Sugarcane gene ScPYL61 and application thereof, overexpression vector and method

Abstract

The invention relates to the technical field of plant biology, in particular to a sugarcane gene ScPYL61 as well as application, an overexpression vector and a method thereof. The nucleotide sequence of the sugarcane gene ScPYL61 is as shown in SEQ ID No.1 (Sequence Identifier Number 1). According to the invention, 63 PYL gene family members are totally identified from a sugarcane No.1 variety in a sugarcane drought-resistant cultivated species, and the only one gene ScPYL61 which is high in expression and is induced by PEG and ABA is identified, and the gene activates an ABA signal, promotes stomata closing and root system elongation under drought stress by depending on an ABA-ScPYL61-ScPP2C49-ScSnRK2s molecular module, and enhances the drought resistance of plants. The identification of sugarcane PYL gene family members and the functional analysis of ScPYL61 in the invention lay a theoretical foundation for drought-resistant genetic breeding of sugarcane and provide an effective candidate gene.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, and in particular to a sugarcane gene ScPYL61 and an application, an overexpression vector and a method thereof. Background Art

[0002] Drought is one of the natural disasters that destroy agricultural production, and the losses it causes each year far exceed those caused by other disasters (Gowtham et al., 2022). As global warming accelerates the terrestrial water cycle, the incidence of agricultural droughts in many regions has increased significantly. It is expected that drought disasters will become more common, 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 ethanol (Hu et al., 2018). However, most of China's sugarcane is grown in semi-arid or arid areas, and limited water leads to inhibition of growth and tillering, resulting in an annual reduction of more than 60% in sugarcane production (Ferreira et al., 2017). Therefore, the discovery and utilization of drought-tolerant genes and the analysis of their molecular mechanisms are crucial to accelerate the genetic breeding process of sugarcane drought resistance.

[0003] When plants are under water shortage, they will produce and accumulate a large amount of abscisic acid (ABA). As an important plant hormone, ABA plays a vital role in helping plants adapt to a variety of abiotic stresses. At present, the ABA signal transduction pathway has been elucidated in the model plants Arabidopsis and rice, among which PYRABACTIN RESISTANCE1 (PYR1) / PYR1-like (PYL) / regulatory component of 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 by dephosphorylating specific residues, resulting in the blocking of downstream ABA network signals; when plants are in adversity, the intracellular ABA level increases rapidly, PYR1 / PYLs / RCAR1 binds to PP2C, and the kinase activity of SnRK2s is activated, resulting in the activation of downstream ABA signals to cope with adverse stress (Hirayama et al., 2010).

[0004] At present, the "gate-latch-latch" interaction network is a recognized mechanism of ABA signal transduction (Melcher et al., 2009). The ABA receptor PYL protein has a pocket for binding to ABA inside, and there are two conserved domains outside the ABA binding domain, namely the "gate" domain and the "latch" domain (Melcher et al., 2009; Nishimura et al., 2009; Yin et al., 2009). The binding of ABA causes the conformation of the PYL receptor binding pocket to change, that is, the proline residue and the conserved serine residue on the "gate ring" are moved into the "gate ring" of PYL by ABA and squeezed out of the "gate ring" (Yin et al., 2009; Santiago et al., 2009). In addition, the C-terminal α helix slowly moves toward ABA, and the "gate ring" is locked by the "lock ring" through hydrogen bonds and van der Waals forces (Santiago et al., 2009). This not only fixes the ABA molecule in the PYL pocket, but also creates an interaction surface for PP2C, leading to the release of SnRK2s (Melcher et al., 2009). This unique ABA perception mechanism suggests that PYL is essential for ABA signal transduction.

[0005] At present, PYL gene family members have been identified in many plants, with 14, 13, 21, 11 and 40 PYL family members identified in Arabidopsis (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. However, the identification and functional analysis of the sugarcane PYL gene family have not been reported in the prior art. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a sugarcane gene ScPYL61 and its application, overexpression vector and method. The present invention identifies 63 PYL gene family members from the sugarcane No. 1 variety of drought-resistant cultivated sugarcane, and identifies the only gene ScPYL61 that is highly expressed and induced by PEG and ABA. The gene activates ABA signals, promotes stomatal closure and root elongation under drought stress, and enhances plant drought resistance by relying on the ABA-ScPYL61-ScPP2C49-ScSnRK2s molecular module. The identification of sugarcane PYL gene family members and the functional analysis of ScPYL61 in the present invention lay a theoretical foundation for sugarcane drought-resistant genetic breeding and provide an effective candidate gene.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a sugarcane gene ScPYL61, and the nucleotide sequence of the sugarcane gene ScPYL61 is shown as SEQ ID No.1.

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

[0010] The present invention also provides the use of the sugarcane gene ScPYL61 described in the above technical solution or the protein described in the above technical solution in improving the drought resistance of plants.

[0011] Preferably, the plant comprises Arabidopsis thaliana and / or Sugarcane.

[0012] Preferably, the drought resistance of plants is improved by promoting root elongation.

[0013] Preferably, the drought resistance of plants is improved by promoting stomatal closure.

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

[0015] The present invention also provides an overexpression vector, which is obtained by inserting the sugarcane gene ScPYL61 described in the above technical solution into a pGWB418 vector.

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

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

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

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

[0020] The Agrobacterium is Agrobacterium GV3101.

[0021] Beneficial effects of the present invention:

[0022] 1. The members of the sugarcane ABA receptor gene family were identified for the first time, and the drought resistance candidate gene ScPYL61 was screened.

[0023] So far, there are no related research reports on the identification of members of the sugarcane ABA receptor gene family. The present invention uses the ABA receptor PYL in Arabidopsis and rice as a template, and identifies 63 ScPYL members in the drought-resistant sugarcane cultivar Zhongzhe No. 1. The transcriptome data of the drought-resistant variety Zhongzhe No. 1 under drought stress and after ABA treatment are used to mine candidate genes that mediate sugarcane drought resistance by regulating ABA signals. The present invention found that among the 63 ScPYL genes, only ScPYL61 is highly expressed in sugarcane, and has drought differential expression characteristics, and can be up-regulated by drought and ABA at the same time. This gene can enhance the drought resistance of plants by promoting stomatal closure and root elongation under drought stress, indicating that this gene has the application potential of regulating drought resistance. This is the first time that ABA receptor genes have been screened and identified in sugarcane, providing genetic resources for improving sugarcane drought resistance through molecular design breeding or genetic engineering technology in the future.

[0024] 2. For the first time, the molecular mechanism by which the sugarcane ABA-PYL-PP2C-SnRK2 molecular module regulates plant drought resistance by mediating ABA signals was analyzed, providing new ideas for the molecular breeding of sugarcane drought resistance.

[0025] ABA signaling molecular modules have been widely reported to be involved in regulating plant drought resistance in species, but no relevant reports have been found in sugarcane. The present invention identified the ABA receptor ScPYL61 that can participate in the regulation of ABA signaling and drought resistance through systematic analysis, and identified the protein phosphatase ScPP2C49 that interacts with it through library screening, and further mined the protein kinase ScSnRK2.2 / 3 / 6 / 7 that interacts with ScPP2C49. The research results established an ABA-ScPYL61-ScPP2C49-ScSnRK2s molecular module, and analyzed the molecular mechanism by which ScPYL61 mediates plant drought resistance by relying on this molecular module, providing new ideas for the research direction of improving sugarcane drought resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0027] Figure 1 The distribution map of 63 ScPYL genes on chromosome 1 in sugarcane cultivars;

[0028] Figure 2 The heat map of the expression of ScPYL genes with FPKM values ​​> 2 in sugarcane cultivar Zhe No. 1 induced by PEG and ABA; Figure A shows the expression level of ScPYL genes under PEG-simulated drought stress; Figure B shows the expression level of ScPYL genes after ABA induction; the red asterisk indicates the ScPYL61 gene;

[0029] Figure 3 The ScPYL61 gene was induced by PEG and ABA and its RT-qPCR results in different tissues; A and B respectively represent the expression levels of the ScPYL61 gene induced by PEG and ABA at different time points, and C represents the expression level of the ScPYL61 gene in roots, stems and leaves;

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

[0031] Figure 5 Figure 2 is the phenotype of drought resistance regulation of plants after heterologous overexpression of ScPYL61 gene; Figure A shows the expression level of ScPYL61 gene in 6 overexpression lines analyzed by RT-qPCR; Figures B and C show the root length phenotype and root length measurement results of ScPYL61 transgenic Arabidopsis under simulated drought stress in culture medium, respectively; Figure D shows the phenotype of ScPYL61 transgenic Arabidopsis after drought stress; Figure E shows the water loss rate of detached leaves of ScPYL61 transgenic Arabidopsis; Figure F shows the Fv / Fm value of ScPYL61 transgenic Arabidopsis under normal growth and drought stress; Figures G and H show the root length phenotype and root length measurement results of ScPYL61 transgenic Arabidopsis under normal growth and drought stress in nutrient soil, respectively;

[0032] Figure 6 The phenotypes of stomatal pores of ScPYL61 transgenic Arabidopsis leaves under drought stress; Figures A and B respectively represent the closure degree and stomatal width / length ratio of ScPYL61 transgenic Arabidopsis leaves under normal growth and drought stress; Figures C and D represent the expression levels of drought resistance marker genes of ScPYL61 transgenic Arabidopsis leaves under normal growth and drought stress;

[0033] Figure 7 The figure shows the sensitivity of ScPYL61 transgenic Arabidopsis to ABA. Figures A and B show the germination phenotype and germination rate statistics of ScPYL61 transgenic Arabidopsis seeds at different ABA concentrations. Figures C and D show the root length phenotype and data statistics of ScPYL61 transgenic Arabidopsis at different ABA concentrations. Figures E and F show the expression levels of ABA signal marker genes of ScPYL61 transgenic Arabidopsis under normal growth and drought stress.

[0034] Figure 8Schematic diagram of the protein interaction between ScPYL61 and ScPP2C49, a core factor of ABA signal transduction; A, B, and C respectively indicate the interaction between ScPYL61 and ScPP2C49 verified by yeast two-hybrid, dual luciferase complementation imaging, and bimolecular fluorescence complementation experiments; D indicates that ABA can promote the interaction between ScPYL61 and ScPP2C49 verified by yeast two-hybrid;

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

[0036] Fig.10 This is a schematic diagram of ScPYL61 mediating plant drought resistance by regulating the ABA signaling pathway; under normal circumstances, the ABA receptor ScPYL61 gene is at a low expression level, ScPP2C49 interacts with ScSnRK2.2 / 3 / 6 / 7 and inhibits the phosphokinase activity of ScSnRK2s, resulting in a weakening of the ABA signal; under drought stress, a large amount of accumulated ABA promotes the interaction between ScPYL61 and ScPP2C49, and the released ScSnRK2.2 / 3 / 6 / 7 activates the ABA signal by phosphorylating downstream transcription factors, thereby improving plant drought resistance. DETAILED DESCRIPTION

[0037] The present invention provides a sugarcane gene ScPYL61, and the nucleotide sequence of the sugarcane gene ScPYL61 is shown as SEQ ID No.1.

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

[0039] The present invention also provides the use of the sugarcane gene ScPYL61 described in the above technical solution or the protein described in the above technical solution in improving plant drought resistance. In the present invention, the plant preferably includes Arabidopsis and / or sugarcane. The present invention preferably improves plant drought resistance by promoting root elongation. The present invention preferably improves plant drought resistance by promoting stomatal closure. The present invention preferably improves plant drought resistance by activating the abscisic acid signaling pathway.

[0040] The present invention provides an overexpression vector obtained by inserting the sugarcane gene ScPYL61 described in the above technical solution into the pGWB418 vector. The present invention has no special limitation on the method of inserting the sugarcane gene ScPYL61 into the pGWB418 vector, and those skilled in the art can use conventional methods.

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

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

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

[0044] The present invention has no particular limitation on the method for transforming the overexpression vector into Agrobacterium, and those skilled in the art can use conventional methods. In the present invention, Agrobacterium is preferably Agrobacterium GV3101.

[0045] The present invention does not specifically limit the method of transforming the transformant into plants by the floral dipping method, and those skilled in the art can use conventional methods. The present invention preferably uses conventional culture methods after transformation to obtain drought-resistant plants. In the present invention, the plants preferably include Arabidopsis thaliana and / or sugarcane.

[0046] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Unless otherwise specified, the experimental materials, reagents, consumables, etc. involved in the following examples can all be obtained through commercial channels. The primer sequences used in the examples are shown in Table 1.

[0048] Table 1 Primer sequences

[0049]

[0050]

[0051] Example 1

[0052] Identification and expression analysis of ScPYL genes in sugarcane

[0053] 1. Screening and identification of all ScPYL genes in sugarcane chromosomes

[0054] The genome sequence of cultivated sugarcane varieties was downloaded according to the reported literature (Bao et al., 2024), and the genome files of Arabidopsis and rice were obtained from the TAIR and RGAP databases, respectively. In order to identify all members of the PYL gene family in sugarcane, this example downloaded the hidden Markov model file (PF10604) of the PYL specific region from the PFAM database (http: / / pfam.xfam.org / ), and used 14 PYL proteins in Arabidopsis and 13 PYL proteins in rice as reference sequences (Dupeux et al., 2011; Yadav et al., 2020). The sugarcane protein database was searched using the HMMER 3.1 program, and the E value was set to 10 -5 . The 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 ScPYL genes were identified in the sugarcane cultivar Zhongzhe 1, which were unevenly distributed on the sugarcane chromosomes ( Figure 1 ).

[0055] 2. Expression analysis of sugarcane ScPYL genes

[0056] Sugarcane cultivar Zhongzhe No. 1 was planted in the Guangxi University Sugarcane Germplasm Breeding Base (Chongzuo). Sugarcane single bud segments disinfected with carbendazim were planted in nutrient soil, and the light and temperature and humidity conditions were: temperature 28-32°C, humidity 60%, 12h light / 12h dark. 150μM ABA and 25% PEG-6000 solutions were evenly sprayed on the four-leaf stage sugarcane leaves, and 800mL of the corresponding solutions were evenly added to the nutrient soil, and an equal amount of ddH2O was used as a control. After 0, 0.5, 1, 3, 6, and 12h of treatment, young leaves at the same position were collected and quickly placed at -80°C to extract RNA for gene expression analysis and RNA sequencing (RNA-seq). By testing the purity, concentration, and integrity of RNA samples, qualified samples were isolated by Biomarker Biotechnology Co., Ltd. for mRNA, prepared by mRNA library, and sequenced to obtain Mapping Data, and the matching sequences were converted into FPKM values ​​for normalization of gene expression. After removing the genes with FPKM values ​​less than 2 in 63 ScPYLs, TBtools software was used to perform expression analysis and heat map production.

[0057] The results showed that among the 24 ScPYL genes, only ScPYL61 was highly expressed and significantly upregulated by PEG and ABA signals ( Figure 2We then further verified this result by RT-qPCR experiments using specific primers ScPYL61-qPCR-F / R, confirming that ScPYL61 (SEQ ID No.1) can be induced by PEG and ABA signals at different time points ( Figure 3 AB in the middle), and tissue expression pattern analysis showed that ScPYL61 had no tissue expression specificity and was expressed to a certain extent in sugarcane roots, stems, and leaves ( Figure 3 Middle C).

[0058] Example 2

[0059] Subcellular localization of sugarcane ScPYL61 gene and analysis of its autoactivation activity in yeast

[0060] 1. Subcellular localization analysis

[0061] Using the specific primer pair ScPYL61-pGDG-F / R (primer sequences are shown in Table 1), the ClonExpress IIOneStep Cloning Kit (Novozyme) was used to insert the coding sequence of ScPYL61 into the pGDG vector according to the infusion vector construction method, wherein the pGDG vector contains a GFP fluorescent tag. The successfully constructed ScPYL61-pGDG vector was transferred into Agrobacterium GV3101 competent cells by electroporation. ScPYL61-pGDG and pGDG empty vector were mixed with AtHY5-RFP nuclear marker bacterial solution in a ratio of 1:1 by Agrobacterium-mediated infiltration method, and then injected into Nicotiana benthamiana leaves grown for about one month. After the tobacco leaves were treated in the dark for 24 hours and cultured in the light for 24-48 hours, the GFP and RFP signal fluorescence in the tobacco epidermal cells were observed and detected by laser confocal microscopy (Nikon C2-ER).

[0062] The results showed that the GFP fluorescence of ScPYL61 and the RFP fluorescence signal of AtHY5 appeared in the cell nucleus at the same time, so it was speculated that ScPYL61 was a nuclear localized protein ( Figure 4 (A).

[0063] 2. Yeast self-activation activity assay

[0064] The coding sequence of ScPYL61 was inserted into the pGBKT7 vector using the ClonExpress II OneStep Cloning Kit (Novagen) using the specific primer pair ScPYL61-pGBKT7-F / R (primer sequences are shown in Table 1). The ScPYL61-pGBKT7 plasmid and the empty pGADT7 plasmid were co-transformed into the yeast strain Y2HGold, and the positive yeasts were transferred to SD-TL, SD-TLH, and SD-TLH-A+X-α-Gal medium for growth.

[0065] The results showed that ScPYL61-pGBKT7+pGADT7 yeast grew normally in SD-TL medium, but did not grow in SD-TLH and SD-TLH-A+X-α-Gal medium, thus it was inferred that ScPYL61 had no self-activation activity in yeast ( Figure 4 (B).

[0066] Example 3

[0067] Analysis of Drought Resistance of ScPYL61 Transgenic Arabidopsis

[0068] Using the specific primer pair ScPYL61-pGWB418-F / R (primer sequences are shown in Table 1), the ClonExpressIIOne Step Cloning Kit (Novozyme) was used to insert the coding sequence of ScPYL61 into the pGWB418 vector according to the infusion vector construction method to obtain recombinant ScPYL61-pGWB418. The ScPYL61-pGWB418 plasmid was transferred to the Agrobacterium strain GV3101 and transferred to Columbia Arabidopsis thaliana (Col-0) by the floral dip method according to the literature (Clough and Bent, 1998). The harvested T1 generation seeds were screened for single copies and the T2 generation seeds were screened for high expression strains by RT-qPCR ( Figure 5 (A) The T3 generations of OE-1 and OE-2 lines were planted for phenotypic investigation and drought resistance analysis.

[0069] After disinfection and vernalization at 4°C, T3 seeds were sown on 1 / 2MS solid medium containing 0, 100, and 150 mM mannitol, respectively, and transferred to normal light conditions for growth. The root growth status was observed every day and the root length was counted. The results showed that after the seedlings grew vertically for about a week, there was no significant difference in root length between the wild type and transgenic Arabidopsis in the medium containing 0 mM mannitol, but in the medium containing 100 and 150 mM mannitol, the root length of the transgenic Arabidopsis was significantly higher than that of the wild type ( Figure 5BC). The disinfected and vernalized seeds were sown in a soil with a volume ratio of 1:1 between nutrient soil and vermiculite, and the soil weight was kept consistent. The Arabidopsis plants that had grown for about 2-3 weeks were subjected to natural drought treatment, and the growth status of Arabidopsis was observed. The results showed that after 15 days of drought without water, the degree of leaf wilting of wild-type plants was significantly higher than that of transgenic Arabidopsis ( Figure 5 D). The detached leaves of the wild type and transgenic Arabidopsis thaliana with the same growth status were collected and placed in an empty culture dish at room temperature of 25°C. The leaf weight was measured every 0.5 h. The results showed that the water loss rate of the detached leaves of the transgenic Arabidopsis thaliana at different time points was significantly lower than that of the wild type ( Figure 5 At the same time, the chlorophyll fluorescence parameter Fv / Fm value was measured under normal growth state and after drought treatment. The results showed that under normal growth state, there was no significant difference between wild type and transgenic Arabidopsis, but under drought conditions, the Fv / Fm value of transgenic Arabidopsis was significantly higher than that of wild type ( Figure 5 Middle F). The roots of Arabidopsis thaliana were washed, observed and counted after normal growth and drought treatment for 15 days. The results showed that there was no significant difference between wild type and transgenic Arabidopsis thaliana under normal growth conditions, but the root length of transgenic Arabidopsis thaliana was significantly longer than that of wild type under drought conditions ( Figure 5 In summary, ScPYL61 can effectively enhance the drought resistance of plants.

[0070] Example 4

[0071] Observation and determination of stomata in Arabidopsis leaves

[0072] In this example, a laser confocal microscope (Nikon C2-ER) was used to observe the stomatal characteristics of wild-type and transgenic Arabidopsis under normal growth conditions and after 7 days of drought treatment. According to existing literature, the nail polish imprint method (Ye et al, 2014) was used to evenly apply colorless transparent nail polish on the back of Arabidopsis leaves without veins. After natural drying for 10 minutes, the nail polish layer was adhered with transparent tape, and the tape was gently pressed with fingers to make full contact with the nail polish layer. Then the transparent tape was torn off, and 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, but after 7 days of drought treatment, the stomata of both wild-type and transgenic Arabidopsis showed a certain degree of closure, and the degree of stomatal closure of transgenic Arabidopsis was significantly higher than that of wild-type ( Figure 6AB), indicating that under drought conditions, ScPYL61 can enhance plant drought resistance by promoting stomatal closure to reduce leaf water loss. In addition, in this example, RNA was extracted from Arabidopsis leaves under normal growth and 7 days after drought treatment. After reverse transcription into cDNA, the expression levels of drought resistance marker genes AtRD29A, AtRD29B and AtABF1 were detected using specific primers (see Table 1 for details). The results showed that under normal growth conditions, there was no significant difference in gene expression levels between wild-type and transgenic Arabidopsis, but after drought treatment, the expression level of marker genes in transgenic Arabidopsis was significantly higher than that in wild-type ( Figure 6 Medium CD).

[0073] Example 5

[0074] ABA sensitivity assay

[0075] After T3 seeds were sterilized and vernalized at 4°C, they were evenly sown on 1 / 2MS solid culture medium containing 0, 0.1, and 0.5 μM ABA, respectively. The germination of wild-type and transgenic Arabidopsis was observed every day and the germination rate was calculated.

[0076] The results showed that in the medium without ABA, wild-type and transgenic Arabidopsis seeds germinated rapidly and there was no significant difference in germination rate. However, in the medium containing 0.1 and 0.5 μM ABA, the germination rate of wild-type and transgenic Arabidopsis seeds decreased rapidly, and the germination rate of transgenic Arabidopsis seeds was significantly lower than that of wild-type seeds at different time points ( Figure 7 Similarly, T3 seeds were evenly sown on medium containing 0, 0.1, and 0.5 μM ABA and grown vertically, and the root growth of wild-type and transgenic Arabidopsis was observed every day. The results showed that there was no significant difference in root length between wild-type and transgenic Arabidopsis in medium without ABA, but on medium containing 0.1 and 0.5 μM ABA, the root length of transgenic Arabidopsis was significantly lower than that of wild-type ( Figure 7 These results suggest that ScPYL61 can enhance the sensitivity of plants to ABA.

[0077] In addition, this example uses specific primers (see Table 1 for details) to detect the expression levels of ABA pathway marker genes AtHAB1, AtABI2 and AtSnRK2.2. The results show that under normal growth conditions, there is no significant difference in gene expression levels between wild-type and transgenic Arabidopsis, but after drought treatment, the expression levels of marker genes in transgenic Arabidopsis are significantly higher than those in wild-type ( Figure 7 EF). In summary, it was shown that ScPYL61 could enhance plant drought resistance by activating the ABA signaling pathway.

[0078] Example 6

[0079] Yeast two-hybrid (Y2H), dual luciferase complementation imaging (LCI), and bimolecular fluorescence complementation (BiFC) assays to verify protein interactions

[0080] 1. Y2H experiment

[0081] The coding sequence of ScPYL61 was inserted into the pGBKT7 vector using the specific primer pair ScPYL61-pGBKT7-F / R (primer sequence is shown in Table 1). The ScPP2C49 gene (SEQ ID No. 49) was cloned from the cDNA of Zhongzhue No. 1 variety using the specific primer pair ScPP2C49-pGADT7-F / R (primer sequence is shown in Table 1), and the ScPP2C49-pGADT7 vector was constructed according to the infusion vector construction method using the ClonExpress II One Step Cloning Kit (Norwezan). 1 μg of ScPYL61-pGBKT7 plasmid and 1 μg of ScPP2C49-pGADT7 plasmid were co-transformed into the yeast strain Y2HGold with reference to the instruction manual of the yeast two-hybrid kit of Clontech, and the yeasts that tested positive were transferred to SD-TL, SD-TLH, and SD-TLH-A+X-α-Gal medium for growth.

[0082] After 3 days of culture, the results showed that ScPYL61-pGBKT7+ScPP2C49-pGADT7 yeast could grow normally on SD-TLH and SD-TLH-A+X-α-Gal medium ( Figure 8 After adding 0 and 50 μM ABA to different SD culture media, the yeast growth status was observed. The results showed that ABA can promote the interaction between ScPYL61 and ScPP2C49 ( Figure 8 (middle D).

[0083] 2. LCI experiment

[0084] Using the specific primer pairs ScPYL61-nLUC-F / R and ScPP2C49-cLUC-F / R (primer sequences are shown in Table 1), the ClonExpress II One Step Cloning Kit (Novozyme) was used to construct ScPYL61 and ScPP2C49 into pCAMBIA-nLUC and pCAMBIA-cLUC vectors, respectively, according to the infusion vector construction method. The constructed positive plasmid and the empty vector were transformed into Agrobacterium GV3101 competent cells, respectively. According to the combination of ScPYL61-nLUC+ScPP2C49-cLUC, nLUC+ScPP2C49-cLUC, ScPYL61-nLUC+cLUC, and nLUC+cLUC, the volume ratio of Agrobacterium liquid was 1:1 and injected into tobacco leaves. After culturing tobacco in the dark for 24 hours and then in the light for 48 hours, luciferase reaction substrate was prepared: 1 mM Luciferin, 0.2% Triton X-100, 100-200 μL of the reaction solution was evenly applied to the back of tobacco leaves, and after reacting in the dark for 10 minutes, a live imager (NightSHADE LB 985) was used to observe and collect fluorescence signals.

[0085] The results showed that only the ScPYL61-nLUC+ScPP2C49-cLUC combination showed strong fluorescence under the imaging instrument ( Figure 8 (B).

[0086] 3. BiFC experiment

[0087] Using the specific primer pairs ScPYL61-nYFP-F / R and ScPP2C49-cYFP-F / R (primer sequences are shown in Table 1), the ClonExpress II One Step Cloning Kit (Novozyme) was used to construct ScPYL61 and ScPP2C49 into ScPYL61-nYFP and ScPP2C49-cYFP vectors, respectively, according to the infusion vector construction method. The constructed positive plasmid and the empty vector were transformed into Agrobacterium GV3101 competent cells, respectively. According to the combination of ScPYL61-nYFP+ScPP2C49-cYFP, nYFP+ScPP2C49-cYFP, and ScPYL61-nYFP+cYFP, the volume ratio of Agrobacterium liquid was 1:1 and injected into tobacco leaves. After the tobacco leaves were treated in the dark for 24 hours and cultured in the light for 24-48 hours, the YFP signal fluorescence in the 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 8In summary, our results suggest that ScPYL61 can interact with the ABA signal repressor ScPP2C49, that their interaction occurs in the nucleus, and that ABA can promote their interaction.

[0088] Example 7

[0089] ScPP2C49 interacts with multiple ScSnRK2 proteins

[0090] Using the specific primer pair ScSnRK2s-pGBKT7-F / R (primer sequences are shown in Table 1), different ScSnRK2 genes were cloned from the cDNA of Zhongzhe No. 1 variety to construct the ScSnRK2s-pGBKT7 vector. According to the instruction manual of the yeast two-hybrid kit of Clontech, 1 μg of ScSnRK2s-pGBKT7 plasmid and 1 μg of empty pGADT7 plasmid were co-transformed into the yeast strain Y2HGold, and the positive yeasts were transferred to SD-TL, SD-TLH, and SD-TLH-A+X-α-Gal medium for growth.

[0091] The results showed that only ScSnRK2.8-pGBKT7+pGADT7 yeast could grow normally on SD-TLH and SD-TLH-A+X-α-Gal medium, while other yeast combinations did not grow, indicating that ScSnRK2.8 has yeast self-activation activity, while ScSnRK2.2 / 3 / 6 / 7 has no yeast self-activation activity ( Fig. 9 A). Subsequently, 1 μg of ScSnRK2.2 / 3 / 6 / 7-pGBKT7 plasmid and 1 μg of ScPP2C49-pGADT7 plasmid were co-transformed into the yeast strain Y2HGold according to the instruction manual of the yeast two-hybrid kit of Clontech. The results showed that all ScSnRK2s-pGBKT7+ScPP2C49-pGADT7 could grow in SD-TLH and SD-TLH-A+X-α-Gal medium, indicating that ScSnRK2.2 / 3 / 6 / 7 interacted with ScPP2C49 ( Fig. 9 (middle B).

[0092] The nucleotide sequence of the ScSnRK2.2 gene is shown as SEQ ID No.50.

[0093] The nucleotide sequence of the ScSnRK2.3 gene is shown as SEQ ID No.51.

[0094] The nucleotide sequence of the ScSnRK2.6 gene is shown as SEQ ID No.52.

[0095] The nucleotide sequence of the ScSnRK2.7 gene is shown in SEQ ID No.53.

[0096] The nucleotide sequence of the ScSnRK2.8 gene is shown in SEQ ID No.54.

[0097] Based on the above research results, the present invention identified ScPYL61 as an ABA receptor gene in sugarcane that can respond 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 content of ABA, ScPYL61 cannot recruit ScPP2C49, so ScPP2C49 hinders its activation of downstream ABA signals by interacting with ScSnRK2.2 / 3 / 6 / 7; under drought stress, ABA accumulates in large quantities, and the ABA-ScPYL61 complex recruits ScPP2C49, inhibiting the interaction between ScPP2C49 and ScSnRK2.2 / 3 / 6 / 7. The released ScSnRK2s then activate ABA signals to improve plant drought resistance ( Fig.10 In the future, it is expected that ScPYL61 will be overexpressed and introduced into the main sugarcane varieties to improve drought resistance and create new sugarcane materials with high drought resistance, high yield and high quality.

[0098] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

[0099] References:

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Claims

1. A sugarcane gene ScPYL61, characterized in that: The nucleotide sequence of the sugarcane gene ScPYL61 is shown in SEQ ID No.

1.

2. The protein encoded by the sugarcane gene ScPYL61 according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. Use of the sugarcane gene ScPYL61 according to claim 1 or the protein according to claim 2 in improving plant drought resistance.

4. The use according to claim 3, characterized in that: The plants include Arabidopsis thaliana and / or Sugarcane.

5. The use according to claim 3, characterized in that: Improve plant drought resistance by promoting root elongation.

6. The use according to claim 3, characterized in that: Improves plant drought resistance by promoting stomatal closure.

7. The use according to claim 3, characterized in that: Improving plant drought resistance by activating the abscisic acid signaling pathway.

8. An overexpression vector, characterized in that: The sugarcane gene ScPYL61 according to claim 1 is inserted into the pGWB418 vector to obtain the sugarcane gene ScPYL61.

9. A method for obtaining drought-resistant plants, characterized in that: The following steps are involved: 1) transforming the overexpression vector according to claim 8 into Agrobacterium to obtain transformed bacteria; 2) The transformed bacteria obtained in step 1) are transformed into plants by floral dipping method to obtain drought-resistant plants.

10. The acquisition method according to claim 9, characterized in that: The plants include Arabidopsis thaliana and / or Sugarcane; The Agrobacterium is Agrobacterium GV3101.

Images