Sugarcane scjaz3 gene and application thereof

Abstract

This invention discloses a sugarcane ScJAZ3 This invention relates to genes and their applications, specifically in the field of gene technology. The nucleotide sequence of this gene is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2. This invention discloses for the first time... ScJAZ3 The gene has a dual function: negatively regulating drought resistance in plants and positively regulating plant growth and development. Overexpression ScJAZ3 It can significantly promote cell elongation and plant growth, while weakening drought resistance. ScJAZ3 Able to interact with JA pathway transcription factors ScbHLH93 It interacts physically with the GA pathway inhibitor protein ScGAI, thereby coordinating the balance between growth and stress resistance. This invention also provides a recombinant expression vector containing this gene, a host cell, and its application in regulating plant drought resistance and / or growth and development, as well as methods for promoting plant growth or improving plant drought resistance and molecular breeding methods for cultivating plants that balance growth and stress resistance.

Description

Technical Field

[0001] This invention belongs to the field of gene technology, and particularly relates to a sugarcane ScJAZ3 Genes and their applications. Background Technology

[0002] In recent years, with the intensification of global climate change, drought has become one of the major abiotic stress factors limiting crop yield and quality. Sugarcane, as the world's most important sugar crop and potential energy crop, is mainly grown on dry, sloping land lacking irrigation, and this is also the case in many of my country's major sugarcane producing areas. Drought stress severely affects the jointing growth and sugar accumulation of sugarcane, leading to a significant decrease in yield and sugar content. Therefore, identifying key drought-resistant genes in sugarcane and analyzing their molecular regulatory networks is of significant strategic importance for breeding high-yielding and highly drought-resistant new sugarcane varieties.

[0003] In the long course of evolution, plants have developed a sophisticated regulatory mechanism to trade off between growth and development and abiotic resistance in order to adapt to complex and ever-changing environments. The plant hormone network plays a central role in this process. Among them, the jasmonic acid (JA) signaling pathway plays a crucial role in plant responses to abiotic stresses such as drought and pests, while gibberellins (GA) are primarily responsible for promoting cell elongation and plant growth. Numerous studies have shown that when plants encounter stresses such as drought, they typically activate stress-resistance signals such as JA while inhibiting growth-promoting signals such as GA, exhibiting a strategy of "growth stagnation in exchange for survival." This antagonistic relationship between growth and stress resistance has become a significant bottleneck in traditional breeding, making it difficult to simultaneously achieve high yields and high resistance.

[0004] JAZ (jasmonate zim-domain) proteins are core negative regulators in the JA signaling pathway. Under normal growth conditions, JAZ proteins in the cell nucleus inhibit the expression of downstream stress-resistance genes by interacting with various transcription factors (such as members of the bHLH family). When plants are under stress, leading to elevated JA levels, JAZ proteins are recognized by the COI1 receptor and degraded through ubiquitination, releasing the repressed transcription factors and activating the stress response. Meanwhile, previous studies have suggested that JAZ proteins may also participate in cross-interactions with other hormone signaling pathways (such as DELLA proteins in the GA pathway), thereby synergistically regulating plant growth and stress resistance. However, in sugarcane with its complex polyploid genome, the functional differentiation of JAZ gene family members remains unclear. In particular, the identification and molecular mechanism analysis of key JAZ genes that simultaneously coordinate drought resistance and jointing growth in sugarcane are still lacking. Discovering these pleiotropic genes that not only participate in stress response but also positively regulate plant growth has great application value for breaking through the bottleneck of "high resistance often accompanied by low yield" in traditional breeding and achieving "stable yield and stress resistance" through molecular design breeding. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing technologies and provide a method derived from sugarcane. ScJAZ3 Genes and their applications. Specifically, the technical problems to be solved by this invention include: identifying and isolating a key gene that can simultaneously regulate plant drought resistance and growth and development, analyzing its molecular mechanism, and providing a set of molecular breeding strategies based on this gene that can be used to balance the plant's "growth-defense" trade-off and break through the bottleneck of "high yield but no stress resistance, and stress resistance but low yield" in traditional breeding.

[0006] To achieve the above objectives, the present invention provides a separated sugarcane ScJAZ3 The gene, the nucleotide sequence of its coding region as shown in SEQ ID No. 1, or the amino acid sequence encoded by it as shown in SEQ ID No. 2.

[0007] The present invention also provides a sugarcane comprising the above-mentioned sugarcane ScJAZ3 Recombinant gene expression vectors.

[0008] The present invention also provides a sugarcane comprising the above-mentioned sugarcane ScJAZ3 The host cell of the gene or the above-mentioned recombinant expression vector.

[0009] The present invention also provides the above-mentioned sugarcane ScJAZ3 The application of genes, the aforementioned recombinant expression vectors, or the aforementioned host cells in regulating plant drought resistance and / or growth and development.

[0010] The present invention also provides a method for promoting plant growth and / or increasing biomass, comprising overexpressing the above-mentioned sugarcane in the plant. ScJAZ3 Gene.

[0011] Furthermore, the overexpression is achieved by transforming the aforementioned recombinant expression vector into a plant and expressing it.

[0012] The present invention also provides a method for improving plant drought resistance, including inhibiting endogenous drought in plants. ScJAZ3 Expression of a gene or its homologous gene.

[0013] Furthermore, the plant in question is sugarcane.

[0014] This invention also provides a molecular breeding method for cultivating plants that balance growth and stress resistance, by regulating the sugarcane through molecular marker-assisted selection or genetic engineering. ScJAZ3 The expression level of genes or their homologs is used to balance plant growth and development with drought resistance.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention is the first to clone and identify [a specific organism] from sugarcane. ScJAZ3This invention provides the complete nucleotide and amino acid sequences of the gene, filling a gap in functional studies of sugarcane JAZ family members and providing important genetic resources for sugarcane molecular breeding. This invention reveals for the first time... ScJAZ3 The gene has the dual function of negatively regulating plant drought resistance and positively regulating plant growth and development, breaking the traditional view that JAZ protein is only a negative regulator of stress resistance. It proposes a new regulatory model of "one factor with multiple effects" and provides a theoretical basis for understanding the trade-off mechanism of plant "growth-defense".

[0016] This invention has demonstrated through extensive experiments that heterologous overexpression in Arabidopsis thaliana is effective. ScJAZ3 This gene significantly promotes cell elongation and plant growth, manifested as increased plant height, hypocotyl elongation, and increased cell area. It also activates the gibberellin signaling pathway and the expression of genes related to cell wall cellulose synthesis. However, overexpression of this gene significantly weakens the plant's drought resistance, manifested as increased leaf wilting, higher water loss rate, increased relative conductivity and malondialdehyde content, decreased proline accumulation, and exacerbated photosynthetic system damage after drought treatment. Furthermore, the expression of key drought resistance marker genes and jasmonic acid signaling pathway genes is suppressed. Transcriptome and protein interaction analyses further revealed… ScJAZ3 Capable of interacting with JA pathway transcription factors ScbHLH93 It interacts physically with the GA pathway inhibitory protein ScGAI, thereby simultaneously regulating two signaling pathways.

[0017] Based on the above functions, this invention provides a complete technical solution for bidirectional application: through overexpression ScJAZ3 It can promote plant growth and increase biomass, and is suitable for high-yield breeding under conditions of sufficient water and fertilizer. This invention also provides a recombinant expression vector containing the gene, host cells and molecular breeding methods, which can be applied to a variety of plants such as sugarcane, Arabidopsis thaliana, rice, corn, sorghum or wheat, and has broad industrial applicability and good application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 sugarcane ScJAZ3 Gene expression patterns and tissue expression analysis induced by PEG and MeJA treatments. A: PEG-6000 treatment at different time points. ScJAZ3 Relative expression levels; B: Methyl jasmonate (MeJA) treatment at different time points ScJAZ3Relative expression level; C: ScJAZ3 Baseline expression levels of genes in sugarcane root, stem, and leaf tissues.

[0020] Figure 2 This study analyzed the degradation, subcellular localization, and yeast autoactivation of ScJAZ3 protein. A: Dual-luciferase reporter gene assay to detect MeJA-induced ScJAZ3 protein degradation phenotype; B: Subcellular localization and fluorescence co-localization analysis of the ScJAZ3-GFP fusion protein in the epidermal cells of *Nicotiana benthamiana* leaves; C: Measurement of transcriptional autoactivation activity of ScJAZ3 in yeast.

[0021] Figure 3 For overexpression ScJAZ3 Phenotypic and physiological analysis of gene-mediated drought resistance in Arabidopsis thaliana. A: Transgenic Arabidopsis thaliana lines (OE-1 and OE-4) ScJAZ3 A: Expression level detection; B: Plant phenotypes of wild-type (WT) and transgenic Arabidopsis thaliana before and after drought treatment; C: Statistical analysis of water loss rate of detached leaves; D: Root phenotypes under normal and drought conditions; E: Statistical analysis of taproot length under normal and drought conditions; F: Relative conductivity measurement results under drought stress; G: Proline content measurement results under drought stress; H: Malondialdehyde (MDA) content measurement results under drought stress; I: Detection of relative expression levels of drought resistance marker genes before and after drought.

[0022] Figure 4 Under drought stress ScJAZ3 Effects on chlorophyll fluorescence parameters in Arabidopsis thaliana. A: Chlorophyll fluorescence imaging of wild-type and transgenic Arabidopsis thaliana; B: Statistical analysis of maximum photochemical efficiency (Fv / Fm); C: Statistical analysis of non-photochemical quenching coefficient (NPQ); D: Statistical analysis of fluorescence decay ratio (Rfd).

[0023] Figure 5 for ScJAZ3 Transcriptome analysis of transgenic Arabidopsis thaliana in response to drought and JA signaling. A: Volcano plot of differentially expressed genes; B: KEGG pathway enrichment analysis of differentially expressed genes; C: GO enrichment analysis of downregulated differentially expressed genes; D: ScJAZ3 Venn diagram alignment of differentially regulated genes with known drought-responsive genes; E: ScJAZ3 Venn diagrams comparing differentially regulated genes with known MeJA response genes; F: heatmap of expression patterns of differentially regulated genes related to drought response and JA signaling; G: RT-qPCR validation of genes related to the JA pathway.

[0024] Figure 6 for ScJAZ3 Correlation analysis between gene expression levels and sugarcane germplasm drought tolerance index (DRI).

[0025] Figure 7 For overexpression ScJAZ3 Phenotypic analysis of genes promoting vegetative growth and cell elongation in Arabidopsis thaliana. A: Plant height phenotype during the reproductive stage under normal growth conditions; B: Plant height data statistics; C: Hypocotyl elongation phenotype during the seedling stage; D: Hypocotyl length statistics; E: Microscopic observation of hypocotyl epidermal cells; F: Hypocotyl epidermal cell length statistics.

[0026] Figure 8 for ScJAZ3 Analysis of pathways related to GA signaling and cell wall synthesis. A: Phenotypic analysis of total chlorophyll extracted from plant leaves under normal conditions; B: Determination of total chlorophyll content; C: GO enrichment analysis of differentially expressed genes; D: Venn diagram comparison of differentially expressed genes and PAC (paclobutrazol) response genes; E: Heatmap of expression patterns of differentially expressed genes related to cell wall biosynthesis and growth; F: RT-qPCR detection results of key genes in the GA pathway.

[0027] Figure 9 This study validates the protein-protein interaction between ScJAZ3 and ScbHLH93, a key transcription factor in the JA signaling pathway. A: Yeast two-hybrid (Y2H) experimental results; B: Three-dimensional structure and molecular docking prediction of the ScJAZ3-ScbHLH93 protein complex; C: In vivo validation results using dual-luciferase complementarity imaging (LCI); D: Bimolecular fluorescence complementarity (BiFC) co-localization validation results.

[0028] Figure 10 This study validates the protein-protein interaction between ScJAZ3 and the GA signaling pathway inhibitor ScGAI. A: Three-dimensional structure and molecular docking prediction of the ScJAZ3-ScGAI protein complex; B: Yeast two-hybrid (Y2H) experimental validation results; C: In vivo validation results using dual-luciferase complementarity imaging (LCI). Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0031] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0032] The primers involved in this invention are as follows: ScJAZ3-F / R F(SEQ ID No.3):ATGGAGAGGGATTTCCTG R(SEQ ID No.4):TCAGAGTAAGATCTGCAG ScJAZ3-qPCR-F / R F(SEQ ID No.5):CTTGCCCGATCTTTGGA R(SEQ ID No.6):AGCACTGCCTACGGTGTC ScJAZ3-pGreenII 0800-F / R F(SEQ ID No.7):CTTGATATCGAATTCCTGCAGATGGAGAGGGATTTCCTG R(SEQ ID No.8):CGCTCTAGAACTAGTGGATCCTCAGAGTAAGATCTGCAG ScJAZ3-pGDG-F / R F(SEQ ID No.9):GTCCGGACTCAGATCTCGAGATGGAGAGGGATTTCCTG R(SEQ ID No.10):GACTGCAGAATTCGAAGCTTTCAGAGTAAGATCTGCAG ScJAZ3-pGBKT7-F / R F(SEQ ID No.11):ATGGAGGCCGAATTCATGGAGAGGGATTTCCTG R(SEQ ID No.12):GCAGGTCGACGGATCCTTTCAGAGTAAGATCTGCAG ScJAZ3-pGWB418-F / R F(SEQ ID No.13):GAAGACTTGAACGGTAGCATGGAGAGGGATTTCCTG R(SEQ ID No.14):ATCGGGGAAATTCGAGCTCTATCAGAGTAAGATCTGCAG ScJAZ3-pCHANGE-CLuc-F / R F(SEQ ID No.15):GGCGGTACCCGGGATCCAATGGAGAGGGATTTCCTG R (SEQ ID No.16):AAGCTCTGCAGGTCGACTCAGAGTAAGATCTGCAG ScJAZ3-pXY104-F / R F (SEQ ID No.17):CAGGTACCCGGGGATCCATGGAGAGGGATTTCCTG R (SEQ ID No.18):GCGCTGCCCGCGTCGACGAGTAAGATCTGCAGTTT Example 1 Sugarcane ScJAZ3 Gene induction expression patterns and tissue-specific analysis This embodiment aims to explore sugarcane. ScJAZ3 The expression dynamics of the gene under simulated drought (PEG-6000) and methyl jasmonate (MeJA) treatments, and its basal expression level in different sugarcane tissues.

[0033] Using young leaves of the sugarcane cultivar "Zhongzhe No. 1" as material, total RNA was extracted using the TRIzol method, and cDNA was obtained by reverse transcription. Specific primers ScJAZ3-F / R were designed based on predicted JAZ family gene sequences from the sugarcane transcriptome database, and cDNA was obtained by RT-PCR amplification. ScJAZ3 The complete open reading frame (ORF) of the gene was obtained. Sequencing results showed that the full-length ORF was 1281 bp (as shown in SEQ ID No. 1) and encoded 426 amino acids (as shown in SEQ ID No. 2). Protein structure prediction analysis indicated that the ScJAZ3 protein contains a conserved JAZ domain at the C-terminus and a ZIM domain in the middle, belonging to a typical JAZ family protein.

[0034] SEQ ID No. 1: SEQ ID No. 2: MERDFLGVIDRAGKGGGEVVVEDSRTEPADNPAAAQWQFPAKSGAAPAFMSFRTAREEGSEEFSISGFRPAGDAFDGIKKQSSLPVISQHQQRQFGLSSSQVTAQQ YHAAAHVGQRLQGMDYSTAALHQLPGGSRLVQPLSVRHPAPFNQANLMVRSQSFHNGTGIHSRNQQPFTMSNGFGGSTVGVYGARNPRNQTSTQLTIFYNGSVNVFD NVPVDKAKELMMLASRASIPSPPSASHKPDSPISAPAKVNVPEVFPARQIVIQKPEPSVPHLSSTSSPIPIVPQVVTLSRSTSHCTTEACGSKPAVQQPVAAPISQATSSQPLATTSAAAVTPRAVPQARKASLARFLEKRKERVTSIEPYPTSKSPLESSDTVGSASAPTKSSSTDIAPVSNNGTEPVRLGQPSNISFSSEVCPSTKLQILL Using the sugarcane cultivar "Zhongzhe No. 1" as the experimental material, single-bud segments of sugarcane disinfected with carbendazim were sown in nutrient soil and cultured in an artificial climate chamber at a temperature of 28–32℃, relative humidity of 60%, and a 12-hour light / 12-hour dark cycle until the four-leaf stage. The seedling leaves were then uniformly sprayed with 25% PEG-6000 solution (simulating drought stress) and 100 μM methyl jasmonate (MeJA), respectively, with an equal amount of ddH2O as a blank control. Young leaves from the same location were collected at 0, 1, 3, 6, and 12 h after treatment and rapidly stored at -80℃ for total RNA extraction and subsequent gene expression analysis and transcriptome sequencing (RNA-seq).

[0035] Total RNA was extracted from each sample using the TRIzol method. After genomic DNA contamination was removed by DNase I digestion, real-time quantitative PCR (RT-qPCR) analysis was performed using specific primers ScJAZ3-qPCR-F / R. The sugarcane housekeeping gene ScGAPDH was used as an internal control. ^ The relative gene expression level is calculated using the (-ΔΔCt) method.

[0036] The results show that ( Figure 1 (A and B): Treatment with PEG-6000 can significantly inhibit ScJAZ3 Expression levels continued to decline with prolonged treatment time; however, MeJA treatment significantly induced expression. ScJAZ3Expression levels peaked 12 h after treatment. These results suggest that this gene may be involved in the cross-regulation of sugarcane drought stress and the jasmonic acid signaling pathway.

[0037] Organizational expression pattern analysis shows that ( Figure 1 (C) ScJAZ3 It is expressed in the basic form in sugarcane roots, stems, and leaves, with no obvious tissue specificity, and the expression level is slightly higher in leaves.

[0038] Example 2 Sugarcane ScJAZ3 Subcellular localization and self-activation analysis of genes This embodiment verifies the degradation characteristics, cellular localization, and transcriptional activity of ScJAZ3 protein through dual-luciferase reporter gene assays, subcellular localization observations, and yeast self-activation activity detection.

[0039] (I) Dual-luciferase reporter gene assay Using the specific primer ScJAZ3-pGreenII 0800-F / R, the ClonExpress II one-step cloning kit was used to clone... ScJAZ3 The coding region sequence was inserted into the pGreenII 0800-LUC vector to construct a recombinant reporter plasmid. The recombinant plasmid was transformed into Agrobacterium GV3101 competent cells and injected into leaves of one-month-old *Nicotiana benthamiana* using the Agrobacterium infiltration method. After 24 h of dark culture, the tobacco was cultured under light for 24–48 h. Six hours before fluorescence observation, 100 μM MeJA was sprayed on the right side of the leaf, while water was sprayed on the left side as a control. The luciferase (LUC) activity signal was detected and captured using an IVIS Lumina LT plant imaging system.

[0040] The results show that ( Figure 2 The fluorescence intensity in the MeJA-treated region (A) was significantly lower than that in the control region, indicating that MeJA treatment can induce the degradation of ScJAZ3 protein, which is consistent with its typical characteristics as a negative regulator of the jasmonic acid signaling pathway.

[0041] (II) Subcellular localization analysis Using the specific primer ScJAZ3-pGDG-F / R, ScJAZ3 The coding region (excluding the stop codon) was cloned into the pGDG vector carrying a GFP tag to construct the ScJAZ3-GFP fusion expression vector. The recombinant plasmid was introduced into Agrobacterium GV3101 via electroporation, and then mixed with a 1:1 volume ratio of a nuclear localization marker vector (AtHY5-RFP) carrying a red fluorescent tag. This mixture was then injected into tobacco leaves. After 24 h of dark culture, the cells were transferred to light culture for 24–48 h, and the fluorescence signal in the tobacco epidermal cells was observed using a laser confocal microscope.

[0042] The results show that ( Figure 2 (B) ScJAZ3 The GFP green fluorescence of ScJAZ3 and the RFP red nuclear labeling fluorescence of AtHY5 completely overlapped in the cell nucleus, confirming that ScJAZ3 is a nuclear localized protein, consistent with the function characteristics of transcription-regulated proteins.

[0043] (III) Yeast self-activation activity detection Using specific primers ScJAZ3-pGBKT7-F / R, ScJAZ3 The coding region was cloned into the pGBKT7 vector. The recombinant plasmid ScJAZ3-pGBKT7 and the empty vector pGBKT7 were transformed into yeast strain Y2HGold, and the positive clones were inoculated onto SD-Trp+X-α-Gal deficient solid medium for culture.

[0044] The results show that ( Figure 2 Yeast cells transformed with ScJAZ3-pGBKT7 (C) colonized the medium and grew but did not turn blue, indicating that... ScJAZ3 It exhibits no transcriptional self-activation activity in yeast, making it suitable for subsequent yeast two-hybrid (Y2H) screening experiments.

[0045] Example 3 Heterologous overexpression ScJAZ3 weakening the plant's drought resistance This embodiment systematically evaluates Arabidopsis thaliana overexpression lines at the phenotypic, physiological, and molecular levels. ScJAZ3 The impact on plant drought resistance.

[0046] (one) ScJAZ3 Obtaining Arabidopsis thaliana overexpression lines Amplification using specific primers ScJAZ3-pGWB418-F / R ScJAZ3 The coding sequence was inserted into the plant binary overexpression vector pGWB418 using seamless cloning technology. After transforming Agrobacterium GV3101, the recombinant plasmid was used to infect Arabidopsis thaliana (Col-0) using the flower-dipping method. T1 generation seeds were screened for resistance, and T2 generation seeds were identified by RT-qPCR to obtain homozygous overexpression lines with significantly increased expression levels. Two high-expression lines, OE-1 and OE-4, were selected. Figure 3 Subsequent phenotypic and functional analyses were conducted on A).

[0047] (ii) Overexpression ScJAZ3 Effects on drought resistance phenotype and physiological indicators in Arabidopsis thaliana After vernalization, T3 generation seeds were sown on 1 / 2 MS medium and transplanted into nutrient soil after 7 days of growth (16 h / 8 h light / dark, 22°C). Three-week-old wild-type (WT) and transgenic lines (OE-1, OE-4) were subjected to 15 days of drought stress treatment.

[0048] The results show that ( Figure 3 (B) After drought treatment, the wilting and yellowing of leaves in the transgenic lines were significantly more severe than in the wild type. Depth of water loss from detached leaves showed (…). Figure 3 (C) During the water loss process from 0 to 6 hours, the water loss rate of the transgenic lines was consistently higher than that of the WT lines. After 6 hours in vitro, the water loss rate of the transgenic lines was significantly higher than that of the WT lines, indicating that their leaves had poor water retention capacity. The plants were removed from the soil and the roots were washed. The length of the taproot from the base to the tip was measured. The results showed ( Figure 3 Under normal water conditions (D and E), there was no significant difference in root length between WT and transgenic lines; under drought stress, the elongation of the taproot of transgenic plants was severely inhibited and was significantly shorter than that of WT.

[0049] To thoroughly assess drought resistance physiological status, the following indicators were measured: Relative conductivity: Electrolyte leakage from leaves was measured using a conductivity meter. After drought, the relative conductivity of the transgenic lines was significantly higher than that of the WT lines. Figure 3 The presence of F indicates that the cell membrane is more severely damaged.

[0050] Proline and malondialdehyde (MDA): Measured using appropriate assay kits, there was no difference between the two under normal conditions; after drought treatment, the accumulation of proline, which plays a role in osmotic regulation, was significantly lower in transgenic lines than in WT, while the content of MDA, which reflects the degree of membrane lipid peroxidation, was significantly higher than that in WT. Figure 3 (G and H).

[0051] Expression of drought resistance marker genes: RNA was extracted from leaves before and after treatment, and RT-qPCR results showed that ( Figure 3 (I) After drought, the expression levels of key drought-resistant positive regulatory genes such as AtRD22, AtNCED3, and AtLEA4 in transgenic Arabidopsis thaliana were significantly lower than those in WT.

[0052] In summary, overexpression ScJAZ3 Genes significantly weaken the plant's drought resistance.

[0053] Example 4: Drought stress ScJAZ3 Effects on Arabidopsis thaliana chlorophyll fluorescence parameters This embodiment is intended to evaluate ScJAZ3 Effects of overexpression on photosynthetic system function in plants under drought stress.

[0054] Three-week-old WT and transgenic Arabidopsis seedlings were subjected to 15 days of drought stress treatment. Plant images were collected before and after treatment using a chlorophyll fluorescence imaging system (IMAGING-PAM), and key photosynthetic physiological indicators were measured: maximum quantum yield of photosystem II (Fv / Fm), non-photochemical quenching coefficient (NPQ), and fluorescence decay ratio (Rfd).

[0055] The results showed that ( Figure 4 In sections A and B), under normal water supply (before drought), the three fluorescence parameters of WT and transgenic lines showed no significant difference; however, after drought treatment, the Fv / Fm, NPQ, and Rfd values ​​of transgenic Arabidopsis were significantly lower than those of WT. This indicates that overexpression... ScJAZ3 Drought stress exacerbates the damage to the plant's photosystem II (PSII) response center, weakens the plant's ability to protect itself through heat dissipation, and leads to a significant decline in photosynthetic performance and potential vitality.

[0056] Example 5 ScJAZ3 Suppressing the expression of key genes involved in drought and JA signaling This embodiment reveals, from the whole genome level, the following information through transcriptome sequencing analysis. ScJAZ3 The molecular basis for overexpression leading to reduced drought resistance in plants.

[0057] WT and 15 days after drought treatment ScJAZ3 Samples were taken from rosette leaves of Arabidopsis thaliana overexpressing the gene (with normal watering as a control), and RNA-seq transcriptome sequencing was performed using the Illumina platform by Biomarker Biotechnology Co., Ltd. Differentially expressed genes (DEGs) were screened using |log2(Fold Change)| ≥ 1 and FDR < 0.05 as the threshold. Compared with WT, a total of 4080 DEGs were identified in the overexpression lines, of which 1973 were downregulated and 2107 were upregulated. Figure 5 (A). KEGG enrichment analysis showed that these genes were significantly enriched in "plant hormone signal transduction" and "MAPK signaling pathway" ( ). Figure 5 (B); GO enrichment analysis showed that the differentially expressed genes were mainly involved in biological processes such as "JA-mediated signaling pathway regulation", "response to water", and "hormonal response". Figure 5 (C) (Note) ScJAZ3 It is a core node integrating adversity and hormonal signals. Venn diagram comparative analysis further shows ( Figure 5 (D and E) ScJAZ3The regulated DEGs overlap significantly with known drought-induced / repressed genes and MeJA-induced / repressed genes. Through analysis of downregulated genes, it was found that the expression levels of several key drought-resistant positive regulatory genes (such as atCOR47, AtLEA5, zinc finger transcription factor AtZAT10, and ABA receptor AtPYL5 / 6) were significantly downregulated in transgenic lines. Figure 5 Meanwhile, the transcriptional levels of key genes involved in JA biosynthesis (such as AtPLIP2, AtPLIP3, and AtOPR1) were also significantly suppressed. Figure 5 (G). The above results elucidate the whole-genome level. ScJAZ3 The molecular basis for overexpression leading to reduced drought resistance in plants.

[0058] Example 6 ScJAZ3 Correlation analysis between gene expression levels and sugarcane drought resistance index This embodiment is illustrated by... ScJAZ3 Correlation analysis of gene expression levels and drought resistance index of different sugarcane germplasms was conducted to verify... ScJAZ3 Genes can negatively regulate drought resistance in natural sugarcane populations.

[0059] Thirty-three sugarcane germplasms with different drought resistance characteristics were selected. Leaf samples were collected at the four-leaf stage and quickly stored at -80 °C for total RNA extraction. Total RNA was extracted from each sample using the TRIzol method. After genomic DNA contamination was removed by DNase I digestion, real-time quantitative PCR (RT-qPCR) analysis was performed using specific primers ScJAZ3-qPCR-F / R to analyze sugarcane housekeeping genes. ScGAPDH As an intrinsic parameter, the 2^(-ΔΔCt) method is used for calculation. ScJAZ3 Relative gene expression levels. Under the same field conditions, each sugarcane germplasm was subjected to drought stress treatment, and the drought tolerance index (DRI, determined according to the "Evaluation Procedure for Drought Tolerance of Sugarcane Varieties" document

[2025] No. 23 issued by the China Agricultural Association) was calculated based on its growth performance and various physiological indicators. The higher the DRI value, the stronger the drought tolerance of the germplasm. The relative expression levels of each sugarcane germplasm were then calculated. ScJAZ3 Linear regression analysis was performed on the relative expression levels and the corresponding DRI values.

[0060] The results show that ( Figure 6 ), ScJAZ3 A significant negative correlation was found between the relative expression levels of the gene and the drought tolerance index (DRI) of sugarcane. Linear regression analysis showed that the coefficient of determination R0 was significantly lower than that of the gene. 2 The p-value was 0.1825, and the statistical p-value was 0.0132, indicating that the negative correlation was statistically significant. Among sugarcane germplasm with higher DRI values ​​(i.e., stronger drought resistance), ScJAZ3Transcript abundance of the gene was generally maintained at a low level. Meanwhile, in sugarcane germplasm with low DRI values ​​(i.e., sensitivity to drought), ScJAZ3 The abundance of gene transcripts increased significantly, showing a large accumulation.

[0061] The above results indicate that ScJAZ3 Gene expression levels are negatively correlated with sugarcane's drought tolerance.

[0062] Example 7 Overexpression ScJAZ3 Promote plant growth and development This embodiment verifies the phenotype of Arabidopsis thaliana by observing plant height during the reproductive growth period and hypocotyl elongation during the seedling stage. ScJAZ3 It promotes plant growth.

[0063] Seedlings cultured on 1 / 2 MS solid medium for 7 days were transplanted into soil and cultured under standard conditions until 6 weeks of age. For evaluation... ScJAZ3 The effect on the reproductive growth period of plants was investigated by measuring plant height.

[0064] The results show that ( Figure 7 In both A and B, the main stem height of the overexpression lines (OE-1 and OE-4) was significantly higher than that of the WT (WT average plant height was about 28 cm).

[0065] To verify its growth-promoting effect during the seedling stage, the hypocotyls of each line grown on 1 / 2 MS medium for 5 days were observed and microscopically measured. The results showed that ( Figure 7 In C and D), the hypocotyl length of OE-1 and OE-4 was significantly longer than that of WT. Further microscopic cytological observation showed ( Figure 7 In the E and F transgenic lines, the hypocotyl cell length was also significantly greater than that of the WT line.

[0066] The above phenotypic and statistical data demonstrate that heterologous overexpression of sugarcane ScJAZ3 Genes can not only promote plant cell elongation, but also significantly promote the overall longitudinal growth and biomass accumulation of plants, indicating that they play an important positive regulatory role in plant growth and development.

[0067] Example 8: ScJAZ3 activates GA signaling and cell wall synthesis-related genes This embodiment analyzes the molecular mechanism by which ScJAZ3 promotes plant growth from physiological, transcriptomic, and gene expression levels.

[0068] (a) Determination of total chlorophyll content Normally, excessive activation of gibberellin (GA) signaling in plants leads to a relative decrease in chlorophyll content. To preliminarily verify whether the growth-promoting phenotype of ScJAZ3 is related to the GA signaling pathway, total chlorophyll was extracted from leaves of 4-week-old Arabidopsis thaliana, and absorbance was measured at 652 nm. The results showed ( Figure 8 In the A and B lines, the total chlorophyll content of the transgenic lines was significantly lower than that of the WT line, suggesting that the GA pathway may be activated.

[0069] (II) Growth Regulation and GA Signal Transcription Level Analysis Based on the transcriptome data obtained in Example 5, GO functional enrichment analysis was performed on the "upregulated" DEGs in the overexpression lines. The results showed that ( Figure 8 In the C-cell model, upregulated genes were significantly enriched in cell wall biosynthesis, cellulose synthesis, microtubule-related motility, and hormone-mediated signaling pathways—all pathways promoting cell and tissue growth. A Venn diagram comparison was performed between ScJAZ3-regulated DEGs and DEGs treated with paclobutrazol (PAC, a GA synthesis inhibitor). Figure 8 (D) It was found that the ScJAZ3-regulated gene network for promoting growth partially overlaps with the GA signaling pathway. Heatmap analysis further clarified ( Figure 8 In the E1000 strain, the expression levels of several key genes encoding cellulose synthase (AtCSLG1 / 2 / 3, AtCESA9) were significantly upregulated. RT-qPCR validation experiments showed that... Figure 8 Compared with WT, the expression of AtGA20OX2, a key gene for GA biosynthesis, was significantly upregulated in the overexpression lines (F), while the expression of the GA receptor gene AtGID1C and the core negative regulatory repressors of the GA signaling pathway, AtRGL3 and AtRGA1, was significantly downregulated.

[0070] The above results indicate that ScJAZ3 can promote plant cell elongation and individual growth by promoting GA synthesis, relieving GA signal inhibition, and synergistically activating downstream cell wall cellulose synthesis.

[0071] Example 9: ScJAZ3 physically interacts with the core proteins ScbHLH93 and ScGAI of the JA / GA signaling pathway. This embodiment utilizes various protein interaction verification techniques to elucidate the molecular basis of ScJAZ3's simultaneous negative regulation of drought resistance and positive regulation of growth and development. The protein-protein interaction relationships between ScbHLH93, a key transcription factor in the JA signaling pathway, and ScGAI, a core repressor of the GA signaling pathway, and ScJAZ3 were verified.

[0072] (I) Yeast Two-Hybrid (Y2H) Experiment The amplified ScbHLH93 and ScGAI The genes were constructed into the pGADT7 vector and co-transformed with the ScJAZ3-pGBKT7 plasmid constructed in Example 2 into yeast strain Y2HGold. Positive clones were plated on different auxotrophic media. After 3 days of culture, the results showed ( Figure 9 China A, Figure 10 (B) Yeast strains containing the combinations of ScJAZ3+ScbHLH93 and ScJAZ3+ScGAI were able to grow normally on the defect-deficient media (SD-LTH-A+X-α-Gal or SD-LT-H+X-α-Gal). This preliminarily demonstrates the direct interaction between ScJAZ3 and ScbHLH93 and ScGAI.

[0073] (II) Analysis of protein three-dimensional structure and molecular docking The three-dimensional structures of the three proteins were predicted using AlphaFold 3, and visualization and docking analysis were performed using PyMol software. Complex simulation models showed that ScJAZ3 can form a stable hydrogen bond network with corresponding residues on the surface of ScbHLH93 through multiple amino acid residues (Arg11, Lys14, Glu18, and Ser24). Figure 9 Similarly, dense hydrogen bond interactions involving up to 17 residues are predicted between ScJAZ3 and ScGAI (B); Figure 9 (A)

[0074] (III) Dual-luciferase complementary imaging (LCI) experiment Will ScJAZ3 Constructed into the pCAMBIA-cLUC vector, ScbHLH93 Agrobacterium and ScGAI were constructed into the pCAMBIA-nLUC vector, respectively. Agrobacterium-mediated co-injection into tobacco leaves, followed by dark / light incubation and application of the reaction substrate (1 mM Luciferin, 0.2% Triton X-100). In vivo imaging revealed… Figure 9 C in the middle Figure 10 Strong fluorescence signals were elicited in the injection sites of the combination of ScJAZ3-cLUC+ScbHLH93-nLUC and ScJAZ3-cLUC+ScGAI-nLUC, confirming protein-protein interactions in living plants.

[0075] (iv) Bimolecular Fluorescence Complementary (BiFC) Experiment Will ScJAZ3 Connected to the cYFP vector, ScbHLH93 Agrobacterium, co-expressing the two plasmids, was injected into tobacco leaves at a 1:1 ratio after being ligated into the nYFP vector. Laser confocal microscopy revealed... Figure 9In the middle D), restored YFP yellow fluorescence signal was observed only in the nuclei of cells co-injected with ScJAZ3-cYFP+ ScbHLH93-nYFP.

[0076] The above experimental results confirm that ScJAZ3 can act as a core bridging protein, binding with core transcription factors of the JA pathway in the cell nucleus. ScbHLH93 It physically binds to ScGAI, the core inhibitor of the GA pathway. ScJAZ3 On the one hand, by combining and inhibiting ScbHLH93 On the one hand, it may block drought resistance response (weaken drought resistance); on the other hand, it may remove the restriction on growth by binding to ScGAI (promote development), thereby finely regulating the "drought resistance-growth" trade-off of plants under adversity.

[0077] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of isolated sugarcane ScJAZ3 Genes, characterized by, The nucleotide sequence of its coding region is shown in SEQ ID No. 1, or the amino acid sequence it encodes is shown in SEQ ID No.

2.

2. Containing the sugarcane as described in claim 1 ScJAZ3 Recombinant gene expression vectors.

3. Containing the sugarcane as described in claim 1 ScJAZ3 The host cell of the gene or the recombinant expression vector of claim 2.

4. The sugarcane according to claim 1 ScJAZ3 The application of genes, the recombinant expression vector of claim 2, or the host cell of claim 3 in regulating plant drought resistance and / or growth and development.

5. A method for promoting plant growth and / or increasing biomass, characterized in that, Includes overexpression of the sugarcane of claim 1 in plants. ScJAZ3 Gene.

6. The method according to claim 5, characterized in that, The overexpression is achieved by transforming the recombinant expression vector of claim 2 into a plant and expressing it.

7. A method for improving plant drought resistance, characterized in that, Including inhibiting endogenous substances in plants ScJAZ3 Expression of a gene or its homologous gene.

8. The method according to any one of claims 5-7, characterized in that, The plant in question is sugarcane.

9. A molecular breeding method for cultivating plants that balance growth and stress resistance, characterized in that, The sugarcane described in claim 1 can be regulated by molecular marker-assisted selection or genetic engineering. ScJAZ3 The expression level of genes or their homologs is used to balance plant growth and development with drought resistance.

Images