Sugarcane CONSTANS4 gene and application method thereof

Abstract

The invention belongs to the technical field of plant genes, discloses a sugarcane CONSTANS4 gene and an application method thereof, discloses the function of CO4 in sugarcane for the first time, and expands the research range of the CONSTANS4 gene in higher plants. The method realizes accurate editing of CO4, improves the adaptability of sugarcane varieties, avoids the influence of precocity on sugar yield in a low-latitude region, accelerates growth in a high-latitude region, and improves the harvesting efficiency. According to the method, the high-sugar-accumulation sugarcane variety is cultivated, sugar metabolism genes such as SUSY, INV and HK are regulated and controlled through CO4, the sucrose accumulation amount is increased by 10%-15%, and economic benefits are remarkably improved. A new sugarcane molecular breeding strategy is developed, gene editing and molecular marker selective breeding (MAS) are combined, and the sugarcane breeding period is greatly shortened from 12-15 years to 6-8 years. Through cloning, functional verification and breeding application of the sugarcane CONSTANS4 gene, the genetic barrier of photoperiod regulation and sugar accumulation is broken through, a new thought is provided for efficient and accurate breeding of sugarcane, and the sugarcane CONSTANS4 gene has important agricultural production value.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant gene technology, and particularly relates to a sugarcane CONSTANS4 gene and an application method thereof. Background Art

[0002] Sugarcane (Saccharumspp.) is an important sugar crop globally, and its growth and development are significantly affected by photoperiod. The CONSTANS (CO) gene family plays a key role in the regulation of flowering by photoperiod in plants. Currently, the research on the sugarcane CONSTANS4 (ScCO4) gene is relatively limited. Existing studies have shown that the expression of the ScCO4 gene can weaken the activity of the viral silencing suppressor P1 and reduce the content of the P1 protein, thereby enhancing the virus resistance of plants. However, existing research mainly focuses on the impact of the ScCO4 gene on virus resistance, and its functions in sugarcane growth and development, flowering regulation, and yield formation have not been deeply explored. In addition, the expression pattern and regulatory mechanism of the ScCO4 gene under different environmental conditions also lack systematic research.

[0003] Existing Technology: Research on Photoperiod Regulation and CONSTANS Gene Family in Sugarcane

[0004] Sugarcane (Saccharumspp.) is one of the most important sugar crops globally, and its growth, development, and sugar yield are jointly affected by photoperiod, gene regulation, and environmental factors. In existing research, the CONSTANS (CO) gene family has been confirmed to be the core regulatory factor of plant photoperiod signal transduction, mainly regulating flowering in photoperiod response, sugar accumulation, and growth and development.

[0005] The CONSTANS4 (CO4) gene is one of the members of the CONSTANS family, and it has been found to have functions of photoperiod regulation, promoting flowering, and increasing biomass in plants such as Arabidopsis thaliana, rice, and maize. However, the research on the function of the CO4 gene in sugarcane and its role in photoperiod response, sugar accumulation, and growth regulation is still scarce.

[0006] Through the above analysis, the problems and defects existing in the prior art are as follows:

[0007] (1) The problem of sugarcane's sensitivity to photoperiod:

[0008] Existing sugarcane varieties are prone to premature ripening in low-latitude regions (short-day conditions), which affects sugar yield; in high-latitude regions (long-day conditions), they may delay ripening, reducing the harvesting efficiency.

[0009] The lack of gene resources that can precisely regulate photoperiod response limits the ability of sugarcane varieties to adapt to different growth environments.

[0010] (2) The mechanism of sugar accumulation regulation is unclear:

[0011] Sugarcane is a typical crop with high sugar accumulation. However, the sugar accumulation process involves a complex gene regulation network, and it is not clear whether the CONSTANS4 gene directly or indirectly participates in this process.

[0012] The existing sugarcane breeding mainly relies on conventional selection and empirical hybridization, lacking a precise breeding strategy based on gene regulation.

[0013] (3) Long breeding cycle and complex genetic background:

[0014] Sugarcane is an allopolyploid (>100 chromosomes) with a complex genotype, making it difficult for traditional marker-assisted selection breeding (MAS) to accurately lock in the key genes controlling photoperiod and sugar accumulation.

[0015] The existing gene editing and transgenic technologies are limited in the application of sugarcane, lacking functional verification and application methods for the CONSTANS4 gene. Summary of the Invention

[0016] In view of the problems existing in the prior art, the present invention provides a sugarcane CONSTANS4 gene and its application method.

[0017] The present invention is implemented as follows. A method for applying a sugarcane CONSTANS4 gene, the method for applying the sugarcane CONSTANS4 gene comprising the following steps:

[0018] Step 1: Cloning and sequence analysis of the sugarcane CO4 gene;

[0019] Step 2: Expression analysis of the sugarcane CO4 gene;

[0020] Step 3: Functional verification of the sugarcane CO4 gene;

[0021] Step 4: Application of the sugarcane CO4 gene in breeding;

[0022] Among them, the functional verification and breeding application improve the traits of sugarcane by regulating the expression level and functional structure of the CONSTANS4 gene;

[0023] The protection scope of the application method covers but is not limited to the complete implementation of the above steps. Any genetic operation directly or indirectly utilizing the sugarcane IAA gene and its functional domain belongs to the protection scope, specifically including:

[0024] (1) Technical solutions for changing the expression level of the CONSTANS4 gene through gene editing, homologous recombination or epigenetic regulation;

[0025] (2) A genetic transformation vector containing the functional domain of the CONSTANS4 gene and its recombinant products;

[0026] (3) A method for transforming sugarcane callus based on the regulation of the CONSTANS4 gene;

[0027] (4) Trait performances such as improved plant type, enhanced stress resistance, or increased sugar accumulation achieved through the dosage effect of the CONSTANS4 gene;

[0028] Regardless of whether the steps are fully followed in the technical implementation process or a partial combination of steps is selectively implemented, any achievements in improving sugarcane traits based on the functional regulation of the CONSTANS4 gene fall within the scope of protection of the claims.

[0029] Furthermore, the cloning and sequence analysis of the sugarcane CO4 gene:

[0030] Extract the total RNA from the leaves of sugarcane seedlings, and use nucleic acid amplification technology, including SMARTer RACE cDNA amplification technology, to clone the full-length sequence of the CO4 gene;

[0031] Use sequence alignment tools for analysis, including NCBI BLAST and MEGA11, to perform sequence alignment, construct a phylogenetic tree, and analyze the evolutionary relationship of CO4 among different species;

[0032] Predict the conserved domain of CO4 through MEME and Pfam, and analyze its functional characteristics.

[0033] Furthermore, the expression analysis of the sugarcane CO4 gene includes:

[0034] Detect the transcriptional level of the IAA gene in different tissues or under different environmental conditions of sugarcane,

[0035] The environmental conditions include at least one of photoperiod, temperature, and humidity;

[0036] Use qRT-PCR to analyze the expression level of CO4 in different tissues of sugarcane;

[0037] Conduct photoperiod treatment experiments to analyze the changes in the transcriptional level of CO4 under different photoperiod conditions.

[0038] Furthermore, the different tissues of sugarcane: roots, stems, leaves, flowers;

[0039] Conduct photoperiod treatment experiments: short day 6 - 12 h vs. long day 15 - 24 h.

[0040] Furthermore, the functional verification of the sugarcane CO4 gene:

[0041] Construct the CO4 overexpression OE and gene editing technology, including the CRISPR / Cas9 technology and vectors, and transform them into Arabidopsis thaliana, sugarcane callus and rice, and detect the effects of CO4 on the flowering period, sugar accumulation and biomass;

[0042] Verify whether CO4 directly binds to the FTFLOWERINGLOCUST or SUSY sucrose synthase promoter to regulate its transcription through yeast one-hybrid Y1H and EMSA experiments.

[0043] Furthermore, the application of the sugarcane CO4 gene in breeding:

[0044] Use CRISPR / Cas9 gene editing to disrupt the negative regulatory region of CO4 and cultivate sugarcane varieties with high sugar accumulation at low latitudes;

[0045] Through molecular marker-assisted selection, including MAS molecular marker technology, selective breeding, and screening of CO4 high-expression lines to improve breeding efficiency;

[0046] In greenhouse and field trials, conduct long-term evaluations of photoperiod response, sugar content and yield, and finally obtain new sugarcane varieties adapted to different latitudes with a 10%-15% increase in sugar yield.

[0047] Another object of the present invention is to provide a sugarcane CONSTANS4 gene application system for implementing the application method of the sugarcane CONSTANS4 gene. The sugarcane CONSTANS4 gene application system includes:

[0048] A gene cloning module for cloning the sugarcane CONSTANS4 gene and performing sequence analysis;

[0049] Clone the sugarcane CONSTANS4 gene using RT-PCR and RACE (rapid amplification of cDNA ends technology), and perform sequence alignment and conserved domain analysis;

[0050] A gene expression analysis module for detecting the spatio-temporal expression pattern of the CONSTANS4 gene;

[0051] Used to analyze the expression pattern of CO4 in different tissues (leaves, stems, roots) and different photoperiod conditions by qRT-PCR and RNA-Seq;

[0052] A gene function verification module for regulating the expression of the CONSTANS4 gene through gene editing or transgenic technology and verifying its function;

[0053] Used to construct CO4 overexpression and gene editing technology, including CRISPR / Cas9, gene knockout vectors, and perform transgenic verification in Arabidopsis thaliana, sugarcane and rice respectively;

[0054] A molecular mechanism analysis module, which is used to combine molecular interaction experiments, including yeast one-hybrid Y1H, chromatin immunoprecipitation ChIP, and dual-luciferase reporter assays, to study the regulatory effects of CO4 on the photoperiod signal genes FT, Hd3a, and the sugar metabolism genes SUSY, INV, HK;

[0055] A breeding application module, which is used to cultivate new sugarcane varieties with broader photoperiod adaptability and stronger sugar accumulation ability through gene editing technologies, including CRISPR / Cas9 gene editing, RNAi interference, and MAS molecular marker selection.

[0056] Another object of the present invention is to provide a computer device, characterized in that the computer device includes a memory and a processor, the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the application method of the sugarcane CONSTANS4 gene.

[0057] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, and when the computer program is executed by a processor, the processor executes the steps of the application method of the sugarcane CONSTANS4 gene.

[0058] Another object of the present invention is to provide an information data processing terminal, which is used to implement the sugarcane CONSTANS4 gene application system.

[0059] Combined with the above technical solutions and the solved technical problems, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0060] This study intends to clarify the role of the ScCO4 gene in sugarcane growth, development, and flowering regulation by cloning and functionally verifying the ScCO4 gene. Specifically, overexpression and RNA interference vectors of the ScCO4 gene will be constructed and transformed into model plants such as sugarcane protoplasts or Arabidopsis thaliana to observe their phenotypic changes. At the same time, real-time fluorescence quantitative PCR (RT-qPCR) technology will be used to analyze the expression of the ScCO4 gene in different tissues and different environmental conditions.

[0061] Through the above research, it is expected to reveal the specific functions and molecular mechanisms of the ScCO4 gene in sugarcane growth, development, and flowering regulation, providing new gene resources and theoretical basis for sugarcane variety improvement. In addition, in-depth understanding of the regulatory network of the ScCO4 gene helps to formulate more effective sugarcane cultivation management strategies and improve the yield and quality of sugarcane.

[0062] The present invention provides a cloning, functional identification and breeding application strategy of the sugarcane CONSTANS4 (CO4) gene, analyzes the role of the CO4 gene in photoperiod regulation and sugar accumulation through molecular biology means, and establishes an efficient gene editing and transgenic technology system to achieve precise improvement of sugarcane breeding.

[0063] Compared with the prior art, the present invention has the following significant advantages in photoperiod regulation, sugar accumulation and breeding application:

[0064] For the first time, it reveals the function of CO4 in sugarcane and expands the research scope of the CONSTANS4 gene in higher plants.

[0065] Achieve precise editing of CO4, improve the adaptability of sugarcane varieties, avoid premature ripening affecting sugar yield in low-latitude regions, accelerate growth in high-latitude regions, and improve harvesting efficiency.

[0066] Cultivate sugarcane varieties with high sugar accumulation. By regulating sugar metabolism genes such as SUSY, INV, and HK through CO4, the sucrose accumulation can be increased by 10%-15%, significantly enhancing economic benefits.

[0067] Develop a new sugarcane molecular breeding strategy, combining gene editing + marker-assisted selection breeding (MAS), and greatly shorten the sugarcane breeding cycle (from 12 - 15 years to 6 - 8 years).

[0068] Through the cloning, functional verification and breeding application of the sugarcane CONSTANS4 gene, the present invention breaks through the genetic barriers of photoperiod regulation and sugar accumulation, provides new ideas for efficient and precise sugarcane breeding, and has important agricultural production value. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 It is a flow chart of the application method of the sugarcane CONSTANS4 gene provided by the embodiment of the present invention.

[0070] Figure 2 It is a flow chart of the cloning and sequence analysis method of the sugarcane CO4 gene provided by the embodiment of the present invention.

[0071] Figure 3 It is a structural block diagram of the application system of the sugarcane CONSTANS4 gene provided by the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0072] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0073] Such as Figure 1As shown in the figure, a method for applying the sugarcane CONSTANS4 gene provided by an embodiment of the present invention includes the following steps:

[0074] S101: Cloning and sequence analysis of the sugarcane CO4 gene;

[0075] S102: Expression analysis of the sugarcane CO4 gene;

[0076] S103: Functional verification of the sugarcane CO4 gene;

[0077] S104: Application of the sugarcane CO4 gene in breeding.

[0078] As Figure 2 shown in the figure, cloning and sequence analysis of the sugarcane CO4 gene provided by an embodiment of the present invention:

[0079] S201, Extract the total RNA of sugarcane seedling leaves, and clone the full-length sequence of the CO4 gene using the SMARTer RACE cDNA amplification technology;

[0080] S202, Use NCBI BLAST and MEGA11 for sequence alignment, construct a phylogenetic tree, and analyze the evolutionary relationship of CO4 among different species;

[0081] S203, Predict the conserved domains of CO4 through MEME and Pfam, and analyze its functional characteristics.

[0082] Expression analysis of the sugarcane CO4 gene provided by an embodiment of the present invention:

[0083] Use qRT-PCR to analyze the expression level of CO4 in different tissues of sugarcane;

[0084] Conduct a photoperiod treatment experiment to analyze the transcriptional level changes of CO4 under different photoperiod conditions.

[0085] Different tissues of sugarcane provided by an embodiment of the present invention: roots, stems, leaves, flowers;

[0086] Conduct a photoperiod treatment experiment: short-day 10h vs. long-day 16h.

[0087] Functional verification of the sugarcane CO4 gene provided by an embodiment of the present invention:

[0088] Construct CO4 overexpression (OE) and CRISPR / Cas9 knockout (KO) vectors, and transform them into Arabidopsis thaliana, sugarcane callus, and rice, and detect the effects of CO4 on the flowering period, sugar accumulation, and biomass;

[0089] Verify whether CO4 directly binds to the promoters of FT (FLOWERING LOCUST) or SUSY (sucrose synthase) and regulates their transcription through yeast one-hybrid (Y1H) and EMSA experiments.

[0090] Application of the sugarcane CO4 gene provided in the embodiments of the present invention in breeding:

[0091] Use CRISPR / Cas9 gene editing to disrupt the negative regulatory region of CO4 and cultivate sugarcane varieties with high sugar accumulation at low latitudes;

[0092] Through MAS molecular marker-assisted selection breeding, screen CO4 highly expressed lines to improve breeding efficiency;

[0093] In greenhouse and field trials, conduct long-term evaluations on photoperiod response, sugar content, and yield, and finally obtain new sugarcane varieties adapted to different latitudes with a 10%-15% increase in sugar yield.

[0094] As Figure 3 shown, a sugarcane CONSTANS4 gene application system provided in the embodiments of the present invention includes:

[0095] A gene cloning module for cloning the sugarcane CONSTANS4 gene using RT-PCR and RACE (rapid amplification of cDNA ends technology), and performing sequence alignment and conserved domain analysis;

[0096] A gene expression analysis module for analyzing the expression patterns of CO4 in different tissues (leaves, stems, roots) and under different photoperiod conditions using qRT-PCR and RNA-Seq;

[0097] A gene function verification module for constructing CO4 overexpression and CRISPR / Cas9 gene knockout vectors and performing transgenic verification in Arabidopsis, sugarcane, and rice respectively;

[0098] A molecular mechanism analysis module for studying the regulatory effects of CO4 on photoperiod signal genes (FT, Hd3a) and sugar metabolism genes (SUSY, INV, HK) by combining yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays;

[0099] A breeding application module for cultivating new sugarcane varieties with broader photoperiod adaptability and stronger sugar accumulation ability through CRISPR / Cas9 gene editing, RNAi interference, and MAS (molecular marker selection).

[0100] The system first extracts and reverse transcribes mRNA from sugarcane samples using RT-PCR and RACE techniques, and amplifies the full-length cDNA of the CONSTANS4 gene. Subsequently, the obtained sequences are aligned and analyzed for conserved domains to confirm the integrity and functional structure of the gene, laying a molecular foundation for subsequent functional studies and the analysis of regulatory mechanisms.

[0101] In the gene expression analysis module, highly sensitive detection techniques such as qRT-PCR and RNA-Seq are used to systematically detect the expression levels of the sugarcane CONSTANS4 gene in different tissues (such as leaves, stems, roots) and under different photoperiod conditions. Through quantitative analysis, the expression dynamics and spatio-temporal distribution of the CO4 gene in photoperiod regulation and other physiological processes are revealed, providing important clues for subsequent functional verification.

[0102] In the gene function verification module, overexpression and CRISPR / Cas9 gene knockout vectors of CO4 are constructed, and transgenic operations are achieved in Arabidopsis, sugarcane, and rice. By comparing the differences in growth, development, and physiological indicators between transgenic plants and wild types, the specific roles of CO4 in regulating photoperiod and sugar metabolism are clarified, providing direct evidence for revealing its biological functions.

[0103] The molecular mechanism analysis module uses yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays to study the regulatory network of CO4 on key photoperiod signal genes (FT, Hd3a) and sugar metabolism genes (SUSY, INV, HK), and analyzes its transcriptional regulatory mechanism. Based on these research results, the breeding application module combines strategies such as CRISPR / Cas9 gene editing, RNAi interference, and MAS to breed new sugarcane varieties with broader photoperiod adaptability and stronger sugar accumulation ability, achieving seamless docking from basic molecular mechanisms to actual breeding applications.

[0104] Specific implementation of the present invention:

[0105] 1. System structure

[0106] The system structure of this study mainly includes the following aspects:

[0107] Gene cloning and sequence analysis:

[0108] Clone the sugarcane CONSTANS4 gene using RT-PCR and RACE (rapid amplification of cDNA ends technology), and perform sequence alignment and conserved domain analysis.

[0109] Gene expression analysis:

[0110] Use qRT-PCR and RNA-Seq to analyze the expression patterns of CO4 in different tissues (leaves, stems, roots) and under different photoperiod conditions.

[0111] Verification of gene function:

[0112] Construct overexpression and CRISPR / Cas9 gene knockout vectors of CO4, and conduct transgenic verification in Arabidopsis, sugarcane, and rice respectively.

[0113] Analysis of molecular mechanism:

[0114] Combined with yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays to study the regulatory effects of CO4 on photoperiod signal genes (FT, Hd3a) and sugar metabolism genes (SUSY, INV, HK).

[0115] Breeding application strategy:

[0116] Through CRISPR / Cas9 gene editing, RNAi interference, and MAS (molecular marker selection), cultivate new sugarcane varieties with a wider photoperiod adaptability and stronger sugar accumulation ability.

[0117] 2. Method steps

[0118] Step 1: Cloning and sequence analysis of sugarcane CO4 gene

[0119] Extract total RNA from sugarcane seedling leaves, and clone the full-length sequence of the CO4 gene using SMARTer RACE cDNA amplification technology.

[0120] Use NCBI BLAST and MEGA11 for sequence alignment, construct a phylogenetic tree, and analyze the evolutionary relationship of CO4 among different species.

[0121] Predict the conserved domains of CO4 through MEME and Pfam, and analyze its functional characteristics.

[0122] Step 2: Expression analysis of sugarcane CO4 gene

[0123] Use qRT-PCR to analyze the expression levels of CO4 in different tissues (roots, stems, leaves, flowers) of sugarcane.

[0124] Conduct photoperiod treatment experiments (short day 10h vs. long day 16h), and analyze the transcriptional level changes of CO4 under different photoperiod conditions.

[0125] Step 3: Verification of sugarcane CO4 gene function

[0126] Construct overexpression (OE) and CRISPR / Cas9 knockout (KO) vectors of CO4, and transform them into Arabidopsis, sugarcane callus, and rice, and detect the effects of CO4 on flowering period, sugar accumulation, and biomass.

[0127] Verify whether CO4 directly binds to the promoters of FT (FLOWERING LOCUST) or SUSY (sucrose synthase) and regulates their transcription through yeast one-hybrid (Y1H) and EMSA experiments.

[0128] Step 4: Application of sugarcane CO4 gene in breeding

[0129] Use CRISPR / Cas9 gene editing to disrupt the negative regulatory region of CO4 and cultivate sugarcane varieties with high sugar accumulation at low latitudes.

[0130] Through MAS molecular marker-assisted selection breeding, screen for high-expression lines of CO4 to improve breeding efficiency.

[0131] In greenhouse and field trials, conduct long-term evaluations of photoperiod response, sugar content, and yield, and finally obtain new sugarcane varieties adapted to different latitudes with a 10%-15% increase in sugar yield.

[0132] Example 1: Cloning and expression pattern analysis of sugarcane CONSTANS4 (ScCO4) gene

[0133] 1. Experimental materials and methods

[0134] Material preparation

[0135] Select sugarcane variety ROC22 as the experimental material and collect samples of different tissues (leaves, stems, roots, flowers).

[0136] Set different photoperiod treatment groups (short day 10h / long day 16h) to analyze the expression changes of ScCO4 under different photoperiod conditions.

[0137] RNA extraction and cDNA synthesis

[0138] Use TRIzol method to extract total RNA from different tissues of sugarcane, and use DNase to remove genomic DNA.

[0139] Use SMARTer RACE technology for 5' and 3' end amplification to obtain the full-length cDNA sequence of ScCO4 gene.

[0140] Sequence analysis

[0141] Use NCBI BLAST for homologous alignment analysis, construct a phylogenetic tree, and explore the evolutionary relationship of ScCO4 in different plants.

[0142] Use MEME software to predict the conserved domain of ScCO4, and analyze its protein molecular structure and subcellular localization.

[0143] Expression pattern analysis

[0144] The expression levels of ScCO4 in different tissues were analyzed by qRT-PCR (quantitative real-time PCR).

[0145] Through the photoperiod treatment experiment (short day 10h vs. long day 16h), the expression changes of ScCO4 under different photoperiod conditions were detected, and the relative expression levels were calculated (ΔΔCt method).

[0146] RNA-Seq (transcriptome sequencing) was used to further analyze the possible downstream regulatory genes of ScCO4 and construct a preliminary gene regulatory network.

[0147] 2. Research results

[0148] The expression level of the ScCO4 gene was the highest in leaves, followed by stems and roots, and relatively low in flowers.

[0149] Under short-day conditions, the expression level of the ScCO4 gene was significantly up-regulated, indicating that it may be involved in the growth regulation process induced by short days.

[0150] RNA-Seq analysis showed that ScCO4 may participate in the flowering regulation of sugarcane by regulating the FT (FLOWERING LOCUS T) and Hd3a genes.

[0151] In this example, the sugarcane ScCO4 gene was successfully cloned and identified, and its expression pattern in different tissues and photoperiod response characteristics were clarified, providing a theoretical basis for subsequent functional research.

[0152] Example 2: Overexpression and CRISPR / Cas9 gene knockout study of the sugarcane ScCO4 gene

[0153] 1. Experimental materials and methods

[0154] Construct ScCO4 overexpression and knockout vectors

[0155] Design the CRISPR / Cas9 knockout vector for the ScCO4 gene, and select the sgRNA target sites near the PAM sequence for gene editing.

[0156] Construct the 35S:ScCO4 overexpression vector, clone the sugarcane ScCO4 gene into the pCAMBIA1301 vector, and introduce the vector into sugarcane callus by Agrobacterium-mediated transformation.

[0157] Screening and molecular detection of transgenic sugarcane

[0158] Positive transformants were obtained by screening with hygromycin resistance (Hygromycin), and the integration of the transgene was verified by PCR and Southern blot.

[0159] RT-qPCR and Western blot were used to confirm the expression level of the ScCO4 gene in transgenic lines.

[0160] Phenotypic analysis

[0161] ScCO4 overexpression lines, knockout lines and wild type (WT) were planted in the greenhouse and field, and the growth rate, stem length, chlorophyll content, sugar accumulation and flowering time were measured.

[0162] The contents of sucrose, glucose and fructose in sugarcane juice were determined by high performance liquid chromatography (HPLC) to analyze the effect of ScCO4 on sugar metabolism.

[0163] Transcriptome and metabolome analysis

[0164] RNA-Seq was used to compare the transcriptome changes of WT, overexpression lines and knockout lines to screen for downstream genes that ScCO4 might regulate.

[0165] Metabolome analysis was performed by LC-MS (liquid chromatography-mass spectrometry) to detect changes in key metabolites in the carbon metabolism pathway.

[0166] 2. Research results

[0167] ScCO4 overexpression lines:

[0168] The plant height increased by 15%, the stem diameter thickened, and the chlorophyll content increased.

[0169] The sucrose content in sugarcane juice was 12%-15% higher than that of WT, and the fructose and glucose contents decreased, indicating that ScCO4 might promote sugar accumulation.

[0170] The flowering time was 7-10 days earlier than that of WT, indicating that ScCO4 might promote flowering through the FT and Hd3a pathways.

[0171] ScCO4 knockout lines (CRISPR / Cas9 editing):

[0172] The growth rate slowed down, and the plant height was 12% lower than that of WT.

[0173] The sucrose content in sugarcane juice was 8%-10% lower than that of WT, indicating that ScCO4 might be closely related to sugar metabolism.

[0174] The flowering time was 10 days later than that of WT, further confirming that ScCO4 is involved in flowering regulation.

[0175] In this example, through the overexpression and gene knockout of the ScCO4 gene, its key role in sugarcane growth and development, sugar accumulation, and photoperiod regulation was verified. The research results show that ScCO4 can be used as an important target gene for sugarcane molecular breeding, providing a new strategy for the improvement of sugarcane photoperiod adaptability and high-sugar breeding.

[0176] The technical achievements of the present invention are mainly applied to the field of sugarcane molecular breeding, and are particularly suitable for the cultivation and improvement of new high-sugar sugarcane varieties. Through the research on the overexpression and knockout of the ScCO4 gene, genetic engineering products for sugarcane growth regulation, enhanced sugar accumulation, and improved photoperiod adaptability can be developed. These technical achievements can be widely applied to agricultural biotechnology enterprises for the production of transgenic sugarcane seeds or plants, and then extended to fields such as sugarcane cultivation, sugar industry production, and related biomass energy development.

[0177] Based on the research results of the present invention, a series of transgenic sugarcane varieties with the ScCO4 gene as the core regulatory factor can be developed, and these varieties show excellent performance in growth rate, sucrose content, and flowering time. In addition, a molecular marker detection kit and related agricultural biotechnology service products based on the ScCO4 regulatory mechanism can be developed to provide accurate gene screening tools for sugarcane breeding, thus promoting the application and popularization of efficient breeding technologies in agricultural production.

[0178] Through the overexpression experiment of the ScCO4 gene, the transgenic plants showed excellent phenotypes such as a 15% increase in plant height, thicker stems, and increased chlorophyll content. At the same time, the sucrose content in sugarcane juice increased by 12%-15% compared with the wild type, and the flowering time was advanced by 7-10 days. These results indicate that ScCO4 plays a positive role in promoting growth, increasing sugar accumulation, and regulating flowering. In contrast, the CRISPR / Cas9 knockout lines of ScCO4 showed a decrease in growth rate, a 12% decrease in plant height, an 8%-10% decrease in sucrose content, and a 10-day delay in flowering time, further verifying the key regulatory role of ScCO4 in sugarcane growth and development and sugar metabolism.

[0179] Transcriptome and metabolome analyses of transgenic and knockout lines using advanced technologies such as RNA-Seq and LC-MS showed that significant changes occurred in the expression of downstream genes regulated by ScCO4 and the levels of key metabolites, confirming its regulatory effect on the carbon metabolism pathway and photoperiod regulation. These experimental evidences at the molecular and metabolic levels fully prove the technical effects of the present invention, not only providing a strong theoretical basis for ScCO4 as a sugarcane molecular breeding target, but also laying a solid technical foundation for the development and promotion of related products.

[0180] Through the overexpression and knockout studies of the ScCO4 gene, this invention systematically reveals the important role of this gene in aspects such as sugarcane growth regulation, enhanced sugar accumulation, and photoperiod adaptability, and develops a new high-sugar sugarcane variety through genetic engineering technology. Experimental data shows that the overexpression of the ScCO4 gene significantly increases the growth rate of sugarcane, with the plant height of transgenic sugarcane increasing by 15%, while the stem diameter thickens and the chlorophyll content increases, showing a better growth state. In contrast, sugarcane plants with the ScCO4 gene knocked out using CRISPR / Cas9 show a reduced growth rate and a 12% decrease in plant height, further verifying the key role of ScCO4 in promoting sugarcane growth. These results not only confirm the importance of ScCO4 in sugarcane growth regulation but also provide new gene targets for molecular breeding.

[0181] In terms of enhancing sugar accumulation, the research results of this invention also show significant technical effects. The sucrose content in the sugarcane juice of the ScCO4 gene overexpression lines is 12%-15% higher than that of the wild type, indicating that this gene plays a role in promoting sugar accumulation during the sugar metabolism process. In the lines with the ScCO4 gene knocked out, the sucrose content decreases by 8%-10%, further verifying the function of ScCO4 as a sugar accumulation regulatory factor. This discovery provides a new molecular basis for the breeding of high-sugar sugarcane and can also be used for variety improvement in the sugar industry production to increase the economic value of sugarcane and improve sugar yield.

[0182] In terms of photoperiod adaptability and flowering regulation, this invention has also made significant breakthroughs. Experimental data shows that the flowering time of the ScCO4 overexpression lines is 7-10 days earlier than that of the wild type, while the lines with ScCO4 knocked out show a 10-day delay in flowering time. This indicates that the ScCO4 gene can affect the flowering process of sugarcane by regulating the photoperiod sensing pathway. Using this characteristic, it is possible to cultivate sugarcane varieties suitable for early or late maturity according to different planting regions and market demands, thereby improving the adaptability of sugarcane cultivation and providing suitable breeding strategies for different climate zones.

[0183] Further research at the molecular and metabolic levels, through transcriptome and metabolome analysis of transgenic lines using RNA-Seq and LC-MS technologies, found that ScCO4 regulates the expression of multiple genes related to carbon metabolism pathways and affects the levels of various key metabolites. These data fully prove the core role of ScCO4 in sugarcane carbon metabolism regulation and provide a solid scientific basis for using this gene for sugarcane molecular breeding. The research results of this invention can be widely applied to agricultural biotechnology enterprises for the production of transgenic sugarcane varieties, molecular marker detection kits, and other biological breeding products, ultimately promoting technological progress and industrial upgrading in fields such as sugarcane cultivation, sugar industry production, and biomass energy development.

[0184] Effect Table of ScCO4 Gene Experimental Data

[0185]

[0186] The above is only a specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent replacement, and improvement made within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for applying the sugarcane CONSTANS4 gene, characterized in that: The sugarcane CONSTANS4 gene application method comprises the following steps: Step 1: Cloning and sequence analysis of sugarcane CO4 gene; Step 2: Expression analysis of sugarcane CO4 gene; Step 3: Functional verification of sugarcane CO4 gene; Step 4: Application of sugarcane CO4 gene in breeding; Among them, the functional verification and breeding application achieve sugarcane trait improvement by regulating the expression level and functional structure of the CONSTANS4 gene; The protection scope of the application method includes but is not limited to the complete implementation of the above steps. Any genetic manipulation that directly or indirectly utilizes the sugarcane IAA gene and its functional domains falls within the scope of protection, including: (1) Technical solutions for changing the expression level of CONSTANS4 gene through gene editing, homologous recombination or epigenetic regulation; (2) a genetic transformation vector comprising the functional domain of the CONSTANS4 gene and its recombinant product; (3) Sugarcane callus transformation method based on CONSTANS4 gene regulation; (4) Trait expression of improved plant type, enhanced stress resistance, or increased sugar accumulation achieved through the CONSTANS4 gene dosage effect; Regardless of whether the described sequence of steps is fully followed during the technical implementation process, or whether a partial combination of steps is selectively implemented, all sugarcane trait improvement results achieved based on the functional regulation of the CONSTANS4 gene fall within the scope of protection of the claims.

2. The sugarcane CONSTANS4 gene application method according to claim 1, characterized in that: Cloning and sequence analysis of the sugarcane CO4 gene: Total RNA was extracted from leaves of sugarcane seedlings, and the full-length sequence of CO4 gene was cloned using nucleic acid amplification technology, including SMARTerRACEcDNA, amplification technology; Sequence alignment tools, including NCBIBLAST and MEGA11, were used to align sequences, construct phylogenetic trees, and analyze the evolutionary relationships of CO4 among different species; The conserved structural domains of CO4 were predicted by MEME and Pfam, and its functional characteristics were analyzed.

3. The sugarcane CONSTANS4 gene application method according to claim 1, characterized in that: The expression analysis of the sugarcane CO4 gene includes: Detect the transcription level of IAA gene in different sugarcane tissues or under different environmental conditions. The environmental conditions include at least one of photoperiod, temperature, and humidity; qRT-PCR was used to analyze the expression level of CO4 in different tissues of sugarcane; Photoperiod treatment experiments were performed to analyze the changes in transcription levels of CO4 under different photoperiod conditions.

4. The sugarcane CONSTANS4 gene application method according to claim 3, characterized in that: The different tissues of sugarcane: roots, stems, leaves, and flowers; A photoperiod treatment experiment was conducted: short day 6-12h vs. long day 15-24h.

5. The sugarcane CONSTANS4 gene application method according to claim 1, characterized in that: Functional verification of the sugarcane CO4 gene: Construct CO4 overexpression OE and gene editing technology, including CRISPR / Cas9 technology, vectors, and transform them into Arabidopsis, sugarcane callus and rice to detect the effects of CO4 on flowering time, sugar accumulation and biomass; Yeast one-hybrid Y1H and EMSA experiments were used to verify whether CO4 directly binds to the FTFLOWERINGLOCUST or SUSY sucrose synthase promoter to regulate its transcription.

6. The sugarcane CONSTANS4 gene application method according to claim 1, characterized in that: Application of the sugarcane CO4 gene in breeding: CRISPR / Cas9 gene editing was used to destroy the CO4 negative regulatory region and cultivate low-latitude high-sugar accumulation sugarcane varieties; Through molecular marker-assisted selection, including MAS molecular marker technology, selective breeding, screening of CO4 high-expression strains, and improving breeding efficiency; In greenhouse and field trials, long-term evaluations were conducted on photoperiod response, sugar content and yield, ultimately resulting in a new sugarcane variety that is suitable for planting at different latitudes and has a 10%-15% increase in sugar yield.

7. A sugarcane CONSTANS4 gene application system for implementing the sugarcane CONSTANS4 gene application method according to any one of claims 1 to 6, characterized in that: The sugarcane CONSTANS4 gene application system comprises: Gene cloning module, used to clone the sugarcane CONSTANS4 gene and perform sequence analysis; The sugarcane CONSTANS4 gene was cloned by RT-PCR and RACE (rapid amplification of cDNA ends), and the sequence alignment and conserved domain analysis were performed. Gene expression analysis module, used to detect the spatiotemporal expression pattern of CONSTANS4 gene; Used to analyze the expression pattern of CO4 in different tissues (leaves, stems, roots) and under different photoperiod conditions using qRT-PCR and RNA-Seq; Gene function verification module, used to regulate CONSTANS4 gene expression and verify its function through gene editing or transgenic technology; Used to construct CO4 overexpression and gene editing technology, including CRISPR / Cas9, gene knockout vectors, and conduct transgenic verification in Arabidopsis, sugarcane and rice; The molecular mechanism analysis module is used to combine molecular interaction experiments, including yeast single-hybrid Y1H, chromatin immunoprecipitation ChIP and dual luciferase reporter experiments to study the regulatory effect of CO4 on photoperiod signal genes FT, Hd3a and sugar metabolism genes SUSY, INV, HK; The breeding application module is used to cultivate new sugarcane varieties with wider photoperiod adaptability and stronger sugar accumulation capacity through gene editing technology, including CRISPR / Cas9 gene editing, RNAi interference and MAS molecular marker selection.

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