Application of ZmPOD44 gene or biological material containing ZmPOD44 gene

Abstract

The present invention belongs to the field of plant molecular biological breeding, and specifically relates to the application of ZmPOD44 gene or biomaterial containing ZmPOD44 gene. The present invention identifies the corn peroxidase ZmPOD44 gene, which has the highest expression in leaves and strongly responds to drought stress. Under drought stress, corn overexpressing ZmPOD44 gene has higher germination rate and survival rate, more developed root system and significantly improved drought resistance. It is also found that the content of active oxygen in overexpressed plants is significantly reduced, the activity of antioxidant enzymes is increased, and the overexpression of ZmPOD44 gene significantly improves the ability of corn to remove active oxygen. In addition, the lignification degree of corn leaves overexpressing ZmPOD44 gene is deeper. These results show that ZmPOD44 gene may affect the drought resistance of corn by promoting the synthesis and lignification degree of lignin and regulating the accumulation of active oxygen in plants.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biological breeding, and in particular relates to the application of ZmPOD44 gene or biological materials containing ZmPOD44 gene. Background Art

[0002] Corn is a major food crop and cash crop. Lack of water not only affects the growth and development of corn, but also seriously restricts its yield. Drought stress can slow down or even stop the growth of corn plants. In terms of corn plant morphology, the leaves become smaller, the stems become thinner, and the roots become shallower. In addition, under drought stress, corn will close its stomata to reduce water evaporation, thereby inhibiting photosynthesis and reducing corn yield. At this time, corn plants will produce a stress response and adjust the expression level of genes to adapt to drought. Recent studies have found that the expression of the ZmWRKY106 gene is strongly induced by drought stress and resists drought stress through the ABA signaling pathway. Under drought stress, the ZmHsf28 gene can downregulate the expression of jasmonic acid (JA) and abscisic acid (ABA) biosynthesis genes, ROS scavenging genes and other drought-related genes, reducing the drought resistance of the plant. Silencing it can significantly improve the drought tolerance of corn. In addition, changes in hormone levels and signal transduction levels can also promote the drought resistance of corn plants. Studies have shown that ZmERF21 is an ethylene response factor that can directly regulate hormones (ethylene and abscisic acid) and Ca signal transduction to enhance drought resistance in corn plants under drought conditions. Exogenous melatonin can inhibit the accumulation of H2O2, promote the removal of ROS, and reduce oxidative damage in corn leaves.

[0003] Peroxidase is a very important antioxidant enzyme in organisms and has many physiological functions. When plants are subjected to abiotic stresses such as salt, cold and drought, peroxidase will remove the excess reactive oxygen produced by the stress to protect plant cells and reduce oxidative damage to cells. Studies have found that overexpressing ZmSAG39 corn will increase the activity of peroxidase under drought stress, thereby reducing H2O2 and O2 - The content of AtPrx17 is increased, which enhances the drought resistance of corn. On the other hand, peroxidase also plays an important role in the biosynthesis of lignin and the lignification process. The lignin content of AtPrx double mutants (AtPrx2 / AtPrx25, AtPrx2 / AtPrx71, AtPrx25 / AtPrx71) is lower than that of single mutants. AtPrx17 is directly regulated by AGL15 and plays an important role in regulating the formation of age-dependent lignified tissue.

[0004] Nowadays, people have found that POD genes in different plants have functions such as cold resistance, salt tolerance, drought resistance, regulation of anthocyanin biosynthesis, and regulation of lignin biosynthesis, but the function of the ZmPOD44 gene in corn has not yet been studied. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defect in the prior art that the function of the ZmPOD44 gene in corn has not been studied, and to provide the application of the ZmPOD44 gene or a biological material containing the ZmPOD44 gene.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0007] The present invention provides the use of ZmPOD44 gene or a biological material containing ZmPOD44 gene in any of the following:

[0008] a. Application in improving drought resistance of corn;

[0009] b. Application in breeding drought-resistant corn;

[0010] c. Application in increasing the lignin content of corn;

[0011] The nucleotide sequence of the ZmPOD44 gene is shown in SEQ ID NO:3.

[0012] In some technical solutions of the present invention, the amino acid sequence of the protein encoded by the ZmPOD44 gene is shown in SEQ ID NO:4.

[0013] In some technical solutions of the present invention, the biomaterial includes any one of the following:

[0014] d1. An expression cassette containing the ZmPOD44 gene;

[0015] d2. A recombinant vector containing the ZmPOD44 gene or a recombinant vector containing the expression cassette;

[0016] d3. A recombinant microorganism containing the ZmPOD44 gene, or a recombinant microorganism containing the expression cassette, or a recombinant microorganism containing the recombinant vector;

[0017] d4. A transgenic cell line containing the ZmPOD44 gene, or a transgenic cell line containing the expression cassette;

[0018] d5. A transgenic plant tissue containing the ZmPOD44 gene, or a transgenic plant tissue containing the expression cassette;

[0019] d6. A transgenic plant organ containing the ZmPOD44 gene, or a transgenic plant organ containing the expression cassette.

[0020] In some technical solutions of the present invention, the original vector of the recombinant vector is selected from any one of the plant expression vectors pCAMBIA3301, pCAMBIA1300, pCAMBIA2300, and pUbi1390.

[0021] In some technical solutions of the present invention, the starting strain of the recombinant microorganism includes Escherichia coli or Agrobacterium.

[0022] The present invention also provides a method for improving drought resistance of corn, comprising: increasing the expression level of the ZmPOD44 gene in corn plants to obtain drought-resistant plants.

[0023] In some technical solutions of the present invention, this is achieved by introducing the ZmPOD44 gene into the target plant.

[0024] The present invention also provides a method for increasing the lignin content in corn, comprising: increasing the expression level of the ZmPOD44 gene in corn plants to obtain plants with high lignin content.

[0025] In some technical solutions of the present invention, this is achieved by introducing the ZmPOD44 gene into the target plant.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] In the present invention, the maize inbred line "B73" was used as the experimental material, and four abiotic stresses were applied to maize at the three-leaf stage, and the ZmPOD44 gene, which was upregulated under drought stress, was screened out. The gene was expressed at the highest level in maize leaves and responded strongly to drought stress. By observing the phenotypic changes and physiological and biochemical index changes of the overexpression strains and gene-edited strains, the application of the overexpression ZmPOD44 gene in improving the drought resistance of maize was found.

[0028] In the present invention, under drought stress, the ZmPOD44 gene overexpressing corn plants showed higher germination rate and survival rate, had stronger root system than wild type and gene-edited plants, and significantly improved drought tolerance. - The reduction in content and the increase in SOD, POD and CAT activities indicate that overexpression of the ZmPOD44 gene improves the ability of corn plants to eliminate reactive oxygen species and enhances the drought resistance of corn.

[0029] The present invention found through tissue-specific expression that the ZmPOD44 gene is expressed in roots, stems and leaves, with the highest expression level in leaves, and the strongest response to drought, followed by ABA stress and salt stress. This may indicate that it functions in multiple tissues and may respond to different signal pathways in different tissues or under different conditions.

[0030] Through bioinformatics prediction, the results showed that ZmPOD44 protein is a secreted protein with a signal peptide fragment and a transmembrane domain, indicating that ZmPOD44 protein will move across the membrane to the extracellular space to function. The results of subcellular localization verified this speculation. The protein was transiently expressed in tobacco leaves and finally localized on the cell wall. In this experiment, ZmPOE1, which interacts with ZmPOD44, was predicted through the STRING website. The two genes were connected to the pGBKT7 and pGADT7 vectors for yeast two-hybrid experiments, and the interaction between ZmPOD44 and ZmPOE1 was confirmed by experiments. This shows that the two proteins have some kind of correlation in the growth and development process of corn or flower development, and this function needs to be further verified in subsequent experiments.

[0031] The present invention performs phenotypic identification on ZmPOD44 gene overexpressing corn plants, gene-edited plants and wild-type plants, and finds that the overexpressing plants have higher germination rate and survival rate under drought stress than the wild-type and gene-edited plants, and the root system of the overexpressing plants is also more developed, which significantly improves the drought resistance of the corn plants under drought stress.

[0032] The present invention uses NBT, DAB staining and index determination methods to confirm that the overexpressed corn leaves contain lower ROS levels than wild-type and gene-edited plants, and the antioxidant enzyme activity and proline and chlorophyll levels are higher, and the malondialdehyde content is lower. Under drought stress, the overexpression of this gene improves the ability of corn to scavenge reactive oxygen species, thereby improving the drought resistance of corn. Therefore, it is speculated that the ZmPOD44 gene may be involved in the ROS signaling pathway in response to drought stress.

[0033] The present invention found that the leaves of corn with overexpression of ZmPOD44 contained higher levels of lignin, cellulose and hemicellulose, and toluidine blue staining proved that the degree of lignification in the overexpression leaves was higher than that of wild-type and gene-edited plants. Therefore, it is speculated that the ZmPOD44 gene is involved in the synthesis and lignification process of corn lignin. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The expression analysis results of ZmPOD44 gene under four abiotic stress treatments are shown in Figure 2. Figure 1 A is the expression analysis result of ZmPOD44 gene under drought stress; Figure 1B is the expression analysis result of ZmPOD44 gene under ABA stress; Figure 1 C is the expression analysis result of ZmPOD44 gene under cold stress; Figure 1 D is the expression analysis result of ZmPOD44 gene under salt stress.

[0035] Figure 2 It is the SDS-PAGE electrophoresis diagram of the recombinant protein; wherein, M represents 180kDa Marker; N represents protein induced by empty vector bacterial solution; and bands 1-9 represent proteins induced by 0-8h bacterial solution.

[0036] Figure 3 This is a diagram showing the drought resistance analysis results of the ZmPOD44 gene in Escherichia coli; Figure 3 A represents the analysis result of the control group; Figure 3 B represents the drought resistance analysis results of the drought treatment group.

[0037] Figure 4 This is a diagram showing the subcellular localization results of ZmPOD44 protein.

[0038] Figure 5 This is a diagram showing the toxicity verification results of the bait vector.

[0039] Figure 6 This is the self-activation verification result diagram of the bait vector.

[0040] Figure 7 This is a diagram showing the verification results of the interaction between ZmPOD44 and ZmPOE1.

[0041] Figure 8 This is the result of measuring the germination rate of transgenic corn under drought stress; Figure 8 A represents the phenotype diagram of corn germination; Figure 8 B represents the statistical bar chart of corn germination rate.

[0042] Fig. 9 This is the result of measuring the root length of transgenic corn under drought stress; Fig. 9 A represents the maize root length phenotype; Fig. 9 B represents the statistical bar chart of corn root length.

[0043] Fig.10 This is the result of the rehydration experiment of genetically modified corn; Fig.10 A represents the maize rehydration phenotype diagram; Fig.10 B represents a bar graph of corn survival rate.

[0044] Fig.11 For transgenic corn plants O2 - And H2O2 detection result diagram; Among them, Fig.11A represents the NBT staining result; Fig.11 B represents the DAB staining result; Fig.11 The C represents O2 - Assay histogram; Fig.11 D represents the bar graph of H2O2 content determination.

[0045] Fig.12 This is the result of the test of physiological and biochemical indicators of drought resistance of transgenic corn; Fig.12 A represents the results of SOD activity determination of transgenic corn; Fig.12 B represents the results of POD activity assay of transgenic corn;

[0046] Fig.12 C represents the results of the CAT activity assay of transgenic corn; Fig.12 D represents the result of determination of proline content in transgenic corn; Fig.12 E represents the result of determination of chlorophyll content in transgenic corn; Fig.12 F represents the results of measuring the malondialdehyde content in transgenic corn.

[0047] Fig.13 The results of toluidine blue staining and lignin content determination of different corn plants at a scale of 50 μm are shown; Fig.13 A represents the toluidine blue staining results of different corn plants; Fig.13 B represents the results of the determination of lignin content in different corn plants; Fig.13 C represents the results of the determination of cellulose content in different corn plants; Fig.13 D represents the results of the determination of hemicellulose content in different corn plants. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0049] In the present invention, corn inbred line "B73" was used as experimental material. T2 generation corn seeds, CRISPR gene-edited plants (KO), overexpression plants (OE), and wild-type plants (WT) were placed in germination boxes and treated with water and 8% PEG6000 aqueous solution to simulate drought treatment, and the germination rate at 7d was calculated. There were 30 corn seeds in each line in each group of treatments. In addition, WT seeds, OE seeds, and KO seeds were sown in the soil of the greenhouse. The growth conditions were 25°C / 16°C (day / night), humidity was controlled at about 60%, and the circadian rhythm was 16h / 8h. When the corn grew to the three-leaf stage, it was subjected to natural drought treatment for 10d, rehydrated for 3d, and then photographed to observe the growth of corn leaves and roots, and the survival rate and root length of corn were counted. The three-leaf stage WT, OE, and KO corn grown in the greenhouse were subjected to drought stress using 12% PEG6000 aqueous solution. After 12 hours, the middle of the second leaf of the corn was taken for physiological and biochemical indicators and qRT-PCR determination.

[0050] The results showed that the ZmPOD44 gene was expressed at the highest level in leaves and responded strongly to drought stress. Subcellular localization results showed that the ZmPOD44 protein was localized on the cell wall. Yeast two-hybrid experiments verified that ZmPOD44 interacted with ZmPOE1. Under drought stress, corn overexpressing the ZmPOD44 gene had higher germination rate and survival rate, more developed root system, and significantly increased drought tolerance. It was also found that the content of reactive oxygen in overexpressed plants was significantly reduced, the activity of antioxidant enzymes was increased, and the overexpression of the ZmPOD44 gene significantly improved the ability of corn to scavenge reactive oxygen. In addition, more lignin, cellulose, and hemicellulose were found in overexpressed corn, and toluidine blue staining results showed that the lignification of overexpressed corn leaves was deeper. These results indicate that the ZmPOD44 gene may affect the drought resistance of corn by promoting the synthesis of lignin and the degree of lignification and regulating the accumulation of reactive oxygen in plants.

[0051] Example 1: Expression of maize ZmPOD44 gene under abiotic stress

[0052] (1) The maize inbred line "B73" was used as the experimental material. Four abiotic stresses were applied to maize at the three-leaf stage: 12% PEG6000, 100 mM ABA, 4°C, and 100 mM NaCl. Leaf samples were collected at 0, 2, 4, 8, 12, 24, and 48 h for each stress, frozen in liquid nitrogen, and then stored at -80°C for use.

[0053] Four abiotic stresses, 12% PEG6000, 100 mM ABA, 4°C, and 100 mM NaCl, were applied to maize at the three-leaf stage. RNA samples were extracted and qRT-PCR was performed to analyze the effects of abiotic stress on ZmPOD44 gene expression. Figure 1As shown in Figures AD, all four stresses can increase the expression of the ZmPOD44 gene, among which the ZmPOD44 gene responds most strongly to drought stress.

[0054] Embodiment 2:

[0055] 1. Obtaining transgenic plants

[0056] In order to obtain overexpression and gene-edited corn, the present invention uses "B73" corn cDNA as a template, uses specific primers POD44-F / R, amplifies the ZmPOD44 gene, and constructs it between the BglⅡ and BstEⅠ sites of the pCAMBIA3301 overexpression vector through seamless cloning technology. The specific primer sequences and the amplified ZmPOD44 gene sequences are shown in Table 1. The gene editing vector was constructed by Weimi Company. Transgenic plants were obtained by Agrobacterium transformation, and PCR verification and sequencing verification were performed to screen out the overexpression ZmPOD44 gene strains and gene-edited strains.

[0057] Table 1 Specific primer sequences and ZmPOD44 gene

[0058]

[0059]

[0060]

[0061] 2. Prokaryotic expression analysis of maize ZmPOD44 gene

[0062] The full length CDS sequence of the ZmPOD44 gene was cloned using specific primers, and the prokaryotic expression vector pET-22b-ZmPOD44 was constructed by seamless cloning. The prokaryotic expression vector was transferred into BL21 Escherichia coli. When OD600 = 0.6, 1 mM IPTG was added to induce the expression of ZmPOD44 protein, and 2 mL of samples were collected every 1 hour before and after the addition of IPTG induction. After treatment, SDS-PAGE electrophoresis was performed to observe the expression of ZmPOD44 protein. The results are shown in Figure 2 As shown, the target protein band was successfully observed in the molecular range of 48-35 kDa, which was consistent with the expected protein size.

[0063] 3. Analysis of drought tolerance of maize ZmPOD44 gene in BL21 Escherichia coli

[0064] Prepare 5 mL of BL21 (pET-22b-ZmPOD44) and BL21 (pET-22b) bacterial solution, and expand to 50 mL when the bacterial solution OD600 is 0.6. Then add IPTG with a final concentration of 1 mM and continue to culture at 29°C for 16 hours. After the culture is completed, the bacterial solution OD 600 Adjusted to 0.6.

[0065] The bacteria were inoculated into LB culture medium containing 0.5 M mannitol at a ratio of 1:25, cultured at 37°C, 180 rpm, and the OD of the culture medium was measured every 2 hours. 600 Finally, a growth curve is drawn.

[0066] The results are as follows Figure 3 A and Figure 3 As shown in B, under normal treatment, there was no significant difference in the growth of BL21(pET-22b) and BL21(pET-22b-ZmPOD44). Under drought stress, the growth of both bacteria was inhibited, but the growth inhibition rate of BL21(pET-22b) was significantly higher than that of BL21(pET-22b-ZmPOD44). Therefore, the expression of the ZmPOD44 gene can improve the drought resistance of Escherichia coli.

[0067] 4. Subcellular localization of maize ZmPOD44 protein

[0068] Bioinformatics prediction results showed that ZmPOD44 protein is a secretory protein with a signal peptide fragment and a transmembrane domain, indicating that ZmPOD44 protein will move across the membrane to the extracellular space to exert its function.

[0069] The present invention uses the specific primers 1300-ZmPOD44-F and 1300-ZmPOD44-R shown in Table 2 to clone the full length CDS sequence of the ZmPOD44 gene, constructs the subcellular localization vector pCAMBIA1300-eGFP-ZmPOD44 by seamless cloning, transfers the subcellular localization vector into GV3101 Agrobacterium, and selects Nicotiana benthamiana at the 6-leaf stage. The Agrobacterium is injected into the tobacco leaves by injection, and after culturing under weak light for 72 hours, the localization of the ZmPOD44 protein is observed under a laser confocal microscope. The microscopic examination results are as follows: Figure 4 As shown, it was shown that ZmPOD44 protein was localized on the cell wall, which was consistent with the prediction results.

[0070] Table 2 Cloning primers

[0071]

[0072] 5. Yeast two-hybrid assay

[0073] The STRING website was used to predict the interacting protein ZmPOE1 of ZmPOD44 protein, and the full-length CDS sequences of ZmPOD44 and ZmPOE1 genes were cloned using specific primers. The bait vector pGBKT7-ZmPOD44 and the prey vector pGADT7-ZmPOE1 were constructed by seamless cloning. They were used for toxicity verification, self-activation verification, and protein interaction verification.

[0074] (1) Toxicity verification

[0075] The constructed bait vector pGBKT7-ZmPOD44 plasmid and the bait empty vector pGBKT7 plasmid were respectively transferred into AH109 yeast cells, spread on SD / -Trp medium, and cultured at 29°C for 48 hours. The number and size of single colonies on the two culture media were observed to determine whether the bait vector pGBKT7-ZmPOD44 was toxic.

[0076] The results are as follows Figure 5 As shown, it was observed that there was no significant difference in the growth density and size of the two yeasts. After culturing in YPDA culture medium at 29°C for 48 hours, the OD600 of the two bacterial solutions was determined by a UV spectrophotometer to be around 0.8, indicating that the bait vector pGBKT7-ZmPOD44 was not toxic.

[0077] (2) Self-activation verification

[0078] The constructed bait vector pGBKT7-ZmPOD44 plasmid and the prey empty vector pGADT7 plasmid were co-transfected into AH109 yeast cells, spread on SD / -Trp-Leu medium, and cultured at 29°C for 48 hours. A single colony was picked and cultured in YPDA culture medium. When the OD600 of the bacterial solution was measured at about 0.6, the positive control, negative control and experimental group were spotted on SD / -Trp-Leu medium, SD / -Trp-Leu-Ade-His medium and SD / -Trp-Leu-Ade-His-X-α-Gal medium. Four parallels were set up for each experimental group, and the growth status of the experimental group was observed to determine whether the bait vector pGBKT7-ZmPOD44 has self-activation activity.

[0079] The results are as follows Figure 6 As shown, it was observed that all three groups of yeast grew on SD / -Trp-Leu medium with no significant difference in growth potential, while on SD / -Trp-Leu-Ade-His and SD / -Trp-Leu-Ade-His-X-α-Gal medium, the experimental groups did not grow or turn blue like the negative control. Therefore, the bait vector pGBKT7-ZmPOD44 did not have self-activation activity.

[0080] (3) Protein interaction verification

[0081] The constructed bait vector pGBKT7-ZmPOD44 plasmid and prey vector pGADT7-ZmPOE1 plasmid were co-transfected into AH109 yeast cells, spread on SD / -Trp-Leu medium, and cultured at 29°C for 48 hours. Then, a single colony was picked and cultured in YPDA culture medium. When the OD600 of the bacterial solution was measured at about 0.6, the positive control, negative control and experimental group were spotted on SD / -Trp-Leu medium, SD / -Trp-Leu-Ade-His medium and SD / -Trp-Leu-Ade-His-X-α-Gal medium. Four parallels were set up for each experimental group, and the growth conditions of the experimental groups were observed to determine whether there was an interaction between the ZmPOD44 protein and the ZmPOE1 protein.

[0082] The results are as follows Figure 7 As shown, it was observed that all three groups of yeast grew on SD / -Trp-Leu medium with no significant difference in growth potential. However, on SD / -Trp-Leu-Ade-His and SD / -Trp-Leu-Ade-His-X-α-Gal medium, the experimental groups grew well and the colony color turned blue, indicating that there was an interaction between the ZmPOD44 protein and the ZmPOE1 protein.

[0083] 6. Phenotypic analysis of overexpression and gene-edited plants

[0084] In order to explore the tolerance of transgenic corn to drought stress, WT, OE, and KO seeds were placed in germination boxes and treated with water and 8% PEG6000 aqueous solution, respectively, and the germination rate at 7 days was counted. The seeds were then planted in a greenhouse, and the growth conditions were a temperature of 25℃ / 16℃ (day / night), humidity controlled at about 60%, and a circadian rhythm of 16h / 8h. When they grew to the three-leaf stage, they were subjected to 10 days of natural drought treatment, and the changes in their leaf and root lengths were compared. After rehydration for 3 days, the survival rate of corn plants was observed and counted.

[0085] The results are as follows Figure 8 A and Figure 8 As shown in B, there was no significant difference in the germination of WT, OE, and KO seeds under water treatment. The germination rate of OE seeds treated with 8% PEG6000 aqueous solution was higher than that of WT and KO seeds, while the germination rate of KO seeds was the lowest.

[0086] like Fig. 9 A and Fig. 9 As shown in B, there was no significant difference in root growth among WT plants, OE plants, and KO plants that were not subjected to drought. The root growth of OE plants in the drought group was more developed and longer than that of WT plants and KO plants, while the roots of KO plants were the shortest.

[0087] like Fig.10 A Fig.10 As shown in B, after 10 days of drought, WT and KO plants showed dried and curled leaves, while OE plants showed slightly wilted leaves. After rehydration, only about 40% and 60% of KO and WT plants survived, while nearly 80% of OE plants could be recovered. In summary, overexpression of the ZmPOD44 gene increased the germination rate of corn, and transgenic corn plants had stronger root systems, higher survival ability, and enhanced drought tolerance of corn plants.

[0088] In summary, it was found that the overexpression plants had higher germination rate and survival rate under drought stress than the wild type and gene-edited plants, and the root system of the overexpression plants was also more developed, which significantly improved the drought resistance of corn plants under drought stress.

[0089] 7. Nitro blue tetrachloride (NBT) staining experiment and O2- content determination and 3,3'-diaminobenzidine (DAB) staining experiment and H2O2 content determination

[0090] Reactive oxygen is a class of molecules with high oxidative properties, such as superoxide anions and hydrogen peroxide, which can participate in immune responses in cells and kill invading pathogens. However, when reactive oxygen accumulates too much, it will cause oxidative stress and cause cell damage. The dynamic balance of reactive oxygen generation and removal is very important. Therefore, the present invention explores the ability of corn overexpressing the ZmPOD44 gene to remove reactive oxygen.

[0091] In order to investigate the O2 - The present invention used NBT dye to dye the second leaf of WT, OE and KO plants at the three-leaf stage under water and 12% PEG6000 aqueous solution to simulate drought for 3 hours, and then placed the leaves in anhydrous ethanol for heating and decolorization. After the decolorization was completed, photos were taken and the staining of the corn leaves was observed. Then, O2 - Assay kit for quantitative analysis of O2 - content.

[0092] In order to explore the accumulation of H2O2 in transgenic corn under drought stress, the present invention used DAB dye to dye the second leaves of WT, OE and KO plants at the three-leaf stage under water and 12% PEG6000 aqueous solution to simulate drought overnight, and then placed the leaves in anhydrous ethanol for heating and decolorization. After decolorization, photos were taken and the staining of the corn leaves was observed. Then, the H2O2 content kit was used to quantitatively analyze the H2O2 content.

[0093] NBT and DAB staining results are as follows Fig.11As shown in AD, under drought stress, the leaves of the OE strain were the lightest stained, while the leaves of the KO strain were the darkest stained, which indicated that the content of active oxygen in the leaves of the OE strain was lower than that in the WT and KO strains. - The results of the staining showed that there was less O2 in the leaves of OE strain corn. - and H2O2, O2 in maize leaves of KO strain - and H2O2 content is the highest.

[0094] In summary, the NBT, DAB staining and index determination methods confirmed that the overexpressed corn leaves contained lower ROS levels than the wild-type and gene-edited plants, and the antioxidant enzyme activity and proline and chlorophyll levels were higher, and the malondialdehyde content was lower. Under drought stress, the overexpression of the ZmPOD44 gene increased the ability of corn to scavenge reactive oxygen species, thereby improving the drought tolerance of corn. Therefore, it is speculated that ZmPOD44 may be involved in the ROS signaling pathway in response to drought stress.

[0095] 8. Determination of Physiological and Biochemical Indicators Related to Drought Resistance of Transgenic Corn

[0096] Physiological and biochemical indicators can digitally display the growth status of plants, and can also better understand the function of the ZmPOD44 gene. The present invention selects to measure the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) of transgenic corn under water and 12% PEG6000 aqueous solution to simulate drought, and the content of malondialdehyde (MDA), proline (Pro), and chlorophyll. The indicators are measured using a kit.

[0097] The results are as follows Fig.12 As shown in AF in Figure 1, under normal conditions, the contents of WT, OE and KO plants were relatively consistent, with no significant differences. However, under drought stress, OE plants contained less malondialdehyde and more proline and chlorophyll than WT and KO plants. This indicates that under drought stress, OE plants have better growth conditions and stronger drought tolerance. The results of the determination of catalase, peroxidase and superoxide dismutase activities are shown in the figure. After drought stress, the activities of the three antioxidant enzymes in OE plants were significantly increased compared with those in WT and KO plants, which is consistent with the results of staining and reactive oxygen content determination.

[0098] In summary, overexpression of the ZmPOD44 gene can improve the ability of transgenic corn plants to scavenge reactive oxygen species and improve the drought resistance of transgenic corn.

[0099] 9. Leaf toluidine blue staining and lignin content determination

[0100] Lignin can not only enhance cell strength, but also promote the activity of antioxidant enzymes and remove excessive free radicals in plants. In order to explore the changes in lignin content caused by the ZmPOD44 gene, the second leaf of WT, OE, and KO plants at the three-leaf stage was stained with toluidine blue, and then examined under a microscope to observe the structure and composition of the xylem of the corn leaves. The lignin, cellulose, and hemicellulose contents of transgenic corn leaves were determined using a kit.

[0101] The results are as follows Fig.13 A and Fig.13 As shown in B, the contents of lignin, cellulose, and hemicellulose in the leaves of OE plants increased significantly, while those in the KO lines decreased significantly. Subsequently, the leaves were stained with toluidine blue to observe the degree of lignification of the leaves of transgenic corn plants. The lignified part was blue-green, the phloem was blue-purple, and the other parts were light blue-green. The staining results showed that the lignification degree of corn leaves in the OE line was the highest, and the blue-green part was more than that of WT and KO plants. These results indicate that overexpression of the ZmPOD44 gene promotes the synthesis of lignin and the lignification process in corn plants.

[0102] All data of the present invention were subjected to three biological replicates. One-way ANOVA was performed on the data using SPSS (SPSS Inc., Chicago, IL, USA) software to confirm the differences between the treated data. When P < 0.05 (*) or P < 0.01 (**), it was considered to be significantly different.

[0103] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0104] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. ZmPOD44 Gene or contains ZmPOD44 The use of genetic biological material in any of the following: a. Application in improving drought resistance of corn; b. Application in breeding drought-resistant corn; c. Application in increasing the lignin content of corn; Said ZmPOD44 The nucleotide sequence of the gene is shown in SEQ ID NO: 3; The above applications are achieved through overexpression ZmPOD44 Genetically accomplished.

2. The use according to claim 1, characterized in that The biological material includes any of the following: d1. Containing ZmPOD44 expression cassette of the gene; d2. Containing the ZmPOD44 Recombinant vectors of genes; d3. Containing the ZmPOD44 Genetically recombinant microorganisms; d4. Containing ZmPOD44 Genetically modified cell lines.

3. The use according to claim 2, characterized in that The biological material also includes any of the following: A recombinant vector containing the expression cassette; A recombinant microorganism containing the expression cassette; A transgenic cell line containing the expression cassette.

4. The use according to claim 2, characterized in that The original vector of the recombinant vector is selected from any one of the plant expression vectors pCAMBIA3301 and pCAMBIA1300.

5. The use according to claim 2, characterized in that The starting strain of the recombinant microorganism includes Escherichia coli or Agrobacterium.

6. A method for improving drought resistance of corn, characterized in that: include: Improve the claim 1 ZmPOD44 The expression level of the gene in corn plants is increased to obtain drought-resistant plants.

7. The method according to claim 6, characterized in that By applying the ZmPOD44 This is achieved by introducing genes into corn.

8. A method for increasing the lignin content in corn, characterized in that: include: Improve the claim 1 ZmPOD44 The expression level of the gene in corn plants results in plants with high lignin content.

9. The method according to claim 8, characterized in that By applying the ZmPOD44 This is achieved by introducing genes into corn.

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