Application of ZmPYL11 gene in improving drought resistance of plants

Abstract

This invention provides an application of the ZmPYL11 gene in improving plant drought resistance. The invention discovers that the ZmPYL11 gene can significantly improve the drought resistance of maize. By overexpressing the ZmPYL11 gene in maize, this invention successfully constructed maize plants with stronger drought resistance. The constructed ZmPYL11 overexpressing plants showed higher relative leaf water content after drought treatment than the wild type, greater sensitivity of leaf stomata to abscisic acid (ABA)-induced stomatal closure, and reduced water loss rate in detached leaves, resulting in significantly enhanced drought resistance. This gene has broad application potential and market value in agricultural planting, particularly in improving plant drought resistance and cultivating drought-resistant transgenic crops.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of a ZmPYL11 gene in improving plant drought resistance. Background Technology

[0002] With global climate change, drought and water scarcity have become one of the main factors restricting crop growth and yield. For a long time, plants have evolved molecular mechanisms to respond to drought stress. Plants transduce drought stress signals in two ways: ABA-dependent (abscisic acid) and ABA-independent signal transduction pathways. ABA receptors PYR1 / PYLs / RCARs are upstream signal regulators in the ABA signaling pathway, capable of recognizing ABA signals and initiating signal transduction processes, playing multiple roles in plant growth, development, and stress response. In Arabidopsis, 14 PYLs have been identified. PYLs exhibit functional redundancy in ABA signal sensing; mutations in a single pyl gene often lack obvious phenotypes. However, mutants of multiple genes (pyr1pyl1, pyl2, pyl4, pyl5, pyl8) have shown ABA insensitivity in both growth and stomatal development. Thirteen gene families (PYLs) were predicted in the rice genome. Overexpression of rice PYL3, PYL5, PYL9, or PYL11 all improved drought resistance, leading to hypersensitivity to ABA and inhibiting seed germination and seedling growth. Overexpression of wheat PYL4 increased wheat yield under drought stress. Thirteen gene family members, ZmPYL1-13, homologous to Arabidopsis PYR1 / PYLs were identified in the maize genome. The expression patterns of these 13 members of the ZmPYL gene family varied in different developmental stages and tissues of maize. ZmPYL8 and ZmPYL9 showed the highest expression levels in maize kernels, while expression levels were lower in other parts. ZmPYL10 and ZmPYL11 showed high expression levels throughout the entire plant, indicating that different ZmPYLs may have functional specificity.

[0003] The function of PYLs in wheat and rice and their response to drought have been reported to some extent, but the function of ZmPYLs genes in maize is rarely reported. Summary of the Invention

[0004] This invention provides an application of the ZmPYL11 gene in improving plant drought resistance. This invention found that the ZmPYL11 gene can significantly improve the drought resistance of maize. Furthermore, this invention utilizes the Agrobacterium-mediated method to overexpress the ZmPYL11 gene in maize, successfully constructing maize plants with stronger drought resistance.

[0005] In a first aspect, the present invention provides an application of ZmPYL11 protein in improving plant drought resistance, wherein the amino acid sequence of the ZmPYL11 protein is at least one of the following:

[0006] (1) As shown in any one of SEQ ID NO.3-4;

[0007] (2) A protein derived from any one of SEQ ID NO. 3-4 and retaining the function of the amino acid sequence shown in any one of SEQ ID NO. 3-4 by substitution, deletion and / or addition of one or more amino acid residues;

[0008] (3) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in any one of SEQ ID NO.3-4 and has the same function as a protein;

[0009] (4) An amino acid sequence obtained by attaching a tag, restriction site and / or linking a peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences in (1)-(3).

[0010] This invention provides an application of the ZmPYL11 gene in improving plant drought resistance, wherein the nucleotide sequence of the ZmPYL11 gene is at least one of the following:

[0011] (1) As shown in any of SEQ ID NO.1-2;

[0012] (2) A nucleotide sequence that expresses the same functional protein by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence shown in any one of SEQ ID NO.1-2;

[0013] (3) A nucleotide sequence that hybridizes with any of the sequences shown in SEQ ID NO. 1-2 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE or 0.1×SSC solution containing 0.1% SDS and washing the membrane with the solution.

[0014] (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in any one of SEQ ID NO.1-2 and expresses the same functional protein.

[0015] The ZmPYL11 gene is coded as Zm00001eb204180 in Zea mays (B73_RefGen_v5), Zm00001d053396 in Zea mays (B73_RefGen_v4), and GRMZM2G048733 in Zea mays (B73_RefGen_v3). The ZmPYL11 gene's Genomic DNA consists of 3322 bases and has two transcripts. This gene has three exons and two introns. Since the same DNA sequence in maize can produce different transcripts that translate into different proteins, the different transcripts produced by this sequence are shown in SEQ ID NO. 1-2. Specifically, the amino acid sequence of the protein translated from the sequence shown in SEQ ID NO. 1 is shown in SEQ ID NO. 3, and the amino acid sequence of the protein translated from the sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO. 4.

[0016] Secondly, this invention provides a method for improving the drought resistance of plants, specifically by increasing the expression level of the ZmPYL11 gene in plants, the method including but not limited to:

[0017] (1) Transformation of plants into vectors overexpressing the ZmPYL11 gene using genetic engineering techniques; or

[0018] (2) Select and modify the promoter that regulates the expression of the ZmPYL11 gene to be a strong promoter through genetic engineering; or

[0019] (3) Increase the copy number of the gene encoding ZmPYL11 in plant chromosomes through genetic engineering; or

[0020] (4) Introducing enhancers through genetic engineering;

[0021] The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO.1-2.

[0022] Preferably, a plasmid containing the ZmPYL11 gene is transformed into plants and then self-pollinated. In some embodiments of the present invention, Agrobacterium-mediated transformation of plant callus tissue is specifically used, and primary transgenic plants are obtained through differentiation culture, followed by self-pollination for breeding.

[0023] Thirdly, the present invention provides an application of the ZmPYL11 gene, or the protein encoded thereon, or biological material containing the gene, or the method thereof in the cultivation of drought-resistant plants.

[0024] The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO. 1-2; or

[0025] The amino acid sequence of the protein is shown in any one of SEQ ID NO. 3-4.

[0026] The present invention also provides the application of the ZmPYL11 gene, or the protein encoded thereon, or biological material containing the gene, or the method thereof in increasing the water content of plant seedlings under drought conditions.

[0027] The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO. 1-2; or

[0028] The amino acid sequence of the protein is shown in any one of SEQ ID NO. 3-4.

[0029] The present invention also provides the application of the ZmPYL11 gene, or the protein encoded thereon, or biological material containing the gene, or the method thereof in improving the sensitivity of plants to abscisic acid.

[0030] The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO. 1-2; or

[0031] The amino acid sequence of the protein is shown in any one of SEQ ID NO. 3-4.

[0032] This invention has found that overexpression of the ZmPYL11 gene in plants can increase the plant's sensitivity to abscisic acid, reduce the stomatal opening of plants after abscisic acid treatment, thereby reducing water evaporation and enhancing the plant's drought resistance.

[0033] The present invention also provides the application of the ZmPYL11 gene, or the protein encoded thereon, or biological material containing the gene, or the method thereof in reducing the water loss rate of detached plant leaves.

[0034] The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO. 1-2; or

[0035] The amino acid sequence of the protein is shown in any one of SEQ ID NO. 3-4.

[0036] This invention found that the water loss rate of detached leaves of plants overexpressing the ZmPYL11 gene was significantly lower than that of wild-type plants, indicating that the ZmPYL11 gene improves the drought resistance of plants mainly due to the slower water loss rate.

[0037] The present invention also provides the application of the ZmPYL11 gene, or the protein encoded thereon, or biological material containing the gene, or the method thereof in the improvement of plant germplasm resources.

[0038] The nucleotide sequence of the ZmPYL11 gene is shown in any one of SEQ ID NO. 1-2; or

[0039] The amino acid sequence of the protein is shown in any one of SEQ ID NO. 3-4.

[0040] Preferably, the plant mentioned in the above application or method is corn, wheat, Chinese cabbage, rapeseed, broccoli, spinach, lettuce, kale, celery, or water spinach, with corn being the most preferred.

[0041] The beneficial effects achieved by this invention include:

[0042] This invention discovers that the ZmPYL11 gene can significantly improve the drought resistance of maize. By overexpressing the ZmPYL11 gene in maize, this invention successfully constructed maize plants with stronger drought resistance. After drought treatment, the relative water content of the leaves of ZmPYL11 overexpressing seedlings was 29.25%, 29.66%, and 31.03% higher than that of the wild type, respectively. The stomata of the leaves were more sensitive to the process of stomatal closure promoted by abscisic acid (ABA) than those of the wild type. The average stomatal opening after ABA treatment was 2.44 μm, 2.25 μm, and 2.44 μm, respectively, which were reduced by 31.46%, 36.80%, and 31.46% compared with the wild type. The water loss rate of detached leaves was reduced, and the drought resistance was significantly enhanced. This mechanism has important significance in the transportation and preservation of various leafy vegetables. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0044] Figure 1 This is the detection result of the expression level of ZmPYL11 overexpressing plants in Example 1 of this invention.

[0045] Figure 2 This is a phenotypic result diagram of ZmPYL11 overexpressing plants and wild-type plants under drought treatment in Example 2 of the present invention.

[0046] Figure 3 This is a graph showing the relative water content of leaves of ZmPYL11 overexpressing plants and wild-type plants under drought treatment in Example 2 of this invention.

[0047] Figure 4 This is a diagram showing the aboveground dry weight of ZmPYL11 overexpressing plants and wild-type plants under drought treatment in Example 2 of this invention.

[0048] Figure 5 This is a graph showing the changes in stomatal aperture of ZmPYL11 overexpressing plants and wild-type plants under ABA treatment in Example 3 of this invention.

[0049] Figure 6 This is a graph showing the water loss rate of detached leaves of ZmPYL11 overexpressing plants and wild-type plants under drought treatment in Example 4 of this invention. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. The pBCXUN vector recombinant vector used in this invention was provided by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.

[0051] Example 1: Preparation of ZmPYL11 overexpressing maize plants

[0052] 1. Construction and detection of ZmPYL11 gene overexpression vector

[0053] Total RNA was extracted from B73 maize (Zea mays L.), and cDNA was obtained by reverse transcription. Using the cDNA as a template, the CDS region of the target gene ZmPYL11 was amplified using upstream and downstream primers and a high-fidelity enzyme. To facilitate subsequent protein experiments, a DNA sequence with a MYC tag was fused to the CDS region of ZmPYL11 (SEQ ID No. 5). The ZmPYL11 gene DNA sequence with the MYC tag was inserted into the pBCXUN vector using the restriction endonuclease XcmⅠ. This recombinant vector expressed the protein shown in (Seq ID No. 6).

[0054] 2. Identification of transgenic maize plants overexpressing the gene

[0055] The constructed pBCXUN recombinant expression vector containing the target gene ZmPYL11 (with the maize Ubiquitin promoter) was transformed into Agrobacterium EHA105. The transgenic vector was then transferred into the immature embryo of the maize inbred line ND101 (ZmCRK1 negatively regulates maize's response to drought stress by phosphorylating plasma membrane H+-ATPase ZmMHA2. New Phytologist, 2024.8) via Agrobacterium-mediated transgenic operation. Dedifferentiation formed callus tissue, and redifferentiation into organs and tissues was then performed to obtain T0 generation transgenic plants.

[0056] Transgenic seedlings of generation T0 were obtained by screening with Basta resistance medium. The T0 plants, identified as having a copy number of 1, were transplanted to a greenhouse at the West Campus of China Agricultural University for self-pollination to obtain T1 generation transgenic seeds. T1 generation transgenic plants were then self-pollinated to obtain T2 generation transgenic seeds. T2 generation transgenic plants were then self-pollinated again to obtain T3 generation transgenic seeds. Positive transgenic plants were identified using PCR amplification in each generation, and strict self-pollination was performed to obtain the next generation of seeds.

[0057] To detect the expression of the ZmPYL11 gene in transgenic lines, RNA was extracted from the leaves of the recipient ND101 and three transgenic lines OE#1, OE#2, and OE#3 seedlings, and reverse transcribed into corresponding cDNA. Quantitative real-time PCR was then performed to detect the expression level (primers are shown below, results are in [link to results]). Figure 1 Subsequent experiments were conducted using seeds from the T3 generation transgenic homozygous line.

[0058] Primer-F: 5'-GGAACATCGAGATCGGTAGCG-3' (Seq ID No. 7).

[0059] Primer-R: 5'-ATGGAGGAGTAGTTCTGCAGCC-3' (Seq ID No. 8).

[0060] Results analysis:

[0061] Depend on Figure 1 It can be seen that the ZmPYL11 gene was successfully transferred into maize plants and integrated into the genomic DNA, and can be stably passed down under self-pollination conditions.

[0062] Example 2: Application of ZmPYL11 overexpressing plants under drought stress

[0063] 1. Soil drought treatment and phenotypic detection of ZmPYL11 overexpressing plants during seedling stage

[0064] Mix imported soil, peat moss, and vermiculite in a 1:1:1 ratio and fill each small pot with 170 g of soil. Sow 4 seeds of wild-type and ZmPYL11 overexpression plants evenly in each pot. Thin the seedlings when they reach the one-leaf-one-heart stage, leaving 3 plants of relatively uniform size and condition per pot. When the seedlings reach the two-leaf-one-heart stage, add water to the trays and, after they have fully absorbed the water, transfer all the small pots to another dry tray. Divide the wild-type and overexpression plants into two groups, and then further divide the planted wild-type and overexpression plants into two treatments (normal watering treatment and drought treatment). The normal watering treatment continues normal cultivation, watering regularly, adding water to the trays every 3 days until saturated, and then transferring all the small pots to another dry tray. The drought treatment plants are no longer watered and are subjected to drought stress treatment, with daily observation.

[0065] Photographs were taken and recorded after approximately 7 days of drought treatment (at the three-leaf-one-heart stage). Results are as follows: Figure 2 As shown, Figure 2 The left side shows the normal watering situation, and the right side shows the drought treatment situation.

[0066] 2. Detection of relative water content in leaves during drought treatment in seedling stage

[0067] Observe the condition of seedlings in pots. When the drought phenotype appears, take 5 pots each of overexpressing plants under normal watering and drought conditions, and 5 control plants. Take the third fully expanded leaf from each pot. Leaves from 3 plants per pot constitute one replicate, for a total of 5 replicates. The weight of the leaf is recorded as W1. Then, cut the leaf segment and place it in a 50 mL centrifuge tube, add distilled water, and allow the leaf to absorb water until saturated (approximately 24 hours). Then, wipe the water off the leaf with absorbent paper and weigh the saturated weight, recording it as W2. After weighing, dry the leaf (80℃, 3 days) and record the dry weight as W3. Calculate the relative water content: Relative water content = (W1-W3) / (W2-W3)×100%. The results are as follows: Figure 3 As shown.

[0068] 3. Measurement of aboveground dry weight under drought treatment during the seedling stage

[0069] Observe the condition of the seedlings in the pots. When the drought phenotype appears, take 5 pots each of overexpressing plants under normal watering and drought conditions and control plants. Each pot contains 3 corn plants as one replicate, for a total of 5 replicates. Take the aboveground parts of the corn plants and place them in kraft paper bags. Dry them in an oven at 80 degrees Celsius (about 3 days) and then weigh the dry weight of the aboveground parts.

[0070] Results analysis:

[0071] Depend on Figure 2It can be seen that the leaves of plants overexpressing the ZmPYL11 gene after drought treatment are more spread out compared with the wild type, and they have obvious drought resistance phenotype.

[0072] Depend on Figure 3 It can be seen that under normal watering conditions, there was no significant difference in the relative water content of leaves between ZmPYL11 gene overexpressing plants and wild-type plants. However, under drought treatment, the relative water content of leaves of ZmPYL11 gene overexpressing plants was significantly higher than that of wild-type plants. Specifically, the relative water content of leaves of PYL11OE#1 was 94.56%, PYL11OE#2 was 94.80%, PYL11OE#3 was 95.76%, and the relative water content of leaves of wild-type plants was 73.12%.

[0073] Depend on Figure 4 It can be seen that drought treatment inhibits plant growth, and the degree of inhibition on wild-type plants is greater than that on ZmPYL11 overexpressing plants.

[0074] Example 3: Detection of the effect of exogenous ABA on stomatal aperture in ZmPYL11 overexpressing plants

[0075] Leaves from wild-type and ZmPYL11-overexpressing plants were used as the experimental subjects, with control and experimental groups (treated with 10 μM ABA) respectively. Clear nail polish was applied to the back of maize leaves, and the lower epidermis was peeled off. Microscopic images were taken, and stomatal aperture (expressed as stomatal opening diameter) was counted using ImageJ software.

[0076] The results of the stomatal movement experiment are as follows Figure 5 As shown in the figure. Under control conditions, there was no difference in stomatal aperture among the plants, all around 6.00 μm. Under ABA treatment, the stomatal aperture of the three ZmPYL11 overexpressing plants was smaller than that of the wild type, at 2.44 μm, 2.25 μm, and 2.44 μm, respectively, while the wild type was 3.56 μm. This indicates that ZmPYL11 overexpressing plants are more sensitive to ABA, and that the ZmPYL11 gene responds to the regulation of plant stomatal movement, playing an important role in plant response to drought stress.

[0077] Example 4: Detection of water loss rate in detached leaves of ZmPYL11 overexpressing plants

[0078] Remove the second fully expanded leaf from corn seedlings that have grown normally to stage V2. Immediately weigh the initial fresh weight using an electronic balance, and then weigh the fresh weight again using a 0.01% electronic balance. After placing the seedlings under normal light conditions (27℃, 30%-45% humidity) for 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 hours, weigh them again. Calculate the water loss rate: Water loss rate = (Initial weight - Weight after water loss) / Initial weight × 100%.

[0079] The results of the water loss rate test of the detached leaf are as follows: Figure 6 As shown, the water loss rate of detached leaves of the overexpressing plants was significantly lower than that of the wild type, indicating that the drought-resistant phenotype of the ZmPYL11 overexpressing material is mainly due to the slower water loss rate of the plants.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a ZmPYL11 protein in improving drought resistance in maize, characterized in that, The amino acid sequence of the ZmPYL11 protein is at least one of the following: (1) As shown in SEQ ID NO.4; (2) An amino acid sequence obtained by attaching a tag, restriction site and / or linking a peptide sequence to the N-terminus and / or C-terminus of the amino acid sequence in (1).

2. A method for improving the drought resistance of maize, characterized in that, Methods to increase the expression level of the ZmPYL11 gene in maize include, but are not limited to: (1) The vector overexpressing the ZmPYL11 gene was transferred into maize using genetic engineering techniques; or (2) Select and modify the promoter that regulates the expression of the ZmPYL11 gene to be a strong promoter through genetic engineering; or (3) Increase the copy number of the gene encoding ZmPYL11 in maize chromosomes through genetic engineering; or (4) Introducing enhancers through genetic engineering; The nucleotide sequence of the ZmPYL11 gene is the encoding nucleotide sequence of the amino acid sequence shown in SEQ ID NO.

4.

3. The method according to claim 2, characterized in that, The plasmid containing the ZmPYL11 gene was transformed into maize and then self-pollinated.

4. The application of a ZmPYL11 gene, or the protein encoded thereon, or biological material containing the gene, or the method described in any one of claims 2-3, in the breeding of drought-resistant maize; The amino acid sequence of the protein encoded by the ZmPYL11 gene is shown in SEQ ID NO.

4.

5. The application of a ZmPYL11 gene, or the protein encoded thereon, or biological material containing the gene, or the method described in any one of claims 2-3, in increasing the water content of maize seedlings under drought conditions; The amino acid sequence of the protein encoded by the ZmPYL11 gene is shown in SEQ ID NO.

4.

6. The application of a ZmPYL11 gene, or the protein encoded thereon, or biological material containing the gene, or the method described in any one of claims 2-3, in improving the sensitivity of maize to abscisic acid; The amino acid sequence of the protein encoded by the ZmPYL11 gene is shown in SEQ ID NO.

4.

7. The application of a ZmPYL11 gene, or the protein encoded thereon, or biological material containing the gene, or the method described in any one of claims 2-3, in reducing water loss from detached maize leaves; The amino acid sequence of the protein encoded by the ZmPYL11 gene is shown in any one of SEQ ID NO.

4.

8. The application of a ZmPYL11 gene, or the protein encoded thereon, or biological material containing the gene, or the method described in any one of claims 2-3, in the improvement of maize germplasm resources; The amino acid sequence of the protein encoded by the ZmPYL11 gene is shown in SEQ ID NO.4; The improvement of maize germplasm resources refers to improvements in enhancing maize's drought resistance.

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