A method for efficiently and stably transforming porphyra yezoensis by agrobacterium

Abstract

The application provides a method for efficiently and stably transforming algae strains of Porphyra yezoensis by Agrobacterium mediation, and belongs to the technical field of plant transgenesis. The application uses a mixed suspension of leaf thallus fragments, single spores and leaf thallus seedlings of Porphyra yezoensis as a transformation receptor, and establishes an efficient and stable genetic transformation technology through steps such as optimization of an infection solution and gradient screening culture. The application effectively solves the technical problems of low genetic transformation efficiency, poor repeatability and difficulty in screening of transformants of the existing Porphyra yezoensis, and the transformation efficiency can reach 30% to 40%, and the genetic stability of positive plants is close to 100%, thereby providing a reliable technical platform for gene function verification, trait improvement and molecular breeding of Porphyra yezoensis, and having important scientific research value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant transgenic technology, and in particular relates to a method for efficient and stable genetic transformation of Porphyra yezoensis mediated by Agrobacterium. Background Technology

[0002] Porphyra tenuifolia ( Pyropia yezoensis Porphyra yezoensis is an important economic seaweed with extremely high nutritional and economic value, playing a vital role in aquaculture in coastal areas. With the development of molecular biology techniques, genetic engineering to improve Porphyra yezoensis and cultivate new, stress-resistant, high-yielding, and high-quality varieties has become an important direction for the sustainable development of the seaweed aquaculture industry. Genetic transformation systems are the core technological foundation of genetic engineering breeding. However, due to the unique cell structure of Porphyra yezoensis (complex cell wall composition, difficulty in obtaining protoplasts) and its unique physiological metabolism, existing genetic transformation technologies are limited by the characteristics of the recipient material and the suitability of the transformation system, resulting in significant technical bottlenecks. Currently, genetic transformation methods for Porphyra yezoensis mainly include protoplast electroporation and gene gun methods, but these methods have significant drawbacks: electroporation causes significant damage to protoplasts, resulting in low survival rates and transformation efficiency of only 3%–5%; gene gun methods involve expensive equipment, complex operation, and are prone to multiple copy insertion of the target gene, affecting plant stability; furthermore, existing transformation systems generally suffer from poor applicability of screening marker genes, high false positive rates in transformants, and unstable expression of exogenous genes, severely limiting the development of genetic engineering for Porphyra yezoensis.

[0003] Agrobacterium-mediated genetic transformation (AGM-mediated transformation) technology boasts advantages such as ease of operation, low cost, high single-copy insertion rate of exogenous genes, and stable expression, and has been widely applied to the genetic transformation of terrestrial plants. However, its application in marine algae is still immature. Since *Porphyra yezoensis* is a marine eukaryotic alga, its growth environment (high salinity, low temperature) differs significantly from that of terrestrial plants. Therefore, the infection efficiency, co-culture conditions, and screening systems of *AGM-mediated transformation* cannot be directly adapted from the techniques used for terrestrial plants. Thus, constructing an *AGM-mediated genetic transformation system adapted to the biological characteristics of *Porphyra yezoensis*, and overcoming key technical bottlenecks such as *AGM-mediated transformation* cell compatibility, optimized infection conditions, and efficient specific screening, is of great significance for advancing the molecular breeding process of *Porphyra yezoensis*. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for the efficient and stable genetic transformation of Porphyra yezoensis mediated by Agrobacterium, with a transformation efficiency of 30% to 40% and a genetic stability of nearly 100% for positive plants.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] A method for efficient and stable genetic transformation of Porphyra yezoensis mediated by Agrobacterium tumefaciens includes the following steps: immersing Porphyra yezoensis recipient material in Agrobacterium tumefaciens infection solution for infection, and after infection, transferring it to hygromycin B sterile seawater culture medium for gradient screening to obtain new plants containing the target gene;

[0007] The Porphyra stripes receptor material is a mixed suspension of single spores, tissue fragments, and thallus seedlings;

[0008] The Agrobacterium infection solution contained 100 μM acetylsalicylic acid and OD200. 600 Sterile seawater with a concentration of 0.5;

[0009] The gradient screening consisted of primary screening on 2 mg / L hygromycin B sterile seawater culture medium and secondary screening on 4 mg / L hygromycin B sterile seawater culture medium.

[0010] Preferably, the method for preparing the receptor material is as follows: select the leaf-like body of Porphyra stripes, remove the base and edge tissues, cut the remaining part into small tissue blocks, place them in sterile seawater culture medium containing PES, and incubate at a constant temperature of 15±2℃ for 10~15 days.

[0011] More preferably, the leaf-like body of *Porphyra yezoensis* is a healthy, disease-free 30-40 day old *Porphyra yezoensis* leaf-like body.

[0012] More preferably, each fragment of the small tissue block contains 30 to 60 cells.

[0013] More preferably, the mass concentration of the PES is 2%.

[0014] Preferably, the preparation method of the Agrobacterium infection solution is as follows: Agrobacterium cells containing the target gene are collected, resuspended in an equal volume of 30‰ sterile seawater, acetylsuccinone is added to a final concentration of 100 μM, and the OD of the bacterial solution is adjusted. 600 At 0.5, the expression of Vir region genes was induced by static incubation at room temperature.

[0015] Preferably, during the infection process, a vacuum permeation of -0.08±0.01MPa is applied for 30±5 min, followed by co-culturing in a dark environment at 15±2℃ for 48 h to complete the T-DNA transfer.

[0016] More preferably, after the co-culture is completed, the surface Agrobacterium is thoroughly washed with sterile seawater containing 500 mg / L cefotaxime to remove it, and then subsequent gradient screening is performed.

[0017] Preferably, the initial screening culture lasts for 14 days to eliminate untransformed hygromycin-sensitive plants and collect resistant plants; the secondary screening culture lasts for 7 days to eliminate false positive plants from the initial screening resistant plants and obtain genetically stable homozygous resistant plants.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention leverages the unique asexual reproduction life cycle of *Porphyra yezoensis* to construct a composite recipient system centered on single spores, tissue fragments, and thallus seedlings. This system fully utilizes the high totipotency, strong regenerative capacity, and high metabolic activity of cells during the asexual reproduction stage, not only improving the integration efficiency of the target gene but also enhancing the genetic stability of transformed offspring. It solves the problems of low transformation efficiency and difficult regeneration associated with traditional recipient materials (such as thallus and filamentous tissues), providing a high-quality recipient guarantee for efficient transformation.

[0020] This invention optimizes the composition of the infection solution, thereby increasing the conversion efficiency to 30%~40%, which is significantly higher than the existing electroporation method (about 5%) and gene gun method (about 10%), breaking through the technical bottleneck of low infection efficiency of Agrobacterium in marine algae.

[0021] This invention establishes a two-step gradient screening system for hygromycin, which achieves efficient enrichment of transformants through increasing concentration gradients, significantly reducing the false positive rate. Combining multi-dimensional identification at the DNA level (PCR), protein level (Western blot), and transcription level (qRT-PCR), and supplemented by multi-generation genetic stability testing, the authenticity and stability of positive transformed plants can be ensured, with the genetic stability of positive plants approaching 100%.

[0022] This invention requires no expensive equipment or complex operating procedures. The Agrobacterium-mediated transformation method is low-cost, and the recipient material preparation and screening system is simple and easy to implement, making it suitable for large-scale batch preparation of transgenic algae. Attached Figure Description

[0023] Figure 1 GUS staining results for different receptor materials;

[0024] Figure 2 A statistical graph showing the conversion efficiency of different receptor materials;

[0025] Figure 3 The staining results after 14 days of screening for hygromycin (2 mg / mL) concentration.

[0026] Figure 4 The results of staining screening 7 days after the second screening with hygromycin (4 mg / mL);

[0027] Figure 5 for OE-PyGUS DNA level identification results of overexpression transgenic lines;

[0028] Figure 6 for OE-PyGUS Protein level identification results of overexpression transgenic lines;

[0029] Figure 7 The results show the relative expression levels of the mutants.

[0030] Figure 8 The staining is for single-spore passage. In the figure, A~C: first-generation single spores; D: second-generation single spore thallus; E: third-generation single spore thallus; F: fourth-generation single spore thallus.

[0031] Figure 9 The filaments were stained during passage. In the figure, A: WT; B: mutant. Detailed Implementation

[0032] This invention provides a method for efficiently and stably genetically transforming *Porphyra yezoensis* strains mediated by *Agrobacterium*, comprising the following steps: immersing *Porphyra yezoensis* recipient material in *Agrobacterium* infection solution for infection; after infection, transferring the material to a sterile seawater culture medium containing hygromycin B for gradient screening to obtain new plants containing the target gene.

[0033] Specifically:

[0034] (1) Preparation of receptor materials

[0035] In this invention, the recipient material is a mixed suspension of *Porphyra yezoensis* spores, tissue fragments, and thallus seedlings. A preferred method for preparing the recipient material is as follows: select *Porphyra yezoensis* thallus, remove the base and edge tissues, cut the remaining portion into small tissue blocks, and place them in sterile seawater culture medium containing PES, incubating at 15±2℃ for 10-15 days. More preferably, the *Porphyra yezoensis* thallus are 30-40 days old, healthy, and disease-free. More preferably, each fragment of the small tissue block contains 30-60 cells, more preferably 40-50 cells per fragment. Preferably, the mass concentration of PES in the sterile seawater culture medium is 2%. As one possible implementation, after removing the base and edge tissues, place the central region on a glass slide and cut it into small tissue blocks using a scalpel.

[0036] (2) Preparation of the infiltration solution

[0037] In this invention, the Agrobacterium infection solution contains 100 μM acetylsuccinone and OD. 600 The sterile seawater was 0.5‰. The preferred method for preparing the Agrobacterium infection solution was as follows: Agrobacterium cells containing the target gene were collected, resuspended in an equal volume of 30‰ sterile seawater, and acetylsuccinone was added to a final concentration of 100 μM to adjust the OD of the bacterial solution. 600 To 0.5, Vir region gene expression was induced by static incubation at room temperature; more preferably, Vir region gene expression was induced by static incubation at room temperature for 30 min; even more preferably, Agrobacterium tumefaciens LBA4404 was used as the recipient strain, and a binary expression vector containing the target gene and the selection marker gene was transformed into its competent cells. Positive clones were activated by shaking in LB liquid medium until the bacterial culture reached OD500. 600When the pH reaches 0.6~0.8, centrifuge at 4000 rpm for 5 min, discard the supernatant, and collect the bacterial cells to prepare Agrobacterium infection solution.

[0038] (3) Infection and transformation

[0039] In this invention, the recipient material from step (1) is transferred to the infection solution from step (2) for infection and transformation. Preferably, during the infection process, a vacuum permeation of -0.08±0.01MPa for 30±5min is applied to enhance the cell-to-cell interaction between Agrobacterium and the recipient material. Afterward, the material is co-cultured in a dark environment at 15±2℃ for 48h to complete the T-DNA transfer. Further preferably, after the co-culture is completed, the surface Agrobacterium is thoroughly washed with sterile seawater containing 500mg / L cefotaxime to remove the Agrobacterium, and then subsequent gradient screening is performed.

[0040] (4) Gradient screening culture

[0041] In this invention, the gradient screening consists of primary screening on 2 mg / L hygromycin B sterile seawater medium and secondary screening on 4 mg / L hygromycin B sterile seawater medium. Preferably, the primary screening culture is 14 days to eliminate untransformed hygromycin-sensitive plants, collecting resistant plants. The collected resistant plants are then cultured for 7 days to eliminate false-positive plants, obtaining genetically stable homozygous resistant plants. Further preferably, the gradient screening culture temperature is 15±2℃ and the light intensity is 40 μmol / (m²). 2 The light-dark cycle was 12h:12h. After gradient selection culture was completed, the culture was transferred to normal seawater.

[0042] In this invention, it is preferable to further include:

[0043] (5) Testing and identification

[0044] Positive transformed plants were identified by PCR and Western blot, and their genetic stability was tested.

[0045] The transgenic plants of *Porphyra yezoensis* obtained by the above preparation method of the present invention have higher genetic stability compared with mutants prepared by other methods.

[0046] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] (1) Preparation of receptor materials that can be efficiently converted

[0049] A transformation recipient system based on the asexual reproduction characteristics of *Porphyra yezoensis* was constructed: Healthy *Porphyra yezoensis* thallus was selected as experimental material, mechanically disrupted under aseptic conditions, and then placed in sterile seawater medium containing PES and cultured in a constant temperature incubator at 15℃. Utilizing the high totipotency and strong regenerative capacity of *Porphyra yezoensis* cells during the asexual reproduction stage, the material initiated the single-spore release process on day 5 after mechanical disruption. On day 10, a mixed suspension of single spores, thallus seedlings, and tissue fragments was collected to obtain a highly active transformation recipient material for later use.

[0050] (2) Preparation and transformation of the inoculum

[0051] The pretreated recipient material was transferred to a co-culture medium (based on sterile seawater medium with 100 μM acetylsalicylic acid added). High-pressure suction-assisted infection technique was used to enhance the cell-to-cell interaction between Agrobacterium and the recipient material. After aspiration for 30 ± 5 min, the gas was slowly released and the material was co-cultured in the dark at 15 °C for 48 h. The transformation effect was observed by GUS staining.

[0052] The results are as follows Figure 1 and Figure 2 As shown, single spores, thallus seedlings, and tissue fragments all showed specific blue staining signals, with single spores showing the highest efficiency, close to 88%, followed by fragments and seedlings, with transformation efficiencies of 58% and 29%, respectively. Untreated thallus and fragments did not show positive results.

[0053] (3) Screening and cultivation

[0054] A two-step gradient screening system for hygromycins was established to achieve efficient enrichment and purification of transformation materials.

[0055] Initial screening stage: The co-cultured recipient material was transferred to the initial screening medium (sterile seawater + 2 mg / mL hygromycin) and screened at 15°C and a light intensity of 40 μmol / (m²). 2 •s), light-dark cycle 12h: cultured for 14 days under 12h conditions, staining and screening results are as follows Figure 3 As shown, hygromycin-sensitive plants that have not been transformed are eliminated, and resistant plants are initially collected.

[0056] Secondary screening stage: The resistant plants that survived the initial screening were transferred to secondary screening medium (sterile seawater + 4 mg / mL hygromycin) and cultured for another 7 days. The staining screening results are as follows. Figure 4 As shown, false positive plants were further eliminated to obtain genetically stable homozygous resistant plants.

[0057] (4) Identification of positive plants

[0058] The resistant plants obtained through screening were subjected to multi-level identification to ensure the authenticity and stability of the transformation.

[0059] PCR detection: Genomic DNA was extracted from resistant plants, and GUS reporter gene-specific primers were designed (as shown in Table 1) for PCR amplification. The reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55-60℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; final extension at 72℃ for 10 min. The amplified products were detected by agarose gel electrophoresis. Plants showing the target band were considered candidate positive plants, such as... Figure 5 As shown.

[0060] Table 1. Specific primers used for RT-qPCR analysis

[0061]

[0062] Western blot analysis: Total soluble protein was extracted using a plant protein extraction kit (CoWin Biosciences, China, catalog number CW0885M). After the concentration was determined using a BCA protein quantification kit (Beyotime Biotechnology, China, catalog number P0010), an equal amount of nucleoprotein sample was taken for SDS-PAGE electrophoresis separation. The sample was then transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with TBST buffer containing 8% skim milk powder at room temperature for 1 h to block non-specific binding. The blocked membrane was incubated with rabbit monoclonal primary antibody against β-glucuronidase (GUSB) (1:1000 dilution, ABclonal, China, catalog number A19248) at room temperature for 2 hours. After washing with TBST, it was incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H+L) secondary antibody (1:4000 dilution, ABclonal, China, catalog number AS014) at room temperature for 1 hour. Finally, it was developed using an enhanced chemiluminescence (ECL) detection kit (Boster Biological Technology, USA, catalog number AR1190). Figure 6 As shown.

[0063] Target gene expression analysis: RNA was extracted from thallus cells of different generations, strictly following the instructions of the Omega Plant RNA Mini-Extraction Kit. cDNA synthesis was performed according to the standard procedure of the Novizan HiScript III 1st StrandcDNA Synthesis Kit (including gDNA wiper). Untreated RZ was used as a control in each group, and three biological replicates and three technical replicates were set up in each group to ensure the reliability and reproducibility of the results. Figure 7 The results showed that all samples expressed the target gene.

[0064] (5) Genetic stability test

[0065] To verify the genetic and expression stability of exogenous genes in transgenic offspring, a multi-generation tracking analysis strategy combining single-spore release and continuous filament culture was adopted to detect the genetic fidelity of marker genes in asexual reproduction and sporophyte stages. Figure 8 and Figure 9 The results showed that the GUS staining positivity rate of single spores released from positive transformants reached 100%, and the thallus formed by single spore culture all showed a uniform blue GUS activity signal, indicating that the exogenous gene follows a stable Mendelian inheritance pattern in the gametophyte generation. After four consecutive generations of asexual reproduction of single spores, the expression activity of the target gene did not show any attenuation or silencing. Further culturing the positive transformants to the filamentous stage (sporophyte stage) showed that the cells still showed a specific GUS staining signal, confirming that the exogenous gene maintained stable expression in the sporophyte generation.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for efficiently and stably genetically transforming *Porphyra yezoensis* strains mediated by *Agrobacterium*, characterized in that, The process includes the following steps: immersing the Porphyra yezoensis recipient material in Agrobacterium tumefaciens infection solution for infection, and then transferring it to hygromycin B sterile seawater culture medium for gradient screening to obtain new plants containing the target gene; The *Porphyra yezoensis* receptor material is a suspension of single spores, tissue fragments, and thallus seedlings. The preparation method of the receptor material is as follows: select *Porphyra yezoensis* thallus, remove the basal and marginal tissues, cut the remaining part into small tissue blocks, and place them in sterile seawater culture medium containing PES, incubating at 15±2℃ for 10-15 days; the *Porphyra yezoensis* thallus are healthy, disease-free 30-40 day old *Porphyra yezoensis* thallus; each fragment of the small tissue block contains 30-60 cells; the mass concentration of PES is 2%. The Agrobacterium infection solution contained 100 μM acetylsalicylic acid and OD200. 600 Sterile seawater with a concentration of 0.5% and a pH of 30‰; During the infection process, a vacuum permeation of -0.08±0.01MPa was applied for 30±5 min, followed by co-incubation in a dark environment at 15±2℃ for 48 h to complete T-DNA transfer; The gradient screening consisted of primary screening on 2 mg / L hygromycin B sterile seawater culture medium and secondary screening on 4 mg / L hygromycin B sterile seawater culture medium.

2. The method according to claim 1, characterized in that, The preparation method of the Agrobacterium infection solution is as follows: Agrobacterium cells containing the target gene are collected, resuspended in an equal volume of 30‰ sterile seawater, and acetylsuccinone is added to a final concentration of 100 μM to adjust the OD of the bacterial solution. 600 At 0.5, the expression of Vir region genes was induced by static incubation at room temperature.

3. The method according to claim 1, characterized in that, After the co-culture was completed, the surface Agrobacterium was thoroughly washed with sterile seawater containing 500 mg / L cefotaxime to remove it, and then subsequent gradient screening was performed.

4. The method according to claim 1, characterized in that, After 14 days of initial screening culture, hygromycin-sensitive plants that have not been transformed are eliminated, and resistant plants are collected. After 7 days of secondary screening culture, false positive plants from the initial screening resistant plants are eliminated, and genetically stable homozygous resistant plants are obtained.

Images