Sugarcane gene scjaz10 and application thereof in regulating plant flowering

Abstract

This invention discloses a sugarcane gene ScJAZ10 The sugarcane gene and its application in regulating plant flowering relate to the field of plant biotechnology. ScJAZ10 The nucleotide sequence of the protein is shown in SEQ ID No. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 2. This invention reveals for the first time that the ScJAZ10 protein can act as an atypical transcriptional coactivator, physically interacting with the ScNAC23 transcription factor in the cell nucleus to form a protein complex, synergistically activating the downstream core florigen gene. ScFT Gibberellin (GA) can activate transcription, thereby promoting earlier flowering in plants. ScJAZ10 The ScJAZ10 protein is transcribed and expressed, and its flowering-promoting function depends on the accumulation of endogenous GA. This invention breaks through the traditional understanding that JAZ proteins are only transcriptional repressors, providing new key gene targets and molecular regulatory modules for the genetic improvement of flowering time in sugarcane and other plants, and has significant theoretical value and broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, and particularly relates to a sugarcane gene. ScJAZ10 And its application in regulating plant flowering. Background Technology

[0002] sugar cane( Saccharum Sugarcane (spp.) is a globally important commercial crop, supplying the majority of the world's sugar and serving as a primary raw material for renewable bioethanol. The transition from vegetative to reproductive growth is a crucial physiological process in sugarcane development. In commercial production, flowering is often considered a highly undesirable trait: the transition to flowering consumes large amounts of photosynthetic products, halts the elongation of apical internodes, and severely depletes sucrose reserves stored in the stem, ultimately leading to a sharp decline in sugarcane yield and sugar quality. However, from a breeding perspective, flowering is an indispensable prerequisite for artificial hybridization and the continuous development of superior varieties. Therefore, elucidating the molecular mechanisms of sugarcane flowering transition is of significant strategic importance for the genetic improvement of high-yielding sugarcane varieties, the development of late-flowering or non-heading varieties, and the precise control of flowering time.

[0003] The regulation of plant flowering time is controlled by environmental signals (such as photoperiod and temperature) and endogenous developmental signals. Within this complex regulatory network, FLOWERING LOCUS T ( FT The gene plays a central role; its encoded mobile floral signaling protein is transported from the leaf through the phloem to the shoot apical meristem, triggering flower morphogenesis through interaction with other proteins, and is a key determinant of flowering transition. FT Upstream of genes, numerous transcription factors regulate their expression directly or indirectly, forming a sophisticated and complex flowering regulatory network. Among them, the NAC (NAM, ATAF1 / 2, and CUC2) family of proteins is a plant-specific transcription factor family that typically acts as a key molecular hub during developmental transitions, directly binding to the promoters of florigen genes to drive the flowering transition process. Currently, studies in sugarcane have reported the NAC transcription factor ScNAC23, which participates in gibberellin (GA)-mediated flowering and senescence regulation through direct physical interaction with the DELLA protein ScGAI, playing a positive regulatory role in promoting early flowering. Furthermore, ScNAC23 can also directly bind to… ScFTIP1 The promoter of the gene negatively regulates its transcription and participates in the regulation of flowering time. However, although the function of ScNAC23 as a flowering promoting factor has been preliminarily revealed, its role in regulating the core florigen gene remains unclear. ScFTThe fine regulatory network and the lineage of interacting target molecules in the expression process remain far from being elucidated. In particular, whether other co-regulatory factors are involved in the ScNAC23-mediated transcriptional activation process is a scientific question that urgently needs to be addressed in this field.

[0004] Jasmonic acid (JA), a key plant hormone, plays a crucial role in plant defense responses, growth and development, and reproductive development. Among these, the Jasmonate Zim-Domain (JAZ) protein, as a core regulatory node in the JA signaling pathway, has received widespread attention. JAZ proteins belong to the plant-specific TIFY superfamily and are characterized by two highly conserved functional domains: the ZIM domain at the N-terminus, which mediates dimer formation and interacts with the co-repressor TOPLESS; and the Jas motif at the C-terminus, which binds to downstream transcription factors and mediates JA-dependent protein degradation. In the classic JA signaling model, when JA levels are low, JAZ proteins act as transcriptional repressors, binding to downstream transcription factors and inhibiting their activity, thus suppressing the expression of JA-responsive genes. When JA signaling is activated, JAZ proteins are recognized by the SCFCOI1 ubiquitin ligase complex and degraded via the ubiquitin-proteasome pathway, thereby relieving the inhibition of downstream transcription factors and initiating the expression of downstream genes. Therefore, JAZ proteins are generally considered core transcriptional repressors in plant defense pathways and development. However, a growing body of research indicates that the function of JAZ proteins is not limited to a single role of transcriptional repression. They are also pivotal proteins that coordinate crosstalk of multiple hormone signals and regulate plant vegetative growth and developmental transitions. Some members of the JAZ family may play different modes of action than classical repression in specific tissues and developmental stages.

[0005] Currently, members of the JAZ gene family have been found in Arabidopsis thaliana ( Arabidopsis thaliana ), rice ( Oryza sativa ),cotton( Gossypium spp.), soybean ( Glycine max It has been systematically identified and partially functionally analyzed in various plants, including Arabidopsis thaliana. Regarding flowering regulation, previous studies have reported that the JAZ protein in Arabidopsis thaliana antagonizes the effects of TOE transcription factors through interaction. FTThe transcriptional repression of JAZ1 regulates flowering time, and the conserved interaction mechanism between JAZ1 and NAC42 transcription factors can inhibit the transactivation activity of NAC in various plants. In sugarcane, studies have systematically identified and analyzed the sugarcane JAZ gene family, and found that overexpression of some members (such as ScJAZ1 / 2 and ScJAZ15-1) in transgenic plants can promote flowering, preliminarily indicating that JAZ proteins are involved in the flowering regulation process of sugarcane. However, the specific molecular mechanisms of sugarcane JAZ proteins in the flowering process, especially whether they can break through the functional paradigm of classical transcriptional repressors and directly participate in the co-transcriptional activation of downstream florigen genes as atypical transcriptional coactivators, and thus participate in the transition from vegetative growth to reproductive growth, have not yet been reported. As a highly complex autopolyploid crop, the scarcity of key gene resources for flowering time regulation in sugarcane severely restricts the progress of sugarcane molecular breeding. Therefore, in-depth exploration of key JAZ genes related to sugarcane flowering and analysis of their interaction mechanisms with known flowering regulators have important theoretical significance and application value for improving sugarcane flowering time. Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the scarcity of key gene resources for sugarcane flowering regulation in existing technologies, and the fact that the specific molecular mechanism of JAZ protein in sugarcane flowering regulation, especially its function as an atypical transcriptional coactivator, has not been elucidated. This invention provides a sugarcane gene... ScJAZ10 The invention also provides a recombinant expression vector containing the gene, a recombinant host cell, and a protein-protein interaction module composed of the ScJAZ10 protein and the ScNAC23 transcription factor, which regulates plant flowering, particularly by synergistically activating downstream florigen genes. ScFT Transcription for applications that promote flowering.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an isolated sugarcane gene. ScJAZ10 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0008] This invention also provides sugarcane genes described above. ScJAZ10 The encoded protein has the amino acid sequence shown in SEQ ID No. 2. Experimental verification showed that the ScJAZ10 protein does not possess transcriptional autoactivation activity in the yeast system. However, subcellular localization analysis revealed that ScJAZ10 is a protein specifically located in the cell nucleus, which is consistent with its localization characteristics as a transcription-regulatory protein.

[0009] Secondly, the present invention provides a method comprising the aforementioned sugarcane gene. ScJAZ10 The present invention further provides a recombinant expression vector comprising the recombinant expression vector. The recombinant expression vector and the recombinant host cell can be used to achieve [the desired effect] in a target plant. ScJAZ10 Gene overexpression, or its use in preparing related transgenic plant materials.

[0010] Thirdly, the present invention provides the aforementioned sugarcane gene. ScJAZ10 Or the application of the protein it encodes in regulating plant flowering time. In a specific embodiment, the application is achieved by overexpressing the sugarcane gene in a target plant. ScJAZ10 The protein, or the sugarcane gene, can be used to significantly promote earlier flowering in plants, thereby promoting the transition to flowering. In other foreseeable embodiments, the application may also involve inhibiting or silencing the sugarcane gene in the target plant. ScJAZ10 The expression of the protein may be used to delay plant flowering or to cultivate late-flowering or non-flowering varieties. The plant in question is specifically sugarcane, but the scope of this invention is not limited to sugarcane, but also includes other monocot or dicot crops that require flowering regulation.

[0011] Fourthly, this invention provides an application of a protein interaction module composed of the ScJAZ10 protein (SEQ ID No. 2) and the sugarcane ScNAC23 transcription factor in regulating plant flowering. Experiments have confirmed that the ScJAZ10 protein can directly interact with ScNAC23 within the plant cell nucleus, forming a ScJAZ10-ScNAC23 protein complex. In this complex, ScNAC23 provides DNA-binding activity, responsible for recognizing and binding to the CACG cis-acting element on the promoter of downstream target genes; while ScJAZ10 functions as a transcriptional coactivator, synergistically enhancing the transcriptional activation ability of ScNAC23 on downstream target genes. Studies have shown that this protein interaction module activates downstream core florigen genes through synergistic activation. ScFT Transcription significantly promotes plant flowering.

[0012] This invention also provides the application of the above-mentioned protein interaction module in the breeding of transgenic plant varieties with improved flowering time. By regulating the interaction strength between ScJAZ10 and ScNAC23 or regulating the formation level of this protein complex, the flowering time of plants, especially sugarcane, can be directionally altered, providing precise molecular manipulation targets for breeding superior early-flowering or late-flowering varieties.

[0013] Compared with the prior art, the present invention has the following advantages and technical effects: This invention marks the first successful cloning and identification of a novel JAZ family gene with significant flower-promoting function from sugarcane. ScJAZ10This invention fills a gap in the systematic study of the flowering regulatory functions of sugarcane JAZ gene family members. More importantly, it breaks through the traditional functional paradigm that JAZ proteins are generally recognized in the field as core transcriptional repressors in the JA signaling pathway. For the first time, it reveals that the ScJAZ10 protein can act as an atypical transcriptional coactivator, forming a protein complex with the key flowering regulation transcription factor ScNAC23 to synergistically activate downstream core florigen genes. ScFT The transcription of ScJAZ10. This discovery not only elucidates a novel molecular mechanism by which ScJAZ10 mediates early flowering, but also provides a new paradigm for understanding the cross-talk between the JA signaling pathway and other hormonal signals (especially gibberellin GA signaling). Furthermore, the GA-induced [transmission mechanism] revealed in this invention... ScJAZ10 Expression, ScJAZ10-ScNAC23 complex co-activation ScFT This complete molecular regulatory module provides efficient and specific key gene targets and molecular tools for the precise regulation of flowering time in sugarcane and other crops through genetic engineering and molecular breeding, and has important theoretical value and broad application prospects. Attached Figure Description

[0014] 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.

[0015] Figure 1 Figure A shows the phylogenetic analysis, tissue expression pattern, and protein characterization results of ScJAZ10; Figure B shows the phylogenetic tree analysis of ScJAZ10. ScJAZ10 Figure C shows the expression levels of the gene in the root, stem, leaf, and flower tissues of sugarcane at the four-leaf stage; Figure D shows the self-activation activity assay of ScJAZ10 in the yeast system; Figure D shows the subcellular localization of ScJAZ10 protein in tobacco epidermal cells, with RFP as the nuclear localization marker.

[0016] Figure 2 For overexpression ScJAZ10 Analysis of expression in Arabidopsis thaliana early-flowering phenotypes and sugarcane varieties; Figure A shows... ScJAZ10 Expression levels in non-flowering and flowering sugarcane varieties; Figure B shows the expression levels in wild-type and overexpression lines analyzed by RT-qPCR. ScJAZ10 Gene expression levels; Figures C and D show the flowering phenotypes of wild-type and transgenic Arabidopsis thaliana under long-day conditions, respectively; Figures E and F show the number of days for bolting and flowering and the number of rosette leaves in wild-type and transgenic Arabidopsis thaliana, respectively; Figure G shows the flowering integrator in wild-type and transgenic Arabidopsis thaliana. AtCO , AtFT and AtSOC1 The relative expression level.

[0017] Figure 3 For overexpression ScJAZ10 Transcriptome (RNA-seq) analysis and chlorophyll content determination of Arabidopsis thaliana; Figure A shows the volcano plot of differentially expressed genes (DEGs) in wild-type and transgenic Arabidopsis thaliana; Figures B and C show the GO and KEGG enrichment analysis results of DEGs, respectively; Figure D shows the expression heatmap of representative DEGs such as GA biosynthesis, flowering transition, and cell cycle; Figure E shows the relative expression level of key GA synthesis genes in wild-type and transgenic Arabidopsis thaliana; Figures F and G show the chlorophyll extract and total chlorophyll content determination results of leaves of wild-type and transgenic Arabidopsis thaliana, respectively.

[0018] Figure 4 The results of ScJAZ10 responding to gibberellin (GA) and promoting flowering in a manner dependent on endogenous GA; where Figure A is... ScJAZ10 Figure B shows the relative expression levels of the gene induced by exogenous GA at different time points; Figure C is a schematic diagram of the construction of the dual-luciferase transient expression experimental vector; Figures C and D show the activation of exogenous GA, respectively. ScJAZ10 In vivo luminescence images and fluorescence statistics of the promoter; Figures E and F show the flowering phenotypes and flowering days of wild-type and transgenic Arabidopsis thaliana under Mock and paclobutrazol (PAC) treatments, respectively; Figures G and H show the flowering phenotypes and flowering days of wild-type and transgenic Arabidopsis thaliana under Mock and PAC treatments, respectively. AtFT and AtSOC1 The relative expression level of genes.

[0019] Figure 5 The interaction between ScJAZ10 and ScNAC23 proteins and their synergistic activation of downstream florigen genes ScFT Experimental results of transcription; Figure A shows the interaction between ScNAC23 and ScJAZ10 verified by yeast two-hybrid assay; Figures B and C show the in vivo interaction phenotype and luminescence intensity statistics of the two cells, respectively, verified by dual-luciferase complementation imaging assay; Figure D shows the interaction of the two cells in the cell nucleus verified by bimolecular fluorescence complementation assay; Figure E shows sugarcane... ScFT Figure F shows the predicted pattern of the CACG recognition motif in the promoter region; Figure F shows the structural patterns of the effector and reporter vectors in the dual-luciferase experiment; Figures G and H show the activation of ScNAC23 by individual expression, respectively. ScFT Emission images and fluorescence statistics of the promoter; Figures I and J show the co-expression and co-activation of ScNAC23 and ScJAZ10, respectively. ScFT Emission images and fluorescence statistics of the promoter.

[0020] Figure 6A molecular working model of the synergistic regulation of sugarcane flowering by ScJAZ10 and ScNAC23; GA accumulation promotes... ScJAZ10 The expression of ScJAZ10 and ScNAC23 forms a complex that synergistically activates downstream florigen genes. ScFT The transcription of this process ultimately accelerates the reproductive developmental transition and flowering process of sugarcane. Detailed Implementation

[0021] 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.

[0022] 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.

[0023] 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.

[0024] Example 1 Sugarcane ScJAZ10 Gene cloning, sequence analysis and molecular characterization 1.1 Sugarcane ScJAZ10 Gene cloning and sequence analysis This invention first cloned a new gene from the JAZ family from sugarcane varieties, and named it... ScJAZ10 The complete coding region nucleotide sequence of this gene is shown in SEQ ID No. 1, specifically: ATGGCCAGTCCTCCGTCGACTTCACCAGCGCCGTAGGCGGTACGACGACGAAGCCTCTGACCATATTCTACAACGGCGGCGTCGCCGTGTTCCATCTCCCGCAAGATAAGGCGGAGGCTCTCATGAAGATGGCGGCGGGTGATGAGGACGG CGGAGACGACGGCAGCCGCCACCGGCGGGCAAACCACGGCGAGGAGTTGCTCGCCAAGTTGAGACAAGAGACGATGCCCGTGGCAAGCAAGAGATCTTTGCAGCGCTTCTTCCAGAAGCGCAAGGAGAGTAGTGCAATTGTATACAATTAG.

[0025] ScJAZ10 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2, specifically: MASSSVDFTSAVGGTTTTKPLTIFYNGGVAVFHLPQDKAEALMKMAAGDEDGGDDGSRHRRANHGEELLAKLRQETMPVASKRSLQRFFQKRKESSAIVYN.

[0026] Table 1 below lists the primer sequence information used in each embodiment of the present invention, including primer names, forward primer sequences, and reverse primer sequences. Those skilled in the art can use the primer information listed in Table 1, combined with conventional molecular cloning experimental procedures, to construct various related vectors and detect gene expression.

[0027] Table 1 Primer Sequences 1.2 Sugarcane ScJAZ10 Phylogenetic analysis of genes To clarify the evolutionary position of the ScJAZ10 protein and its phylogenetic relationship with other JAZ family members in other species, sugarcane samples were obtained from public databases. Saccharum officinarum Arabidopsis thaliana ( ) Arabidopsis thaliana ), rice ( Oryza sativa ), sorghum ( Sorghum bicolor ) and corn ( Zea mays The JAZ protein sequence was obtained. A phylogenetic tree was constructed using MEGA11 software via Neighbor-Joining (NJ), with the Bootstrap value set to 1000 replicates to assess the statistical reliability of each branch.

[0028] Phylogenetic analysis results as follows Figure 1 As shown in Figure A, ScJAZ10 is stably clustered with homologous JAZ proteins from sorghum and maize in Group I, indicating that this protein has a high degree of sequence and functional conservation in the evolution of grasses.

[0029] 1.3 Sugarcane ScJAZ10 Tissue-specific expression pattern analysis of genes To explore ScJAZ10 To investigate gene expression distribution in different sugarcane tissues, root, stem, and leaf tissues from sugarcane plants at the four-leaf stage, as well as flower tissues from sugarcane plants at the flowering stage, were collected. Total RNA was extracted from each tissue sample after flash freezing in liquid nitrogen, and first-strand cDNA was obtained through reverse transcription. Using the synthesized cDNA as a template, sugarcane... GAPDH Genes were used as internal controls, and RT-qPCR technology was used for detection. ScJAZ10 Relative expression levels in different organizations.

[0030] The results are as follows Figure 1 As shown in Figure B, ScJAZ10The gene exhibits a constitutive expression pattern in the root, stem, leaf, and flower tissues of sugarcane, but its expression abundance varies significantly among different tissues. The relative expression level is highest in the leaves at the four-leaf stage, significantly higher than in other tissues, suggesting that this gene may play an important biological function in the leaves during the vegetative growth stage of sugarcane.

[0031] 1.4 Assay of yeast self-activation activity of ScJAZ10 protein To determine whether the ScJAZ10 protein possesses transcriptional activation capabilities, its coding sequence was inserted into the pGBKT7 yeast expression vector via homologous recombination to construct the BD-ScJAZ10 fusion expression plasmid. The successfully constructed BD-ScJAZ10 plasmid, along with the positive control plasmid and the negative control empty vector, were transformed into Y2HGold competent yeast cells. The transformed yeast cells were then plated on SD / -Trp auxotrophic medium supplemented with X-α-Gal, and colony growth and color changes were observed.

[0032] The results are as follows Figure 1 As shown in Figure C, yeast cells carrying the BD-ScJAZ10 fusion plasmid grew normally on SD / -Trp medium, indicating that the BD-ScJAZ10 plasmid was successfully transformed into yeast cells and did not produce toxicity to yeast growth. However, its colonies did not show a blue staining reaction, consistent with the phenotype of the negative control, while the colonies of the positive control group showed a distinct blue color. This result indicates that the ScJAZ10 protein does not possess transcriptional autoactivation activity in the yeast system.

[0033] 1.5 Subcellular localization analysis of ScJAZ10 protein To determine the localization of the ScJAZ10 protein in plant cells, the stop codon was first removed. ScJAZ10 The coding sequence was inserted into the pCAMBIA1300-GFP plant expression vector via restriction enzyme digestion and ligation to construct the fusion expression vector 35S::ScJAZ10-GFP, driven by a 35S constitutive promoter, which expresses ScJAZ10 and green fluorescent protein (GFP). This fusion expression vector and the empty vector control were transformed into Agrobacterium GV3101 competent cells using a freeze-thaw method. Subsequently, Agrobacterium cells containing the 35S::ScJAZ10-GFP vector were compared with those carrying the nuclear localization marker gene. AtHY5-RFP Agrobacterium (containing red fluorescent protein) was mixed evenly at a 1:1 volume ratio and injected into Nicotiana benthamiana (using the injection method). Nicotiana benthamiana The lower epidermis of the leaves. After dark treatment and light incubation for 48-72 h, the distribution and co-localization of GFP green fluorescence signal and RFP red fluorescence signal were observed using laser confocal microscopy.

[0034] The results are as follows Figure 1 As shown in Figure D, the green fluorescent signal of 35S::ScJAZ10-GFP is brightly and concentratedly distributed in the nuclear region, and this signal completely overlaps with the red fluorescent signal of the AtHY5-RFP nuclear marker in the nucleus, proving that ScJAZ10 is a typical nuclear localization protein, which is consistent with its spatial localization requirements for functioning as a transcriptional regulator.

[0035] Example 2: Ectopic Overexpression ScJAZ10 Correlation analysis of gene expression in early-flowering Arabidopsis thaliana and sugarcane varieties 2.1 ScJAZ10 Analysis of gene expression levels in sugarcane varieties with different flowering phenotypes To explore ScJAZ10 To investigate the association between gene transcription levels and sugarcane flowering phenotype, a non-flowering sugarcane variety (ZZ1) and two normally flowering sugarcane varieties (16-191 and 1418504) grown to 10 months of age under the same planting conditions were selected. Mature leaves at the same leaf position were collected from each variety, and total RNA was extracted and reverse transcribed into cDNA. Sugarcane... GAPDH This is an internal reference gene, detected using RT-qPCR technology. ScJAZ10 Relative expression levels in leaves of sugarcane varieties with different flowering phenotypes.

[0036] The results are as follows Figure 2 As shown in Figure A, compared to the non-flowering variety ZZ1, ScJAZ10 The relative expression levels were significantly increased in the leaves of the two flowering varieties (16-191 and 1418504), indicating that... ScJAZ10 The transcriptional level of [the substance] is closely positively correlated with the flowering phenotype of sugarcane.

[0037] 2.2 Stable overexpression ScJAZ10 Obtaining and molecularly identifying transgenic Arabidopsis thaliana To further verify ScJAZ10 Biological functions in flowering regulation: The 35S::ScJAZ10-GFP fusion expression vector constructed above was transformed into wild-type (WT) Columbia ecotype Arabidopsis thaliana via Agrobacterium-mediated floral dip. Arabidopsis thaliana Col-0). Harvested T0 generation seeds were screened for resistance on 1 / 2 MS solid medium containing hygromycin. Resistant T1 generation seedlings were transplanted into nutrient soil for further cultivation, and seeds were harvested from individual plants. Continuous self-pollination and hygromycin resistance screening were conducted until homozygous transgenic T3 generation lines were obtained. Total RNA was extracted from leaves of T3 homozygous lines and wild-type Arabidopsis thaliana, and after reverse transcription, RT-qPCR was used to detect RNA in each line. ScJAZ10 The relative expression level of genes.

[0038] The results are as follows Figure 2 As shown in Figure B, it was detected in multiple independent transgenic lines. ScJAZ10 High-level expression of the gene was observed, with two representative high-expression lines (OE-1 and OE-9) showing significantly increased expression levels. These two lines were selected as representatives for subsequent detailed phenotypic analysis, while the wild type was retained as a control.

[0039] 2.3 Overexpression ScJAZ10 Flowering phenotype observation and statistical analysis of transgenic Arabidopsis thaliana Wild-type (WT) Arabidopsis thaliana and ScJAZ10-OE overexpression lines (OE-1 and OE-9) were planted under long-day conditions (16 hours of light / 8 hours of darkness). The growth, development, and flowering of each line were observed and recorded daily.

[0040] The results are as follows Figure 2 As shown in Figures C and D, at different developmental stages, both ScJAZ10-OE overexpression lines exhibited a clearly identifiable early-flowering phenotype compared to the wild type. Statistical analysis further indicated that the ScJAZ10-OE transgenic lines had significantly fewer days from sowing date to the full opening of the first flower compared to the wild type. Figure 2 (Figure E in the middle), and the number of rosette leaves during the bolting and flowering period is also significantly less than that of the wild type ( Figure 2 (See Figure F). All the above results collectively indicate that ectopic overexpression... ScJAZ10 It can significantly promote the transition of Arabidopsis thaliana from vegetative growth to reproductive growth, leading to the emergence of an early flowering phenotype.

[0041] 2.4 Expression level analysis of key integrated genes in the flowering pathway To reveal from the molecular level ScJAZ10 Downstream regulatory mechanisms of early flowering in Arabidopsis thaliana caused by overexpression were investigated. Total RNA was extracted from whole seedlings of 15-day-old wild-type and ScJAZ10-OE overexpressing transgenic Arabidopsis thaliana, and cDNA was synthesized via reverse transcription. The relative expression levels of several key integrative factor genes in the flowering pathway were detected using RT-qPCR. Target genes detected included... CONSTANS ( AtCO ), FLOWERING LOCUS T ( AtFT )and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ( AtSOC1 ).

[0042] The results are as follows Figure 2 As shown in Figure G, in the ScJAZ10-OE overexpression lines, the flowering-promoting factor... AtCO , AtFT and AtSOC1The expression levels of all genes were significantly upregulated compared to the wild type, indicating that ScJAZ10 promotes flowering transition by positively regulating the transcriptional levels of these key integrative genes located at the core of the flowering network.

[0043] Example 3 Overexpression ScJAZ10 Transcriptome (RNA-seq) analysis and chlorophyll content determination of transgenic Arabidopsis thaliana 3.1 Transcriptome sequencing (RNA-seq) and differentially expressed gene screening analysis For a comprehensive analysis ScJAZ10 The effect of overexpression on the global transcriptional level of Arabidopsis was investigated. Total RNA was extracted from whole seedlings of 15-day-old wild-type and ScJAZ10-OE overexpression line (OE-1), with three biological replicates for each sample. RNA samples that passed quality control were then subjected to transcriptome sequencing (RNA-seq). The raw sequencing data were filtered and quality-controlled before being aligned to the Arabidopsis reference genome. Gene expression levels were quantified and differential expression was analyzed using the DESeq2 software package. A strict threshold of FDR ≤ 0.05 and Fold change ≥ 2 (i.e., |log2FC| ≥ 1) was used to screen for differentially expressed genes. Analysis identified 522 significantly differentially expressed genes (DEGs) between the wild-type and ScJAZ10-OE lines, including 269 upregulated genes and 253 downregulated genes. Figure 3 (See Figure A in the middle). GO (Gene Ontology) functional enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis were performed on the differentially expressed genes mentioned above.

[0044] The results are as follows Figure 3 As shown in Figures B and C, these differentially expressed genes were significantly enriched in multiple biological pathways and signaling pathways closely related to growth, development, and hormone responses, including "developmental processes," "reproductive processes," and "plant hormone signal transduction." Further expression heatmap analysis visually demonstrated that in the ScJAZ10-OE overexpression lines, the expression levels of a series of representative genes involved in multiple key categories, including gibberellin (GA) biosynthesis, flowering transition, meristem determination, and cell cycle regulation, underwent significant changes. Key genes positively regulating flowering transition and GA synthesis were significantly upregulated, while flowering inhibitors (such as...) were significantly downregulated. FLOWERING LOCUS C , FLC ; Short Vegetative Phase , SVP The expression level of ) was significantly downregulated. Figure 3(Figure D in the middle). The results of transcriptome analysis were highly consistent with the early flowering phenotype of the overexpressing lines, providing rich transcriptome-level information for further analysis of the downstream regulatory network of ScJAZ10.

[0045] 3.2 Validation of expression levels of key genes in GA biosynthesis Based on the GA signaling and biosynthesis-related pathways enriched in transcriptome data, RT-qPCR was used to target two key rate-limiting enzyme genes in the GA biosynthesis pathway. AtGA20ox1 and AtGA20ox3 The expression level was independently verified.

[0046] The results showed consistency with transcriptome sequencing data. AtGA20ox1 and AtGA20ox3 The relative expression levels of both genes in the ScJAZ10-OE overexpression line were significantly higher than those in the wild type. Figure 3 Figure E shows that overexpression of ScJAZ10 can effectively promote the transcription of key genes in the GA biosynthesis pathway.

[0047] 3.3 Determination of chlorophyll content in transgenic Arabidopsis leaves Activation of glycosidic acid (GA) signaling is often accompanied by a physiological phenotype of decreased chlorophyll content in many plants. To investigate whether ScJAZ10 overexpression induces the aforementioned typical GA physiological effects, rosette leaves were collected from 4-week-old wild-type and ScJAZ10-OE overexpressing lines growing normally under long-day conditions. Equal amounts of leaf tissue were weighed, and chlorophyll was extracted using 95% ethanol. The absorbance of the extracts at wavelengths of 665 nm and 649 nm was measured using a spectrophotometer, and the total chlorophyll content was calculated according to the formula.

[0048] The results are as follows Figure 3 As shown in Figures F and G, the chlorophyll extract from the leaves of the ScJAZ10-OE strain is visibly lighter in color than that of the wild type, and quantitative analysis also shows that its total chlorophyll content is significantly lower than that of the wild type. This physiological phenotype is completely consistent with the typical characteristics exhibited when GA signaling in plants is continuously activated, indirectly confirming the enhancement of endogenous GA signaling in the ScJAZ10-OE strain.

[0049] Example 4: ScJAZ10 promotes flowering in a manner dependent on active gibberellin (GA). 4.1 Sugarcane under exogenous GA treatment ScJAZ10 Gene expression response To explore ScJAZ10To investigate the gene's response pattern to exogenous gibberellin signals, uniformly growing sugarcane seedlings at the four-leaf stage were selected. A 100 μM gibberellin (GA) solution was sprayed evenly onto the surface of all leaves using a sprayer, with an equal volume of deionized water used as a control. Leaf samples were collected at 0 h before treatment and at 20 min, 1 h, 3 h, and 12 h after treatment. Total RNA was extracted after liquid nitrogen flash freezing and detected using RT-qPCR. ScJAZ10 The relative expression levels of genes at different time points.

[0050] The results showed that exogenous GA3 treatment could rapidly and significantly induce ScJAZ10 The transcription of the gene is upregulated, and its expression level begins to increase shortly after GA treatment, showing a sustained trend of induced response. Figure 4 (Figure A in the middle) indicates ScJAZ10 It is a gene that is highly responsive to gibberellin signals.

[0051] 4.2 Gibberellin's effect on ScJAZ10 Activation verification of promoter transcriptional activity To further verify whether gibberellin can directly activate transcription at the transcriptional level ScJAZ10 Gene expression, cloning ScJAZ10 Gene promoter region sequence ( ProScJAZ10 The enzyme was inserted into the pGreenII0800-LUC dual-luciferase reporter vector via enzyme digestion and ligation to construct a vector containing... ScJAZ10 A recombinant vector for expressing the firefly luciferase (LUC) reporter gene was developed, driven by a promoter. This reporter vector was transformed into Agrobacterium GV3101 and injected into tobacco leaves for transient expression. After injection, the tobacco leaves were treated with either a mock treatment (control) or a GA treatment. After 48 hours of culture, the bioluminescent signal in the injection area was observed in vivo using a dual-luciferase reporter assay (DLR), and the relative LUC activity was quantified.

[0052] Carrier construction mode such as Figure 4 As shown in Figure B. The experimental results are as follows. Figure 4 As shown in Figures C and D, compared with the control group treated with Mock, exogenous application of GA significantly enhanced the bioluminescent signal intensity of firefly luciferase in the injected area of ​​tobacco leaves, and the quantitative statistical results of relative LUC activity also showed a significant increase. These results directly demonstrate at the in vivo plant tissue level that gibberellin can activate [gibberellin] at the transcriptional level. ScJAZ10 Transcriptional activity of gene promoters.

[0053] 4.3 Experiment on the inhibition of flowering phenotype in Arabidopsis thaliana by paclobutrazol (PAC) treatment To further confirm whether the ScJAZ10-mediated early flowering regulation process depends on the accumulation of endogenous active gibberellin (GA) in plants, a blocking experiment was conducted using GA biosynthesis inhibitor paclobutrazol (PAC). Wild-type (WT) and ScJAZ10-OE overexpressing Arabidopsis seedlings were transplanted into nutrient soil, and foliar sprays of 50 μM PAC solution or an equal amount of ddH2O (Mock control) were applied periodically. The growth and development status and flowering time of plants in each treatment group were continuously observed and recorded.

[0054] Results of flowering phenotype observation as follows Figure 4 As shown in Figure E, under Mock treatment, the ScJAZ10-OE overexpression lines exhibited their inherent early-flowering phenotype as expected, with bolting and flowering times significantly earlier than the wild type. However, under PAC treatment, the original early-flowering phenotype of the transgenic overexpression lines was completely eliminated, and their flowering time was significantly delayed, showing no significant difference from the flowering time of wild-type plants also treated with PAC. Precise statistical results of the bolting and flowering days for each group are shown below. Figure 4 As shown in Figure F, the difference in flowering days between the PAC-treated ScJAZ10-OE plants and the wild-type plants disappeared. Further molecular-level detection results showed that... Figure 4 (Figures G and H), under the Mock treatment background, in the ScJAZ10-OE line AtFT and AtSOC1 The expression level of this gene was significantly higher than that of the wild type; however, under PAC treatment conditions, the expression level of this gene was significantly lower than that of the wild type. ScJAZ10 Overexpression caused AtFT and AtSOC1 Gene expression upregulation was almost completely suppressed. The combined genetic and molecular biological evidence strongly suggests that the ScJAZ10-mediated regulation of early flowering is strictly dependent on the accumulation and signal transduction of active gibberellins in the plant.

[0055] Example 5: ScJAZ10 and ScNAC23 transcription factors physically interact and synergistically activate downstream florigen genes. ScFT transcription 5.1 Yeast two-hybrid (Y2H) experiment to verify the protein interaction between ScJAZ10 and ScNAC23 To verify whether there is a direct physical interaction between ScJAZ10 and ScNAC23, a key transcription factor regulating sugarcane flowering, protein-protein interaction was detected using a yeast two-hybrid system. ScNAC23The complete coding sequence was cloned into the pGADT7 vector using homologous recombination to construct the AD-ScNAC23 fusion expression vector (prey vector). Simultaneously, the BD-ScJAZ10 fusion expression vector (bait vector) and the AD-ScNAC23 vector were co-transformed into yeast Y2HGold competent cells. The co-transformed yeast cells were plated on SD / -Trp-Leu selective medium to verify successful transduction of both plasmids, and on SD / -Trp-Leu-His-Ade selective medium to detect reporter gene activation.

[0056] The results are as follows Figure 5 As shown in Figure A, yeast cells co-expressing AD-ScNAC23 and BD-ScJAZ10 can grow normally on a two-deficient medium and form colonies normally on a harsh four-deficient selective medium. Furthermore, in medium supplemented with X-α-Gal, the co-transformed yeast colonies exhibit a distinct blue color reaction. These results clearly confirm that the ScJAZ10 protein and the ScNAC23 transcription factor undergo a direct and specific physical interaction within the yeast cell nucleus.

[0057] 5.2 Dual-luciferase complementarity imaging (LCI) experiment to verify protein interactions in plants To verify the interaction between ScJAZ10 and ScNAC23 in living plant cells, firefly luciferase complementation imaging technology was used for detection. Fusion expression vectors of ScNAC23 and the N-terminal fragment (nLUC) of firefly luciferase (ScNAC23-nLUC) and ScJAZ10 and the C-terminal fragment (cLUC) of firefly luciferase (ScJAZ10-cLUC) were constructed. The two Agrobacterium cultures were mixed at a 1:1 volume ratio and co-injected into tobacco leaves. After culturing for 48-72 hours, luciferin substrate was smeared on the abaxial surface of the tobacco leaves, and the bioluminescent signal was observed and its intensity was statistically analyzed using a live-cell imaging system.

[0058] The results are as follows Figure 5 As shown in Figures B and C, strong bioluminescent signals were detected only in the injection sites of tobacco leaves where both ScNAC23-nLUC and ScJAZ10-cLUC fusion proteins were co-expressed, while no specific bioluminescent signals were detected in the injection sites of other negative control combinations. These results further validate the specific interaction between the ScJAZ10 protein and the ScNAC23 transcription factor in living plant cells.

[0059] 5.3 Bimolecular fluorescence complementation (BiFC) assay to verify the localization of interactions in the cell nucleus To further verify whether the interaction between ScJAZ10 and ScNAC23 occurs within the cell nucleus and to visualize the subcellular localization of their interaction, a bimolecular fluorescence complementation assay was used. Fusion expression vectors of ScJAZ10 and the C-terminal fragment of yellow fluorescent protein (cYFP) (ScJAZ10-cYFP) and ScNAC23 and the N-terminal fragment of yellow fluorescent protein (nYFP) (ScNAC23-nYFP) were constructed. Both types of Agrobacterium bacterial cultures were co-injected into tobacco leaves, along with Agrobacterium carrying a nuclear marker gene as a nuclear localization reference. After culturing for 48-72 hours, the distribution of the yellow fluorescence signal of YFP was observed using a laser confocal microscope.

[0060] The results are as follows Figure 5 As shown in Figure D, a strong and concentrated yellow fluorescent signal of YFP was observed in the nuclei of tobacco epidermal cells co-expressing ScJAZ10-cYFP and ScNAC23-nYFP, and this signal completely overlapped with the localization signal of the nuclear marker. This result clearly confirms that the ScJAZ10 protein and the ScNAC23 transcription factor undergo direct physical interaction in the nucleus of living plant cells.

[0061] 5.4 Sugarcane orientin gene ScFT Cis-acting element analysis of promoters Fragrance gene FT It is the most crucial integrative factor in the flowering regulatory network. To explore the downstream direct transcriptional targets of the ScJAZ10-ScNAC23 protein complex, the sugarcane flowering glycogen gene was investigated. ScFT The startup sub-region ( ProScFT Sequence analysis and cis-acting element prediction are performed.

[0062] Analysis results as follows Figure 5 As shown in Figure E, in ScFT The upstream region of the gene's promoter contains several typical NAC transcription factor recognition and binding motifs (CACG-boxes). The arrangement and copy number of these CACG core sequences allow ScNAC23 to directly bind and regulate the gene. ScFT Transcription provides a potential molecular structural basis.

[0063] 5.5 Dual-Luciferase (Dual-LUC) assay to verify the effects of ScNAC23 and ScJAZ10 on... ScFT Co-activation of transcription by promoters To directly verify the effects of ScNAC23 and the ScJAZ10-ScNAC23 protein complex on... ScFT The regulatory role of promoter transcriptional activity was functionally verified using a dual-luciferase transient expression detection system. ProScFT The promoter sequence was cloned into the pGreenII 0800-LUC reporter vector for construction. ProScFT ::LUC report carrier (pGreenII 0800-ProScFT-LUC), and simultaneously respectively ScNAC23 and ScJAZ10 The coding sequence was cloned into the pGreenII 62-SK effector vector to construct the 35S::ScNAC23 and 35S::ScJAZ10 effector vectors. The structural patterns of the effector and reporter vectors are as follows: Figure 5 As shown in Figure F, Agrobacterium containing different combinations of effector and reporter vectors were mixed in a certain proportion and co-injected into tobacco leaves. After culturing for 48-72 hours, bioluminescent signals were observed using an in vivo imaging system, and the relative LUC activity of each combination was quantitatively determined using a DLR detection system.

[0064] When ScNAC23 effector protein is expressed alone (35S::ScNAC23 + ProScFT The ::LUC combination showed a significant increase in bioluminescent signal and relative LUC activity in the injection region compared to the empty effect carrier control group. Figure 5 The G and H diagrams show that the ScNAC23 transcription factor can directly recognize and bind to... ScFT The CACG motif in the promoter is activated as a transcription activator. ScFT Gene transcription.

[0065] Furthermore, when ScNAC23 and ScJAZ10 are expressed together as effector proteins (35S::ScNAC23 + 35S::ScJAZ10 + ... ProScFT The bioluminescent signal intensity and relative LUC activity in the injection region were further significantly enhanced by the ::LUC combination, with an activation fold significantly higher than that of ScNAC23 expressed alone. Figure 5 (See Figures I and J). This result indicates that although the ScJAZ10 protein itself does not possess transcriptional autoactivation activity that directly binds to DNA (see Figure I and Figure J). Figure 1 (Figure C) However, it can act as a transcriptional co-activator, synergistically enhancing the effect of ScNAC23 on downstream target genes by forming a protein complex with the ScNAC23 transcription factor. ScFT The transcriptional activation capacity of the promoter.

[0066] Based on the experimental results of all the above embodiments, this invention proposes a complete molecular pathway model for the regulation of sugarcane flowering, the core working mode of which is as follows: Figure 6 As shown in the working model, the accumulation and transmission of gibberellin (GA) signaling in sugarcane plants primarily promotes... ScJAZ10 Gene transcriptional expression; with increasing ScJAZ10 protein levels, it directly interacts with the key floral regulation transcription factor ScNAC23 in the cell nucleus, forming the ScJAZ10-ScNAC23 transcriptional regulatory complex; this protein complex, as a functional transcriptional regulatory unit, can co-bind to the downstream core florigen gene. ScFT The promoter region of the gene, with ScJAZ10 exerting transcriptional co-activation and ScNAC23 providing DNA binding activity in a synergistic mode, significantly enhances the transcriptional co-activation function. ScFT The level of gene transcriptional activation; with the large accumulation of the florigen protein ScFT and its transport from the leaves to the shoot apex meristem via the vascular system, the sugarcane's transition from vegetative growth to reproductive development is ultimately accelerated, promoting bolting and flowering. This invention discloses... ScJAZ10 The gene and the ScJAZ10-ScNAC23 interaction regulatory module it participates in provide new key gene targets and molecular manipulation tools for the genetic improvement of flowering time regulation in sugarcane and other crops.

[0067] 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 sugarcane gene ScJAZ10 Its characteristics are, The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. A sugarcane gene according to claim 1 ScJAZ10 The encoded protein is characterized by, The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. Contains the sugarcane gene as described in claim 1 ScJAZ10 Recombinant expression vectors.

4. A recombinant host cell containing the recombinant expression vector of claim 3.

5. The sugarcane gene of claim 1 ScJAZ10 Application in regulating plant flowering time.

6. The application of the protein of claim 2 in regulating the flowering time of plants.

7. The application according to claim 5 or 6, characterized in that, The application involves overexpressing the sugarcane gene in plants. ScJAZ10 Or the aforementioned protein, to promote early flowering in plants.

8. The application according to claim 5 or 6, characterized in that, The application involves suppressing or silencing the sugarcane gene in the plant. ScJAZ10 Or the expression of the protein, to delay plant flowering or to breed late-flowering varieties.

9. The application according to claim 5 or 6, characterized in that, The plant in question is sugarcane.

10. The application of the protein-sponge interaction module of claim 2 with the ScNAC23 transcription factor in regulating plant flowering, characterized in that, The protein interaction module co-activates downstream fructose genes. ScFT Transcription promotes plant flowering.

Images