Sugarcane gene ScPYL61, application thereof, overexpression vector and method

Abstract

The present application relates to the field of plant biotechnology, and particularly relates to a sugarcane gene ScPYL61 and application, overexpression vector and method. The nucleotide sequence of the sugarcane gene ScPYL61 is shown as SEQ ID No. 1. In the present application, 63 PYL gene family members are identified from a drought-resistant sugarcane cultivar, Sugarcane No. 1, and only one gene ScPYL61 is identified which is highly expressed and induced by PEG and ABA. The gene activates ABA signal, promotes stomatal closure and root elongation, and enhances drought resistance of plants under drought stress through a molecular module dependent on ABA-ScPYL61-ScPP2C49-ScSnRK2s. The identification of the sugarcane PYL gene family members and the functional analysis of ScPYL61 in the present application lay a theoretical foundation and provide an effective candidate gene for genetic breeding of drought-resistant sugarcane.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, and in particular to a sugarcane gene. ScPYL61 Its applications, overexpression vectors, and methods. Background Technology

[0002] Drought is one of the natural disasters that disrupts agricultural production, causing losses far exceeding those caused by other disasters each year (Gowtham et al., 2022). Due to the accelerated terrestrial water cycle caused by global warming, the incidence of agricultural drought has increased significantly in many regions, and it is projected that drought disasters will become more frequent, more severe, and longer-lasting under future warming conditions. Sugarcane is an important sugar crop and bioenergy crop, producing at least 80% of the world's sugar and 40% of its ethanol (Hu et al., 2018).

[0003] When plants are under water shortage, they produce and accumulate large amounts of abscisic acid (ABA). As an important plant hormone, ABA plays a crucial role in helping plants adapt to various abiotic stresses. Currently, the ABA signal transduction pathway has been elucidated in the model plants Arabidopsis thaliana and rice, among which the regulatory components of PYRABACTIN RESISTANCE1 (PYR1) / PYR1-like (PYL) / ABA receptor (RCAR), protein phosphatase PP2C, and protein kinase SnRK2s are the core components of ABA signal transduction (Melcher et al., 2009; Ma et al., 2009). Under normal growth conditions, the ABA content in cells is low. PP2C binds to SnRK2s and inhibits the kinase activity of SnRK2s through dephosphorylation of specific residues, resulting in the blockage of downstream ABA network signaling. When plants are under stress, the intracellular ABA level rises rapidly. PYR1 / PYLs / RCAR1 binds to PP2C, and SnRK2s kinase activity is activated, leading to the activation of downstream ABA signaling to cope with abiotic stress (Hirayama et al., 2010).

[0004] Currently, the "gate-lock-lock" interaction network is the recognized mechanism of ABA signaling (Melcher et al., 2009). The ABA receptor PYL protein contains a pocket for ABA binding, and two conserved domains, the "gate" and "latch" domains, are located outside the ABA-binding domain (Melcher et al., 2009; Nishimura et al., 2009; Yin et al., 2009). ABA binding causes a conformational change in the PYL receptor binding pocket; proline residues and conserved serine residues on the "gate loop" are moved into the PYL "gate loop" by ABA and expelled from it (Yinet et al., 2009; Santiago et al., 2009). Furthermore, the C-terminal α-helix slowly moves towards ABA, and the "gate loop" is locked by the "locking loop" through hydrogen bonds and van der Waals forces (Santiago et al., 2009). This not only anchors ABA molecules within the pocket of PYL, but also creates an interacting surface for PP2C, leading to the release of SnRK2s (Melcher et al., 2009). This unique ABA-sensing mechanism demonstrates the crucial role of PYL in ABA signal transduction.

[0005] Currently, it has been identified in a variety of plants. PYL Gene family members were identified as 14, 13, 21, 11, and 40 in Arabidopsis thaliana (Dupeux et al., 2011), rice (Yadav et al., 2020), soybean (Zhang et al., 2020), sweet potato (Mathura et al., 2023), and cotton (Liu et al., 2023), respectively. PYL Family members. However, sugarcane is not found in existing technology. PYL Identification and functional analysis of gene families. Summary of the Invention

[0006] To address the above problems, this invention provides a sugarcane gene. ScPYL61 This invention relates to the application, overexpression vectors, and methods of sugarcane, and identified 63 expression vectors in the drought-resistant sugarcane cultivar Zhongzhe No. 1. PYL Gene family members were identified, and the only gene in the family that was highly expressed and induced by PEG and ABA was found. ScPYL61 This gene, through a molecular module dependent on ABA-ScPYL61-ScPP2C49-ScSnRK2s, activates ABA signaling, promotes stomatal closure and root elongation under drought stress, thereby enhancing the plant's drought resistance. In this invention, sugarcane... PYL Identification of gene family members and ScPYL61 Functional analysis laid a theoretical foundation for drought-resistant genetic breeding in sugarcane and provided an effective candidate gene.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a sugarcane gene ScPYL61 The sugarcane gene ScPYL61 The nucleotide sequence is shown in SEQ ID No. 1.

[0009] This invention also provides the sugarcane gene described in the above technical solution. ScPYL61 The encoded protein, the amino acid sequence of which is shown in SEQ ID No. 2.

[0010] This invention also provides the sugarcane gene described in the above technical solution. ScPYL61 Or the application of the proteins described in the above technical solutions in improving plant drought resistance.

[0011] Preferably, the plant includes Arabidopsis thaliana and / or sugarcane.

[0012] Preferably, it improves plant drought resistance by promoting root elongation.

[0013] Preferably, it improves plant drought resistance by promoting stomatal closure.

[0014] Preferably, plant drought resistance is improved by activating the abscisic acid signaling pathway.

[0015] This invention also provides an overexpression vector, comprising the sugarcane gene described in the above technical solution. ScPYL61 It was obtained by inserting it into the pGWB418 vector.

[0016] This invention also provides a method for obtaining drought-resistant plants, comprising the following steps:

[0017] 1) The overexpression vector described in the above technical solution is transformed into Agrobacterium to obtain the transformed bacteria;

[0018] 2) The transforming bacteria obtained in step 1) are transformed into plants by the flower dipping method to obtain drought-resistant plants.

[0019] Preferably, the plant includes Arabidopsis thaliana and / or sugarcane;

[0020] The Agrobacterium is Agrobacterium GV3101.

[0021] The beneficial effects of this invention are:

[0022] 1. For the first time, members of the sugarcane ABA receptor gene family were identified and drought-resistant candidate genes were screened. ScPYL61 .

[0023] To date, there are no reported studies on the identification of members of the sugarcane ABA receptor gene family. This invention uses the ABA receptor PYL from Arabidopsis thaliana and rice as templates to identify 63 receptors in the drought-resistant sugarcane cultivar Zhongzhe No. 1. ScPYL Members. Using transcriptome data from the drought-resistant variety Zhongzhe No. 1 under drought stress and after ABA treatment, this invention identified 63 candidate genes mediating sugarcane drought resistance through the regulation of ABA signaling. ScPYL In genes, only ScPYL61 This gene is highly expressed in sugarcane and exhibits drought-differential expression characteristics, being simultaneously upregulated by drought and ABA. Under drought stress, this gene enhances the plant's drought resistance by promoting stomatal closure and root elongation, indicating its potential for regulating drought resistance. This is the first time an ABA receptor gene has been screened and identified in sugarcane, providing genetic resources for future improvements in sugarcane drought resistance through molecular design breeding or genetic engineering techniques.

[0024] 2. For the first time, the molecular mechanism by which the sugarcane ABA-PYL-PP2C-SnRK2 molecular module regulates plant drought resistance through ABA signaling was elucidated, providing a new approach for molecular breeding of drought-resistant sugarcane.

[0025] ABA signaling molecular modules have been widely reported in various species for their role in regulating drought resistance in plants, but no such reports have been found in sugarcane. This invention systematically identified the ABA receptor ScPYL61, which participates in the regulation of ABA signaling and drought resistance, and identified the interacting protein phosphatase ScPP2C49 through library screening. Furthermore, it identified the protein kinases ScSnRK2.2 / 3 / 6 / 7 that interact with ScPP2C49. The results established an ABA-ScPYL61-ScPP2C49-ScSnRK2s molecular module and elucidated the molecular mechanism by which ScPYL61 mediates plant drought resistance through this module, providing new insights for improving drought resistance in sugarcane. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 63 ScPYL Distribution of genes on chromosome 1 of sugarcane in sugarcane cultivars;

[0028] Figure 2 For sugarcane cultivar Zhe No. 1, the FPKM value is >2. ScPYL Gene expression heatmaps induced by PEG and ABA; where Figure A represents... ScPYLGene expression levels under PEG-simulated drought stress; Figure B represents... ScPYL Gene expression levels after ABA induction; red asterisks indicate ScPYL61 Gene;

[0029] Figure 3 for ScPYL61 Gene induction by PEG and ABA and their RT-qPCR results in different tissues; where A and B represent... ScPYL61 Gene expression levels induced by PEG and ABA at different time points, represented by Figure C. ScPYL61 Gene expression levels in roots, stems, and leaves;

[0030] Figure 4 The results show the subcellular localization and autoactivation activity of ScPYL61 protein; Figure A shows that ScPYL61 protein is localized in the cell nucleus, and RFP is the nuclear localization marker; Figure B shows the autoactivation activity of ScPYL61 in yeast.

[0031] Figure 5 for ScPYL61 The phenotypes regulating drought resistance in plants after heterologous gene overexpression; Figure A shows the RT-qPCR analysis of six overexpression lines. ScPYL61 Gene expression levels; Figures B and C respectively represent... ScPYL61 Transgenic Arabidopsis thaliana root length phenotype and root length measurements under drought stress in culture medium; Figure D represents... ScPYL61 Phenotypes of transgenic Arabidopsis thaliana under drought stress; Figure E represents ScPYL61 Water loss rate of detached leaves of transgenic Arabidopsis thaliana; F-figure represents ScPYL61 Fv / Fm values ​​of transgenic Arabidopsis thaliana under normal growth and drought stress; G and H figures represent... ScPYL61 Root length phenotype and root length measurement results of transgenic Arabidopsis thaliana under normal growth and drought stress in nutrient soil;

[0032] Figure 6 for ScPYL61 Phenotypic features of stomatal cells in transgenic Arabidopsis thaliana leaves under drought stress; Figures A and B represent... ScPYL61 The degree of stomatal closure and stomatal width / length ratio in transgenic Arabidopsis leaves under normal growth and drought stress; Figures C and D represent... ScPYL61 Expression levels of drought resistance marker genes in transgenic Arabidopsis thaliana under normal growth and drought stress;

[0033] Figure 7 for ScPYL61 Determination of ABA sensitivity in transgenic Arabidopsis thaliana; Figures A and B respectively represent ScPYL61 Statistics on germination morphology and germination rate of transgenic Arabidopsis seeds under different ABA concentrations; Figures C and D represent... ScPYL61 Root length phenotypes and statistical data of transgenic Arabidopsis thaliana at different ABA concentrations; Figures E and F represent... ScPYL61 Expression levels of ABA signaling marker genes in transgenic Arabidopsis thaliana under normal growth and drought stress;

[0034] Figure 8 This diagram illustrates the protein-protein interaction between ScPYL61 and ScPP2C49, a core factor in ABA signal transduction. Figures A, B, and C demonstrate the interaction between ScPYL61 and ScPP2C49 as demonstrated by yeast two-hybrid assays, dual-luciferase complementation imaging experiments, and bimolecular fluorescence complementation experiments, respectively. Figure D shows the validation of ABA promoting the interaction between ScPYL61 and ScPP2C49 through yeast two-hybrid assays.

[0035] Figure 9 To verify the interaction between ScPP2C49 and ScSnRK2s in yeast two-hybrid experiments; Figure A shows the verification of the self-activation activity of ScSnRK2.2 / 3 / 6 / 7 / 8 in yeast; Figure B shows the verification of the interaction between ScPP2C49 and ScSnRK2.2 / 3 / 6 / 7 in yeast two-hybrid experiments.

[0036] Figure 10 This is a schematic diagram illustrating how ScPYL61 mediates plant drought resistance by regulating the ABA signaling pathway; under normal conditions, the ABA receptor... ScPYL61 When the gene is expressed at a low level, ScPP2C49 interacts with ScSnRK2.2 / 3 / 6 / 7 and inhibits the phosphokinase activity of ScSnRK2s, resulting in weakened ABA signaling. However, under drought stress, the large accumulation of ABA promotes the interaction between ScPYL61 and ScPP2C49, and the released ScSnRK2.2 / 3 / 6 / 7 activates ABA signaling by phosphorylating downstream transcription factors, thereby improving plant drought resistance. Detailed Implementation

[0037] This invention provides a sugarcane gene ScPYL61 The sugarcane gene ScPYL61 The nucleotide sequence is shown in SEQ ID No. 1.

[0038] This invention also provides the sugarcane gene described in the above technical solution. ScPYL61 The encoded protein, the amino acid sequence of which is shown in SEQ ID No. 2.

[0039] This invention also provides the sugarcane gene described in the above technical solution. ScPYL61Alternatively, the application of the proteins described in the above-mentioned technical solutions in improving plant drought resistance. In this invention, the plant preferably includes Arabidopsis thaliana and / or sugarcane. This invention preferably improves plant drought resistance by promoting root elongation. This invention preferably improves plant drought resistance by promoting stomatal closure. This invention preferably improves plant drought resistance by activating the abscisic acid signaling pathway.

[0040] This invention provides an overexpression vector, comprising the sugarcane gene described in the above technical solution. ScPYL61 The sugarcane gene was obtained by insertion into the pGWB418 vector. This invention relates to the sugarcane gene... ScPYL61 There are no special limitations on the method of insertion into the pGWB418 vector; those skilled in the art can follow conventional methods.

[0041] This invention also provides a method for obtaining drought-resistant plants, comprising the following steps:

[0042] 1) The overexpression vector described in the above technical solution is transformed into Agrobacterium to obtain the transformed bacteria;

[0043] 2) The transforming bacteria obtained in step 1) are transformed into plants by the flower dipping method to obtain drought-resistant plants.

[0044] This invention does not specifically limit the method for transforming the overexpression vector into Agrobacterium; those skilled in the art can use conventional methods. In this invention, Agrobacterium is preferably Agrobacterium GV3101.

[0045] This invention does not specifically limit the method of transforming the transforming bacteria into plants via the flower-dipping method; conventional methods can be used by those skilled in the art. Preferably, this invention uses conventional culture methods after transformation to obtain drought-resistant plants. In this invention, the plants preferably include Arabidopsis thaliana and / or sugarcane.

[0046] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0047] Unless otherwise specified, all experimental materials, reagents, and consumables used in the following examples are commercially available. The primer sequences used in the examples are shown in Table 1.

[0048] Table 1 Primer Sequences

[0049]

[0050] Example 1

[0051] sugar cane ScPYL Gene identification and expression analysis

[0052] 1. All of the chromosomes in sugarcane ScPYL Gene screening and identification

[0053] The genome sequence of cultivated sugarcane was downloaded based on previously reported literature (Bao et al., 2024), and genome files of Arabidopsis thaliana and rice were obtained from the TAIR and RGAP databases, respectively. To identify all members of the PYL gene family in sugarcane, this embodiment downloaded a hidden Markov model file (PF10604) of a specific PYL region from the PFAM database (http: / / pfam.xfam.org / ), using 14 PYL proteins from Arabidopsis thaliana and 13 PYL proteins from rice as reference sequences (Dupeux et al., 2011; Yadav et al., 2020). The sugarcane protein database was searched using the HMMER 3.1 program, with an E value set to 10. -5 Conserved domains of the sugarcane PYL gene family were identified using the NCBI-CDD and PFAM databases, and sequences containing complete Gate (LPA) and Latch (HRL) domains were screened. The chromosomal localization of the sugarcane ScPYL family was visualized using TBtools. The results showed that a total of 63 domains were identified in the sugarcane cultivar Zhe 1. ScPYL Genes are unevenly distributed across the chromosomes of sugarcane. Figure 1 ).

[0054] 2. Sugarcane ScPYL Gene expression analysis

[0055] The sugarcane cultivar Zhongzhe No. 1 was planted at the Sugarcane Germplasm Breeding Base of Guangxi University (Chongzuo). Single bud segments of sugarcane, disinfected with carbendazim, were planted in nutrient soil under the following light and humidity conditions: temperature 28-32℃, humidity 60%, 12 h light / 12 h dark. Sugarcane leaves at the four-leaf stage were uniformly sprayed with 150 μM ABA and 25% PEG-6000 solutions, respectively, and 800 mL of the corresponding solutions were uniformly added to the nutrient soil. An equal amount of ddH2O was used as a control. Young leaves from the same location were collected after 0, 0.5, 1, 3, 6, and 12 h of treatment and rapidly placed at -80℃ for RNA extraction for gene expression analysis and RNA sequencing (RNA-seq). The purity, concentration, and integrity of the RNA samples were tested. Qualified samples were processed by Biomarker Biotechnology Co., Ltd. for mRNA isolation, mRNA library preparation, and sequencing to obtain mapping data. Matching sequences were converted to FPKM values ​​for gene expression normalization. 63 samples were discarded. ScPYL After identifying genes with FPKM values ​​less than 2, their expression was analyzed and heatmaps were generated using TBtools software.

[0056] The results showed that 24 ScPYL Only in genes ScPYL61 High levels of expression were observed, and the expression was significantly upregulated by PEG and ABA signaling. Figure 2 (AB). We then further utilized specific primers. ScPYL61 -qPCR-F / R validated this result using RT-qPCR experiments, confirming that... ScPYL61 (SEQ ID No. 1) can be induced by PEG and ABA signals at different time points. Figure 3 (AB), and tissue expression pattern analysis showed that, ScPYL61 It has no specific expression characteristics and is expressed in sugarcane roots, stems, and leaves. Figure 3 (C)

[0057] Example 2

[0058] sugar cane ScPYL61 Subcellular localization of genes and analysis of yeast self-activation activity

[0059] 1. Subcellular localization analysis

[0060] Using specific primer pairs ScPYL61 -pGDG-F / R (primer sequences are shown in Table 1), using the ClonExpress II OneStep Cloning Kit (Novizan), according to the infusion vector construction method, ... ScPYL61 The coding sequence was inserted into the pGDG vector, which contains a GFP fluorescent tag. The successfully constructed [product / process] was then converted using electroporation. ScPYL61 The pGDG vector was transferred into Agrobacterium GV3101 competent cells. Agrobacterium-mediated infiltration was then used to... ScPYL61 -pGDG and pGDG no-load are respectively with AtHY5 A 1:1 mixture of RFP nucleomarker bacterial culture was injected into tobacco leaves of plants that had been growing for about one month. After 24 hours of dark treatment and 24-48 hours of light culture, the fluorescence of GFP and RFP signals in the tobacco epidermal cells was observed and detected using a laser confocal microscope (Nikon C2-ER).

[0061] The results showed that the GFP fluorescence of ScPYL61 and the RFP fluorescence signal of AtHY5 appeared simultaneously in the cell nucleus, thus suggesting that ScPYL61 is a nuclear localization protein. Figure 4 (A)

[0062] 2. Yeast self-activation activity assay

[0063] Using specific primer pairs ScPYL61-pGBKT7-F / R (primer sequences are shown in Table 1), using the ClonExpress II One Step Cloning Kit (Novizan), according to the infusion vector construction method, ... ScPYL61 The encoded sequence is inserted into the pGBKT7 vector. ScPYL61 The pGBKT7 plasmid and the empty pGADT7 plasmid were co-transformed into yeast strain Y2HGold. The positive yeast strains were then transferred to SD-TL, SD-TLH, and SD-TLH-A+X-α-Gal media for growth.

[0064] The results show ScPYL61 -pGBKT7+pGADT7 yeast grew normally on SD-TL medium, but not on SD-TLH and SD-TLH-A+X-α-Gal medium, thus suggesting that ScPYL61 has no self-activation activity in yeast. Figure 4 (B)

[0065] Example 3

[0066] ScPYL61 Drought resistance analysis of transgenic Arabidopsis thaliana

[0067] Using specific primer pairs ScPYL61 -pGWB418-F / R (primer sequences are shown in Table 1), using the ClonExpress II One Step Cloning Kit (Novizan), according to the infusion vector construction method, ... ScPYL61 The coding sequence was inserted into the pGWB418 vector to obtain recombinant... ScPYL61 -pGWB418. (This will...) ScPYL61 The pGWB418 plasmid was transferred to Agrobacterium strain GV3101, and then transferred into Arabidopsis thaliana (Col-0) using the flower-dipping method, following literature (Clough and Bent, 1998). The harvested T1 generation seeds were screened for single-copy selection, and the T2 generation seeds were screened for high-expression lines using RT-qPCR. Figure 5 Phenotypic investigation and drought resistance analysis were conducted on the T3 generation of two strains, OE-1 and OE-2, in the middle (A).

[0068] After disinfection and vernalization at 4℃, T3 generation seeds were sown on 1 / 2 MS solid medium containing 0, 100, and 150 mM mannitol, respectively, and then transferred to normal light conditions for growth. Root growth was observed and root length was recorded daily. Results showed that after about one week of vertical growth, there was no significant difference in root length between wild-type and transgenic Arabidopsis thaliana in the 0 mM mannitol medium. However, in the mediums containing 100 and 150 mM mannitol, the root length of transgenic Arabidopsis thaliana was significantly longer than that of wild-type. Figure 5 (Middle BC). After disinfection and vernalization, the seeds were sown in a soil mixture of nutrient soil and vermiculite at a volume ratio of 1:1, ensuring consistent soil weight. Arabidopsis plants that had grown for approximately 2-3 weeks were subjected to natural drought treatment, and their growth was monitored. Results showed that after 15 days of drought without water, the wild-type plants exhibited significantly greater leaf wilting than the transgenic Arabidopsis plants. Figure 5 (D). Ex vivo leaves from the same leaf position of wild-type and transgenic Arabidopsis thaliana with consistent growth status were collected and placed in empty culture dishes at 25℃. Leaf weight was measured every 0.5 hours. The results showed that the water loss rate of ex vivo transgenic Arabidopsis thaliana leaves was significantly lower than that of wild-type leaves at different time points. Figure 5 (E). Simultaneously, the chlorophyll fluorescence parameter Fv / Fm was measured under normal growth conditions and after drought treatment. The results showed no significant difference between wild-type and transgenic Arabidopsis under normal growth conditions, but the Fv / Fm value of transgenic Arabidopsis was significantly higher than that of wild-type under drought conditions. Figure 5 (F). The roots of Arabidopsis thaliana under normal growth and drought treatment for 15 days were washed, observed, and statistically analyzed. Results showed no significant difference between wild-type and transgenic Arabidopsis thaliana under normal growth conditions, but under drought conditions, the root length of transgenic Arabidopsis thaliana was significantly longer than that of wild-type. Figure 5 (Gross GH). In summary, ScPYL61 It can effectively enhance the drought resistance of plants.

[0069] Example 4

[0070] Observation and measurement of stomata on Arabidopsis leaves

[0071] In this embodiment, laser confocal microscopy (Nikon C2-ER) was used to observe the stomatal characteristics of wild-type and transgenic Arabidopsis thaliana leaves under normal growth conditions and after 7 days of drought treatment. Based on existing literature, the nail polish imprint method (Ye et al., 2014) was used. Colorless transparent nail polish was evenly applied to the veinless areas on the underside of Arabidopsis thaliana leaves. After air drying for 10 minutes, the nail polish layer was removed with transparent tape. The tape was then gently pressed to ensure full contact between the tape and the nail polish layer, and the tape was peeled off. The degree of stomatal closure was observed under a microscope, and the length and width of 30 stomata were randomly calculated using ImageJ software. The results showed that under normal growth conditions, there was no significant difference in the degree of stomatal opening between wild-type and transgenic Arabidopsis thaliana. However, after 7 days of drought treatment, both wild-type and transgenic Arabidopsis thaliana exhibited a certain degree of stomatal closure, and the degree of stomatal closure in transgenic Arabidopsis thaliana was significantly higher than that in wild-type. Figure 6 (AB) indicates that under drought conditions, ScPYL61 Drought resistance in plants can be enhanced by reducing leaf water loss through promoting stomatal closure. Furthermore, in this embodiment, RNA was extracted from leaves of Arabidopsis thaliana after normal growth and 7 days of drought treatment. After reverse transcription into cDNA, drought resistance marker genes were detected using specific primers (see Table 1). AtRD29A , AtRD29B and AtABF1 The expression levels of the marker genes were analyzed, and the results showed that under normal growth conditions, there was no significant difference in gene expression levels between wild-type and transgenic Arabidopsis thaliana. However, after drought treatment, the expression levels of the marker genes in transgenic Arabidopsis thaliana were significantly higher than those in wild-type. Figure 6 Medium CD).

[0072] Example 5

[0073] ABA sensitivity assay

[0074] After disinfection and vernalization at 4℃, T3 generation seeds were evenly sown onto 1 / 2MS solid medium containing 0, 0.1, and 0.5 μM ABA, respectively. The germination of wild-type and transgenic Arabidopsis thaliana was observed daily and the germination rate was counted.

[0075] The results showed that in ABA-free medium, wild-type and transgenic Arabidopsis seeds germinated rapidly with no significant difference in germination rate. However, in mediums containing 0.1 and 0.5 μM ABA, the germination rate of both wild-type and transgenic Arabidopsis seeds decreased rapidly, and at different time points, the germination rate of transgenic Arabidopsis seeds was significantly lower than that of wild-type seeds. Figure 7(AB). Similarly, T3 generation seeds were evenly sown on media containing 0, 0.1, and 0.5 μM ABA, respectively, and allowed to grow vertically. Root growth of wild-type and transgenic Arabidopsis was observed daily. Results showed no significant difference in root length between wild-type and transgenic Arabidopsis in ABA-free media, but the root length of transgenic Arabidopsis was significantly shorter than that of wild-type in media containing 0.1 and 0.5 μM ABA. Figure 7 (middle CD). These results indicate ScPYL61 It can increase the plant's sensitivity to ABA.

[0076] In addition, this embodiment used specific primers (see Table 1 for details) to detect ABA pathway marker genes. AtHAB1 , AtABI2 and AtSnRK2.2 The expression levels of the marker genes were analyzed, and the results showed that under normal growth conditions, there was no significant difference in gene expression levels between wild-type and transgenic Arabidopsis thaliana. However, after drought treatment, the expression levels of the marker genes in transgenic Arabidopsis thaliana were significantly higher than those in wild-type. Figure 7 EF). In summary, this indicates ScPYL61 Plant drought resistance can be enhanced by activating the ABA signaling pathway.

[0077] Example 6

[0078] Yeast two-hybrid (Y2H), dual-luciferase complementation (LCI), and bimolecular fluorescence complementation (BiFC) assays were used to verify protein interactions.

[0079] 1. Y2H Experiment

[0080] Using specific primer pairs ScPYL61 -pGBKT7-F / R (primer sequences are shown in Table 1), ScPYL61 The coding sequence was inserted into the pGBKT7 vector. Specific primer pairs were used. ScPP2C49 -pGADT7-F / R (primer sequences shown in Table 1) were cloned from the cDNA of Zhongzhe No. 1 variety. ScPP2C49 The gene (SEQ ID No. 49) was constructed using the ClonExpress II One Step Cloning Kit (Novizan) according to the infusion vector construction method. ScPP2C49 -pGADT7 vector. Following the instructions of the Clontech yeast two-hybrid kit, add 1 μg... ScPYL61 -pGBKT7 plasmid and 1 μg ScPP2C49 The -pGADT7 plasmid was co-transformed into yeast strain Y2HGold, and the positive yeast strains were transferred to SD-TL, SD-TLH, and SD-TLH-A+X-α-Gal media for growth.

[0081] After 3 days of cultivation, the results showed ScPYL61 -pGBKT7+ ScPP2C49 -pGADT7 yeast can grow normally on SD-TLH and SD-TLH-A+X-α-Gal media. Figure 8 (A). Yeast growth was observed after adding 0 and 50 μM ABA to different SD media. The results showed that ABA promoted the interaction between ScPYL61 and ScPP2C49. Figure 8 (D).

[0082] 2. LCI Experiment

[0083] Using specific primer pairs ScPYL61 -nLUC-F / R and ScPP2C49 -cLUC-F / R (primer sequences are shown in Table 1), using the ClonExpress II One Step Cloning Kit (Novizan), according to the infusion vector construction method, ... ScPYL61 and ScPP2C49 The positive plasmids were constructed into the pCAMBIA-nLUC and pCAMBIA-cLUC vectors, respectively. The constructed positive plasmids and empty vectors were then transformed into Agrobacterium GV3101 competent cells, respectively. ScPYL61 -nLUC+ ScPP2C49 -cLUC、nLUC+ ScPP2C49 -cLUC、 ScPYL61 The combination of -nLUC+cLUC and nLUC+cLUC was injected into tobacco leaves at a Agrobacterium-to-liquid volume ratio of 1:1. After tobacco was cultured in the dark for 24 h and then under light for 48 h, a luciferase reaction substrate was prepared: 1 mM Luciferin and 0.2% Triton X-100. 100-200 μL of the reaction solution was evenly spread on the back of the tobacco leaves. After reacting in the dark for 10 min, the fluorescence signal was observed and collected by photographing using a live imaging system (NightSHADE LB 985).

[0084] The results showed that only ScPYL61 -nLUC+ ScPP2C49 -cLUC combination showed strong fluorescence under imaging ( Figure 8 (B)

[0085] 3. BiFC Experiment

[0086] Using specific primer pairs ScPYL61 -nYFP-F / R and ScPP2C49-cYFP-F / R (primer sequences are shown in Table 1), using the ClonExpress II One Step Cloning Kit (Novizan), according to the infusion vector construction method, ... ScPYL61 and ScPP2C49 The positive plasmids were constructed into the ScPYL61-nYFP and ScPP2C49-cYFP vectors, respectively. The constructed positive plasmids and empty vectors were then transformed into Agrobacterium GV3101 competent cells, respectively. ScPYL61 -nYFP+ ScPP2C49 -cYFP、nYFP+ ScPP2C49 -cYFP、 ScPYL61 The combination of -nYFP and cYFP was injected into tobacco leaves at a 1:1 volume ratio of Agrobacterium tumefaciens to bacterial culture. After 24 hours of dark treatment and 24-48 hours of light culture, the fluorescence of YFP signals in tobacco epidermal cells was observed and detected using a laser confocal microscope (Nikon C2-ER). The results showed that ScPYL61 and ScPP2C49 were co-localized in the cell nucleus. Figure 8 (D). In summary, our results suggest that ScPYL61 can interact with the ABA signaling inhibitor ScPP2C49, and that this interaction occurs in the cell nucleus, with ABA promoting the interaction.

[0087] Example 7

[0088] ScPP2C49 interacts with multiple ScSnRK2 proteins.

[0089] Using specific primer pairs ScSnRK2s -pGBKT7-F / R (primer sequences are shown in Table 1), clone different cDNAs from the Zhongzhe No. 1 variety. ScSnRK2 Genes, construction ScSnRK2s -pGBKT7 vector. Following the instructions of the Clontech yeast two-hybrid kit, add 1 μg... ScSnRK2s -pGBKT7 plasmid and 1 μg empty pGADT7 plasmid were co-transformed into yeast strain Y2HGold. The positive yeast strains were then transferred to SD-TL, SD-TLH, and SD-TLH-A+X-α-Gal media for growth.

[0090] The results show that only ScSnRK2.8 The pGBKT7+pGADT7 yeast can grow normally on SD-TLH and SD-TLH-A+X-α-Gal media, while other yeast combinations do not grow, indicating that ScSnRK2.8 has yeast self-activation activity, while ScSnRK2.2 / 3 / 6 / 7 do not. Figure 9(A). Subsequently, following the instructions of the Clontech yeast two-hybrid kit, 1 μg of ScSnRK2.2 / 3 / 6 / 7-pGBKT7 plasmid and 1 μg of ScPP2C49-pGADT7 plasmid were co-transformed into yeast strain Y2HGold. The results showed that all ScSnRK2s-pGBKT7+ScPP2C49-pGADT7 plasmids could grow on SD-TLH and SD-TLH-A+X-α-Gal media, indicating that ScSnRK2.2 / 3 / 6 / 7 and ScPP2C49 interact ( Figure 9 (B)

[0091] The nucleotide sequence of the ScSnRK2.2 gene is shown in SEQ ID No. 50.

[0092] The nucleotide sequence of the ScSnRK2.3 gene is shown in SEQ ID No. 51.

[0093] The nucleotide sequence of the ScSnRK2.6 gene is shown in SEQ ID No. 52.

[0094] The nucleotide sequence of the ScSnRK2.7 gene is shown in SEQ ID No. 53.

[0095] The nucleotide sequence of the ScSnRK2.8 gene is shown in SEQ ID No. 54.

[0096] Based on the above research results, this invention has been identified as... ScPYL61 ScPYL61 is an ABA receptor gene in sugarcane that responds to drought stress signals, and its expression level is rapidly induced by drought stress and ABA. Under normal growth conditions, due to the low background ABA content, ScPYL61 cannot recruit ScPP2C49. Therefore, ScPP2C49 inhibits its activation of downstream ABA signaling by interacting with ScSnRK2.2 / 3 / 6 / 7. Under drought stress, ABA accumulates in large quantities. The ABA-ScPYL61 complex recruits ScPP2C49, inhibiting the interaction between ScPP2C49 and ScSnRK2.2 / 3 / 6 / 7. The released ScSnRK2s then activate ABA signaling, improving the plant's drought resistance. Figure 10 In the future, it is hoped that overexpression will be possible. ScPYL61 By introducing it into the main sugarcane varieties, drought resistance can be improved, creating new sugarcane materials that are highly drought-resistant, high-yielding, and of high quality.

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

[0098] References:

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Claims

1. Overexpression of sugarcane gene ScPYL61 Its application in improving plant drought resistance is characterized by, The sugarcane gene ScPYL61 The nucleotide sequence is shown in SEQ ID No. 1, and the plant is Arabidopsis thaliana or sugarcane.

2. The application according to claim 1, characterized in that, Improve plant drought resistance by promoting root elongation.

3. The application according to claim 1, characterized in that, Plant drought resistance is improved by promoting the closure of leaf stomata.

4. The application according to claim 1, characterized in that, Improve plant drought resistance by activating the abscisic acid signaling pathway.

5. A method for obtaining drought-resistant plants, characterized in that, Includes the following steps: 1) The sugarcane gene in the application described in claim 1 ScPYL61 An overexpression vector was obtained by inserting the vector into the pGWB418 vector, and the overexpression vector was transformed into Agrobacterium to obtain the transformed bacteria; 2) The transforming bacteria obtained in step 1) are transformed into plants by the flower-dipping method to obtain drought-resistant plants; the plants are Arabidopsis thaliana or sugarcane.

6. The acquisition method according to claim 5, characterized in that, The Agrobacterium is Agrobacterium GV3101.

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