Use of a ZmDIR4 gene in the regulation of corn sensitivity to drought

Abstract

The application belongs to the technical field of biological genetic engineering, and particularly relates to application of a ZmDIR4 gene in regulation of drought sensitivity of corn. The application finds through experiments that after the ZmDIR4 gene 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 lignan, and the activation degree of the hormone metabolic pathway (zeatin and abscisic acid) are all significantly higher than those of wild type and overexpression materials, proving that the ZmDIR4 gene has a negative regulation function in drought stress resistance of corn. The ZmDIR4 gene can be used to create new corn germplasm with improved drought resistance, and provides a new idea for corn drought resistance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological gene engineering, and particularly relates to application of a ZmDIR4 gene in regulation of drought sensitivity of maize. BACKGROUND

[0002] Maize is one of the most important food crops in the world, and is of great concern due to its significant adaptability and wide planting area. In maize production, a key challenge is the impact of drought stress, which greatly limits the yield potential. In response to these abiotic stresses, maize plants have evolved complex physiological and molecular response systems. Among them, the role of drought-resistant genes is particularly significant, which plays a key role in regulating plant growth, development process and morphological establishment, and also plays an important role in environmental stress response. Therefore, it is very important to mine maize drought-resistant genes, analyze maize drought-resistant mechanisms, and improve the drought resistance of maize for the realization of food security and the sustainable development goals of future agricultural production. At the same time, mining maize drought-resistant genes has important research foundation and practical significance for using genetic engineering to accelerate maize drought-resistant breeding. SUMMARY

[0003] The purpose of the present application is to provide application of a ZmDIR4 gene in regulation of drought sensitivity of maize, and the ZmDIR4 gene has a negative regulation function in maize drought stress resistance, which provides a new idea for maize drought resistance.

[0004] The present application provides application of a ZmDIR4 gene in regulation of drought sensitivity of maize, and the accession number of the ZmDIR4 gene is Zm00001d034563.

[0005] As a preferred solution, the regulation comprises overexpression of the ZmDIR4 gene to reduce the drought resistance of maize, and inhibition of expression or knockout of the ZmDIR4 gene to improve the drought resistance of maize.

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

[0007] As a preferred solution, the biological material comprises overexpression of the ZmDIR4 gene, and the biological material comprising overexpression of 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.

[0008] As a preferred solution, the biological material comprises inhibition of expression or knockout of the ZmDIR4 gene, and the inhibition method comprises silencing.

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

[0010] The application further provides application of the biological material in maize germplasm improvement or new germplasm creation.

[0011] The application further provides a method for creating drought-resistant maize germplasm, comprising the following steps: knocking down the expression amount of a ZmDIR4 gene in a target maize genome, wherein the accession number of the ZmDIR4 gene is Zm00001d034563.

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

[0013] As a preferred solution, the maize comprises maize B104.

[0014] Beneficial effects: the application provides application of a ZmDIR4 gene in regulation of drought sensitivity of maize. The application finds through experiments that after the ZmDIR4 is knocked out, the dry matter accumulation of mutant maize plants after drought stress, the activity of antioxidant enzymes in the antioxidant metabolic pathway, the accumulation of proline, chlorophyll and lignan, and the activation degree of the hormone metabolic pathway (zeatin and abscisic acid) are all significantly higher than those of wild type and overexpression materials, proving that the ZmDIR4 has a negative regulation function in drought stress resistance of maize. The ZmDIR4 gene provided in the application can be used to create new maize germplasm with improved drought resistance, and provides a new idea for maize drought resistance. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0016] Figure 1 Figures for construction of ZmDIR4 gene overexpression materials and knockout materials in Example 1, wherein A is the amplification result of the ZmDIR4 gene, B is the expression amount of the ZmDIR4 gene of the overexpression material, and C and D are the fragment deletion conditions of the knockout material;

[0017] Figure 2 Figures for phenotypes of overexpression materials and knockout materials under drought stress in Example 2;

[0018] Figure 3Figure 2 is a graph showing the expression of ZmDIR4 gene in different parts of corn under drought stress and the expression of ZmDIR4 gene in the second true leaf of corn under different treatment times, wherein the left graph shows the expression of ZmDIR4 gene in different parts of corn under drought stress, and the right graph shows the expression of ZmDIR4 gene in the second true leaf of corn under different treatment times;

[0019] Figure 4 Figure 4 is a graph showing the fresh weight, dry weight, length of aboveground part, and length of underground part (root length) of overexpression material and mutant material after drought stress treatment, wherein A is the fresh weight, B is the dry weight, C is the length of aboveground part, and D is the length of underground part (root length);

[0020] Figure 5 Figure 5 is a graph showing the subcellular localization of ZmDIR4, wherein the fusion vectors GFP-ZmDIR4, RFP-AtH2B, and RFP-AtWAK-HDEL are transiently expressed in tobacco leaves, the GFP fluorescence is green, the RFP fluorescence is red, and the Merge graph is a superimposed graph of green and red fluorescence;

[0021] Figure 6 Figure 6 is a graph showing the antioxidant capacity detection results of ZmDIR4 gene overexpression material and knockout material, wherein A is the malondialdehyde detection result, B is the hydrogen peroxide detection result, C is the superoxide anion detection result, D is the superoxide dismutase detection result, E is the catalase detection result, and F is the peroxidase detection result;

[0022] Figure 7 Figure 7 is a graph showing the metabolite accumulation detection results of ZmDIR4 gene overexpression material and knockout material, wherein A is the proline detection result, B is the chlorophyll detection result, C is the total lignan detection result, D is the zeatin detection result, and E is the abscisic acid detection result;

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

[0024] Figure 9 Figure 9 is a graph showing the gene relative expression detection results of ZmDIR4 gene overexpression material and knockout material, wherein A is ZmSOD1, B is ZmCAT3, C is ZmPOD1, D is ZmIPT2, E is ZmCKO5, and F is ZmZOG1. DETAILED DESCRIPTION

[0025] The application provides application of a ZmDIR4 gene in regulation of drought sensitivity of corn, wherein the accession number of the ZmDIR4 gene is Zm00001d034563.

[0026] As a specific embodiment, the regulation comprises overexpression of the ZmDIR4 gene, reduction of drought resistance of corn; inhibition of expression of the ZmDIR4 gene or knockout of the ZmDIR4 gene, improvement of drought resistance of corn.

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

[0028] The biological material has the effect of regulating expression amount of the ZmDIR4 gene, such as overexpression of the ZmDIR4 gene, inhibition of expression of the ZmDIR4 gene or knockout of the ZmDIR4 gene. As a specific embodiment, the biological material for overexpression of 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. As an embodiment, the recombinant expression vector containing the ZmDIR4 gene comprises a pBCXUN-ZmDIR4 overexpression vector obtained by inserting the ZmDIR4 gene into a pBCXUN vector. As an embodiment, the recombinant microorganism containing the ZmDIR4 gene comprises an agrobacterium containing the ZmDIR4 gene.

[0029] As a specific embodiment, the biological material for inhibition of expression of the ZmDIR4 gene or knockout of the ZmDIR4 gene comprises a method for inhibition, which comprises silencing. As a specific embodiment, the biological material for knockout of the ZmDIR4 gene takes the sequence shown in SEQ ID NO. 1 as a target. 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 comprises an agrobacterium containing the gene editing vector.

[0030] The application also provides application of the biological material in corn germplasm improvement or new germplasm creation.

[0031] The application also provides a method for creating drought-resistant maize germplasm, comprising the following steps: knocking down the expression amount of a ZmDIR4 gene in a target maize genome, wherein the accession number of the ZmDIR4 gene is Zm00001d034563. In specific embodiments, after the pBUE411-ZmDIR4 gene editing vector is transferred into the Agrobacterium EHA101 strain, the gene editing vector is transformed into the target maize plant by using the Agrobacterium-mediated transgenic method, and the maize with improved drought resistance is obtained. As a specific embodiment, after drought stress treatment, it is found that the fresh weight, dry weight, length of the aboveground part and length of the underground part of the CRISPR knockout material are significantly higher than those of the wild type material and the overexpression material, indicating that ZmDIR4 plays a negative regulation role in the drought stress resistance of maize.

[0032] The application also provides a method for creating a drought-sensitive maize model, comprising the following steps: overexpressing a ZmDIR4 gene in a target maize genome, wherein the accession number of the ZmDIR4 gene is Zm00001d034563. In specific embodiments, after the pBCXUN-ZmDIR4 overexpression vector is transferred into the Agrobacterium GV3101 strain, the overexpression vector is transformed into the target maize plant by using the Agrobacterium-mediated transgenic method, and the drought-sensitive maize model is obtained. As a specific embodiment, the maize includes maize B104.

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

[0034] In order to further illustrate the application, the application of the ZmDIR4 gene in the regulation of the drought sensitivity of maize is described in detail in the embodiments below, but they should not be understood as limiting the scope of protection of the application.

[0035] Unless otherwise specified, the raw materials used in the application have no special requirements and commercially available products known to those skilled in the art can be used.

[0036] Example 1: Obtaining ZmDIR4 gene overexpression material and knockout material

[0037] The present study carried out CRISPR knockout and 35S: overexpression in the background material corn B104 (Yao, M. Y., Li, J., Liu, Z. G., et al. Cloning of maize ZmMPI gene and detection of its overexpression transgenic lines [J]. North China Journal of Agricultural Sciences, 2024, 39(4): 1-9.) to obtain two overexpression materials (OE2 / OE3) and two CRISPR materials with different knockout sites (CR4 / CR7) Figure 1 ), and WT-B104 material as control material. The overexpression material used the vector pBCXUN (Sun, Y. Functional analysis of maize ZmCCR-like5 in salt tolerance [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 ZmDIR4 gene was inserted into the pBCXUN vector (between the two multiple cloning sites of BamHI and SmaI) to obtain the pBCXUN-ZmDIR4 recombinant overexpression vector. The Crispr gene editing vector was pBUE411 (Yan, T. W. Systematic analysis of maize ZmLBD gene family and functional mechanism of ZmMs1, ZmIG1 and ZmIAL1 [D]. Beijing University of Science and Technology, 2024.), and the target sequence was 5'-TTCCTCCACGACACTCTGAG-3' (as shown in SEQ ID NO. 1). The knockout vector primers were F: 5'-GCCGTTCCTCCACGACACTCTGAG-3' (as shown in SEQ ID NO. 4); R: 5'-AAACCTCAGAGTGTCGTGGAGGAA-3' (as shown in SEQ ID NO. 5). The target sequence was constructed into the vector pBUE411 (the multiple cloning site of the vector was BsaI) to obtain the pBUE411-ZmDIR4 recombinant knockout vector (gene editing vector).

[0038] After pBCXUN-ZmDIR4 overexpression vector was transformed into Agrobacterium GV3101 strain, the overexpression vector was transformed into target maize plants by Agrobacterium-mediated transformation method (Qin T, Liu S, Zhang Z, et al. GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the E3 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 pBUE411-ZmDIR4 gene editing vector was transformed into Agrobacterium EHA101 strain, the gene editing vector was transformed into target maize plants by Agrobacterium-mediated transformation method (Qin T, Liu S, Zhang Z, et al. GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the E3 ubiquitin 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 Phenotype analysis of overexpression materials and knockout materials under drought stress

[0040] The maize wild type control material, overexpression material and knockout material (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, under the condition of 16h light and 8h darkness. 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 20% PEG6000 solution prepared with 1 / 2 Hogland.

[0041] The tissue expression pattern analysis 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 leaves of the wild-type corn at different time points (0 h, 1 h, 6 h, 12 h, 18 h, 24 h, 36 h and 48 h) under drought stress were 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, and the experiment analysis was completed according to the method of ChamQ SYBR color qPCR Master Mix kit (Vazyme) instruction and Applied Biosystems fluorescence quantitative PCR analyzer (QuantStudioTM 6 Flex). The results are shown in Figure 3 ZmDIR4 is mainly expressed in corn leaves and is up-regulated by drought induction.

[0042] After three days of drought stress treatment, it was found that the wilting degree of the overexpression lines OE2 and OE3 (overexpression materials obtained in Example 1) was significantly higher than that of B104 and CR4, CR7 (knockout materials C4, C7) (knockout materials obtained in Example 1), and the overall wilting degree of the knockout materials CR4 and CR7 was the lowest Figure 2 . This indicates that ZmDIR4 can reduce the drought tolerance of plants under drought stress. After three days of treatment, it was found that Figure 4Under normal treatment, the differences in growth indicators (plant height, root length, and dry and fresh weight) of the overexpression lines (OE2, OE3), the wild type, and the Crispr knockout lines (C4, C7) were not significant. The plant heights of OE2, OE3, WT, C4, and C7 under normal treatment were 33.1 cm, 32.5 cm, 33.8 cm, 32.5 cm, and 32.7 cm, respectively, and the plant heights of OE2, OE3, WT, C4, and C7 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 the Crispr lines was significantly higher than that of the wild type and the overexpression lines under this treatment. 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, and the root lengths of OE2, OE3, WT, C4, and C7 under drought stress treatment 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 the Crispr lines was significantly higher than that of the wild type and the overexpression lines. Under normal treatment, 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, and the fresh weights of OE2, OE3, WT, C4, and C7 under drought treatment were 2.54 g, 2.44 g, 2.84 g, 3.37 g, and 3.35 g, respectively. The average fresh weight of the Crispr lines was significantly higher than that of the wild type and the overexpression lines. Under normal treatment, the dry weights of OE2, OE3, WT, C4, and C7 were 0.66 g, 0.63 g, 0.62 g, 0.64 g, and 0.61 g, respectively, and the dry weights of OE2, OE3, WT, C4, and C7 under drought treatment were 0.20 g, 0.21 g, 0.28 g, 0.49 g, and 0.44 g, respectively. At this time, the average dry weight of the Crispr lines was significantly higher than that of the wild type and the overexpression lines. Using WT-B104, two overexpression materials, and two mutant materials for drought stress treatment, it was found that the fresh weight, dry weight, aboveground part length, and underground part 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 drought stress resistance in maize.

[0043] Example 3 Subcellular localization of ZmDIR4 gene

[0044] ZmDIR4 (Zm00001d034563) sequence was amplified and cloned into the vector pCAMBIA1305-GFP (Puritech, Cat# 8600612) between the BamHI and Smal multiple cloning sites, AtH2B (At5g22880) sequence and AtWAK-HDEL (At1g21250) sequence were constructed into the vector pCAMBIA1305-RFP (Puritech, Cat# 8604537) between the BamHI and Smal multiple cloning sites, respectively. The constructed vectors were extracted as plasmids, and then transfected into the competent GV3101 Agrobacterium. The mixed liquid was injected into the tobacco leaves which were grown to 4 weeks old. After 36h dark culture, the laser confocal observation was performed and the pictures were taken. The method of constructing the vector for subcellular localization experiment was the same as the reference (Sun Y. Functional analysis of maize ZmCCR-like5 regulating salt tolerance[D]. Yangzhou University, 2023. DOI:10.27441 / d.cnki.gyzdu.2023.000707.), and the method of extracting plasmid was referred to the plasmid extraction kit instruction of Beijing Tiangen Biochemical Technology Co., Ltd. (Cat# DP103). The control protein H2B is a nuclear-localized histone structure (Wozny M, Schattat M H, Mathur N, et al. Color Recovery after Photoconversion of H2B::mEosFP Allows Detection of Increased Nuclear DNA Content in Developing Plant Cells[J]. Plant Physiology, 2012, 158(1): 95-106.), and the WAK-HDEL protein is located in the endoplasmic reticulum. WAK is a cell wall-associated kinase protein (Verica J A, He Z H. 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-Laine A C, et al. Different Plant Cell Types Sequester Different Sets of N-Linked High-Mannose Oligosaccharides[J]. Proc Natl Acad Sci U S A, 1995, 92(25): 1 1878-1 1882.). 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. The Plant Journal, 1997, 11(2): 313-325.) Subcellular localization results show that ZmDIR4 is located in the endoplasmic reticulum structure Figure 5

[0045] Example 4 Oxidative metabolism index detection

[0046] MDA (malondialdehyde), Pro (proline), H2O2 (hydrogen peroxide), O 2- ​The content of superoxide anion in the corn leaves and the activity of SOD (superoxide dismutase), CAT (catalase), and POD (peroxidase) in the corn leaves. The 12-day-old seedlings of 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) was treated with 20% PEG6000 solution prepared with 1 / 2 Hogland. After 3 days of treatment with 20% PEG6000 solution, the second true leaf was taken and detected according to the following method. The index detection was performed using the kit produced by Solabio Company (MDA detection kit, item number BC0025, Pro detection kit, item number BC0295, H2O2 detection kit, item number BC3595, superoxide anion content detection kit, item number BC1295, SOD detection kit, item number BC0175, CAT detection kit, item number BC0205, and POD detection kit, item number BC0095). The method was micro method, and the enzyme label instrument was used for detection. The second fresh leaf of the corn seedling was used for extraction of chlorophyll. 0.1 g of fresh leaf was weighed and extracted. The organic solvent extraction method was used. The extraction reagent (acetone) was purchased from Shanghai Yuanye Company (item number: R30054). Fresh corn leaves were washed with distilled water, the surface water was absorbed, the midrib was removed, about 0.1 g was weighed, and was cut into a mortar or homogenizer. 10 mg of extraction powder (calcium carbonate+quartz sand+buffer salt, the volume ratio of the three was 1:1:1) and 1 mL of extraction solution (anhydrous ethanol and acetone mixed in a volume ratio of 1:2) were added. The mortar was ground thoroughly in the dark condition, and was transferred into a 10 mL test tube. The mortar was washed with extraction solution (anhydrous ethanol and acetone mixed in a ratio of 1:2), and all the washing liquid was transferred into a 10 mL tube. The extraction solution was added to 10 mL, and was immersed for 3 h in the dark condition. The bottom tissue residue was observed to be close to white, indicating complete extraction. If the tissue residue was not completely white, the immersion was continued until the tissue residue was close to white. Then, the extraction solution was filtered with 4 layers of white gauze. After centrifugation, 200 μL of supernatant was detected on the enzyme label instrument. The absorbance values at A663 nm and A645 nm were measured on the enzyme label instrument. The chlorophyll content was calculated according to the formula: total chlorophyll content (mg / g)=(20.2 A645 nm+8.02 A663 nm)×V / (1000×W). (A663 nm and A645 nm represent the absorbance values at wavelengths of 663 nm and 645 nm; V is the volume of the extraction solution, in mL; and W is the fresh weight of the corn, in g). The determination of total lignin content requires weighing 0.1 g of the second corn true leaf frozen in liquid nitrogen. Methanol is used as the lignin extraction solvent, the solid-liquid ratio is 1:20 (g:mL), the ultrasonic extraction instrument power is set to 250 W, and the extraction is performed at 40°C for 1 h.Thereafter, centrifugation was performed to take the supernatant, which was detected by using a total lignan ELISA detection kit (Chemical Book, item number CB21405974). Finally, the absorbance value at 450 nm was determined on an enzyme label instrument, and the content was calculated according to the standard curve regression equation (y=0.235x+0.0239, R. 2 The determination of ABA was performed by using a GC-MS method, and the determination of Zeatin was performed by using an LC-MS method. Both determinations were completed by Shanghai Pisenino Company.

[0047] Reverse transcription qPCR analysis

[0048] After 12-day-old seedlings (wild-type material, overexpression material, knockout material (the overexpression material and the knockout material are materials obtained in Example 1)) were treated with a 20% PEG6000 solution for 3 days (the control material was 1 / 2 Hogland nutrient solution), the second true leaf was taken, total RNA of the corn leaf was extracted by using a TRIZon total RNA extraction kit (DP405) of Tiangen, and then the total RNA of the corn was reverse transcribed into cDNA by using a HiScript III All-in-one RT Supermix (Vazyme, Nanjing, China) kit. RT-qPCR was performed by using a ChamQ SYBR color qPCR Master Mix kit (Vazyme). RT-qPCR analysis was completed by using an Applied Biosystems fluorescence quantitative PCR analyzer (QuantStudioTM6 Flex) of American Applied Biosystems Company. 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] Accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide anion (O 2- ) can damage the cell membrane of the plant, cause oxidative damage, and inhibit the growth of the plant. The results are shown in Table 2. Figure 6As shown in Figures A through F, under normal treatment, the MDA content in the leaves of OE2, OE3, WT, C4, and C7 lines was 148.63 nmol / g, 170.21 nmol / g, 181.28 nmol / g, 155.16 nmol / g, and 160.76 nmol / g, respectively, with no significant difference. However, under stress treatment, the MDA content in the leaves of OE2, OE3, WT, C4, and C7 lines was 756.32 nmol / g, 789.36 nmol / g, 557.63 nmol / g, 323.30 nmol / g, and 310.64 nmol / g, respectively. Under these conditions, the MDA content of the CRISPR lines was significantly lower than that of the wild type and lower than that of the overexpression lines. Under normal treatment, the H2O2 content in the leaves of different plant lines did not differ significantly, being 14.36 μmol / g, 15.02 μmol / g, 15.56 μmol / g, 14.93 μmol / g, and 19.20 μmol / g, respectively. However, after stress treatment, the H2O2 content of the CRISPR lines was significantly lower than that of the wild type and the overexpression lines, being 80.63 μmol / g, 77.20 μmol / g, 49.02 μmol / g, 24.98 μmol / g, and 30.72 μmol / g, respectively. Superoxide anion (O2) 2- The determination of O2 content also showed no significant differences in the leaves of OE2, OE3, WT, C4, and C7 lines under normal treatment, with values ​​of 2.06 μmol / g, 2.16 μmol / g, 2.10 μmol / g, 1.93 μmol / g, and 2.05 μmol / g, respectively. However, under the treatment, the O2 content in the CRISPR lines was significantly higher. 2- The levels of the gene ZmSOD1, a gene responsible for endogenous antioxidant defense, were significantly lower in the treated lines than in the wild type and the overexpression lines, at 9.45 μmol / g, 9.03 μmol / g, 7.31 μmol / g, 5.14 μmol / g, and 5.23 μmol / g, respectively. RT-qPCR analysis showed that under normal treatment, the levels of ZmSOD1, a gene responsible for endogenous antioxidant defense, were significantly lower in the overexpression lines (OE2, OE3), wild type, and Crispr knockout lines (C4, C7). Figure 9 (A), ZmCAT3 ( Figure 9 (B), ZmPOD1 ( Figure 9The relative expression of C) had no significant difference, but after stress treatment, the relative expression of ZmSOD1, ZmCAT3, ZmPOD1 in the leaves of Crispr strain was significantly higher than that in wild type and overexpression strain. We then determined the activity of three key antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), peroxidase (POD). It was found that under normal treatment, the SOD enzyme activity in the leaves of OE2, OE3, WT, C4, C7 strains was 499.25 U / g, 501.25 U / g, 478.70 U / g, 491.85 U / g and 504.70 U / g, respectively, with no significant difference. But under drought treatment, the SOD enzyme activity in the leaves of OE2, OE3, WT, C4, C7 strains increased to 770.23 U / g, 801.87 U / g, 1000.87 U / g, 1557.00 U / g and 1455.65 U / g, respectively, and the SOD enzyme activity of Crispr strain was significantly higher than that of wild type and overexpression strain after treatment; similarly, under normal treatment, the CAT enzyme activity in the leaves of OE2, OE3, WT, C4, C7 strains was 158.26 U / g, 154.36 U / g, 169.35 U / g, 168.86 U / g, 159.23 U / g, respectively, with no significant difference. Under drought treatment, the CAT enzyme activity in the leaves of OE2, OE3, WT, C4, C7 strains increased to 394.36 U / g, 387.25 U / g, 563.47 U / g, 823.56 U / g and 817.36 U / g, respectively, and the CAT enzyme activity of Crispr strain was significantly higher than that of wild type and overexpression strain after treatment; in addition, the POD enzyme activity in the leaves of OE2, OE3, WT, C4, C7 strains under normal treatment was 15134.00 U / g, 14034.00 U / g, 13416.59 U / g, 14249.61 U / g and 16310.73 U / g, respectively, but under treatment, it increased to 22000.44 U / g, 22453.31 U / g, 28042.25 U / g, 43473.42 U / g and 46578.00 U / g, respectively, and the POD enzyme activity of Crispr strain was significantly higher than that of wild type and overexpression strain at this time. The overall drought tolerance was Crispr strain > wild type > overexpression strain, indicating that the knockout of ZmDIR4 could enhance the antioxidant capacity of the plant, reduce the accumulation of ROS, reduce the oxidative damage of cells and improve the drought resistance of the plant.

[0053] Under normal treatment, the ABA content in the leaves of OE2, OE3, WT, C4, C7 strains was 0.23 μg / g, 0.22 μg / g, 0.24 μg / g, 0.23 μg / g and 0.22 μg / g, respectively, with no significant difference. Under drought treatment, the ABA content in the leaves of OE2, OE3, WT, C4, C7 strains increased to 0.45 μg / g, 0.44 μg / g, 0.46 μg / g, 0.45 μg / g and 0.44 μg / g, respectively, and the ABA content of Crispr strain was significantly higher than that of wild type and overexpression strain after treatment. Figure 7The ABA content in the leaves of the OE2, OE3, WT, C4, and C7 lines was 8.35 pg / mg, 7.96 pg / mg, 7.14 pg / mg, 8.26 pg / mg, and 8.44 pg / mg, respectively, with no significant difference. However, under drought stress, plants synthesized large amounts of ABA to resist the stress. At this time, the ABA content in the leaves of the OE2, OE3, WT, C4, and C7 lines increased to 36.85 pg / mg, 39.41 pg / mg, 55.36 pg / mg, 69.26 pg / mg, and 71.32 pg / mg, respectively. The ABA content in the CRISPR lines was significantly higher than that in the wild type and higher than that in the overexpression lines. Subsequently, we determined the key gene ZmSnRK2.6 (…) related to ABA synthesis transduction. Figure 8 D), ZmNCED3 ( Figure 8 (E) and ZmABA1 ( Figure 8 In the normal treatment (F), there was no significant difference in the relative expression levels of these genes among different strains. However, under drought treatment, the relative expression levels of ZmSnRK2.6, ZmNCED3, and ZmABA1 in Crispr strains were significantly higher than those in wild-type plants, which in turn were higher than those in overexpression strains. Zeatin is also one of the most crucial plant hormones for resisting stress, and its synthesis increases after drought stress treatment. Figure 7 As shown in Figure D, under normal treatment, the zeatin content in the leaves of OE2, OE3, WT, C4, and C7 lines were 0.50 μg / g, 0.53 μg / g, 0.57 μg / g, 0.55 μg / g, and 0.56 μg / g, respectively. After drought treatment, the zeatin content in the leaves of OE2, OE3, WT, C4, and C7 lines were 0.82 μg / g, 0.94 μg / g, 1.36 μg / g, and 1.29 μg / g, respectively. At this point, the zeatin content of the CRISPR lines was significantly higher than that of the wild type and higher than that of the overexpression lines. The ZmIPT2 ( Figure 9 D), ZmCKO5 ( Figure 9 (E) and ZmCZOG1 ( Figure 9 We investigated the expression levels of three key maize glycogen synthesis genes (ZmIPT2, ZmCKO5, and ZmCZOG1) in leaves of different maize lines under normal treatment. We found no significant difference in the relative expression levels of ZmIPT2, ZmCKO5, and ZmCZOG1 in the leaves of different lines under normal treatment. However, after drought treatment, the relative expression levels of ZmIPT2, ZmCKO5, and ZmCZOG1 in the CRISPR lines were significantly higher than those in the wild type and higher than those in the overexpression lines. We further measured the relative gene expression levels of drought-related genes and found that under normal treatment, ZmRD21 (… Figure 8 (A), ZmRD29A ( Figure 8 (B) and ZmERD1 ( Figure 8There was no significant difference in the relative expression levels of C4 and C7 lines, but after treatment, the relative expression levels of these three genes in the leaves of C4 and C7 lines were significantly higher than those of WT, which in turn were higher than those of OE2 and OE3 lines. This indicates that under drought conditions, the CRISPR lines exhibit stronger drought tolerance than the wild type, which in turn is stronger than the overexpression lines. This suggests that knockout of the ZmDIR4 gene enhances the synthesis of ABA and zeatin, thereby increasing the plant's drought tolerance, while overexpression of ZmDIR4 weakens the plant's drought tolerance.

[0054] Free proline is a key physiological indicator of plant drought tolerance; proline accumulation can alleviate osmotic pressure on crops under drought stress. Measurements (results as shown) Figure 7 As shown in Figure A, the free proline (Pro) content in the leaves of the OE2, OE3, WT, C4, and C7 lines was 139.24 μg / g, 145.16 μg / g, 141.56 μg / g, 173.41 μg / g, and 165.26 μg / g, respectively, with no significant difference. However, the Pro content in the treatment groups OE2, OE3, WT, C4, and C7 lines was 350.63 μg / g, 376.84 μg / g, 514.41 μg / g, 1233.17 μg / g, and 1162.89 μg / g, respectively. This indicates that the Pro content of the CRISPR lines was significantly higher than that of the wild type under drought treatment, and the wild type was significantly higher than that of the overexpression lines. This suggests that the drought tolerance of the CRISPR lines is stronger than that of the wild type and stronger than that of the overexpression lines.

[0055] like Figure 7 As shown in Figure C, under normal treatment, the total lignan content in the leaves of OE2, OE3, WT, C4, and C7 strains was 120.26 mg / g, 121.45 mg / g, 110.27 mg / g, 90.41 mg / g, and 88.62 mg / g, respectively. Meanwhile, under drought treatment, the total lignan content in the leaves of OE2, OE3, WT, C4, and C7 strains was 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 ( Figure 7Under normal treatment, the chlorophyll contents of OE2, OE3, WT, C4 and C7 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. After drought stress treatment, the chlorophyll contents of OE2, OE3, WT, C4 and C7 were 0.73 mg / g, 0.70 mg / g, 1.00 mg / g, 1.26 mg / g and 1.25 mg / g respectively, and the chlorophyll content of the Crispr strain was significantly higher than that of the wild type and the overexpression strain. These growth indicators showed that under drought stress, the drought tolerance of the strains was Crispr knockout strain > wild type > overexpression strain.

[0057] Therefore, ZmDIR4 has a negative regulation function in drought stress resistance of corn. After 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 lignan, and the activation degree of the hormone metabolic pathway (zeatin and abscisic acid) are significantly higher than those of wild type B104 and overexpression materials.

[0058] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which belong to the protection scope of the present application.

Claims

1. ZmDIR4 The application of genes in regulating the drought sensitivity of maize is characterized by, The ZmDIR4 The gene's accession number is Zm00001d034563; The regulation is to inhibit the ZmDIR4 Gene expression or knockout ZmDIR4 Genes that enhance corn's drought resistance.

2. The application according to claim 1, characterized in that, Using suppression ZmDIR4 Gene expression or knockout ZmDIR4 Biomaterial regulation of genes ZmDIR4 Gene.

3. The application according to claim 2, characterized in that, Knock out the ZmDIR4 The biological material of the gene is targeted with the sequence shown in SEQ ID NO.

1.

4. Knockout ZmDIR4 The application of genetically modified biomaterials in maize germplasm improvement or creation of new maize germplasm is characterized by, The ZmDIR4 The gene's accession number is Zm00001d034563; knocking out the described gene... ZmDIR4 The biological material of the gene is targeted with the sequence shown in SEQ ID NO.

1.

5. A method for creating drought-resistant maize germplasm, characterized in that, Includes the following steps: Knock down the target maize genome ZmDIR4 The expression level of the gene, the ZmDIR4 The gene's accession number is Zm00001d034563.

6. The method according to claim 5, characterized in that, The corn includes corn B104.

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