Use of zmglk36 protein

Abstract

Provided is a use of a ZmGLK36 protein. The use comprises improving drought resistance of crops, promoting nitrogen use efficiency of the crops, and enhancing photosynthesis of the crops. The present invention provides new genetic resources for breeding new crop varieties having enhanced photosynthesis, drought resistance, and nitrogen use efficiency, and is of great significance for future exploration of the way to enhance the crop yield potential through genetic improvement and guarantee national food security.

Description

Uses of a ZmGLK36 protein Technical Field

[0001] This invention relates to the field of agricultural genetic engineering, and more specifically, to the application of a ZmGLK36 gene or its encoded protein in improving crop photosynthesis, drought resistance, and nitrogen use efficiency. Background Technology

[0002] Maize (Zea mays L.) is a vital global food crop. Its widespread geographical distribution makes it susceptible to adverse growing conditions, including water stress and nutrient deficiencies. With extremely arid, dry, and semi-arid lands covering approximately one-third of the global land area, water scarcity and soil nitrogen deficiency threaten global maize production. Nitrogen is a component of all nucleic acids and amino acids, and adequate nitrogen fertilizer is crucial for maize yield potential. Nitrogen is closely related to plant photosynthesis; applying nitrogen fertilizer generally promotes photosynthesis, improves water use efficiency, and can mitigate the effects of drought on plant growth. Although the annual nitrogen consumption of maize production is increasing, only 30%-40% of applied nitrogen fertilizer is absorbed and utilized, and drought exacerbates this poor utilization, as water is essential for nutrient transport from the soil. Nitrogen fertilizer application reduces yield losses caused by drought stress, and adequate irrigation mitigates the impact of low-nitrogen environments on yield. Therefore, nitrogen use efficiency (NUE) and drought resistance are two major traits of concern to researchers and breeders. Currently, a series of transporters, enzymes, and signaling factors related to nitrogen use efficiency and drought resistance have been identified, but there are few reports on genes related to transcriptional regulation.

[0003] Meanwhile, improving photosynthesis is also an important way to further enhance crop yield potential. However, recent studies have shown that while improving crop light energy use efficiency, if water use efficiency is not improved in a coordinated manner, the increase in crop yield will still be difficult to achieve. In some relatively arid regions, studies have found that improving light energy use efficiency without corresponding improvements in water use efficiency not only fails to increase crop yield but may even lead to a decrease. Therefore, the synergistic improvement of both light energy and water use efficiency is also an important guarantee for achieving high light efficiency breeding in the future.

[0004] For crops, yield is a complex trait influenced by multiple factors. Photosynthetic efficiency, water use efficiency, nutrient translocation and absorption efficiency, resistance to abiotic stresses, and pest and disease resistance all affect crop yield. It is noteworthy that these factors do not affect yield independently; they often change simultaneously and even interact. Current research on the synergistic effects of multiple factors largely focuses on transcription factors. Therefore, identifying key transcription factors related to photosynthesis, drought resistance, and nitrogen use, and synergistically regulating other physiological processes, is of great significance for future exploration of genetic improvements to significantly increase crop yield potential and ensure national food security.

[0005] Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned technical problems and provide a use for the ZmGLK36 protein. This invention has found that the ZmGLK36 gene or its encoded protein, or biological materials containing its encoded protein, can improve crop drought resistance, promote crop nitrogen use efficiency, and promote crop photosynthesis.

[0007] To achieve the above objectives, specifically, the present invention provides the application of the ZmGLK36 gene or its encoded protein, or biological materials containing its encoded protein, in improving crop drought resistance.

[0008] The application mentioned above aims to improve the drought resistance of crops throughout their entire growth period.

[0009] The present invention also provides the application of the ZmGLK36 gene or its encoded protein, or biological materials containing its encoded protein, in promoting crop nitrogen use efficiency.

[0010] Furthermore, the present invention provides one or more of the following applications of the ZmGLK36 gene or its encoded protein, or biological materials containing its encoded protein:

[0011] 1) To increase the chlorophyll content in the leaves of the crop ear,

[0012] 2) Increase the aboveground or underground biomass of the plant, and increase the aboveground or underground fresh and dry weight of the plant.

[0013] 3) Promotes taproot elongation and increases the number of seed roots.

[0014] 4) It slows down the decrease in plant height.

[0015] 5) Increase ear length, ear weight, single ear grain weight, 100-grain weight, and / or yield.

[0016] 6) Promotes root development.

[0017] The above-described applications of the present invention are particularly suitable for crops in low-nitrogen environments, and can significantly achieve the effects of (1)-(6) described above.

[0018] This invention provides the application of the ZmGLK36 gene or its encoded protein, or biological materials containing its encoded protein, in promoting crop photosynthesis.

[0019] Furthermore, the promotion of crop photosynthesis includes:

[0020] 1) Delays plant aging.

[0021] 2) Increase the plant's green retention capacity.

[0022] 3) Promotes the upregulation of the green-holding gene SGR1 and / or chloroplast Beta-glucosidase.

[0023] The biological materials mentioned are recombinant expression vectors, plasmids, expression cassettes, transgenic cell lines, transgenic plant tissues, transgenic plant organs, or recombinant microorganisms. The transgenic cell lines, transgenic plant tissues, and transgenic plant organs include those capable of developing into individual plants, and those that cannot.

[0024] Preferably, the ZmGLK36 protein of the present invention has any one of the following amino acid sequences:

[0025] 1) The amino acid sequence shown in SEQ ID NO.2;

[0026] Or 2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2.

[0027] The cDNA of the ZmGLK36 protein described in this invention has any of the following nucleotide sequences:

[0028] 1) The nucleotide sequence shown in SEQ ID NO.1;

[0029] Or 2) The nucleotide sequence encoding a protein with the same function obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.1;

[0030] 3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.1 under strict conditions.

[0031] The crop is a monocotyledonous or dicotyledonous crop; preferably corn, rice, wheat, soybean, sorghum, millet, cotton or barley, and more preferably corn.

[0032] This invention also provides a method for preparing crops with drought resistance, high nitrogen use efficiency, and / or high photosynthetic efficiency. The method involves expressing or overexpressing the ZmGLK36 gene in the crop through transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction. Preferably, the transgenic process includes introducing a recombinant expression vector containing the ZmGLK36 gene into the crop using Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, gene gun, electrophoresis, or Agrobacterium-mediated transformation to obtain transgenic lines.

[0033] Preferably, the crop described in the above method is corn.

[0034] This invention has discovered that the ZmGLK36 gene or its encoded protein, or biological materials containing its encoded protein, contains key transcription factors that are related to photosynthesis, drought resistance, and nitrogen use, and synergistically regulate other physiological processes. These factors can be used to improve crop drought resistance, promote crop nitrogen use efficiency, and enhance crop photosynthesis. This invention provides new genetic resources for breeding new crop varieties with improved photosynthesis, drought resistance, and nitrogen use, and is of great significance for future exploration of the potential to increase crop yield through genetic improvement and for ensuring national food security. Attached Figure Description

[0035] Figure 1 shows the overexpression vector CUB in Embodiment 2 of the present invention;

[0036] Figure 2 shows the Bar test strip detection in the construction of the overexpression vector and the identification of transgenic plants in Example 2 of the present invention, where the left side of Figure 2 is the negative plant and the right side of Figure 2 is the positive plant.

[0037] Figure 3 is a comparison of the expression levels of ZmGLK36 transgenic overexpressing plants and wild-type plants detected by RT-qPCR in Example 2 of this invention;

[0038] Figure 4 shows a comparison of ZmGLK36 expression induced by PEG and low nitrogen in roots and leaves in Example 3 of the present invention; where a is the detection of the transcriptional level of ZmGLK36 in roots at different time points after PEG treatment; b is the detection of the transcriptional level of ZmGLK36 in leaves at different time points after PEG treatment; c is the detection of the transcriptional level of ZmGLK36 in roots at different time points after low nitrogen treatment; d is the detection of the transcriptional level of ZmGLK36 in leaves at different time points after low nitrogen treatment.

[0039] Figure 5 shows the ZmGLK36 promoting isotope in Example 4 of the present invention. 15 A comparison diagram of N transport and absorption; where a represents isotopes within the same time period. 15 Detection and analysis of N in roots of transgenic overexpression lines and wild-type (Zong 31); b represents isotopes within the same time period. 15N was detected and analyzed in leaves of transgenic overexpression lines and wild-type (Zong 31); c represents the isotopic contrast between transgenic overexpression lines and wild-type (Zong 31) at the same time point. 15 N uptake activity analysis; d represents the isotope uptake of transgenic overexpression lines and wild-type (synthesis 31) within the same time period. 15 N transport activity analysis;

[0040] Figure 6 is a comparative analysis of the low-nitrogen hydroponic experiment of B73 and near-isogenic lines in Example 4 of the present invention; a is the phenotypic diagram of low-nitrogen treatment for B73 and near-isogenic lines; b is the aboveground fresh weight analysis of B73 and near-isogenic lines after normal nitrogen and low nitrogen treatment; c is the underground fresh weight analysis of B73 and near-isogenic lines after normal nitrogen and low nitrogen treatment; d is the aboveground dry weight analysis of B73 and near-isogenic lines after normal nitrogen and low nitrogen treatment; e is the underground dry weight analysis of B73 and near-isogenic lines after normal nitrogen and low nitrogen treatment; f is the main root length analysis of B73 and near-isogenic lines after normal nitrogen and low nitrogen treatment; g is the seed root number analysis of B73 and near-isogenic lines after normal nitrogen and low nitrogen treatment.

[0041] Figure 7 is a comparative analysis of the low-nitrogen hydroponic experiment of the transgenic lines in Example 4 of this invention; a is the phenotypic diagram of the transgenic overexpression line and the wild type (Zong 31) under low-nitrogen treatment; b is the aboveground fresh weight analysis of the transgenic overexpression line and Zong 31 after normal nitrogen and low nitrogen treatment; c is the underground fresh weight analysis of the transgenic overexpression line and Zong 31 after normal nitrogen and low nitrogen treatment; d is the aboveground dry weight analysis of the transgenic overexpression line and Zong 31 after normal nitrogen and low nitrogen treatment; e is the underground dry weight analysis of the transgenic overexpression line and Zong 31 after normal nitrogen and low nitrogen treatment; f is the main root length analysis of the transgenic overexpression line and Zong 31 after normal nitrogen and low nitrogen treatment; g is the seed root number analysis of the transgenic overexpression line and Zong 31 after normal nitrogen and low nitrogen treatment.

[0042] Figure 8 is a comparison of the field phenotypic identification of near-isogenic lines and transgenic lines under different nitrogen conditions in Example 4 of the present invention.

[0043] Figure 9 is a comparison of the phenotypic identification of near-isogenic lines and transgenic lines under different nitrogen conditions in the field in Example 4 of the present invention.

[0044] Figure 10 is a comparative analysis of the low nitrogen phenotype of near-isogenic lines in field in Example 4 of the present invention; a) chlorophyll content in the ear-position leaves of near-isogenic lines under different nitrogen levels; b) plant height analysis of near-isogenic lines under different nitrogen levels; c) fresh weight analysis of the aboveground parts of plants of near-isogenic lines under different nitrogen levels; d) N content analysis in the soil under different nitrogen levels; e) N content analysis in the roots of near-isogenic lines under different nitrogen levels; f) N content analysis in the ear-position stems of near-isogenic lines under different nitrogen levels; g) N content analysis in the ear-position leaves of near-isogenic lines under different nitrogen levels.

[0045] Figure 11 is a comparative analysis of the low-nitrogen phenotype of transgenic lines in field in Example 4 of the present invention; a) chlorophyll content in the ear leaves of the transgenic overexpression lines under different nitrogen levels; b) photosynthetic efficiency in the ear leaves of the transgenic overexpression lines under different nitrogen levels; c) fresh weight of the aboveground parts of the transgenic overexpression lines under different nitrogen levels; d) water use efficiency of the ear leaves of the transgenic overexpression lines under different nitrogen levels; e) plant height of the transgenic overexpression lines under different nitrogen levels.

[0046] Figure 12 is a comparative analysis of low-nitrogen yield of transgenic lines in field in Example 4 of the present invention; a is a schematic diagram of the ears of transgenic overexpression lines under different nitrogen levels; b is the analysis of ear length of transgenic overexpression lines under different nitrogen levels; c is the analysis of single ear weight of transgenic overexpression lines under different nitrogen levels; d is the analysis of single ear grain weight of transgenic overexpression lines under different nitrogen levels; e is the analysis of 100-grain weight of transgenic overexpression lines under different nitrogen levels.

[0047] Figure 13 is a comparison diagram of the low nitrogen phenotype identification of the improved hybrid in the field in Example 4 of the present invention; a is a schematic diagram of the improved hybrid and the original hybrid in the field under different nitrogen levels; b is a schematic diagram of the improved hybrid and the original hybrid in the field under different nitrogen levels.

[0048] Figure 14 is a comparative analysis of the low-nitrogen phenotype of the improved hybrid in field in Example 4 of the present invention; a) Analysis of N content in the ear leaf of the improved hybrid and the original hybrid under different nitrogen levels; b) Analysis of chlorophyll content in the ear leaf of the improved hybrid and the original hybrid under different nitrogen levels; c) Analysis of ear length of the improved hybrid and the original hybrid under different nitrogen levels; d) Analysis of single ear weight of the improved hybrid and the original hybrid under different nitrogen levels; e) Analysis of plot yield of the improved hybrid and the original hybrid under different nitrogen levels.

[0049] Figure 15 is a comparison of the field greening phenotype and transcriptome analysis of the transgenic lines in Example 5 of the present invention; a is a schematic diagram of the greening of the transgenic overexpression lines in the field; b is a transcriptome analysis of differentially expressed genes related to chloroplasts in wild-type and transgenic overexpression lines.

[0050] Figure 16 is a comparison of photosynthetic parameters of transgenic lines and near-isogenic lines in Example 5 of the present invention; a is the analysis of photosynthetic efficiency of the spike-side leaves of transgenic overexpression lines and wild-type plants; b is the analysis of chlorophyll content of the spike-side leaves of transgenic overexpression lines and wild-type plants; c and d are the analysis of diurnal variation of photosynthetic efficiency of transgenic overexpression lines and wild-type, B73 and its near-isogenic lines.

[0051] Figure 17 is a comparative diagram of drought resistance identification of near-isogenic lines in the seedling stage in Example 6 of the present invention; a is a schematic diagram of the phenotype of near-isogenic lines after normal watering and drought rehydration; b is the survival rate analysis of near-isogenic lines after drought rehydration; c is the water loss efficiency analysis of near-isogenic lines in detached leaves.

[0052] Figure 18 is a comparative diagram of drought resistance identification of transgenic lines in seedling stage in Example 6 of the present invention; a is a schematic diagram of the phenotypes of transgenic lines and wild type after normal watering and drought re-watering; b is the survival rate analysis of transgenic lines and wild type after drought re-watering; c is the water loss efficiency analysis of detached leaves of transgenic lines and wild type.

[0053] Figure 19 is a comparison of field drought resistance identification of near-isogenic lines in Example 6 of the present invention; a is a schematic diagram of the phenotypes of roots and ears of near-isogenic lines after normal irrigation and drought treatment; b is an analysis of chlorophyll content of ear leaves of near-isogenic lines after normal irrigation and drought treatment; c is an analysis of plot yield of near-isogenic lines after normal irrigation and drought treatment.

[0054] Figure 20 is a comparison diagram of field drought resistance identification of transgenic lines in Example 6 of the present invention; a is a schematic diagram of the phenotype of transgenic and wild-type plants in the field after normal watering and drought treatment; b is a schematic diagram of the phenotype of roots of transgenic and wild-type plants in the field after normal watering and drought treatment.

[0055] Figure 21 is a comparative analysis of the field drought resistance phenotypes of the transgenic lines in Example 6 of the present invention; ad is the analysis of the tasseling period, pollen shedding period, silking period and the interval between male and female flowering (ASI) of the transgenic and wild types after normal irrigation and drought treatment; e is the analysis of the chlorophyll content of the panicle leaves of the transgenic and wild types after normal irrigation and drought treatment; f is the plot yield analysis of the transgenic and wild types after normal irrigation and drought treatment.

[0056] Figure 22 is a comparative analysis of the field drought resistance phenotypes of the improved hybrids in Example 6 of the present invention; a is a schematic diagram of the phenotypes of the ears of the improved hybrids and the original hybrids after normal irrigation and drought treatment; b is an analysis of the chlorophyll content of the ear leaves of the improved hybrids and the original hybrids after normal irrigation and drought treatment; c is an analysis of the grain weight of a single ear of the improved hybrids and the original hybrids after normal irrigation and drought treatment; d is an analysis of the plot yield of the improved hybrids and the original hybrids after normal irrigation and drought treatment. Detailed Implementation

[0057] Embodiments of the present invention will now be described with reference to the accompanying drawings. Elements and features described in one drawing or embodiment of the invention may be combined with elements and features shown in one or more other drawings or embodiments. It should be noted that, for clarity, representations and descriptions of components or processes unrelated to the invention and known to those skilled in the art have been omitted from the drawings and description.

[0058] The present invention will now be further described with reference to the accompanying drawings.

[0059] Example 1: ZmGLK36 gene CDS and protein sequence

[0060] Based on the gene and sequence information of the B73 reference genome, the nucleotide sequence of ZmGLK36 in Qi319 was obtained by amplification using the primers in Table 1. The sequence was compared with the coding sequence of the ZmGLK36 gene in the B73 reference genome. The homology of the nucleic acid sequence was 97.45%, and the homology of the protein was 95.99%.

[0061] Table 1 Primer sequences for genetic transformation vectors

[0062] Among them, the CDS sequence of ZmGLK36 in Qi 319 is SEQ ID NO.1, and the protein sequence is SEQ ID NO.2.

[0063] SEQ ID NO: 1

[0064] SEQ ID NO: 2

[0065] Example 2: Construction of overexpression vector and identification of transgenic plants

[0066] Based on the gene and sequence information of the B73 reference genome, the full-length CDS of ZmGLK36 from Qi 319 was amplified (SEQ ID NO. 1). Using the obtained plasmid as a template, PCR amplification was performed. The CUB vector was linearized by BamHI digestion, and the primer sequences are shown in Table 2. The CUB vector sequence is shown in SEQ ID NO: 5-6. The promoter of this vector is the Ubi promoter. The linearized vector and the gel recovery product containing the CDS with restriction sites and homologous arms were ligated using Infusion recombinase. The ligation product was cultured overnight for 12-16 hours, and positive clones and the size of the insert were identified by PCR. One single clone containing the ZmGLK36 CDS was obtained. After confirming the plasmid sequence, it was transformed into Agrobacterium EHA105 using chemical transformation, and then into maize embryos using transgenic technology to obtain transgenic plants.

[0067] The overexpression vector CUB contains the Bar gene (as shown in Figure 1), so Bar test strips were used for detection first. The results showed that the Bar protein was rapidly expressed in positive plants, displaying two bands, indicating that the Bar test strips could effectively distinguish between positive and negative plants (as shown in Figure 2, where the left side of the red dashed line represents negative plants and the right side represents positive plants). Simultaneously, RT-qPCR was performed on the transgenic overexpression plants and the wild type. The results showed that the expression levels of the two transgenic overexpression plants (OE-1 and OE-2) were higher than the control group Zong 31 (as shown in Figure 3).

[0068] Table 2 Primer sequences for genetic transformation vectors

[0069] Example 3: Discovery and identification of ZmGLK36's role in plant drought resistance and nitrogen utilization.

[0070] Previous studies have found that the maize inbred line Qi 319 exhibits excellent drought resistance and tolerance to low nitrogen. However, considering that most research on the synergistic effects of multiple factors focuses on transcription factors, the existence of transcription factors that synergistically regulate drought resistance and low nitrogen tolerance in maize is currently unknown. Therefore, this study, through transcriptome enrichment of Qi 319 after drought and low nitrogen treatments, discovered that the gene ZmGLK36 was significantly upregulated in both treatments.

[0071] The specific hydroponic method is as follows: (1) Seed disinfection. Select plump corn seeds of uniform size, rinse them once with tap water, rinse them twice with deionized water, soak them in 0.5% NaClO for 10 minutes for surface disinfection, rinse them twice with deionized water, and soak them overnight in saturated CaSO4. (2) Corn germination. Spread the soaked seeds evenly in a tray with moist filter paper at the bottom, with 4 portions of material in each tray, separated by absorbent paper, and cover with a black plastic bag. Place the tray in an artificial culture room to promote germination. When the roots have sprouted to about 2cm, roll the seedlings. Place the seeds 1-2cm from the top of the square filter paper, roll them into a cylindrical shape, and then place them in a bucket containing 2 / 3 volume of deionized water. Cover the bucket with a black plastic bag to block light and allow them to grow. (3) Hydroponic growth. When the above-ground leaves are about 5cm long and green, remove the black plastic bag and continue to cultivate until the two-leaf-one-heart stage. Select seedlings with uniform growth and transfer them to an incubator containing 1 / 2 Hogland nutrient solution (humidity 60%). The day / night temperature is 26±2℃ / 20±2℃, with 14 hours of light and 10 hours of darkness per day. After three days of cultivation in 1 / 2 Hogland nutrient solution, replace it with normal Hogland nutrient solution and change the nutrient solution every three days. (4) Low nitrogen treatment. When the corn seedlings grow to the three-leaf stage, a low nitrogen treatment is carried out. The concentration of N in the normal Hogland nutrient solution in the control group is 4 mmol / L, and N comes from calcium nitrate. In the low nitrogen group experiment, the content of calcium nitrate is reduced, and calcium chloride is added to supplement the concentration of calcium ions. Finally, the concentration of N in the Hogland nutrient solution in the low nitrogen group is 0.04 mmol / L. Change the nutrient solution every three days. (5) PEG simulated drought treatment. When the corn seedlings grow to the three-leaf stage in the normal nutrient solution, replace it with Hogland nutrient solution containing 15% PEG (kg / V) for PEG simulated drought treatment and change the PEG-containing nutrient solution every three days.

[0072] After PEG simulation of drought and low nitrogen (LN) treatment, ZmGLK36 was found to be induced in both roots and leaves (as shown in Figure 4), which indicates that ZmGLK36 responds to drought and low nitrogen stress.

[0073] Example 4: ZmGLK36 promotes nitrogen utilization in plants.

[0074] This embodiment aims to investigate whether ZmGLK36 is beneficial for nitrogen absorption and transport, and can be used to improve nitrogen utilization efficiency (NUE).

[0075] To clarify that ZmGLK36 is beneficial for nitrogen absorption and transport, thereby improving nitrogen utilization efficiency (NUE), this invention first utilizes isotopes 15 N was used for marker detection, and the results showed that the aboveground and underground parts of the transgenic overexpression lines (OE-1 and OE-2) showed similar activity at the same time point. 15The uptake of nitrogen (N) was significantly higher than that of the control line Zong 31 (a transgenic recipient inbred line) (as shown in Figures 5a and 5b). Further studies revealed that the transgenic overexpression plants showed increased uptake of isotopes. 15 The absorption and transport activity of N is significantly higher than that of Synthesis 31 (as shown in Figure 5c and Figure 5d).

[0076] In summary, the ZmGLK36 of this invention participates in the absorption and transport of nitrogen.

[0077] Furthermore, hydroponic experiments showed that the near-isogenic line (B73) ZmGLK36 Under low nitrogen conditions, the fresh and dry weights of the aboveground and underground parts of the plants (OE-1 and OE-2) were significantly higher than those of the controls B73 and Zong31 (as shown in Figures 6 and 7). At the same time, ZmGLK36 contributed to the elongation of the taproot and the increase in the number of seed roots under low nitrogen conditions (as shown in Figures 6 and 7).

[0078] In 2022, field identification of low nitrogen phenotypes was conducted at the Hainan Experimental Base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences. The results showed that the near-isogenic line (B73) exhibited low nitrogen phenotypes. ZmGLK36 Compared with the control (B73), under conditions of 50% nitrogen fertilizer and no nitrogen fertilizer, it can significantly increase the chlorophyll content in the leaves at the ear position, the aboveground biomass of the plant, delay the decrease in plant height and promote root development (as shown in Figures 8, 9 and 10).

[0079] Further identification of the transgenic overexpression lines showed that, compared with the control (Zong 31), the overexpression lines (OE-1 and OE-2) significantly increased chlorophyll content in ear leaves, photosynthetic efficiency, aboveground biomass, delayed plant height reduction, and promoted root development under conditions of 50% nitrogen fertilizer and no nitrogen fertilizer (as shown in Figures 8, 9, and 11). Simultaneously, soil nitrogen content was measured in the field. Under normal fertilization, 50% fertilization, and no fertilization conditions, the soil nitrogen content was 0.083%, 0.061%, and 0.045%, respectively (as shown in d of Figure 10). Field yield assessment results showed that, under normal nitrogen and low nitrogen conditions (no fertilization), the overexpression lines significantly increased ear length, ear weight, single ear grain weight, and 100-grain weight compared with the control (Zong 31) (as shown in Figure 12).

[0080] To further verify whether ZmGLK36 can be applied to breeding, in 2023, at the Langfang Experimental Base of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, Hebei Province, the cross between ZmGLK36 and the improved ZmGLK36 was tested. ZmGLK36 ×Chang 7-2 ZmGLK36Low-nitrogen phenotype identification was conducted. Field identification results showed that the nitrogen content and chlorophyll content in the ear leaves of the improved Zhengdan 958 were significantly higher than those of the unimproved Zhengdan 958 under unfertilized conditions (as shown in Figure 14). Field plot yield measurements showed that, under unfertilized conditions, the improved Zhengdan 958 was significantly higher than the unimproved Zhengdan 958 in ear length, ear weight, and plot yield (as shown in Figures 13 and 14).

[0081] Example 5: ZmGLK36 promotes plant photosynthesis and improves photosynthetic efficiency.

[0082] This embodiment aims to investigate whether ZmGLK36 promotes plant photosynthesis and can improve photosynthetic efficiency.

[0083] To further investigate whether the function of ZmGLK36 is related to photosynthesis, field identification results at the Hainan Experimental Base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, showed that transgenic overexpression plants could delay plant senescence and increase plant chlorophyll retention (as shown in Figure 15). Transcriptome data from the leaves of transgenic overexpression plants showed that chloroplast-related genes involved in photosynthesis were upregulated in the overexpression plants (as shown in Figure 15), such as the chlorophyll retention gene (SGR1) and chloroplast Beta-glucosidase.

[0084] Further light response curve measurements showed that the photosynthetic rate of the ZmGLK36 overexpressing lines in the ear leaves was significantly higher than that of the wild type (Figure 16). Diurnal variation measurements of photosynthesis indicated that the photosynthetic rate of the ZmGLK36 overexpressing lines and near-isogenic lines in the ear leaves was higher than that of the wild type throughout the day (6:00-16:00) (Figure 16). Chlorophyll content measurements in the ear leaves showed that the chlorophyll content in the ZmGLK36 overexpressing lines was significantly higher than that in the wild type (Figure 16).

[0085] Example 6: ZmGLK36 improves the drought resistance of plants

[0086] This embodiment aims to investigate whether ZmGLK36 has drought resistance for plants throughout their entire growth period.

[0087] Drought resistance in maize can be divided into seedling drought resistance and maturity drought resistance. Seedling drought resistance was the first step in the evaluation. The results showed that the survival rate of near-isogenic lines and overexpression lines after drought rehydration was significantly higher than that of the control B73 and Zong31 (as shown in Figures 17 and 18), and the water loss rate of detached leaves of the overexpression lines was significantly lower than that of the control Zong31 (as shown in Figure 18).

[0088] In 2022, drought resistance assessments at maize maturity were conducted at the Hainan Experimental Base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences. Near-isogenic lines showed significantly higher chlorophyll content in ear leaves after drought resistance treatment compared to the control B73 (Figure 19). ZmGLK36 promoted root development after drought resistance treatment (Figure 19). Field plot yield measurements showed no difference in plot yield under normal irrigation conditions, but under drought resistance conditions, the plot yield of near-isogenic lines was significantly higher than that of the control B73 (Figure 19). Transgenic overexpression lines showed stronger root development than the control Zong31 under both normal irrigation and drought resistance conditions (Figure 20), and their average inter-flowering interval (ASI) after drought resistance treatment was lower than that of the control Zong31 (Figure 21). Chlorophyll content in ear leaves and plot yield were higher than those of the control Zong31 under both normal irrigation and drought resistance conditions (Figure 21).

[0089] In summary, ZmGLK36 can improve the drought resistance of maize throughout its entire growth period, thereby increasing yield.

[0090] In 2023, at the experimental base of the Xinjiang Academy of Agricultural Sciences, field drought resistance assessments were conducted on the original hybrid Zhengdan 958 and the improved Zhengdan 958. The results showed that under normal irrigation, the chlorophyll content of the ear leaves, grain weight per ear, and plot yield of the improved Zhengdan 958 were not different from those of the original Zhengdan 958 (Figure 22). Under drought conditions, the chlorophyll content of the ear leaves, grain weight per ear, and plot yield of the improved Zhengdan 958 were higher than those of the unimproved Zhengdan 958 (Figure 22).

[0091] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of the ZmGLK36 gene or its encoded protein, or biological materials containing its encoded protein, in improving crop drought resistance.

2. Application of the ZmGLK36 gene or its encoded protein, or biological materials containing its encoded protein, in promoting crop nitrogen use efficiency.

3. Applications of the ZmGLK36 gene or its encoded protein, or biological materials containing its encoded protein, including any one or more of the following: 1) To increase the chlorophyll content in the leaves of the crop ear, 2) Increase the aboveground or underground biomass of the plant, and increase the aboveground or underground fresh and dry weight of the plant. 3) Promotes taproot elongation and increases seed number. 4) It slows down the decrease in plant height. 5) Increase ear length, ear weight, single ear grain weight, 100-grain weight, and / or yield. 6) Applications in promoting root development.

4. Application of the ZmGLK36 gene or its encoded protein, or biological materials containing its encoded protein, in promoting crop photosynthesis.

5. The application according to any one of claims 1-4, characterized in that, The biological materials mentioned are recombinant expression vectors, plasmids, expression cassettes, transgenic cell lines, transgenic plant tissues, transgenic plant organs, or recombinant microorganisms.

6. The application according to any one of claims 1-5, characterized in that, The ZmGLK36 protein has any of the following amino acid sequences: 1) The amino acid sequence shown in SEQ ID NO.2; Or 2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.

2.

7. The application according to any one of claims 1-6, characterized in that, The cDNA of the ZmGLK36 protein has any of the following nucleotide sequences: 1) The nucleotide sequence shown in SEQ ID NO.1; Or 2) The nucleotide sequence encoding a protein with the same function obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.1; 3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.1 under strict conditions.

8. The application according to any one of claims 1-7, characterized in that, The crop is a monocotyledonous or dicotyledonous crop; preferably corn, rice, wheat, soybean, sorghum, millet, cotton or barley.

9. The application according to claim 8, characterized in that, The crop in question is corn.

10. A method for preparing crops with drought resistance, high nitrogen use efficiency, and / or high photosynthetic efficiency, characterized in that, The ZmGLK36 gene is expressed or overexpressed in crops through transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction methods. Preferably, the transgenic process involves introducing a recombinant expression vector containing the ZmGLK36 gene into crops using Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, gene gun, electrocoagulation, or Agrobacterium-mediated methods to obtain transgenic lines.

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