 and has a characteristic light absorption at 470 nm. One unit of enzyme activity is defined as a change of 0.01 A470 per minute per gram of tissue in each mL of reaction system.

[0031] (2) Catalase (CAT) activity was calculated by measuring the H2O2 content. H2O2 has a characteristic absorption peak at 240 nm. CAT can decompose H2O2, causing the absorbance of the reaction solution at 240 nm to decrease with reaction time. The CAT activity can be calculated based on the rate of change in absorbance. One unit of enzyme activity is defined as the degradation of 1 μM H2O2 per minute by 1 g of tissue in the reaction system.

[0032] (3) Ascorbate peroxidase (APX) content was determined by measuring the oxidation rate of ascorbate (AsA). APX catalyzes the oxidation of ascorbate (AsA) by H2O2 and is the main consumer of AsA in plants. APX activity was calculated by measuring the oxidation rate of AsA. One unit of enzyme activity was defined as 1 μM AsA oxidized per gram of tissue per minute.

[0033] 1.4 Determination of hydrogen peroxide content

[0034] Hydrogen peroxide (H2O2) reacts with titanium sulfate to form a yellow titanium peroxide complex with a maximum absorption wavelength of 415 nm. The H2O2 content in the sample can be estimated by colorimetry.

[0035] 2. Experimental Results

[0036] 2.1 Obtaining and identifying maize zmwip3 knockout lines

[0037] To investigate the function of ZmWIP3, two Zmwip3 knockout seeds, zmwip3-1 and zmwip3-2, with a B73 genetic background, were obtained. Maize seedlings were obtained by culturing the knockout and wild-type lines. DNA was extracted from the leaves of the seedlings, primers were designed before and after the target sequence, and high-fidelity PCR was performed. The products were then sequenced, and the editing site of Zmwip3 was determined by sequence alignment. Figure 1 As shown, zmwip3-1 replaces 7 bases and inserts 2 bases compared to WT, while zmwip3-2 inserts one base. Both result in frameshift mutations and premature termination of translation.

[0038] 2.2 Effects of exogenous methyl jasmonate on the morphology of zmwip3 knockout lines

[0039] like Figure 2 As shown, wild-type B73 and knockout lines zmwip3-1 (ko-1) and zmwip3 (ko-2) were planted in large square pots, from left to right as ko-1, WT and ko-2, with two rows for each line. At the two-leaf stage, maize seedlings with consistent growth were selected and grouped, with 8 plants left for each line. Water was controlled in the drought stress group until the three-leaf stage when severe drought was reached. NW and DS were sprayed with deionized water or 100 μM MeJA. The stems of the drought stress group maize withered and softened 4 days after spraying, and rehydration began. Five days after rehydration, the number of dead maize seedlings no longer increased, and the survival rate after rehydration was counted.

[0040] Phenotypic observation revealed no significant difference in growth among the three lines under normal water conditions. Under the DS+W treatment, the wild-type lines showed better growth, with significant differences observed 5 days after rehydration. The rehydration survival rates for WT, ko-1, and ko-2 were 100%, 87.5%, and 75%, respectively. Under the DS+M treatment, there was no significant difference in growth among the three lines, and the rehydration survival rate was 100% for all three lines. This indicates that the deletion of the ZmWIP3 gene resulted in poorer growth of maize seedlings under drought conditions, while MeJA compensated for this difference, making the phenotypic differences among the three lines under the DS+M treatment insignificant.

[0041] Further measurements were taken of the plant height, stem diameter, and leaf area of ​​maize seedlings 48 hours after treatment. The results are as follows: Figure 3 As shown, all the above-mentioned indicators of maize seedlings under drought stress were lower than those under normal water supply treatment. Under the same treatment, the plant height and leaf area of ​​zmwip3-1 and zmwip3-2 were lower than those of the wild type, but there was no significant difference among the three lines. Under normal water supply conditions, there was no significant difference in stem diameter among the three lines. Under drought stress, the stem diameter of zmwip3-1 and zmwip3-2 was significantly lower than that of the wild type, decreasing by 22.52% and 17.63% respectively under DS+W treatment, and by 14.86% and 8.99% respectively under DS+M treatment. After spraying with MeJA, the plant height, stem diameter, and leaf area under normal water and drought stress were all increased. Under the NW+M treatment, the plant height, stem diameter, and leaf area of ​​zmwip3 increased by 4.69%, 6.76%, and 1.23% respectively compared with NW+W. Under the DS+M treatment, the plant height, stem diameter, and leaf area of ​​zmwip3 increased by 11.03%, 27.53%, and 31.57% respectively compared with DS+W.

[0042] In summary, under normal water supply conditions, the morphology of the zmwip3 knockout lines was no different from that of the wild type. Under drought stress, the plant height and leaf area of ​​the zmwip3 knockout lines were not affected by the reduced expression of the ZmWIP3 gene, but it significantly affected the stem diameter of maize seedlings, making them more susceptible to lodging. MeJA had no significant effect on the growth of maize seedlings of the zmwip3 knockout lines under normal water supply conditions, but it could significantly promote stem diameter and leaf area under drought stress, making their leaf area no different from that of the wild type lines.

[0043] 2.3 Effects of exogenous methyl jasmonate on leaf physiology in zmwip3 knockout lines

[0044] To investigate the effects of exogenous methyl jasmonate on leaf physiology in zmwip3 knockout lines under drought stress, SPAD, RWC, and water loss rate of detached leaves in maize seedlings were measured 48 h after treatment. The results are as follows: Figure 4As shown, all the above-mentioned indicators of maize seedlings under drought stress were lower than those under normal water supply treatment. Under the same treatment, there were no significant differences in SPAD and RWC among the three lines, but the water loss rate of detached leaves differed significantly among drought stress treatments. Under DS+W and DS+M treatments, the water loss rate of the zmwip3 knockout lines was significantly higher than that of the wild type by 38.62% and 30.21%, respectively. After spraying with MeJA, under the NW+M treatment, the SPAD, RWC, and water loss rate of detached leaves of the zmwip3 knockout lines increased by 2.68%, 3.06%, and decreased by 4.17% compared with the control NW+W, respectively. Under the DS+M treatment, the SPAD, RWC, and water loss rate of detached leaves of the zmwip3 knockout lines increased by 4.60%, 10.0%, and decreased by 18.62% compared with the control NW+W, respectively. The results showed that the SPAD and RWC of the zmwip3 knockout lines were less affected by the deletion of the ZmWIP3 gene, resulting in a faster rate of water loss from the leaves and exacerbating the effects of drought stress. MeJA could significantly reduce the rate of water loss in the zmwip3 knockout lines under drought stress and partially alleviate drought damage.

[0045] 2.4 Effect of exogenous methyl jasmonate on the antioxidant capacity of zmwip3 knockout lines

[0046] like Figure 5 As shown, under drought stress, the H2O2 ( Figure 5 A), POD ( Figure 5 B) and APX ( Figure 5 C) All levels were higher than normal water levels, with significant accumulation of H2O2 and APX, increasing by 2.03, 0.40, and 2.08 times, respectively. Under the same treatment, there were no significant differences in H2O2 and APX among the three lines. Under normal water conditions, there were no differences in POD among the three lines. Under drought stress, the POD activity of the zmwip3 knockout lines was significantly lower than that of the wild type, decreasing by 22.94% and 13.73% under DS+W and DS+M treatments, respectively. Under normal water conditions, after MeJA treatment, the H2O2, POD, and APX of the zmwip3 knockout lines increased by 2.13%, 29.85%, and 53.28%, respectively, compared to the sprayed water control. Under drought stress, after MeJA treatment, the H2O2, POD, and APX of the zmwip3 knockout lines decreased by 21.43%, increased by 16.59%, and increased by 14.45%, respectively, compared to the sprayed water control. In summary, the ZmWIP3 knockout strains showed less impact from the ZmWIP3 gene deletion on H2O2 and APX, but significantly reduced POD activity under drought conditions. Spraying MeJA significantly promoted the POD and APX activities of the Zmwip3 knockout strains under normal water conditions, and had a significant effect on H2O2, POD, and APX under drought stress.

[0047] In summary, the maize drought-resistance protein ZmWIP3 provided by this invention has a significant impact on maize drought resistance. Knocking out the ZmWIP3 gene inhibits the expression of the ZmWIP3 protein, reducing the antioxidant capacity of maize under drought stress, accelerating the rate of water loss from maize leaves, resulting in poorer seedling growth, lower plant height and leaf area compared to the wild type, and a significantly lower stem diameter. The ZmWIP3 protein and its encoding gene mimics or activators can serve as positive regulators of drought resistance in maize.

[0048] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Application of ZmWIP3 protein mimic and / or its encoding gene activator in positive regulation of drought resistance in maize.

2. The application according to claim 1, characterized in that, The ZmWIP3 protein mimic and its encoded gene activator are used to enhance the antioxidant capacity of maize under drought stress.

3. The application according to claim 1, characterized in that, The ZmWIP3 protein mimic and its encoded gene activator are used to inhibit the rate of water loss from maize leaves.

4. The application according to claim 1, characterized in that, The ZmWIP3 protein mimic and its encoded gene activator are used to enhance maize plant height and leaf area, and inhibit the decline in maize seedling growth.

5. A formulation for enhancing drought resistance in maize, characterized in that, The formulation comprises the ZmWIP3 protein mimicry and its encoded gene activator as described in claim 1.

6. The formulation according to claim 5, characterized in that, The formulation includes various acceptable dosage forms, including aqueous solutions, emulsions, soluble liquids, water-in-oil emulsions, microemulsions, or suspensions.

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