Method for cloning and transforming fern genes and application thereof

Abstract

The application relates to the technical field of gene cloning, and provides a cloning and transformation method of a fern gene. The cloning and transformation method of the fern gene comprises the following steps: taking a homologous gene sequence of a specific plant as a probe to perform homologous comparison and obtain target gene sequence information; total RNA of a fern plant is extracted and reverse transcribed to synthesize cDNA; the cDNA is taken as a template to perform PCR amplification to obtain a PCR amplification product; the PCR amplification product is subjected to topoisomerase-mediated connection reaction with a primer vector, and is introduced into E. coli to obtain a gene plasmid; the gene plasmid is mixed with a target vector, an enzyme mixture is added to perform recombination reaction, E. coli containing the target expression vector is obtained, and then culture, screening and single clone strain obtaining are performed; the single clone strain is used to infect fern plant material, culture is performed, and transgenic fern material is obtained. The method can improve the success rate of cloning and transformation of the fern gene.

Description

Technical Field

[0001] This application relates to the field of gene cloning technology, and in particular to a method for cloning and transforming fern genes and its application. Background Technology

[0002] Ferns occupy a crucial position in plant evolution, but their molecular biology research has long been limited by inefficient genetic manipulation tools. Traditional gene cloning and transformation methods are mostly developed for model angiosperms (such as Arabidopsis thaliana), and their direct application to ferns often faces problems such as low efficiency and cumbersome procedures. Therefore, developing a gene cloning and transformation method specifically for ferns is crucial for advancing functional genomics research in ferns. Summary of the Invention

[0003] In view of this, this application aims to propose a method for cloning and transforming fern genes, so as to improve the success rate of fern gene cloning and transformation.

[0004] To achieve the above objectives, the technical solution of this application is implemented as follows:

[0005] A method for cloning and transforming a fern gene, the method comprising:

[0006] Using homologous gene sequences of specific plants as probes, homology comparisons are performed in the genomes of ferns to obtain target gene sequence information;

[0007] Using one or more of the gametophytes, sporophytes, or mixed tissues of ferns as materials, total RNA was extracted and reverse transcribed to synthesize cDNA;

[0008] The cDNA was used as a template for PCR amplification to obtain the PCR amplification product.

[0009] The PCR amplification product was ligated with the pENTR / D-TOPO introductory vector using a topoisomerase-mediated ligation reaction. The resulting ligation product was introduced into E. coli, and the pENTR-target gene plasmid was obtained by colony PCR identification and sequencing verification.

[0010] The pENTR-target gene plasmid was mixed with the target vector, and a mixture of LR Clonase II enzymes was added to carry out a recombination reaction. The resulting reaction product was introduced into E. coli to obtain E. coli containing the target expression vector.

[0011] Escherichia coli containing the target expression vector were cultured and screened to obtain monoclonal strains;

[0012] The monoclonal strain was used to infect fern materials and culture them to obtain transgenic fern materials.

[0013] Furthermore, the specific plant includes one of bryophytes, lycophytes, or Arabidopsis thaliana.

[0014] Furthermore, the primers used for reverse transcription are Oligo dT primers;

[0015] The reverse transcriptase used for the reverse transcription is M-MuLV reverse transcriptase.

[0016] Furthermore, the PCR amplification using the cDNA as a template to obtain the PCR amplification product includes:

[0017] Using the cDNA as a template, PCR amplification was performed using high-fidelity DNA polymerase, and the amplification product was purified to obtain the PCR amplification product.

[0018] The amplification products were purified using low-melting-point agarose gel electrophoresis gel cutting and recovery technology.

[0019] Furthermore, the sequencing verification was performed using Sanger sequencing with M13F / R primers.

[0020] Furthermore, the method for culturing Escherichia coli containing the target expression vector employs a three-parental mating method.

[0021] Furthermore, the strains used in the triparental mating method include helper Escherichia coli and Agrobacterium tumefaciens receptor strains.

[0022] Furthermore, the helper Escherichia coli carries a helper plasmid capable of providing conjugation transfer function;

[0023] The Agrobacterium tumefaciens recipient strain used is the GV3101 Agrobacterium tumefaciens recipient strain.

[0024] Compared with related technologies, this application has the following advantages:

[0025] The method for cloning and transforming fern genes in this application, through the synergistic cooperation between steps, smoothly connects the cloning system and the genetic transformation process, jointly ensuring the overall success rate of obtaining transgenic fern materials.

[0026] First, by using homologous gene sequences from specific plants as probes for alignment, the limitation of insufficient genome annotation information in ferns was overcome, improving the success rate of obtaining target gene sequences. Second, gametophytes, sporophytes, or a mixture thereof were selected as RNA extraction materials, fully considering the gene expression characteristics of different life generations of ferns and ensuring the integrity and representativeness of the cDNA template. Based on this, a cloning strategy independent of restriction endonuclease sites was developed by employing topoisomerase-mediated ligation and LR recombination reactions. This not only simplified the operational process but also fundamentally avoided the risk of construction failure due to incomplete restriction enzyme mapping information in fern genes. Finally, single-clone strains were obtained through culture, Agrobacterium transformation, and screening, and used for infection in the final step. This transformation process, tailored to the characteristics of fern materials, smoothly connects the cloning system with the genetic transformation process, improving the success rate of fern gene cloning and transformation.

[0027] This application also proposes an application of the above-mentioned method for cloning and transforming fern genes, and its application in fern gene function research. Attached Figure Description

[0028] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0029] Figure 1 This is a flowchart of the method for cloning and transforming fern genes as described in the embodiments of this application;

[0030] Figure 2 This is a diagram of the gametophyte culture experiment of the water fern Ceratopteris richardii as described in Example 1 of this application;

[0031] Figure 3 This is a diagram of the gametophyte culture experiment of the water fern Ceratopteris richardii as described in Example 1 of this application. Detailed Implementation

[0032] To make the technical solution and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0033] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0034] For items where specific conditions are not specified in this application, conventional conditions or conditions recommended by the manufacturer of the equipment used shall apply. For items where the manufacturer of the reagents or instruments used is not specified, conventional products that can be purchased commercially shall be used. As for the technical means or processes involved, if specific conditions are not specified, they shall be carried out in accordance with the existing methods in the relevant field.

[0035] The embodiments of the first aspect of this application provide a method for cloning and transforming fern genes, which can improve the success rate of fern gene cloning and transformation.

[0036] In related technologies, due to the large genome size, numerous repetitive sequences, and rich content of secondary metabolites such as polysaccharides and polyphenols in ferns, directly applying RNA extraction and cDNA synthesis protocols used for angiosperms often yields low-quality templates. Furthermore, traditional restriction enzyme ligation cloning methods rely on restriction endonuclease sites, resulting in lengthy and time-consuming procedures, and extremely low success rates for fern genes lacking detailed restriction maps. In the genetic transformation stage, conventional liquid nitrogen freeze-thaw or electroporation methods for transforming Agrobacterium are inefficient for large plasmids or specific strains; while traditional Agrobacterium-mediated transformation of ferns also suffers from low transformation efficiency and numerous chimeras. These shortcomings collectively make fern gene cloning and transformation a technological bottleneck restricting the development of functional genomics in this field.

[0037] In view of this, in order to overcome the shortcomings of related technologies, the method for cloning and transforming fern genes in this embodiment combines... Figure 1 In terms of overall design, it includes:

[0038] Step S1: Using the homologous gene sequence of a specific plant as a probe, perform homologous alignment in the genome of ferns to obtain the target gene sequence information.

[0039] The specific plant in step S1 above may preferably include, for example, one of bryophytes, lycophytes, or Arabidopsis thaliana.

[0040] It is worth noting that homologous gene sequences refer to gene sequences that originate from a common ancestral DNA sequence in different species or the same genome, and usually have similar structures or functions.

[0041] Bryophytes are the closest non-vascular plant relatives of ferns among terrestrial plants, sharing many common basic characteristics of terrestrial life. At the same time, as model organisms, bryophytes have complete genetic information and are a ready-made probe library.

[0042] Lycophytes are one of the earliest divergent branches among extant terrestrial plants and the basal group of all vascular plants. Their genomes evolve very slowly and have a highly stable structure. Using them as probes to compare homologous genes in ferns yields a higher success rate.

[0043] Arabidopsis thaliana was the first plant genome to be fully sequenced. The functions, domains, interacting proteins, and expression patterns of almost all of its genes have been precisely analyzed, and it is often used for gene function research.

[0044] Step S2: Using one or more of the gametophytes, sporophytes, or mixed tissues of ferns as materials, extract total RNA and reverse transcribe it to synthesize cDNA.

[0045] The gametophyte is the structure in a plant that is specifically responsible for producing gametes (sperm and egg cells) during its life process; the cells of the gametophyte contain only one set of chromosomes. The sporophyte is mainly responsible for producing fertilized eggs; the roots, stems, and leaves of ferns all belong to the sporophyte.

[0046] The primers used for reverse transcription in step S2 above are preferably, for example, Oligo dT primers.

[0047] The reverse transcriptase used for reverse transcription is preferably, for example, M-MuLV reverse transcriptase.

[0048] It's worth noting that Oligo dT primers are short, single-stranded DNA fragments composed of a string of deoxythymidine nucleotides (dT). Their core function is to utilize the poly(A) tail at the 3' end of eukaryotic mRNA for complementary pairing. During reverse transcription to synthesize cDNA, after Oligo dT binds to the poly(A) tail of mRNA, reverse transcriptase uses the mRNA as a template to synthesize the first cDNA strand starting from the 3' end. Compared to random primers, Oligo dT primers can more specifically reverse transcribe only mRNA with a poly(A) tail from total RNA.

[0049] M-MuLV reverse transcriptase (Moloney Murine Leukemia Virus Reverse Transcriptase) is an RNA-dependent DNA polymerase isolated from Moloney murine leukemia virus. It is one of the most commonly used enzymes in molecular biology for synthesizing cDNA, possessing RNA-dependent DNA polymerase activity and the ability to degrade the RNA template in RNA-DNA hybrid strands. In this embodiment, its core function is to synthesize complementary first-strand cDNA using RNA as a template, starting with primers.

[0050] Step S3: Perform PCR amplification using cDNA as a template to obtain PCR amplification products.

[0051] In step S3 above, PCR amplification is performed using the cDNA as a template to obtain PCR amplification products. A preferred embodiment may include, for example:

[0052] Using cDNA as a template, PCR amplification was performed using high-fidelity DNA polymerase, and the amplification products were purified to obtain the PCR amplification products.

[0053] Among them, the preferred method for purifying the amplification products is, for example, low-melting-point agarose gel electrophoresis gel cutting and recovery technology.

[0054] It is worth noting that high-fidelity DNA polymerases are a class of DNA polymerases with 3'→5' exonuclease activity in molecular biology. In this embodiment, the preferred high-fidelity DNA polymerase is, for example, NEB Q5 polymerase.

[0055] Low-melting-point agarose gel electrophoresis gel extraction and recovery technology is used to separate and purify specific DNA fragments from agarose gels. Compared to conventional agarose, low-melting-point agarose has a lower melting and freezing point, which allows the gel to be melted and the DNA released under mild conditions without denaturing the DNA.

[0056] Step S4: Perform a topoisomerase-mediated ligation reaction between the PCR amplification product and the pENTR / D-TOPO introductory vector, and introduce the resulting ligation product into E. coli. The pENTR-target gene plasmid is obtained by colony PCR identification and sequencing verification.

[0057] The sequencing verification in step S4 above, as a preferred implementation, can be, for example, Sanger sequencing using M13F / R primers. Sequencing the target fragment cloned into the pENTR / D-TOPO vector using M13F / R primers is a standard method for verifying the correctness and orientation of the inserted fragment sequence.

[0058] Step S5: Mix the pENTR-target gene plasmid with the target vector, add LR Clonase II enzyme mixture to carry out recombination reaction, and introduce the obtained reaction product into Escherichia coli to obtain Escherichia coli containing the target expression vector.

[0059] It is worth noting that the function of LR Clonase II enzyme is to catalyze the in vitro recombination reaction between gene plasmids and target vectors, thereby rapidly and efficiently transferring the target gene into different expression vectors.

[0060] The target vector in step S5 above can preferably be, for example, one of the promoter-GFP-HDEL reporter vector or RNAi vector.

[0061] The promoter-GFP-HDEL reporter vector is preferably, for example, the pMDC204 vector. The RNAi vector is preferably, for example, the pH7GWIWG2(I) vector.

[0062] Step S6: After culturing the Escherichia coli containing the target expression vector and transforming it with Agrobacterium, the bacteria are screened to obtain Agrobacterium monoclonal strains.

[0063] In step S6 above, the Escherichia coli containing the target expression vector is cultured. As a preferred embodiment, the three-parent mating method can be used for culturing.

[0064] Specifically, the three-parent mating method is a method that utilizes the conjugation of three types of bacteria (donor, helper bacteria, and recipient) to efficiently introduce the target plasmid into recipient bacteria that are difficult to transform.

[0065] The strains used in the three-parent mating method may preferably include, for example, helper Escherichia coli and Agrobacterium tumefaciens receptor strains.

[0066] Specifically, the helper Escherichia coli preferably carries, for example, a helper plasmid that can provide conjugation transfer function.

[0067] For example, EC282 Escherichia coli can be used as an auxiliary Escherichia coli.

[0068] The preferred recipient strain of Agrobacterium tumefaciens can be, for example, Agrobacterium tumefaciens recipient strain GV3101.

[0069] In summary, the fern gene cloning and transformation method of this embodiment overcomes the limitation of insufficient genome annotation information in ferns by using homologous gene sequences of specific plants as probes for comparison, thus improving the success rate of obtaining target gene sequences. Simultaneously, the selection of gametophytes, sporophytes, or a mixture thereof as RNA extraction materials fully considers the gene expression characteristics of different life generations of ferns, ensuring the integrity and representativeness of the cDNA template. Furthermore, by employing topoisomerase-mediated ligation and LR recombination reactions, a cloning strategy that does not rely on restriction endonuclease sites can be formed. This not only simplifies the operation process but also fundamentally avoids the risk of construction failure due to incomplete fern gene restriction map information.

[0070] Ultimately, by culturing and transforming with Agrobacterium, and screening to obtain monoclonal strains, and using them in the final step of infection and other synergistic processes, the cloning system and genetic transformation process were smoothly connected, jointly ensuring the overall success rate of obtaining transgenic fern materials.

[0071] An embodiment of the second aspect of this application provides an application of the above-mentioned method for cloning and transforming fern genes.

[0072] A preferred application of the above-mentioned technologies is in fern gene function research.

[0073] Example 1

[0074] Example 1 illustrates the cloning of the full-length coding sequence of CrTARGET1, a gene related to the reproductive development of the water fern (Ceratopteris richardii), and the construction of its RNAi silencing vector. The specific steps are as follows:

[0075] Step S1: In the Arabidopsis thaliana genome database (TAIR), a gene AtTARGET1, which is known to be involved in pollen tube guidance, was retrieved. Using the predicted amino acid sequence of this gene as a probe, tBLASTn homology was performed in the Phytozome (Ceratopteris richardii v2.0) database, obtaining a highly homologous candidate sequence, named CrTARGET1. Based on the obtained genome and predicted mRNA sequence, specific primers for amplifying its full-length CDS were designed using Primer Premier 6.0 software.

[0076] The primers were specifically validated using the IDT PrimerQuest tool to confirm that they produced only a single band in the target genome.

[0077] Step S2: Take approximately 100 mg each of mature sporophytes (including rhizomes, vegetative leaves, and young sporophytes) and independently growing gametophytes (heart-shaped prothallus stage) from wild-type water fern (ecotype: Hn-n), and grind them thoroughly in liquid nitrogen. Extract total RNA using Plant RNA Purification Reagent (Invitrogen) according to the manufacturer's recommended protocol (including on-column digestion with Dnase I). NanoDrop microspectrophotometer analysis showed that the A260 / A280 ratio of the extracted total RNA was 2.01, and the A260 / A230 ratio was 2.12, indicating good RNA purity. 1.2% agarose denaturing gel electrophoresis revealed clear 28S rRNA and 18S rRNA bands with no significant degradation.

[0078] Use 2 μg of total RNA as a template, add 1 μL of Oligo dT primer (50 μM, NEB), and add DEPC-treated water to a final volume of 10 μL. Denature at 65°C for 5 min, then immediately incubate on ice. Next, add the M-MuLV reverse transcriptase reaction mixture (containing 1×M-MuLV reaction buffer, 0.5 mM dNTP mixture, 10 units of RNase inhibitor, and 100 units of M-MuLV reverse transcriptase, NEB), for a total reaction volume of 20 μL. Incubate at 42°C for 60 min, then inactivate at 80°C for 5 min to obtain the first-strand cDNA. Store the obtained cDNA product at -20°C for later use.

[0079] Step S3: Using the cDNA synthesized in step S2 as a template, perform PCR amplification. The PCR reaction system is 50 μL, containing: 1×Q5 reaction buffer (NEB), 0.2 mM dNTP mixture, 0.5 μM forward primer CrTARGET1-F, 0.5 μM reverse primer CrTARGET1-R, 1 μL cDNA template, 0.5 μL Q5 high-fidelity DNA polymerase (NEB, 2 U / μL), and add sterile ultrapure water to a final volume of 50 μL. PCR reaction program: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 62℃ annealing for 20 s, 72℃ extension for 90 s (estimated based on Q5 polymerase extension rate of approximately 30 s / kb), for a total of 35 cycles; final extension at 72℃ for 5 min.

[0080] Five μL of PCR product was analyzed by 1.0% agarose gel electrophoresis. The results showed a bright main band at approximately 1.5 kb, consistent with the expected CDS length, with no nonspecific amplification bands. The remaining 45 μL of PCR product was separated by electrophoresis on a low-melting-point agarose gel (Lonza SeaPlaque, 1.0%). The gel block containing the target band was rapidly excised under long-wavelength UV transillumination. The gel was then excised and recovered using the Monarch DNA Gel Extraction Kit (NEB), with a final elution volume of 20 μL. The purified product concentration was determined to be 42 ng / μL by micro-spectrophotometry.

[0081] Step S4: Take 2 μL (about 84 ng) of the purified PCR product and mix it with 0.5 μL of pENTR / D-TOPO entry vector (ThermoFisher, 15-20 ng / μL). Add 0.5 μL of salt solution (1.2 M NaCl, 0.06 M MgCl2) and add sterile ultrapure water to a total reaction volume of 3 μL. After gently mixing, incubate at room temperature (23℃) for 15 min to carry out the topoisomerase-mediated ligation reaction.

[0082] All 3 μL of ligation product was transformed into 50 μL of chemocompetent *E. coli* TOP10 cells using a heat shock method: ice bath for 30 min, heat shock at 42°C for 45 s, ice bath for 2 min, 250 μL of SOC liquid medium was added, and the cells were cultured at 37°C with shaking at 200 rpm for 1 h. 100 μL of the bacterial culture was evenly spread on LB agar plates containing kanamycin (50 μg / mL) and incubated upside down at 37°C for 16 h.

[0083] The following day, 12 single-clone colonies were picked from the plate and rapid screening was performed using M13F / R universal primers. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 100 s, for a total of 25 cycles; final extension at 72℃ for 7 min. 5 μL of the colony PCR product was analyzed by 1.0% agarose gel electrophoresis. The results showed that 10 clones amplified bands of approximately 1.7 kb (including upstream and downstream sequences of the vector), and were identified as positive clones.

[0084] Three positive clones were selected and inoculated into 5 mL of LB liquid medium containing kanamycin (50 μg / mL) and cultured overnight at 37°C and 220 rpm. 2 mL of the bacterial culture was used to extract the plasmid using the Monarch Plasmid Miniprep Kit (NEB). Using the extracted plasmid as a template, Sanger sequencing was performed using M13F / R primers. The sequencing chromatograms were assembled and analyzed using Sequencher software and compared with the predicted CDS sequence of CrTARGET1 obtained in step S1. The sequence identity was 100%, and the insertion direction was correct, confirming the acquisition of the correct introductory clone plasmid, named pENTR-CrTARGET1. The verified TOP10 E. coli bacterial culture containing pENTR-CrTARGET1 was mixed with 25% sterile glycerol and stored at -80°C.

[0085] Step S5: Using the Gateway LR recombination reaction system, the target gene CDS fragment was transferred from the entry vector pENTR-CrTARGET1 to the RNAi target vector pH7GWIWG2(I) (provided by VIB-Gent University, containing the CaMV 35S promoter-driven hairpin structure and hygromycin-resistant plant selection markers).

[0086] The LR recombination reaction system was as follows: 150 ng of pENTR-CrTARGET1 initiator plasmid, 150 ng of pH 7 GWIWG2(I) target vector, 1 μL of LR Clonase II enzyme mixture (Thermo Fisher), and TE buffer (pH 8.0) to a total reaction volume of 5 μL. After gentle mixing, the mixture was incubated at 25°C for 16 h. After the reaction was complete, 0.5 μL of proteinase K solution (provided with the LR Clonase II kit) was added, and the reaction was terminated by incubation at 37°C for 10 min.

[0087] 2 μL of the reaction product was used to transform 50 μL of *E. coli* TOP10 chemocompetent cells using the heat shock method. The transformed cells were then plated on LB agar plates containing spectinomycin (100 μg / mL) and incubated at 37°C for 16 h. Eight single clones were picked from the plates and colony PCR was performed using the internal gene primers CrTARGET1-F and CrTARGET1-R. The results showed that all eight clones amplified the target band of approximately 1.5 kb, indicating that the target gene had been successfully recombined into the target vector.

[0088] To further verify the structural integrity of the recombinant, three colony-positive PCR clones were cultured and plasmids were extracted. Restriction endonuclease Hind III was used for digestion analysis. The expected characteristic band sizes were approximately 3.2 kb, 2.1 kb, and 1.4 kb, respectively. Agarose gel electrophoresis results were completely consistent with expectations, confirming the correct construction of the recombinant expression vector. The plasmid was named pH7GWIWG2(I)-CrTARGET1-RNAi.

[0089] Step S6: Using the three-parental mating method, the constructed expression vector pH7GWIWG2(I)-CrTARGET1-RNAi was introduced into the Agrobacterium tumefaciens GV3101 recipient strain (denoted as AG038).

[0090] The strains required for triparental mating and their culture conditions are as follows:

[0091] donor bacteria Top 10 E. coli containing pH7GWIWG2(I)-CrTARGET1-RNAi LB + spectinomycin (100 μg / mL) 37℃, 220rpm, 12h Support bacteria EC282 Escherichia coli (carrying helper plasmid) LB + kanamycin (50 μg / mL) 37℃, 220 rpm, 12h Receptor bacteria GV3101 Agrobacterium tumefaciens receptor strain LB + Rifampin (50 μg / mL) + Gentamicin (30 μg / mL) 28℃, 220 rpm, 24h

[0092] Take 200 μL of each of the three bacterial suspensions and aseptically transfer them to the same 1.5 mL centrifuge tube. Mix thoroughly, then use a sterile inoculation loop to draw a square area of ​​approximately 1 cm × 1 cm on the surface of an LB agar plate without any antibiotics. Incubate the plate in a 30 °C incubator for 48 h.

[0093] After cultivation, an appropriate amount of bacterial cells was scraped from the square area using a sterile inoculation loop and spread onto LB agar plates containing triple antibiotics (rifampin 50 μg / mL, gentamicin 30 μg / mL, spectinomycin 100 μg / mL) using a 10-fold serial dilution method. The plates were then incubated upside down at 30°C for 48 h. Under these screening conditions, rifampin and gentamicin maintained the growth screening pressure of the GV3101 recipient bacteria, while spectinomycin screened for successfully conjugated strains containing the target expression vector. Results showed that at a dilution of 10... -3 On the plate, approximately 50 morphologically uniform monoclonal colonies were obtained.

[0094] Twelve single-clone colonies were randomly selected from the screening plate and identified by colony PCR using primers specific to the hygromycin resistance gene sequence carried by the vector. All 12 clones amplified specific bands of the expected length (approximately 1.0 kb), confirming that the expression vector pH7GWIWG2(I)-CrTARGET1-RNAi had been successfully introduced into Agrobacterium GV3101, with a positive rate of 100%. One positive clone was selected and named GV3101-pH7GWIWG2(I)-CrTARGET1-RNAi, and stored at -80°C with a final concentration of 25% glycerol.

[0095] Step S7: Activate the preserved GV3101-pH7GWIWG2(I)-CrTARGET1-RNAi strain by streaking it onto an LB agar plate containing triple antibiotics and incubate at 30°C for 48 h. Pick single colonies and inoculate them into 10 mL of LB liquid medium containing the same antibiotics, incubating at 30°C and 220 rpm for approximately 16 h. Transfer the cells to 50 mL of fresh LB liquid medium containing antibiotics at a 1:50 ratio and continue culturing until the OD600 value is approximately 0.8 (logarithmic growth phase). Collect the cells by centrifugation at 5000 rpm for 10 min, resuspend them in an equal volume of infection buffer (1 / 2 MS salt solution containing 100 μM acetylsalicylic acid, pH 5.6), adjust the OD600 value to 0.6, and incubate at room temperature (23°C) for 2 h to induce Vir region gene expression.

[0096] Wild-type water fern (ecotype: Hn-n) spores were germinated in 1 / 2 MS sugar-free liquid medium. After about 10 days, young gametophytes in the 4-8 cell development stage or early heart-shaped prothallium stage were collected as transformation recipient materials.

[0097] The above gametophyte material was immersed in pre-induced Agrobacterium infection solution and gently shaken (about 60 rpm) for 30 min. After infection, excess bacterial solution on the surface of the gametophytes was absorbed with sterile filter paper, and the gametophytes were transferred to co-culture medium (1 / 2 MS salt solution, 1% sucrose, 0.8% agar, 100 μM acetylsyl syringone, pH 5.6) and incubated in the dark at 25°C for 3 days.

[0098] After co-culturing, the gametophytes were rinsed three times with sterile water containing cefotaxime sodium (250 μg / mL), 5 min each time, to thoroughly remove any attached Agrobacterium. After rinsing, the gametophytes were blotted dry with sterile filter paper and transferred to selection and regeneration medium (1 / 2 MS salt solution, 1% sucrose, 0.8% agar, 250 μg / mL cefotaxime sodium, 25 μg / mL hygromycin B, pH 5.8) and cultured at 25°C under a 16 h light / 8 h dark light cycle. The selection and regeneration medium was changed every two weeks with the same formulation.

[0099] After 6-8 weeks of continuous screening and culture, some gametophyte materials were observed to remain green and differentiate normally into sporophytes and young leaves on the screening regeneration medium, indicating a potential transformation event. In contrast, wild-type untransformed control gametophytes completely browned and died under the same screening conditions. The preliminary transformation efficiency was calculated to be approximately 5% (number of transformation events / number of initially infected gametophytes). Genomic DNA was extracted from the hygromycin-resistant plants, and PCR detection was performed using hygromycin resistance gene-specific primers (Hyg-F / R, sequence same as in step six). All samples amplified the expected band of approximately 1.0 kb, preliminarily confirming them as positive transgenic *Aquaticus* lines. These transgenic plants will be used for subsequent analysis of the CrTARGET1 gene silencing efficiency and observation of reproductive development phenotypes. Results are as follows... Figure 2 and Figure 3 The diagram shown is an experimental illustration of gametophyte culture of the water fern Ceratopteris richardii.

[0100] Example 2

[0101] Example 2 describes the cloning of the promoter of the Ceratopteris richardii CrTARGET1 gene and the construction of the promoter-driven GFP-HDEL reporter vector. The specific steps are as follows:

[0102] Step S1: Based on the genomic sequence information of the CrTARGET1 gene identified in Example 1, extract a sequence region approximately 2000 bp upstream of its start codon ATG as a candidate promoter sequence. Use Primer Premier 6.0 software to design specific primers for amplifying this promoter region.

[0103] The primers were specifically validated using the IDT PrimerQuest tool to confirm that they produced only a single band in the target genome.

[0104] Step S2: Take approximately 200 mg of mature sporophytes and young sporophytes from wild-type water fern and grind them thoroughly in liquid nitrogen. Genomic DNA was extracted using the Plant DNA Extraction Kit (Qiagen DNeasy Plant Mini Kit) according to the manufacturer's recommended protocol. NanoDrop microspectrophotometry showed that the extracted genomic DNA had an A260 / A280 ratio of 1.85 and an A260 / A230 ratio of 2.08, indicating good DNA purity. 0.8% agarose gel electrophoresis revealed intact genomic DNA bands with a molecular weight greater than 10 kb and no significant degradation.

[0105] Step S3: Using the genomic DNA extracted in Step S2 as a template, perform PCR amplification. The PCR reaction system is 50 μL, containing: 1×Q5 reaction buffer (NEB), 0.2 mM dNTP mixture, 0.5 μM forward primer CrPRO-F, 0.5 μM reverse primer CrPRO-R, 100 ng genomic DNA template, 0.5 μL Q5 high-fidelity DNA polymerase (NEB, 2 U / μL), and add sterile ultrapure water to a final volume of 50 μL. PCR reaction program: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 60 s, for a total of 35 cycles; final extension at 72℃ for 5 min.

[0106] Five μL of PCR product was analyzed by 1.0% agarose gel electrophoresis. The results showed a bright main band at approximately 2.0 kb, consistent with the expected promoter region length, with no nonspecific amplification bands. The remaining 45 μL of PCR product was separated by electrophoresis on a low-melting-point agarose gel (Lonza SeaPlaque, 1.0%). The gel block containing the target band was rapidly excised under long-wavelength UV transillumination. The gel was then excised and recovered using the Monarch DNA Gel Extraction Kit (NEB), with a final elution volume of 20 μL. The purified product concentration was determined to be 38 ng / μL by micro-spectrophotometry.

[0107] Step S4: Take 2 μL (about 76 ng) of the purified PCR product and mix it with 0.5 μL of pENTR / D-TOPO entry vector (ThermoFisher, 15-20 ng / μL). Add 0.5 μL of salt solution (1.2M NaCl, 0.06M MgCl2) and add sterile ultrapure water to a total reaction volume of 3 μL. After gently mixing, incubate at room temperature (23℃) for 15 min to carry out the topoisomerase-mediated ligation reaction.

[0108] All 3 μL of ligation product was transformed into 50 μL of chemocompetent *E. coli* TOP10 cells using a heat shock method: ice bath for 30 min, heat shock at 42°C for 45 s, ice bath for 2 min, 250 μL of SOC liquid medium was added, and the cells were cultured at 37°C with shaking at 200 rpm for 1 h. 100 μL of the bacterial culture was then evenly spread onto LB agar plates containing kanamycin (50 μg / mL) and incubated upside down at 37°C for 16 h.

[0109] The following day, ten single-clone colonies were picked from the plate and rapid colony PCR screening was performed using M13F / R universal primers. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 120 s, for a total of 25 cycles; final extension at 72℃ for 7 min. 5 μL of the colony PCR product was analyzed by 1.0% agarose gel electrophoresis. The results showed that eight clones amplified bands of approximately 2.2 kb (including upstream and downstream sequences of the vector), and were identified as positive clones.

[0110] Three positive clones were selected and inoculated into 5 mL of LB liquid medium containing kanamycin (50 μg / mL) and cultured overnight at 37°C and 220 rpm. 2 mL of the bacterial culture was used to extract plasmids using the Monarch Plasmid Miniprep Kit (NEB). Using the extracted plasmid as a template, Sanger sequencing was performed using M13F / R primers. The sequencing chromatograms were assembled and analyzed using Sequencher software and compared with the CrTARGET1 promoter candidate sequence extracted in step S1. The sequence identity was 100%, and the insertion direction was correct, confirming the acquisition of the correct introductory cloning plasmid, named pENTR-CrPRO. The verified TOP10 E. coli bacterial culture containing pENTR-CrPRO was mixed with 25% sterile glycerol and stored at -80°C.

[0111] Step S5: Using the Gateway LR recombination reaction system, the promoter sequence was transferred from the entry vector pENTR-CrPRO to the promoter analysis target vector pMDC204 (containing the GFP-HDEL reporter gene and hygromycin resistance plant selection markers, provided by the Arabidopsis thaliana biological resource center ABRC).

[0112] The LR recombination reaction system was as follows: 150 ng of pENTR-CrPRO initiator plasmid, 150 ng of pMDC204 target vector, 1 μL of LRClonase II enzyme mixture (Thermo Fisher), and TE buffer (pH 8.0) was added to a total reaction volume of 5 μL. After gentle mixing, the mixture was incubated at 25°C for 16 h. After the reaction was completed, 0.5 μL of proteinase K solution was added, and the reaction was terminated by incubation at 37°C for 10 min.

[0113] Two μL of the reaction product was used to transform 50 μL of *E. coli* TOP10 chemocompetent cells using the heat shock method. The transformed cells were then plated on LB agar plates containing kanamycin (50 μg / mL) and incubated at 37°C for 16 h. Six single clones were picked from the plates and colony PCR was performed using the promoter forward primer CrPRO-F and the GFP reverse primer. The results showed that all six clones amplified the target band of approximately 2.7 kb (containing the promoter and a portion of the GFP sequence), indicating that the promoter had been successfully recombined into the target vector and fused with the GFP-HDEL reporter gene.

[0114] To further verify the structural integrity of the recombinant, three colony-positive PCR clones were cultured and plasmids were extracted. Restriction endonuclease Sac I was used for digestion analysis. The expected characteristic band sizes were approximately 5.8 kb and 2.1 kb, respectively. Agarose gel electrophoresis results were completely consistent with expectations, confirming the correct construction of the recombinant expression vector. The plasmid was named pMDC204-CrPRO-GFP-HDEL.

[0115] Step S6: Using the three-parental mating method, the constructed expression vector pMDC204-CrPRO-GFP-HDEL was introduced into the Agrobacterium tumefaciens GV3101 recipient strain.

[0116] The strains required for triparental mating and their culture conditions are as follows:

[0117] donor bacteria Top 10 Escherichia coli containing pMDC204-CrPRO-GFP-HDEL LB + kanamycin (50 μg / mL) 37℃, 220 rpm, 12h Support bacteria EC282 Escherichia coli (carrying helper plasmid) LB + kanamycin (50 μg / mL) 37℃, 220 rpm, 12h Receptor bacteria GV3101 Agrobacterium tumefaciens receptor strain LB + Rifampin (50 μg / mL) + Gentamicin (30 μg / mL) 28℃, 220 rpm, 24h

[0118] Take 200 μL of each of the three bacterial suspensions and aseptically transfer them to the same 1.5 mL centrifuge tube. Mix thoroughly, then use a sterile inoculation loop to draw a square area of ​​approximately 1 cm × 1 cm on the surface of an LB agar plate without any antibiotics. Incubate the plate in a 30 °C incubator for 48 h.

[0119] After incubation, an appropriate amount of bacterial cells was scraped from the square area using a sterile inoculation loop and spread onto LB agar plates containing triple antibiotics (rifampin 50 μg / mL, gentamicin 30 μg / mL, kanamycin 50 μg / mL) using a 10-fold serial dilution method. The plates were then incubated upside down at 30°C for 48 h. Under these screening conditions, rifampin and gentamicin maintained the growth screening pressure for the GV3101 recipient bacteria, while kanamycin screened for successfully conjugated strains containing the target expression vector. The results showed that approximately 45 morphologically uniform single colonies were obtained on plates with a dilution factor of 10⁻³.

[0120] Ten single-clone colonies were randomly selected from the screening plate, and colony PCR identification was performed using primers specific to the hygromycin resistance gene sequence carried by the vector. All ten clones amplified specific bands of the expected length (approximately 1.0 kb), confirming that the expression vector pMDC204-CrPRO-GFP-HDEL had been successfully introduced into Agrobacterium GV3101, with a positive rate of 100%. One positive clone was selected and named GV3101-pMDC204-CrPRO-GFP-HDEL, added to a final concentration of 25% glycerol, and stored at -80℃.

[0121] Step S7: Activate the preserved GV3101-pMDC204-CrPRO-GFP-HDEL strain by streaking it onto an LB agar plate containing triple antibiotics and incubate at 30°C for 48 h. Pick single clones and inoculate them into 10 mL of LB liquid medium containing the same antibiotics, incubating at 30°C and 220 rpm for approximately 16 h. Transfer the cells to 50 mL of fresh LB liquid medium containing antibiotics at a 1:50 ratio and continue culturing until the OD600 value is approximately 0.8 (logarithmic growth phase). Collect the bacterial cells by centrifugation at 5000 rpm for 10 min, resuspend them in an equal volume of infection buffer (1 / 2 MS salt solution containing 100 μM acetylsylcholine, pH 5.6), adjust the OD600 value to 0.6, and incubate at room temperature (23°C) for 2 h to induce Vir region gene expression.

[0122] Wild-type water fern (ecotype: Hn-n) spores were germinated in 1 / 2 MS sugar-free liquid medium. After about 10 days, young gametophytes in the 4-8 cell development stage or early heart-shaped prothallium stage were collected as transformation recipient materials.

[0123] The above gametophyte material was immersed in pre-induced Agrobacterium infection solution and gently shaken (approximately 60 rpm) for 30 min. After infection, excess bacterial solution was absorbed from the surface of the gametophytes using sterile filter paper, and the gametophytes were transferred to co-culture medium (1 / 2 MS salt solution, 1% sucrose, 0.8% agar, 100 μM acetylsyl syringone, pH 5.6) and incubated in the dark at 25°C for 3 days.

[0124] After co-culturing, the gametophytes were rinsed three times with sterile water containing cefotaxime sodium (250 μg / mL), 5 min each time, to thoroughly remove any attached Agrobacterium. After rinsing, the gametophytes were blotted dry with sterile filter paper and transferred to selection and regeneration medium (1 / 2 MS salt solution, 1% sucrose, 0.8% agar, 250 μg / mL cefotaxime sodium, 25 μg / mL hygromycin B, pH 5.8) and cultured at 25°C under a 16 h light / 8 h dark light cycle. The selection and regeneration medium was changed every two weeks with the same formulation.

[0125] After continuous screening and culture for 6-8 weeks, transgenic *Adiantum capillus-veneris* gamete systems resistant to hygromycin were obtained on screening regeneration medium. Genomic DNA was extracted from the resistant plants, and PCR detection was performed using primers specific to the hygromycin resistance gene. Expected bands of approximately 1.0 kb were amplified in all cases, confirming them as positive transgenic *Adiantum capillus-veneris* lines.

[0126] Positive transgenic *Adiantum capillus-veneris* gametophytes were examined under a fluorescence stereomicroscope to observe the GFP green fluorescence signal (excitation wavelength 488 nm, emission wavelength 507 nm). The results showed that the GFP fluorescence signal was mainly concentrated in the cells surrounding the archegonia (female reproductive organs) and the meristematic region of young sporophytes, while almost no fluorescence signal was observed in vegetative cells and the marginal cells of the thallus. As a control, no GFP fluorescence signal was detected in *Adiantum capillus-veneris* gametophytes transformed with only the pMDC204 empty vector (without promoter insertion) under the same conditions. These results indicate that the CrTARGET1 gene promoter has significant reproductive tissue- and meristematic tissue-specific expression-driving activity.

[0127] As can be seen from Examples 1 and 2 above, the fern gene cloning and transformation method of this application can complete the construction and verification of the entry cloning plasmid from the target gene in about 2-3 weeks, complete the LR recombination construction of the RNAi expression vector in 1 week, and combine the three-parent mating method and Agrobacterium-mediated gametophyte transformation to obtain screened and verified transgenic aquatic fern materials in about 3 months, thus improving the efficiency and success rate of cloning and transformation.

[0128] Furthermore, the fern gene cloning and transformation method described in this application is also applicable to the cloning and functional analysis of gene regulatory sequences such as promoters. By flexibly combining the Gateway cloning system's entry vector with different types of target vectors, it is possible to quickly switch between various functional research objectives, such as gene coding sequence cloning and promoter activity analysis, without changing the basic experimental procedures.

[0129] The above descriptions are merely some embodiments of this application and are not intended to limit this application. The technical features or structures in the foregoing different embodiments can be arbitrarily combined to form other specific technical solutions as needed. For those skilled in the art, this application can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of the claims of this application.

Claims

1. A method for cloning and transforming genes in fern plants, characterized in that, The method includes: Using homologous gene sequences of specific plants as probes, homology comparisons are performed in the genomes of ferns to obtain target gene sequence information; Using one or more of the gametophytes, sporophytes, or mixed tissues of ferns as materials, total RNA was extracted and reverse transcribed to synthesize cDNA; The cDNA was used as a template for PCR amplification to obtain the PCR amplification product. The PCR amplification product was ligated with the pENTR / D-TOPO introductory vector using a topoisomerase-mediated ligation reaction. The resulting ligation product was then introduced into Escherichia coli. The pENTR-target gene plasmid was obtained by colony PCR identification and sequencing verification. The pENTR-target gene plasmid was mixed with the target vector, and a mixture of LR Clonase II enzymes was added to carry out a recombination reaction. The resulting reaction product was introduced into E. coli to obtain E. coli containing the target expression vector. Escherichia coli containing the target expression vector were cultured and screened to obtain monoclonal strains; The monoclonal strain was used to infect fern materials and culture them to obtain transgenic fern materials.

2. The method for cloning and transforming fern genes according to claim 1, characterized in that: The specific plant includes one of the following: bryophytes, lycophytes, or Arabidopsis thaliana.

3. The method for cloning and transforming fern genes according to claim 1, characterized in that: The primers used for reverse transcription are Oligo dT primers; The reverse transcriptase used for the reverse transcription is M-MuLV reverse transcriptase.

4. The method for cloning and transforming fern genes according to claim 1, characterized in that, The PCR amplification product obtained by using the cDNA as a template includes: Using the cDNA as a template, PCR amplification was performed using high-fidelity DNA polymerase, and the amplification product was purified to obtain the PCR amplification product. The purification of the amplification product is carried out using a low-melting-point agarose gel electrophoresis gel cutting and recovery technique.

5. The method for cloning and transforming fern genes according to claim 1, characterized in that: The sequencing verification was performed using Sanger sequencing with M13F / R primers.

6. The method for cloning and transforming fern genes according to claim 1, characterized in that: The method for culturing Escherichia coli containing the target expression vector employs a three-parent mating method.

7. The method for cloning and transforming fern genes according to claim 6, characterized in that: The strains used in the triparental mating method include helper Escherichia coli and Agrobacterium tumefaciens recipient strains.

8. The method for cloning and transforming fern genes according to claim 7, characterized in that: The helper E. coli carries a helper plasmid that can provide conjugation transfer function; The Agrobacterium tumefaciens recipient strain used is the GV3101 Agrobacterium tumefaciens recipient strain.

9. The application of the method for cloning and transforming fern genes as described in any one of claims 1-8, characterized in that: Application of the cloning and transformation methods in fern gene function research.

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