Application of ZmDIR4 gene in regulation of drought sensitivity of corn

Abstract

The invention belongs to the technical field of biological genetic engineering, and particularly relates to application of a ZmDIR4 gene in regulation and control of corn drought sensitivity. Experiments show that after the ZmDIR4 gene is knocked out, the dry matter accumulation, the activity of antioxidant enzyme in an antioxidant metabolic pathway, the accumulation of proline, chlorophyll and lignan and the activation degree of hormone metabolic pathways (zeatin and abscisic acid) of a mutant corn plant after drought stress are all obviously higher than those of a wild type and an over-expression material; it is proved that the ZmDIR4 gene has a negative regulation function in corn drought stress. The ZmDIR4 gene disclosed by the invention can be used for creating a new corn germplasm with improved drought resistance, and a new thought is provided for the drought resistance of the corn.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering, and in particular relates to the application of ZmDIR4 gene in regulating the drought sensitivity of corn. Background Art

[0002] Maize is one of the most important food crops in the world and has attracted much attention due to its remarkable adaptability and wide planting areas. In maize production, a key challenge is the impact of drought stress, which greatly limits the yield potential. In order to cope with these abiotic stresses, maize plants have evolved complex physiological and molecular response systems. Among them, the role of drought-resistant genes is particularly significant. These drought-resistant genes play a key role in regulating plant growth, developmental processes and morphological construction, and also play an important role in environmental stress response. Therefore, it is very important to explore maize drought-resistant genes, analyze maize drought-resistant mechanisms, and improve maize drought resistance in order to achieve food security and sustainable development goals of future agricultural production. At the same time, exploring maize drought-resistant genes has an important research basis and practical significance for accelerating maize drought-resistant breeding using genetic engineering. Summary of the invention

[0003] The purpose of the present invention is to provide an application of the ZmDIR4 gene in regulating the sensitivity of corn to drought. The ZmDIR4 gene has a negative regulatory function in corn drought stress, providing a new idea for corn drought resistance.

[0004] The invention provides application of ZmDIR4 gene in regulating the drought sensitivity of corn. The accession number of the ZmDIR4 gene is Zm00001d034563.

[0005] As a preferred solution, the regulation includes overexpressing the ZmDIR4 gene to reduce the drought resistance of corn; inhibiting the expression of the ZmDIR4 gene or knocking out the ZmDIR4 gene to improve the drought resistance of corn.

[0006] The present invention also provides a biological material capable of regulating the expression amount of the ZmDIR4 gene, and the accession number of the ZmDIR4 gene is Zm00001d034563.

[0007] As a preferred embodiment, it includes biological materials overexpressing the ZmDIR4 gene, and the biological materials overexpressing the ZmDIR4 gene include one or more of a primer set for amplifying the ZmDIR4 gene, a recombinant expression vector containing the ZmDIR4 gene, and a recombinant microorganism containing the ZmDIR4 gene.

[0008] As a preferred embodiment, it comprises biological materials for inhibiting the expression of the ZmDIR4 gene or knocking out the ZmDIR4 gene, and the inhibition method comprises silencing.

[0009] As a preferred solution, the biological material for knocking out the ZmDIR4 gene uses the sequence shown in SEQ ID NO.1 as a target.

[0010] The invention also provides the application of the biological material in improving corn germplasm or creating new germplasm.

[0011] The present invention also provides a method for creating drought-resistant corn germplasm, comprising the following steps: knocking down the expression level of the ZmDIR4 gene in the target corn genome, wherein the accession number of the ZmDIR4 gene is Zm00001d034563.

[0012] The present invention also provides a method for creating a drought-sensitive corn model, comprising the following steps: overexpressing the ZmDIR4 gene in the target corn genome, wherein the accession number of the ZmDIR4 gene is Zm00001d034563.

[0013] As a preferred embodiment, the corn includes corn B104.

[0014] Beneficial effects: The present invention provides an application of the ZmDIR4 gene in regulating the sensitivity of corn to drought. The present invention found through experiments that when ZmDIR4 was knocked out, the dry matter accumulation of the mutant corn plants after drought stress, the activity of antioxidant enzymes in the antioxidant metabolic pathway, the accumulation of proline, chlorophyll, and lignans, and the activation degree of the hormone metabolic pathway (zeatin and abscisic acid) were significantly higher than those of the wild type and overexpression materials, proving that ZmDIR4 has a negative regulatory function in corn drought stress. The ZmDIR4 gene of the present invention can be used to create new corn germplasm with improved drought resistance, providing a new idea for corn drought resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0016] Figure 1 The diagram is a construction diagram of the ZmDIR4 gene overexpression material and the knockout material in Example 1, wherein A is the ZmDIR4 gene amplification result, B is the ZmDIR4 gene expression level of the overexpression material, and C and D are the deletion conditions of the knockout material fragments;

[0017] Figure 2 This is the phenotype diagram of the overexpression material and the knockout material under drought stress in Example 2;

[0018] Figure 3The expression of ZmDIR4 gene in different parts of corn under drought stress and at different treatment times in Example 2, wherein the left figure shows the expression of ZmDIR4 gene in different parts of corn under drought stress, and the right figure shows the expression of ZmDIR4 gene in the second true leaf of corn at different treatment times;

[0019] Figure 4 The statistics of fresh weight, dry weight, above-ground length, and underground length (root length) of the overexpression material and mutant material in Example 2 after drought stress treatment, wherein A is fresh weight, B is dry weight, C is above-ground length, and D is underground length (root length);

[0020] Figure 5 The subcellular localization result of ZmDIR4 in Example 3, the fusion vectors GFP-ZmDP4, RFP-AtH2B and RFP-AtWAK-HDEL were transiently expressed in tobacco leaves, GFP fluorescence was green, RFP fluorescence was red, and the Merge image was a superposition of green and red fluorescence;

[0021] Figure 6 The antioxidant capacity test results of the ZmDIR4 gene overexpression material and the knockout material in Example 4, wherein A is the malondialdehyde test result, B is the hydrogen peroxide test result, C is the superoxide anion test result, D is the superoxide dismutase test result, E is the catalase test result, and F is the peroxidase test result;

[0022] Figure 7 The metabolite accumulation test results of the ZmDIR4 gene overexpression material and the knockout material in Example 4, wherein A is the proline test result, B is the chlorophyll test result, C is the total lignan test result, D is the zeatin test result, and E is the abscisic acid test result;

[0023] Figure 8 The relative expression detection results of the ZmDIR4 gene overexpression material and knockout material in Example 4, wherein A is ZmRD21, B is ZmDREB2A, C is ZmERD1, D is ZmSnRK2.6, E is ZmNCED3, and F is ZmABA1;

[0024] Fig. 9 These are the relative expression detection results of the ZmDIR4 gene overexpression material and knockout material in Example 4, wherein A is ZmSOD1, B is ZmCAT3, C is ZmPOD1, D is ZmIPT2, E is ZmCKO5, and F is ZmZOG1. DETAILED DESCRIPTION

[0025] The invention provides application of ZmDIR4 gene in regulating the drought sensitivity of corn. The accession number of the ZmDIR4 gene is Zm00001d034563.

[0026] As a specific implementation manner, the regulation includes overexpressing the ZmDIR4 gene to reduce the drought resistance of corn; inhibiting the expression of the ZmDIR4 gene or knocking out the ZmDIR4 gene to improve the drought resistance of corn.

[0027] The present invention also provides a biological material capable of regulating the expression amount of the ZmDIR4 gene, and the accession number of the ZmDIR4 gene is Zm00001d034563.

[0028] The biomaterial of the present invention has the effect of regulating the expression amount of the ZmDIR4 gene, such as overexpressing the ZmDIR4 gene, inhibiting the expression of the ZmDIR4 gene or knocking out the ZmDIR4 gene. As a specific embodiment, the biomaterial overexpressing the ZmDIR4 gene includes one or more of a primer set for amplifying the ZmDIR4 gene, a recombinant expression vector containing the ZmDIR4 gene, and a recombinant microorganism containing the ZmDIR4 gene. As an embodiment, the recombinant expression vector containing the ZmDIR4 gene includes inserting the ZmDIR4 gene into a pBCXUN vector to obtain a pBCXUN-ZmDIR4 overexpression vector. As an embodiment, the recombinant microorganism containing the ZmDIR4 gene includes Agrobacterium containing the ZmDIR4 gene.

[0029] As a specific embodiment, a biological material that inhibits the expression of the ZmDIR4 gene or knocks out the ZmDIR4 gene is included, and the method of inhibition includes silencing. As a specific embodiment, the biological material for knocking out the ZmDIR4 gene targets the sequence shown in SEQ ID NO.1. The nucleotide sequence of the target is shown in SEQ ID NO.1: 5'-TTCCTCCACGACACTCTGAG-3'. As an embodiment, the knockout vector is a pBUE411 gene editing vector, and the recombinant microorganism includes Agrobacterium containing the gene editing vector.

[0030] The invention also provides the application of the biological material in improving corn germplasm or creating new germplasm.

[0031] The present invention also provides a method for creating drought-resistant corn germplasm, comprising the following steps: knocking down the expression of the ZmDIR4 gene in the target corn genome, the accession number of the ZmDIR4 gene being Zm00001d034563. In a specific embodiment, the present invention transfers the pBUE411-ZmDIR4 gene editing vector into the Agrobacterium EHA101 strain, and then uses an Agrobacterium-mediated transgenic method to transform the gene editing vector into the target corn plant to obtain corn with improved drought resistance. As a specific embodiment, after drought stress treatment, it was found that the fresh weight, dry weight, above-ground length, and underground length of the CRISPR knockout material were significantly higher than those of the wild-type material and the overexpression material, indicating that ZmDIR4 plays a negative regulatory role in corn drought stress.

[0032] The present invention also provides a method for creating a drought-sensitive corn model, comprising the following steps: overexpressing the ZmDIR4 gene in the target corn genome, wherein the accession number of the ZmDIR4 gene is Zm00001d034563. In a specific embodiment, the present invention transfers the pBCXUN-ZmDIR4 overexpression vector into the Agrobacterium GV3101 strain, and then uses an Agrobacterium-mediated transgenic method to transform the overexpression vector into the target corn plant to obtain a drought-sensitive corn model. As a specific embodiment, the corn includes corn B104.

[0033] As a specific embodiment, when ZmDIR4 is knocked out, the dry matter accumulation of the mutant corn plants after drought stress, the activity of antioxidant enzymes in the antioxidant metabolic pathway, the accumulation of proline, chlorophyll, and lignans, and the activation degree of the hormone metabolic pathway (zeatin and abscisic acid) are all significantly higher than those of the wild type and overexpression materials, proving that ZmDIR4 has a negative regulatory function in corn drought stress. Therefore, the ZmDIR4 gene can be used to create new corn germplasm with improved drought resistance, providing a new idea for corn drought resistance.

[0034] To further illustrate the present invention, the application of the ZmDIR4 gene provided by the present invention in regulating the drought sensitivity of corn is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products known to those skilled in the art can be used.

[0036] Example 1 Obtaining ZmDIR4 gene overexpression materials and knockout materials

[0037] In this study, CRISPR knockout and 35S:: overexpression were performed in the background material maize B104 (Yao Mengyao, Li Juan, Liu Zhigang, et al. Cloning of maize ZmMPI gene and detection of its overexpression transgenic lines [J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 1-9.) to obtain two overexpression materials (OE2 / OE3) and two CRISPR materials with knockout at different sites (CR4 / CR7) ( Figure 1 ), WT-B104 material was used as control material. The overexpression material used was vector pBCXUN (Sun Yu. Functional analysis of ZmCCR-like5 regulating salt tolerance in maize [D]. Yangzhou University, 2024.), and the primers were ZmDIR4-BamHI+: 5'-CCCGTGCTCCTCCGCCTCGTCGCTGTCCTGCTCG-3' (as shown in SEQ ID NO.2); ZmDIR4-SmaI-: 5'-GGGAGCTCGCCGTCGTCGGCGGCCGCGGCGCGT-3' (as shown in SEQ ID NO.3). The ZmDI R4 gene was inserted into the pBCXUN vector (inserted between the two multiple cloning sites of BamHI and SmaI) to obtain the pBCXUN-ZmDIR4 recombinant overexpression vector. The Crispr gene editing vector is pBUE411 (Yan Tingwei. Systematic analysis of the maize ZmLBD gene family and functional mechanism research of ZmMs1, ZmlG1 and ZmlALI [D]. Beijing University of Science and Technology, 2024.), the target sequence is 5'-TTCCTCCACGACACTCTGAG-3' (as shown in SEQ ID NO.1), and the knockout vector primers are F: 5'-GCCGTTCCTCCACGACACTCTGAG-3' (as shown in SEQ ID NO.4); R: 5'-AAACCTCAGAG TGTCGTGGAGGAA-3' (as shown in SEQ ID NO.5). The target sequence was constructed into the vector pBUE411 (the insertion vector multi-cloning site is BsaI), and the pBUE411-ZmDIR4 recombinant knockout vector (gene editing vector) was obtained.

[0038] After the pBCXUN-ZmDIR4 overexpression vector was transferred into the Agrobacterium GV3101 strain, the overexpression vector was transformed into the target corn plant using the Agrobacterium-mediated transgenic method (Qin T, Liu S, Zhang Z, et al. GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting theE3 ubiquitin ligase activity of GhPUB17[J]. Plant molecular biology, 2019, 99(4-5): 379-393. DOI: 10.1007 / s11103-019-00824-y.), and two overexpression materials (OE2 and OE3) were obtained. After the pBUE411-ZmDIR4 gene editing vector was transferred into the Agrobacterium EHA101 strain, the gene editing vector was transformed into the target corn plant using the Agrobacterium-mediated transgenic method (Qin T, Liu S, Zhang Z, et al. GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the E3ubiquitin ligase activity of GhPUB17[J]. Plant molecular biology, 2019, 99(4-5): 379-393. DOI: 10.1007 / s11103-019-00824-y.), and two knockout CRISPR materials (CR4 and CR7) were obtained.

[0039] Example 2 Phenotypic analysis of overexpression materials and knockout materials under drought stress

[0040] The corn wild-type control material, overexpression material and knockout material (the overexpression material and knockout material are the materials obtained in Example 1) were cultured to the three-leaf stage in an artificial climate chamber at 28°C during the day and 21°C at night, with a photoperiod of 16 h light and 8 h dark. The control group (wild-type control material, overexpression material and knockout material) was treated with 1 / 2 Hogland nutrient solution, and the drought stress group (wild-type control material, overexpression material and knockout material) was treated with 1 / 2 Hogland prepared 20% PEG6000 solution.

[0041] The tissue expression pattern and drought-induced expression analysis of ZmDIR4 were performed. Different parts (roots, stems, leaves, pollen and silk) of wild-type corn in the control group were taken, and the second flat true leaf from the seedling stage of wild-type corn in the drought stress group at different time points (0h, 1h, 6h, 12h, 18h, 24h, 36h and 48h) was taken. The primers ZmDIR4-RT-qPCR-F: 5'-GCACTTCTTCCTCCACGACACTCT-3' (as shown in SEQ ID NO.6); ZmDIR4-RT-qPCR-R: 5'-ACGTTGTACTCTATGATGGCGTCG-3' (as shown in SEQ ID NO.7) were used to complete the experimental analysis according to the instructions of the ChamQ SYBR color qPCR Master Mix kit (Vazyme) and the Applied Biosystems fluorescent quantitative PCR analyzer (QuantStudioTM 6Flex). The results are shown in the figure. Figure 3 As shown, ZmDIR4 is dominantly expressed in maize leaves and its expression is upregulated by drought.

[0042] After three days of drought stress treatment, it was found that the wilting degree of the overexpression strains OE2 and OE3 (overexpression materials obtained in Example 1) was significantly stronger than that of the three strains B104 and CR4 and CR7 (C4 and C7) (knockout materials obtained in Example 1), among which the overall wilting degree of the knockout materials CR4 and CR7 was the lowest ( Figure 2 This suggests that ZmDIR4 can reduce the drought tolerance of plants under drought stress. After three days of treatment, it was found that ( Figure 4(A-D) There were no significant differences in growth indicators (plant height, root length, and dry and fresh weight) between overexpression strains (OE2, OE3), wild type, and Crispr knockout strains (C4, C7) under normal treatment. The plant heights of OE2, OE3, WT, C4, and C7 strains under normal treatment were 33.1 cm, 32.5 cm, 33.8 cm, 32.5 cm, and 32.7 cm, respectively, while the plant heights of OE2, OE3, WT, C4, and C7 strains under drought stress treatment were 20.4 cm, 20.9 cm, 23.4 cm, 28.1 cm, and 27.8 cm, respectively. The average plant height of Crispr strains under this treatment was significantly higher than that of wild type. Under normal treatment, the root lengths of OE2, OE3, WT, C4, and C7 were 25.9 cm, 26.2 cm, 25.7 cm, 26.4 cm, and 25.4 cm, respectively, while the root lengths of OE2, OE3, WT, C4, and C7 in the drought stress treatment group were 30.9 cm, 30.3 cm, 33.4 cm, 37.6 cm, and 37.5 cm, respectively. At this time, the average root length of Crispr strains was significantly The fresh weights of OE2, OE3, WT, C4, and C7 were 3.89 g, 3.90 g, 3.97 g, 3.93 g, and 3.83 g, respectively, under normal treatment, while the fresh weights of OE2, OE3, WT, C4, and C7 in the drought treatment group were 2.54 g, 2.44 g, 2.84 g, 3.37 g, and 3.35 g, respectively. The average fresh weight of Crispr strains was significantly higher than that of wild type and overexpression strains. In the overexpression strains; under normal treatment, the dry weights of OE2, OE3, WT, C4, and C7 strains were 0.66g, 0.63g, 0.62g, 0.64g, and 0.61g, respectively, while the dry weights of OE2, OE3, WT, C4, and C7 strains in the drought treatment group were 0.20g, 0.21g, 0.28g, 0.49g, and 0.44g, respectively. At this time, the average dry weight of Crispr strains was significantly higher than that of wild-type and overexpression strains. Using WT-B104, 2 overexpression materials, and 2 mutant materials for drought stress treatment, it was found that the fresh weight, dry weight, aboveground length, and underground length of CRISPR knockout materials were significantly higher than those of wild-type materials and overexpression materials, indicating that ZmDIR4 plays a negative regulatory role in maize drought stress resistance.

[0043] Example 3 Subcellular localization of ZmDIR4 gene

[0044] The ZmDIR4 (Zm00001d034563) sequence was amplified, cloned and constructed into the vector pCAMBIA1305-GFP (Prudin Biotech, Cat. No. 8600612) (between the BamHI and SmaI multiple cloning sites), and the AtH2B (At5g22880) sequence and AtWAK-HDEL (At1g21250) sequence were respectively constructed into the vector pCAMBIA1305-RFP (Prudin Biotech, Cat. No. 8604537) (between the BamHI and SmaI multiple cloning sites). After the vector was constructed, the plasmid was extracted and transfected into the competent GV3101 Agrobacterium. The prepared mixture was injected into tobacco leaves grown to 4 weeks old. After the tobacco was cultured in the dark for 36 hours, laser confocal observation and photography were performed. The method for constructing the subcellular localization experimental vector is the same as the reference of this article (Sun Yu. Functional analysis of maize ZmCCR-like5 regulating salt tolerance [D]. Yangzhou University, 2023. DOI: 10.27441 / d.cnki.gyzdu.2023.000707.), and the plasmid extraction method refers to the instructions of the plasmid extraction kit of Beijing Tiangen Biotechnology Co., Ltd. (Cat. No. DP103). The localization control protein H2B is a nuclear localized histone structure (WOZNYM, SCHATTAT MH, MATHUR N, et al. Color Recovery after Photoconversion of H2B::mEosFP Allows Detection of Increased NuclearDNA Content in Developing Plant Cells[J]. Plant Physiology, 2012, 158(1):95-106.), WAK-HDEL protein is localized in the endoplasmic reticulum, WAK is a cell wall-associated kinase protein (VERICA JA, HE ZH. The Cell Wall-Associated Kinase (WAK) and WAK-Like Kinase Gene Family[J]. Plant Physiology, 2002, 129(2):455-459.), and HDEL is a special endoplasmic reticulum retention polypeptide signal (GOMORD V, DENMAT L, FITCHETTE- et al. The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER [J]. The Plant Journal, 1997, 11 (2): 313-325.). Subcellular localization results showed that ZmDIR4 was localized in the endoplasmic reticulum structure ( Figure 5 ).

[0045] Example 4 Oxidative Metabolism Index Detection

[0046] This study determined the content of MDA (malondialdehyde), Pro (proline), H2O2 (hydrogen peroxide), O 2-The content of superoxide anion in corn leaves, as well as the activities of three enzymes in corn leaves: SOD (superoxide dismutase), CAT (catalase), and POD (peroxidase). The 12-day-old seedlings, the control group (wild-type control material, overexpression material, and knockout material) were treated with 1 / 2 Hogland nutrient solution, and the drought stress group (wild-type control material, overexpression material, and knockout material) were treated with 1 / 2 Hogland prepared 20% PEG6000 solution. After 3 days of treatment with 20% PEG6000 solution, the second true leaf was taken and tested according to the following method. All index tests were performed using kits produced by Solebow (the MDA test kit is BC0025, the Pro test kit is BC0295, the H2O2 test kit is BC3595, the superoxide anion content test kit is BC1295, the SOD test kit is BC0175, the CAT test kit is BC0205, and the POD test kit is BC0095). The method is the micro-method, and the microplate reader is used for detection. The chlorophyll extraction uses the second fresh leaf of the corn seedling, cuts and weighs 0.1g of fresh leaves for extraction. The method uses the organic solvent extraction method, and the extraction reagent (acetone) is purchased from Shanghai Yuanye Company (item number: R30054). Take fresh corn leaves, wash them with distilled water, dry the surface water, remove the midrib, weigh about 0.1g, cut them into pieces and put them in a mortar or homogenizer. Add extraction powder (calcium carbonate + quartz sand + buffer salt, the volume ratio of the three is 1:1:1) (10 mg) and 1 mL of extraction solution (anhydrous ethanol and acetone are mixed in a volume ratio of 1:2), grind thoroughly in the dark, and transfer to a 10 mL test tube. Rinse the mortar with the extraction solution (anhydrous ethanol and acetone are mixed in a volume ratio of 1:2), transfer all the washing solution to a 10 mL tube, make up to 10 mL with the extraction solution, and extract in the dark for 3 hours. If the tissue residue at the bottom is close to white, the extraction is complete. If the tissue residue is not completely white, continue to extract until the color of the tissue residue is close to white. Then filter with a 4-layer white gauze filter, centrifuge the filtrate, and take 200 μL of supernatant for detection on an enzyme marker. The absorbance at A663nm and A645nm was measured on an ELISA instrument, and the chlorophyll content was calculated using the formula: total chlorophyll (mg / g) = (20.2A645nm + 8.02A663nm) × V / (1000 × W) (A663nm, A645nm represent the absorbance at wavelengths of 663nm and 645nm; V is the volume of the extracts in different treatments, in mL; W is the weight of the fresh corn leaves, in g). The determination of the total lignan content requires weighing 0.1 g of the second corn true leaf frozen in liquid nitrogen, using methanol as the lignan extraction solvent, with a solid-liquid ratio of 1:20 (g:mL), setting the ultrasonic extraction instrument power to 250W, and extracting at 40°C for 1h.The supernatant was then centrifuged and tested using a plant total lignans ELISA kit (Chemical Book, catalog number CB21405974). Finally, the absorbance at 450 nm was measured on an ELISA reader, and the content was calculated based on the standard curve regression equation (y=0.235x+0.0239, R 2 =0.996). ABA was determined by GC-MS, and Zeatin was determined by LC-MS. Both determinations were completed by Shanghai Paisono Company.

[0047] Reverse transcription qPCR analysis

[0048] After 12-day-old seedlings (wild-type materials, overexpression materials, knockout materials (overexpression materials and knockout materials are materials obtained in Example 1)) were treated with 20% PEG6000 solution for 3 days (the control material was 1 / 2 Hogland nutrient solution), the second true leaf was taken, and the total RNA of corn leaves was extracted using the Tiangen TRIZon total RNA extraction kit (DP405), and then the total RNA of corn was reverse transcribed into cDNA using the HiScript Ⅲ Allin-one RT Supermix (Vazyme, Nanjing, China) kit. RT-qPCR was performed using the ChamQ SYBR color qPCR Master Mix kit (Vazyme). RT-qPCR analysis was performed using the Applied Biosystems fluorescent quantitative PCR analyzer (QuantStudioTM6Flex) of Applied Biosystems, USA. The RT-qPCR primers are shown in Table 1, and ZmACTIN1 was used as an internal reference gene.

[0049] Table 1 RT-qPCR primers

[0050]

[0051]

[0052] Malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide anion (O 2- ) will damage the plant cell membrane, cause oxidative damage, and inhibit plant growth. Figure 6As shown in A to F, under normal treatment, the MDA content in the leaves of OE2, OE3, WT, C4, and C7 lines were 148.63nmol / g, 170.21nmol / g, 181.28nmol / g, 155.16nmol / g, and 160.76nmol / g, respectively, and the difference was not significant. Under stress treatment, the MDA content in the leaves of OE2, OE3, WT, C4, and C7 lines was 756.32nmol / g, 789.36nmol / g, 557.63nmol / g, 323.30nmol / g, and 310.64nmol / g, respectively. At this time, the MDA content of Crispr strains was significantly lower than that of wild type and overexpression strains. Under normal treatment, the H2O2 content in the leaves of plants of different strains was not significantly different, which were 14.36μmol / g, 15.02μmol / g, 15.56μmol / g, 14.93μmol / g and 19.20μmol / g, respectively. However, after stress treatment, the H2O2 content of Crispr strain plants was significantly lower than that of wild type and overexpression strain, which were 80.63μmol / g, 77.20μmol / g, 49.02μmol / g, 24.98μmol / g and 30.72μmol / g, respectively. Superoxide anion (O 2- ) content in leaves of OE2, OE3, WT, C4, and C7 strains showed no significant difference under normal treatment, which were 2.06 μmol / g, 2.16 μmol / g, 2.10 μmol / g, 1.93 μmol / g, and 2.05 μmol / g, respectively. However, under treatment, the O content of Crispr strains increased. 2- The content was significantly lower than that of wild type and overexpression strains after treatment, which were 9.45μmol / g, 9.03μmol / g, 7.31μmol / g, 5.14μmol / g and 5.23μmol / g respectively. RT-qPCR analysis showed that under normal treatment, the endogenous antioxidant defense gene ZmSOD1 ( Fig. 9 Middle A), ZmCAT3( Fig. 9 Middle B), ZmPOD1( Fig. 9There was no significant difference in the relative expression of ZmSOD1, ZmCAT3, and ZmPOD1 in the leaves of the Crispr strains after stress treatment, but the relative expression of ZmSOD1, ZmCAT3, and ZmPOD1 in the leaves of the Crispr strains were significantly higher than those of the wild type and the overexpression strains. We then measured the activities of three key antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). It was found that under normal treatment, the SOD enzyme activities in the leaves of OE2, OE3, WT, C4, and C7 strains were 499.25U / g, 501.25U / g, 478.70U / g, 491.85U / g, and 504.70U / g, respectively, with no significant difference. However, under drought treatment, the SOD enzyme activity in the leaves of OE2, OE3, WT, C4 and C7 strains increased to 770.23U / g, 801.87U / g, 1000.87U / g, 1557.00U / g and 1455.65U / g, respectively. The SOD enzyme activity of Crispr strain plants was significantly higher than that of the wild type and the overexpression strain after treatment. Similarly, under normal treatment, the CAT enzyme activity in the leaves of OE2, OE3, WT, C4 and C7 strains was 158.26U / g, 154.36U / g, 169.35U / g, 168.86U / g and 159.23U / g, respectively, with no significant difference. Under drought treatment, the CAT enzyme activities in the leaves of OE2, OE3, WT, C4, and C7 strains increased to 394.36U / g, 387.25U / g, 563.47U / g, 823.56U / g, and 817.36U / g, respectively. The CAT enzyme activities of Crispr strains were significantly higher than those of wild type and overexpression strains after treatment. In addition, the POD enzyme activities in the leaves of OE2, OE3, WT, C4, and C7 strains were 1 The POD enzyme activity of Crispr strains was significantly higher than that of wild type and overexpression strains. The overall drought resistance was Crispr strains> wild type> overexpression strains, indicating that the knockout of ZmDIR4 can enhance the antioxidant capacity of plants, reduce the accumulation of ROS, alleviate the oxidative damage to cells, and improve the drought resistance of plants.

[0053] Under normal treatment, the ABA content in leaves of OE2, OE3, WT, C4, and C7 lines ( Figure 7E) were 8.35pg / mg, 7.96pg / mg, 7.14pg / g, 8.26pg / mg and 8.44pg / mg, respectively. The difference was not significant at this time. Under drought stress, the plants synthesized a large amount of ABA hormone to resist adverse stress. At this time, the ABA content in the leaves of OE2, OE3, WT, C4, and C7 strains increased to 36.85pg / mg, 39.41pg / mg, 55.36pg / mg, 69.26pg / mg and 71.32pg / mg, respectively. The ABA content of Crispr strains was significantly higher than that of wild-type and overexpressed strains. We then determined the key gene ZmSnRK2.6 ( Figure 8 ZhongD), ZmNCED3( Figure 8 Middle E) and ZmABA1 ( Figure 8 In Figure F), under normal treatment, there was no significant difference in the relative expression of these genes in different strains, but under drought treatment, the relative expression of ZmSnRK2.6, ZmNCED3 and ZmABA1 in Crispr strains was significantly higher than that in wild type and overexpression strains. Zeatin is also one of the key plant hormones for plants to resist stress. After drought stress treatment, the synthesis of this hormone will increase. Figure 7 As shown in D, under normal treatment, the zeatin content in the leaves of OE2, OE3, WT, C4, and C7 lines were 0.50μg / g, 0.53μg / g, 0.57μg / g, 0.55μg / g, and 0.56μg / g, respectively. After drought treatment, the zeatin content in the leaves of OE2, OE3, WT, C4, and C7 lines were 0.82μg / g, 0.94μg / g, 1.36μg / g, and 1.29μg / g, respectively. At this time, the zeatin content of the Crispr line plants was significantly higher than that of the wild type and the overexpression line. After measuring ZmIPT2( Fig. 9 Middle D), ZmCKO5( Fig. 9 Middle E) and ZmCZOG1 ( Fig. 9 F) These three key genes for zeatin biosynthesis, we found that under normal treatment, there was no significant difference in the relative expression of ZmIPT2, ZmCKO5 and ZmCZOG1 in the leaves of different strains, but after drought treatment, the relative expression of ZmIPT2, ZmCKO5 and ZmCZOG1 in the Crispr strain was significantly higher than that in the wild type and the overexpression strain. We continued to measure the relative gene expression of drought response-related genes and found that under normal treatment, ZmRD21( Figure 8 Middle A), ZmRD29A( Figure 8 Middle B) and ZmERD1 ( Figure 8There was no significant difference in the relative expression of the three genes in C), but the relative expression of the three genes in the leaves of C4 and C7 strains after treatment was significantly higher than that of WT and OE2 and OE3 strains. This shows that under drought conditions, the drought tolerance of the Crispr strain is stronger than that of the wild type and the overexpression strain. This shows that the knockout of the ZmDIR4 gene can enhance the synthesis of ABA and zeatin, thereby enhancing the drought tolerance of the plant, while the overexpression of ZmDIR4 will weaken the drought tolerance of the plant.

[0054] Free proline is a significant physiological indicator for measuring plant drought tolerance. The accumulation of proline can reduce the osmotic pressure on crops under drought stress. Figure 7 As shown in A), the content of free proline (Pro) in the leaves of OE2, OE3, WT, C4, and C7 strains was 139.24μg / g, 145.16μg / g, 141.56μg / g, 173.41μg / g, and 165.26μg / g, respectively. The difference was not significant at this time. In the treatment group, the Pro content of OE2, OE3, WT, C4, and C7 strains was 350.63μg / g, 376.84μg / g, 514.41μg / g, 1233.17μg / g, and 1162.89μg / g, respectively, indicating that the Pro content of Crispr strain plants was significantly higher than that of wild type under drought treatment, and the wild type was significantly higher than that of overexpression strains. This shows that the drought resistance of Crispr strains is stronger than that of wild type and overexpression strains.

[0055] like Figure 7 As shown in C, under normal treatment, the total lignan contents in the leaves of OE2, OE3, WT, C4 and C7 lines were 120.26 mg / g, 121.45 mg / g, 110.27 mg / g, 90.41 mg / g and 88.62 mg / g, respectively. Under drought treatment, the total lignan contents in the leaves of OE2, OE3, WT, C4 and C7 lines were 205.78 mg / g, 212.36 mg / g, 177.45 mg / g, 125.21 mg / g and 126.48 mg / g, respectively.

[0056] The determination of chlorophyll content also showed that ( Figure 7In B), under normal treatment, the chlorophyll contents of OE2, OE3, WT, C4, and C7 strains were 1.79 mg / g, 1.85 mg / g, 1.83 mg / g, 1.93 mg / g, and 1.79 mg / g, respectively, and the difference was not significant at this time. After drought stress treatment, the chlorophyll contents of OE2, OE3, WT, C4, and C7 strains were 0.73 mg / g, 0.70 mg / g, 1.00 mg / g, 1.26 mg / g, and 1.25 mg / g, respectively. The chlorophyll content of Crispr strain plants was significantly higher than that of wild type and overexpression strains. These growth indicators show that under drought stress, the drought tolerance among strains is Crispr knockout strains> wild type> overexpression strains.

[0057] It can be seen that ZmDIR4 has a negative regulatory function in corn drought stress. When ZmDIR4 is knocked out, the dry matter accumulation of mutant corn plants after drought stress, the activity of antioxidant enzymes in the antioxidant metabolic pathway, the accumulation of proline, chlorophyll, and lignans, and the activation level of the hormone metabolic pathway (zeatin and abscisic acid) are significantly higher than those of the wild type B104 and the overexpression material.

[0058] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. Application of ZmDIR4 gene in regulating drought sensitivity of corn, characterized in that: The accession number of the ZmDIR4 gene is Zm00001d034563.

2. The use according to claim 1, characterized in that: The regulation includes overexpressing the ZmDIR4 gene to reduce the drought resistance of corn; inhibiting the expression of the ZmDIR4 gene or knocking out the ZmDIR4 gene to improve the drought resistance of corn.

3. A biomaterial capable of regulating the expression of ZmDIR4 gene, characterized in that: The accession number of the ZmDIR4 gene is Zm00001d034563.

4. The biomaterial according to claim 3, characterized in that The biological material overexpressing the ZmDIR4 gene comprises one or more of a primer set for amplifying the ZmDIR4 gene, a recombinant expression vector containing the ZmDIR4 gene, and a recombinant microorganism containing the ZmDIR4 gene.

5. The biomaterial according to claim 3, characterized in that The invention comprises biological materials for inhibiting the expression of the ZmDIR4 gene or knocking out the ZmDIR4 gene, wherein the inhibition method comprises silencing.

6. The biomaterial according to claim 5, characterized in that The biological material for knocking out the ZmDIR4 gene uses the sequence shown in SEQ ID NO.1 as a target.

7. Use of the biological material according to any one of claims 3 to 6 in improving corn germplasm or creating new germplasm.

8. A method for creating drought-resistant corn germplasm, characterized in that: The following steps are involved: The expression level of the ZmDIR4 gene in the target corn genome is knocked down, and the accession number of the ZmDIR4 gene is Zm00001d034563.

9. A method for creating a drought-sensitive corn model, characterized in that: The following steps are involved: The ZmDIR4 gene is overexpressed in the target maize genome, and the accession number of the ZmDIR4 gene is Zm00001d034563.

10. The method according to claim 8 or 9, characterized in that: The corn includes corn B104.

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