Method for transforming sugarcane protoplasts

Abstract

The application discloses a method for transforming sugarcane protoplasts, which comprises the following steps: preparing a sugarcane protoplast suspension; and introducing a recombinant plasmid into the sugarcane protoplasts by PEG mediation; wherein the recombinant plasmid comprises an expression cassette driven by a promoter element selected from one of a ZmUbi promoter, a 35S promoter, a CmYLCV promoter, a MAS+CmYLCV composite promoter or a CmYLCV+MAS composite promoter. The method of the application can be used for the construction of a sugarcane protoplast transient expression system, the evaluation of promoter activity, the expression of target proteins, the expression of reporter genes, the study of gene functions and the expression of gene editing elements.

Description

Technical Field

[0001] This application relates to the field of plant biotechnology, and more specifically, to a method for transforming sugarcane protoplasts. Background Technology

[0002] sugar cane( Saccharum spp. Sugarcane is a globally important sugar and bioenergy crop, widely cultivated in tropical and subtropical regions. Due to its high heterology, complex high-ploidy genome structure, and long breeding cycle, traditional sugarcane breeding methods face problems such as low efficiency, long cycles, and difficulty in genetic improvement. In recent years, with the development of genetic engineering and synthetic biology, protoplasts, as a cell wall-less plant cell form, have become an important tool for plant gene function research, gene editing, cell fusion, and transient expression.

[0003] However, existing technologies for isolating, culturing, and transforming sugarcane protoplasts still have room for improvement in terms of protoplast yield, activity retention, culture regeneration capacity, and efficiency of genetic manipulation. Summary of the Invention

[0004] This application addresses the problems existing in current sugarcane protoplast isolation and transformation systems, such as unstable protoplast yield and cell viability, unsatisfactory exogenous DNA transformation efficiency, and a lack of efficient promoter elements suitable for sugarcane protoplasts. It provides a method for transforming sugarcane protoplasts. This method can be used for transient expression of sugarcane protoplasts, promoter activity evaluation, reporter gene expression, target protein expression, gene function studies, and gene editing element expression.

[0005] This application provides a method for transforming sugarcane protoplasts, which includes the following steps: Preparation of sugarcane protoplast suspension; The recombinant plasmid was introduced into the sugarcane protoplast using PEG-mediated delivery. The recombinant plasmid includes an expression cassette driven by a promoter element, wherein the promoter element is selected from one of the ZmUbi promoter, 35S promoter, CmYLCV promoter, MAS+CmYLCV composite promoter, or CmYLCV+MAS composite promoter.

[0006] Alternatively, the expression cassette is used to drive the expression of at least one of a reporter gene, a target protein-coding gene, and a gene editing element.

[0007] Alternatively, the preparation of the sugarcane protoplast suspension includes: Obtain sugarcane seedlings, including two- to three-month-old tissue-cultured sugarcane seedlings / or sugarcane seedlings obtained by soil cultivation from sugarcane stem segments with buds; Take the tender stem and / or leaf sheath portion of the sugarcane seedling; Cut the tender stems and / or leaf sheaths into strips; The strips were placed in an enzymatic hydrolysate, and after being vacuum-treated, they were subjected to enzymatic hydrolysis in a shaker under light-protected conditions. After removing impurities, collect the free sugarcane protoplasts.

[0008] Furthermore, after collecting the free sugarcane protoplasts, the free sugarcane protoplasts are resuspended in MMG solution to obtain a sugarcane protoplast suspension for conversion.

[0009] Alternatively, the tissue-cultured sugarcane seedlings are obtained by inducing differentiation from sugarcane callus.

[0010] Furthermore, the PEG-mediated introduction of the recombinant plasmid into the sugarcane protoplasts comprises: adding 100–200 μL of sugarcane protoplast suspension, 5–30 μg of the recombinant plasmid, and PEG solution to the transformation reaction system, mixing thoroughly, and then treating under light-protected conditions at room temperature; wherein the cell number of the sugarcane protoplasts is 6.25 × 10³ to 1 × 10³. 5 indivual.

[0011] Alternatively, in the transformation reaction system, the volume of the sugarcane protoplast suspension is 200 μL, and the number of sugarcane protoplast cells is 2.5 × 10⁻⁶. 4 ~5×10 4 The amount of the recombinant plasmid used is 20-30 μg; the volume of the PEG solution added is 220 μL.

[0012] Alternatively, when the recombinant vector includes the ZmUbi promoter fragment, the primer sequences for amplifying the ZmUbi promoter fragment are shown in SEQ ID No. 2 and SEQ ID No. 3; When the recombinant vector includes the 35S promoter fragment, the primer sequences for amplifying the 35S promoter fragment are shown in SEQ ID No. 5 and SEQ ID No. 6; When the recombinant vector includes the CmYLCV promoter fragment, the primer sequences for amplifying the CmYLCV promoter fragment are shown in SEQ ID No. 8 and SEQ ID No. 9; When the recombinant vector includes the MAS+CmYLCV complex promoter fragment, the DNA sequence of the MAS+CmYLCV complex promoter is shown in SEQ ID No. 10, and the primer sequences for constructing the MAS+CmYLCV complex promoter fragment are shown in SEQ ID No. 11 and SEQ ID No. 12. When the recombinant vector includes a CmYLCV+MAS complex promoter fragment, the DNA sequence of the CmYLCV+MAS complex promoter is shown in SEQ ID No. 13, and the primer sequences for constructing the CmYLCV+MAS complex promoter fragment are shown in SEQ ID No. 14 and SEQ ID No. 15.

[0013] Furthermore, after introducing the recombinant plasmid into the sugarcane protoplast using PEG-mediated transformation, the cell state of the sugarcane protoplast is confirmed, and the transformation rate and / or expression of the recombinant plasmid are detected by reporter gene assay.

[0014] Further, alternatively, the reporter gene includes a fluorescent protein gene; after introducing the recombinant plasmid into the sugarcane protoplast using PEG-mediated induction, the sugarcane protoplast is cultured in the dark for 24 h, and the fluorescence signal intensity of the reporter gene and / or the proportion of fluorescently positive cells are confirmed by fluorescence microscopy.

[0015] The beneficial technical effects of the technical solutions provided in this application include: The sugarcane protoplasts obtained using the method described in this application exhibit better activity, thereby increasing the conversion rate of sugarcane. Furthermore, it was discovered that a composite promoter containing both the MAS promoter and the CmYLCV promoter can be efficiently expressed in sugarcane, making it possible to construct a high-expression transformation system in sugarcane. This provides technical support for gene function research, gene editing, single-cell omics analysis, and the breeding of superior varieties in sugarcane.

[0016] Additional aspects and advantages of this application will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this application. Attached Figure Description

[0017] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 The above is a fluorescence image of sugarcane protoplasts driven by the 35S promoter in an embodiment of this application. Figure 2 The above is a fluorescence image of sugarcane protoplasts driven by the ZmUbi promoter in an embodiment of this application. Figure 3 The above is a fluorescence image of sugarcane protoplasts driven by the CmYLCV promoter in an embodiment of this application. Figure 4 The image shows the fluorescence of sugarcane protoplasts in the treatment group using the CmYLCV promoter, 30ug of plasmid, and 100ul of protoplasts in the embodiments of this application. Figure 5 The image shows the fluorescence of sugarcane protoplasts in the treatment group using the CmYLCV promoter, 5 μg of plasmid, and 200 μL of protoplasts in the embodiments of this application. Figure 6 The image shows the fluorescence of sugarcane protoplasts in the treatment group using the CmYLCV promoter, 10ug of plasmid, and 200ul of protoplasts in the embodiments of this application. Figure 7 The image shows the fluorescence of sugarcane protoplasts in the treatment group using the CmYLCV promoter, 20ug of plasmid, and 200ul of protoplasts in the embodiments of this application. Figure 8 The image shows the fluorescence of sugarcane protoplasts in the treatment group using the CmYLCV promoter, 30 μg of plasmid, and 200 μL of protoplasts in the embodiments of this application.

[0018] Figure 9 This is a comparison diagram showing the fluorescence effects of MAS+CmYLCV promoter, CmYLCV+MAS promoter, and CmYLCV promoter driving GFP expression in sugarcane protoplasts in the embodiments of this application. Detailed Implementation

[0019] The embodiments of this application are described below with reference to the accompanying drawings. It should be understood that the embodiments described below with reference to the accompanying drawings are exemplary descriptions for explaining the technical solutions of the embodiments of this application, and do not constitute a limitation on the technical solutions of the embodiments of this application.

[0020] Those skilled in the art will understand that, unless specifically stated otherwise, the terms "described" and "the" as used herein may also include plural forms. It should be further understood that the term "comprising" as used in the specification of this application means the presence of the stated features, integers, steps, operations, elements, and / or components, but does not exclude other features, information, data, steps, operations, elements, components, and / or combinations thereof supported by the art. The term "and / or" as used herein refers to at least one of the items defined by the term; for example, "A and / or B" can be implemented as "A," or as "B," or as "A and B."

[0021] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0022] Materials and Methods 1. Carrier Construction This invention uses GFP as a reporter gene and constructs various promoter-driven GFP expression vectors to compare the expression-driving ability of different promoters and promoter combinations in sugarcane protoplasts. Specifically, LQP-RP-11ZmUbi-GFP, LQP-RP-1235S-GFP, and LQP-RP-CmYLCV-GFP are the basic promoter expression vectors driven by the ZmUbi promoter, 35S promoter, and CmYLCV promoter, respectively, for comparing the expression effects of different basic promoters in sugarcane protoplasts. The ZmUbi promoter fragment is shown in SEQ No. 1; the 35S promoter fragment is shown in SEQ No. 4; and the CmYLCV promoter fragment is shown in SEQ No. 7.

[0023] ZmUbi promoter (Zea mays Ubiquitin promoter, maize ubiquitin gene promoter), derived from maize ( Zeamays The upstream regulatory region of ubiquitin genes typically contains the first exon and the first intron of the gene. It is a constitutive strong promoter, especially applicable to monocotyledonous plants.

[0024] The 35S promoter (Cauliflower Mosaic Virus 35S) is derived from the upstream sequence of the cauliflower mosaic virus (CaMV) genome that directs 35S RNA synthesis. It comprises the region from -941 to +208 (core approximately 343 bp) and is a constitutively strong promoter, the most widely used promoter in plant genetic engineering. It is efficiently and stably expressed in almost all tissues and developmental stages of dicotyledonous plants (tobacco, cotton, soybean, tomato, etc.); however, its activity is significantly reduced in monocotyledonous plants (rice, maize, wheat), and high-frequency inactivation and gene silencing are observed.

[0025] The CmYLCV promoter (Cestrum yellow leaf curling virus promoter) is derived from Cestrum yellow leaf curling virus (CmYLCV)—a geminivirus—and is a viral constitutive promoter. It can activate constitutive expression of GUS or GFP in Arabidopsis, tobacco, maize, and rice. In transgenic wheat, it drives strong GUS expression in various tissues other than pollen and roots. Compared to the 35S promoter, CmYLCV performs better in monocotyledonous plants and is one of the preferred viral promoters for cereal transformation.

[0026] The GFP reporter gene (Green Fluorescent Protein reporter gene) is derived from the Victoria multituberculate jellyfish (…). Aequoreavictoria This protein contains 238 amino acids and has a β-barrel structure. When excited by 395 nm / 475 nm blue light, it emits green fluorescence at 508–509 nm, exhibiting autofluorescence without any exogenous substrates or cofactors. It is commonly used in the following scenarios: transgenic positive screening, protein subcellular localization, bimolecular fluorescence complementation for detecting protein interactions, and in CRISPR / dCas9 systems to coordinate with the Pol III promoter to drive sgRNA.

[0027] Based on this, to further evaluate the effect of combining the MAS element with the CmYLCV promoter on the expression-driving ability of exogenous genes, MAS (Mannopine Synthase) is derived from Agrobacterium tumefaciens (…). Agrobacterium tumefaciens The T-DNA region of the Ti plasmid contains two genes, mas1' and mas2', which encode mannopine synthase and catalyze the synthesis of mannopine. The 479 bp spacer between mas1' and mas2' is the MAS bidirectional promoter. The standalone CmYLCV promoter is a constitutive promoter, and its persistent expression makes it difficult to regulate. However, after introducing the MAS element to form a composite promoter, the composite promoter becomes an inducible promoter, capable of being regulated by multiple stimulatory factors. The MAS element contains multiple positive regulatory elements, such as an A / T-rich region (enhancing transcription), an O element (octopine synthesis-related enhancer), and three palindromic structures (a, a', β) (essential for maintaining high activity). When these elements are tandemly linked with the CmYLCV core promoter, a synergistic effect is produced. MAS is derived from the Agrobacterium Ti plasmid and naturally possesses the characteristic of high-efficiency expression in both dicotyledonous and monocotyledonous plants. When combined with CmYLCV, it compensates for the insufficient expression of CmYLCV in some dicotyledonous plants. The MAS promoter is short (~479 bp) and compact in structure, making it less prone to silencing due to methylation. The overall stability of the composite promoter is superior to that of a single strong promoter.

[0028] This invention employs the Gibson assembly method to ligate the MAS element and the CmYLCV promoter into GFP reporter vectors in different arrangements, obtaining MAS+CmYLCV-GFP (SEQ ID No. 10) and CmYLCV+MAS-GFP (SEQ ID No. 13) expression vectors, respectively. Specifically, MAS+CmYLCV-GFP indicates that the MAS element is located upstream of the CmYLCV promoter, and CmYLCV+MAS-GFP indicates that the MAS element is located downstream of the CmYLCV promoter.

[0029] The aforementioned GFP expression vectors were all used in PEG-mediated transient transformation experiments of sugarcane protoplasts. PEG-mediated transient protoplast transformation utilizes high concentrations of PEG (polyethylene glycol) in Ca2+. 2+ Under high pH conditions, the permeability of the protoplast cell membrane of decelled plants temporarily increases, allowing exogenous DNA to enter the cell through a "molecular bridge" mechanism, achieving rapid gene expression within 6 to 48 hours.

[0030] In this embodiment, the LQP-RP-11 ZmUbi-GFP, LQP-RP-12 35S-GFP, and LQP-RP-CmYLCV-GFP expression vectors were used to compare the expression effects of different basic promoters in the optimized sugarcane protoplast transformation system; the MAS+CmYLCV-GFP and CmYLCV+MAS-GFP expression vectors were used to further compare the effects of different arrangements of MAS elements and CmYLCV promoters on the transient expression effect of GFP after determining the optimal protoplast transformation conditions.

[0031] After transformation, GFP fluorescence signals were observed using a fluorescence microscope. The intensity of the GFP fluorescence signal, the proportion of fluorescently positive cells, and the state of sugarcane protoplast cells were used as indicators to evaluate the expression-driving ability of different promoters or promoter combinations. Primers used for vector construction are shown in Table 1.

[0032] Table 1 Primers used in vector construction

[0033] 2. Protoplast isolation and transformation The protoplasts used in this application were derived from the sugarcane varieties Zhonggui Sugar No. 42 and Xintai Sugar No. 22.

[0034] 2.1 Sugarcane seedling cultivation 2.1.1 Tissue Culture Seedling Culture Healthy sugarcane plants free from obvious pests and diseases were selected as test materials. Young sugarcane tissue was used as explants, cleaned and surface-sterilized, then aseptically cut into tissue blocks and inoculated into callus propagation medium. The callus propagation medium was based on MS medium, supplemented with sucrose, agar, and plant growth regulators to promote callus proliferation. After inoculation, the culture materials were placed at 25±2℃ to induce callus formation, and well-grown callus tissue was subcultured for propagation. The callus propagation stage lasted approximately 4 weeks.

[0035] The callus tissue obtained from propagation was transferred to a differentiation and regeneration medium. This medium was based on MS medium with added sucrose, agar, and plant growth regulators to promote shoot differentiation. After transfer, the tissue was cultured at 25±2℃ under 12–16 h of light per day, allowing the callus tissue to gradually turn green and form adventitious shoots. The differentiation and regeneration stage lasted approximately 4 weeks.

[0036] After adventitious buds have formed, they are cut off and transferred to a rooting medium. The rooting medium is based on MS medium or 1 / 2 MS medium, with added plant growth regulators to promote root formation. During the culture process, young roots gradually form at the base of the adventitious buds and further develop into sugarcane seedlings with root systems. The rooting stage lasts approximately 2–3 weeks.

[0037] Sugarcane seedlings with established root systems are transferred to a stem elongation medium. This medium is based on MS medium or a modified MS medium, with the addition of low concentrations of plant growth regulators or none at all. During the culture process, the sugarcane seedling stems gradually elongate, the leaves unfold, and the plants become more robust. The stem elongation stage lasts approximately 2–4 weeks. After about three months of this culture process, sugarcane tissue culture seedlings suitable for subsequent protoplast isolation and transformation experiments are obtained.

[0038] 2.1.2 Soil-based seedling cultivation Healthy sugarcane plants with plump stems, no obvious pests or diseases, and intact buds were selected from the field as test materials. After harvesting, the outer leaf sheaths and surface attachments of the sugarcane stems were removed, and they were rinsed thoroughly with clean water. The rinsed sugarcane stems were then placed in a cool, ventilated place to dry.

[0039] Cut the sugarcane stalks into segments with buds according to the node position, retaining one intact bud on each segment. The segments should be 4–6 cm long. Keep the cut clean and avoid damaging the buds during the cutting process.

[0040] Take a seedling pot, add commercial potting soil, gently press it down to level it, and water it to thoroughly moisten the soil. Then, place the sugarcane stem segment with buds horizontally in the potting soil, with the buds facing upwards or at an angle, and cover it with 1-2 cm of potting soil. After planting, water it again as needed to keep the potting soil moist.

[0041] The planted sugarcane stem segments were placed in a culture environment of 25–32℃ under natural diffused light. During the culture period, the moisture content of the nutrient soil and the bud germination were observed regularly, and water was added as needed according to the dryness of the substrate to keep the nutrient soil moist but not waterlogged. After about two weeks of culture, the sugarcane buds sprouted to form new shoots, and gradually grew young roots and new leaves, yielding soil-grown sugarcane seedlings. The obtained soil-grown sugarcane seedlings can be used for subsequent protoplast preparation, transformation, treatment, sampling, or phenotypic observation experiments.

[0042] 2.2 Protoplast Isolation Take sugarcane seedlings that are two to three months old, including sugarcane tissue culture seedlings and / or sugarcane soil culture seedlings. Take the tender stems and leaf sheaths of the sugarcane, cut off the roots and leaves of the seedlings, wipe off the surface moisture of the stems, and cut them into strips about 0.5 mm wide with a new blade.

[0043] The cut strips were added directly to the enzymatic hydrolysate, and vacuum treatment was carried out for 30 minutes at a vacuum pump reading of 0.6 to ensure that the enzymatic hydrolysate fully infiltrated the plant tissue. After vacuum treatment, the container containing the strips and enzymatic hydrolysate was placed in a light-proof shaker and enzymatically hydrolyzed for 5 hours at 26°C and 25 rpm to release sugarcane protoplasts.

[0044] After enzymatic hydrolysis, filter out the hydrolysate. Add approximately 15 mL of W5 solution to the container each time, and shake horizontally for about 10 seconds to elute and release the protoplasts. Filter the eluent containing protoplasts into a 50 mL round-bottom centrifuge tube using a 70 μm nylon membrane. Repeat the above elution process four times.

[0045] The filtered protoplast suspension was centrifuged horizontally at 200 g for 5 min to precipitate the protoplasts. After centrifugation, the supernatant was discarded, and the protoplasts were resuspended in W5 solution. The resuspended protoplasts were combined into the same centrifuge tube and incubated at 4°C for 30 min.

[0046] After cold treatment, the protoplasts were centrifuged at 200 g for 5 min at room temperature, and the supernatant was discarded. Then, an appropriate amount of MMG solution (MMG = MES + MgCl2 + mannitol (Glucose)) was added to resuspend the protoplasts, and they were counted using a hemocytometer. Simultaneously, the morphology and cell state of the protoplasts were observed under a microscope. Based on the counting results and the needs of subsequent transformation experiments, MMG solution was added to adjust the volume of the protoplast suspension, obtaining a sugarcane protoplast suspension for PEG-mediated transformation.

[0047] 2.3 Protoplast Transformation Add the recombinant plasmid to a 2 mL round-bottom centrifuge tube. For single-plasmid transformation, the amount of recombinant plasmid used can be 5–30 μg; in some embodiments, the amount of recombinant plasmid used is 30 μg. If multiple plasmids are used simultaneously, the amount of each plasmid used can be 5 μg.

[0048] Add sugarcane protoplast suspension to a centrifuge tube containing the recombinant plasmid, then add freshly prepared PEG solution, and quickly and gently invert to mix. The volume of sugarcane protoplast suspension added is 100–200 μL, and the volume of PEG solution added is 120 μL. After mixing, place the reaction system at room temperature in the dark for 15 min to induce the introduction of the recombinant plasmid into the sugarcane protoplast.

[0049] To optimize the PEG-mediated transformation conditions of sugarcane protoplasts, this application established gradients for different protoplast volumes, cell numbers, and plasmid dosages. Specifically, the protoplast volumes were set to 100 μL and 200 μL; the cell number was set to 1 × 10⁻⁶ cells / mL. 5 5×10 4 2.5×10 4 1.25×10 4 And 6.25×10³; the amount of plasmid used was set to 5 μg, 10 μg, 20 μg and 30 μg.

[0050] After PEG-induced transformation, slowly add 1 mL of W5 solution to the reaction system and gently invert to mix and terminate the transformation reaction. Then, centrifuge horizontally at 100 g for 5 min at room temperature and discard the supernatant. Resuspend the protoplasts in 1 mL of W5 solution. If for fluorescence observation, transfer the resuspended protoplasts to 24-well or 48-well plates, adding 1 mL to each well of the 24-well plate and 500 μL to each well of the 48-well plate. If for protoplast genomic DNA extraction, place the round-bottom centrifuge tubes horizontally in an EP tube rack and incubate in the dark. Observe and photograph GFP fluorescence after 24 h of incubation; after 48 h of incubation, genomic DNA can be extracted.

[0051] The reagent formulations used in each of the above steps can be found in Table 2 below: Table 2. Some reagent formulations used in sugarcane protoplast isolation and transformation

[0052] 3. Optimization of protoplast transformation system and verification of promoter expression effect 3.1 Optimization of sugarcane protoplast transformation conditions To establish an efficient PEG-mediated transformation system suitable for sugarcane protoplasts, this application optimized protoplast volume, cell number, and plasmid dosage. Specifically, 100 μL and 200 μL protoplast treatment groups were set up, and different cell number gradients were set in different treatment groups, including 1×10⁻⁶ cells / cells. 5 5×10 4 2.5×10 4 1.25×10 4 and 6.25×10 3 Cells; and different plasmid dosage gradients were set, including 5 μg, 10 μg, 20 μg and 30 μg.

[0053] After PEG-mediated transformation, each treatment group was cultured in the dark under the same conditions for 24 h, and GFP expression was observed using a fluorescence microscope. The results showed that when the plasmid dosage was 20–30 μg, the protoplast volume was 200 μL, and the cell number was 2.5 × 10⁻⁶ cells / mL, the expression of GFP was optimal. 4 ~5×10 4 At this time, the sugarcane protoplasts showed strong fluorescence signals, a high proportion of fluorescently positive cells, and relatively stable protoplast cell states. Therefore, the plasmid dosage was 20–30 μg, the protoplast volume was 200 μL, and the cell number was 2.5 × 10⁻⁶. 4 ~5×104 These were determined to be the optimal conditions for PEG-mediated transformation of sugarcane protoplasts. Fluorescence detection results for treatment groups with different plasmid amounts, protoplast volumes, and cell numbers are shown below. Figures 4 to 8 As shown.

[0054] 3.2 Comparison of the expression effects of different basic promoters After determining the optimal conditions for PEG-mediated transformation of sugarcane protoplasts, this application further utilized this optimized system to compare the expression-driving capabilities of different basic promoters in sugarcane protoplasts. Specifically, 35S-GFP, ZmUbi-GFP, and CmYLCV-GFP expression vectors were introduced into sugarcane protoplasts, respectively. All treatment groups used the same protoplast source, cell number, protoplast volume, plasmid amount, PEG treatment time, and culture conditions.

[0055] After transformation, protoplasts were cultured in the dark for 24 h, and GFP fluorescence signals were observed using a fluorescence microscope. The results showed differences in fluorescence intensity and the proportion of fluorescently positive cells driven by different promoters. The GFP fluorescence signal driven by the CmYLCV promoter was stronger than that driven by the 35S and ZmUbi promoters, indicating that the CmYLCV promoter has a strong ability to drive exogenous gene expression in sugarcane protoplasts. This suggests that the CmYLCV promoter can be considered a preferred promoter element for transient expression, reporter gene expression, target protein expression, and gene editing element expression in sugarcane protoplasts. The fluorescence detection results of GFP expression driven by the 35S, ZmUbi, and CmYLCV promoters are shown below. Figures 1 to 3 As shown.

[0056] 3.3 Comparison of MAS element and CmYLCV promoter combination methods Based on the determination of the optimal conditions for PEG-mediated transformation of sugarcane protoplasts and the demonstration that the CmYLCV promoter can efficiently drive GFP expression in sugarcane protoplasts, this application further combines the MAS element with the CmYLCV promoter to construct MAS+CmYLCV-GFP and CmYLCV+MAS-GFP expression vectors to evaluate the effects of different arrangements of the MAS element and the CmYLCV promoter on the transient expression of exogenous genes.

[0057] Specifically, under the optimized PEG-mediated transformation conditions of sugarcane protoplasts, the MAS+CmYLCV-GFP and CmYLCV+MAS-GFP expression vectors were introduced into sugarcane protoplasts, respectively. Both treatment groups used the same protoplast source, protoplast volume, cell number, plasmid amount, PEG induction time, and culture conditions. After transformation, the protoplasts were cultured in the dark for 24 h, and the GFP fluorescence signal was observed using a fluorescence microscope.

[0058] The results showed that both the MAS+CmYLCV and CmYLCV+MAS composite promoters could drive GFP expression in sugarcane protoplasts, indicating that the combination of MAS elements and CmYLCV promoters still possesses the ability to drive exogenous gene expression. Further comparison of the fluorescence expression effects of the two combinations confirmed that the arrangement of the MAS elements and CmYLCV promoters influences their expression-driving ability. Therefore, the MAS+CmYLCV and CmYLCV+MAS composite promoters can serve as candidate composite promoter elements in sugarcane protoplast transient expression systems for target protein expression, reporter gene expression, or gene editing element expression. The fluorescence detection results of GFP expression driven by the MAS+CmYLCV and CmYLCV+MAS composite promoters are shown below. Figure 9 As shown. Figure 9 The fluorescence intensity shown should be understood as a total reflection of the conversion rate of the composite promoter and the fluorescence intensity of a single cell. Further experiments are needed to confirm the controllable energy efficiency of the composite promoter.

[0059] In summary, this application provides a method for transforming sugarcane protoplasts, comprising the following steps: preparing a sugarcane protoplast suspension; introducing a recombinant plasmid into the sugarcane protoplasts using PEG-mediated transformation; wherein the recombinant plasmid includes an expression cassette driven by a promoter element, the promoter element being selected from one of the following: ZmUbi promoter, 35S promoter, CmYLCV promoter, MAS+CmYLCV composite promoter, or CmYLCV+MAS composite promoter.

[0060] The method described in this application yields sugarcane protoplasts with relatively intact morphology and stable cell state. Bright-field observations show that the obtained sugarcane protoplasts are mostly nearly round or round with clear cell outlines and relatively few cell fragments. GFP fluorescence observations indicate that GFP fluorescence signals can be observed in sugarcane protoplasts after PEG-mediated treatment, demonstrating that the exogenous recombinant plasmid can be introduced into sugarcane protoplasts and achieve transient expression. By optimizing protoplast volume, cell number, plasmid dosage, PEG treatment conditions, purification method, and centrifugation conditions, this application establishes a transient transformation system suitable for sugarcane protoplasts.

[0061] Furthermore, this application validated the expression-driving ability of different promoters and promoter combinations in sugarcane protoplasts through transient GFP reporter gene expression. The results showed that the CmYLCV promoter can efficiently drive exogenous gene expression in sugarcane protoplasts, and the MAS+CmYLCV and CmYLCV+MAS composite promoters can also drive exogenous gene expression. Therefore, the methods and promoter elements provided in this application can be used for transient expression in sugarcane protoplasts, promoter screening, reporter gene expression, target protein expression, and gene editing element expression.

[0062] Those skilled in the art will understand that the steps, measures, and solutions in the various operations, methods, and processes discussed in this application can be alternated, modified, combined, or deleted. Furthermore, other steps, measures, and solutions in the various operations, methods, and processes discussed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted. Furthermore, steps, measures, and solutions in related technologies that are similar to those disclosed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted.

[0063] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0064] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0065] The above description is only a partial implementation of this application. It should be noted that for those skilled in the art, other similar implementation methods based on the technical concept of this application, without departing from the technical concept of this application, also fall within the protection scope of the embodiments of this application.

Claims

1. A method for transforming sugarcane protoplasts, characterized in that, It includes the following steps: Preparation of sugarcane protoplast suspension; The recombinant plasmid was introduced into the sugarcane protoplast using PEG-mediated delivery. The recombinant plasmid includes an expression cassette driven by a promoter element, wherein the promoter element is selected from one of the ZmUbi promoter, 35S promoter, CmYLCV promoter, MAS+CmYLCV composite promoter, or CmYLCV+MAS composite promoter.

2. The method as described in claim 1, characterized in that, The expression cassette is used to drive the expression of at least one of a reporter gene, a target protein encoding gene, and a gene editing element.

3. The method as described in claim 1, characterized in that, The preparation of the sugarcane protoplast suspension includes: Obtain sugarcane seedlings, including two- to three-month-old tissue-cultured sugarcane seedlings / or sugarcane seedlings obtained by soil cultivation from sugarcane stem segments with buds; Take the tender stem and / or leaf sheath portion of the sugarcane seedling; Cut the tender stems and / or leaf sheaths into strips; The strips were placed in an enzymatic hydrolysate, and after being vacuum-treated, they were subjected to enzymatic hydrolysis in a shaker under light-protected conditions. After removing impurities, collect the free sugarcane protoplasts.

4. The method as described in claim 3, characterized in that, After collecting the free sugarcane protoplasts, the free sugarcane protoplasts were resuspended in MMG solution to obtain a sugarcane protoplast suspension for use in transformation.

5. The method as described in claim 3, characterized in that, The tissue-cultured sugarcane seedlings were obtained by inducing differentiation from sugarcane callus tissue.

6. The method as described in claim 1, characterized in that, The PEG-mediated introduction of recombinant plasmids into sugarcane protoplasts comprises: adding 100–200 μL of sugarcane protoplast suspension, 5–30 μg of the recombinant plasmid, and PEG solution to the transformation reaction system, mixing thoroughly, and then treating under light-protected conditions at room temperature; wherein the cell number of the sugarcane protoplasts is 6.25 × 10³ to 1 × 10³. 5 indivual.

7. The method as described in claim 6, characterized in that, In the transformation reaction system, the volume of the sugarcane protoplast suspension is 200 μL, and the number of sugarcane protoplast cells is 2.5 × 10⁻⁶. 4 ~5×10 4 The amount of the recombinant plasmid used is 20-30 μg; the volume of the PEG solution added is 220 μL.

8. The method as described in claim 1, characterized in that, When the recombinant vector includes the ZmUbi promoter fragment, the primer sequences for amplifying the ZmUbi promoter fragment are shown in SEQ ID No. 2 and SEQ ID No. 3; When the recombinant vector includes the 35S promoter fragment, the primer sequences for amplifying the 35S promoter fragment are shown in SEQ ID No. 5 and SEQ ID No. 6; When the recombinant vector includes the CmYLCV promoter fragment, the primer sequences for amplifying the CmYLCV promoter fragment are shown in SEQ ID No. 8 and SEQ ID No. 9; When the recombinant vector includes the MAS+CmYLCV complex promoter fragment, the DNA sequence of the MAS+CmYLCV complex promoter is shown in SEQ ID No. 10, and the primer sequences for constructing the MAS+CmYLCV complex promoter fragment are shown in SEQ ID No. 11 and SEQ ID No.

12. When the recombinant vector includes a CmYLCV+MAS complex promoter fragment, the DNA sequence of the CmYLCV+MAS complex promoter is shown in SEQ ID No. 13, and the primer sequences for constructing the CmYLCV+MAS complex promoter fragment are shown in SEQ ID No. 14 and SEQ ID No.

15.

9. The method as described in claim 1, characterized in that, After introducing the recombinant plasmid into the sugarcane protoplasts using PEG-mediated transformation, the cell status of the sugarcane protoplasts is confirmed, and the transformation rate and / or expression of the recombinant plasmid are detected by reporter gene assay.

10. The method as described in claim 9, characterized in that, The reporter gene includes a fluorescent protein gene; after the recombinant plasmid is introduced into the sugarcane protoplast using PEG-mediated induction, the sugarcane protoplast is cultured in the dark for 24 h, and the fluorescence signal intensity of the reporter gene and / or the proportion of fluorescent positive cells are confirmed by fluorescence microscopy.

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