A vacuum-osmosis-mediated transient transformation method for mulberry trees based on Agrobacterium tumefaciens.

Abstract

This invention discloses a method for transient in vitro transformation of mulberry seedlings based on vacuum permeation mediated by Agrobacterium tumefaciens. Specifically, the method involves: culturing Agrobacterium tumefaciens containing the target gene using a shaking incubator, collecting the bacterial cells, and then resuspending them in a permeation buffer to obtain the Agrobacterium infection solution; first, immersing mulberry tissue in the permeation buffer and sonicating it to create micropores; then, immersing the mulberry tissue in the Agrobacterium infection solution for vacuum permeation infection; wherein the permeation buffer is: sterile double-distilled water containing 10 mM MES, 10 mM MgCl2, 50–200 μmol / L acetylsylgenone (AS), pH 5.6. This invention, based on the Agrobacterium vacuum permeation infection method, overcomes the problem of low injection infection efficiency caused by leaf vein tissue. Combined with ultrasonic treatment, it establishes a rapid and efficient transient transformation system for mulberry tissue culture seedlings, enabling rapid, efficient, and large-scale functional verification of mulberry genes.

Description

Technical Field

[0001] This invention belongs to the field of mulberry genetic transformation technology, specifically relating to a method for in vitro transient transformation of mulberry trees based on vacuum permeation mediated by Agrobacterium tumefaciens. Background Technology

[0002] Mulberry belongs to the family Moraceae and the genus *Morus*. Morus Mulberry is a perennial woody tree, a high-quality natural feed for silkworms, and also a traditional medicinal plant in my country. Its fruit (mulberry), roots, stems, leaves, and processing byproducts all have high medicinal value and health benefits. Therefore, the mulberry tree is an important ecological and economic forest tree. However, the traditional silkworm farming model, characterized by an emphasis on silkworms and a neglect of mulberry cultivation, has resulted in weak basic research on mulberry trees and lagging research on gene function, severely restricting the diversified development and multi-purpose comprehensive utilization of mulberry trees. With the development of Sichuan mulberry (… Morus notabilis Schneid. (He et al., 2013) and white mulberry ( Morus alba The completion of the genome analysis of mulberry (L.) (Jiao et al., 2020) and the batch generation of mulberry transcriptome sequencing data mark the entry of mulberry into the post-genomic era. A large number of mulberry genes and their functions urgently need to be verified and identified. Therefore, establishing a rapid and efficient genetic transformation system is an important technical means to achieve batch functional identification of genes.

[0003] Hirano et al. (1985) first confirmed that mulberry trees could be infected by Agrobacterium. Subsequently, many researchers carried out a large number of transgenic studies on mulberry trees. However, to this day, a mature and stable transgenic system for mulberry trees has not yet been established. At present, there are four main methods of genetic transformation of mulberry trees: (1) Leaf disc transformation method; Machii et al. (1990) first transferred the GUS gene and NPTII gene into mulberry trees based on Agrobacterium-mediated leaf disc transformation method and successfully obtained plants with GUS enzyme activity. Wu Chengcang et al. (1992) transferred the silkworm antimicrobial peptide B gene (artificially synthesized) and GUS gene into mulberry trees through leaf disc transformation method and obtained resistant callus tissue after screening. Tan Jianzhong et al. (1999, 2001) transformed the soybean globulin gene A1aB1b into the leaf disc and shoot tip of mulberry trees respectively through leaf disc transformation method. After kanamycin resistance screening culture, A1aB1b transgenic positive plants were obtained. Shalini et al. (2008) and Manaswini et al. (2011) successfully introduced exogenous genes into Indian mulberry 'K2' using Agrobacterium-mediated leaf disc transformation, and both obtained transgenic positive plants. (2) Gene gun bombardment method; In addition to the traditional Agrobacterium-mediated plant tissue culture transformation method, gene gun technology has also been applied in mulberry transgenic research. Machii et al. (1996) successfully introduced the GUS gene into mulberry suspension callus tissue using gene gun bombardment, but ultimately failed to obtain transgenic plants. Wang Hongli et al. (2003) successfully introduced the rice cysteine ​​protease inhibitor gene ( OC (3) Plant transformation method: The plant transformation method uses the stem tip or axillary bud of the plant as the recipient material and transforms it by injecting Agrobacterium. Lu Xiaoping et al. (2004) first reported the use of Agrobacterium to infect the axillary bud of mulberry after it was punctured by a needle, and identified the phenotypic abnormal mulberry buds that grew from the infected site, confirming that the target gene was successfully introduced. Lin Tianbao et al. (2018) injected Agrobacterium carrying GUS and Kan genes into the winter buds of mulberry. After GUS staining, PCR detection and kanamycin resistance screening, it was confirmed that the GUS gene had been successfully transferred into mulberry. (4) Pollen tube pathway method: The pollen tube pathway method is to directly inject DNA solution into the ovary after the plant is pollinated. Using the pollen tube that elongates during fertilization, the foreign gene is transferred into the fertilized egg cell and further integrated into the genome, and finally a transgenic individual is obtained. Li Zhengang et al. (2014) used the pollen tube pathway method combined with Agrobacterium-mediated transformation to transform mulberry trees, and the transformed plants were identified by PCR. phy CIn addition, Zhang Dayan et al. (2015) explored the effects of buffer solution, Agrobacterium silencing concentration, transformation time, and mulberry variety on the instantaneous transformation efficiency of mulberry leaves based on Agrobacterium-mediated injection, and preliminarily constructed an Agrobacterium-mediated instantaneous transformation system for mulberry leaves. Li Ruixue et al. (2018) reported an Agrobacterium-mediated virus-induced gene silencing (VIGS) transformation system and successfully transformed mulberry leaves into mulberry leaves. PDS Gene silencing causes leaves to turn white.

[0004] Currently, research on mulberry genetic transformation still faces the following problems: (1) Low efficiency and regeneration rate of stable genetic transformation. Mulberry is a perennial woody plant, making it difficult to establish a mature regeneration system, and it is highly dependent on genotype; (2) Low efficiency of transient transformation. Existing Agrobacterium-mediated transient transformation of mulberry living leaves all use the injection infection method. Due to the thinness of mulberry leaves and the presence of many veins, the efficiency of injection infection and Agrobacterium transformation is low, making it impossible to achieve rapid, efficient, and large-scale gene function verification. Summary of the Invention

[0005] To address the problems in existing technologies, this invention aims to establish a rapid and efficient instantaneous transformation system for mulberry tissue culture seedlings, so as to achieve rapid, efficient, and large-scale functional verification of mulberry genes and promote the rapid development of basic research on mulberry.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a method for in vitro transient transformation of mulberry trees based on vacuum permeation mediated by Agrobacterium tumefaciens, the specific steps of which are as follows: Step 1: After shaking and culturing Agrobacterium tumefaciens containing the target gene, collect the bacterial cells and resuspend them in osmotic buffer to obtain Agrobacterium infection solution; wherein, the osmotic buffer is specifically: sterile double-distilled water containing 10 mM MES, 10 mM MgCl2, 50~200 μmol / L acetylsylgenone (AS), and pH 5.6.

[0007] Step 2: First, immerse the mulberry isolated tissue in a permeation buffer and sonicate it to create micropores. Then, immerse the mulberry isolated tissue in Agrobacterium infection solution for vacuum permeation infection.

[0008] Furthermore, in the above-mentioned in vitro transient transformation method of mulberry trees, the Agrobacterium tumefaciens can be Agrobacterium GV3101, Agrobacterium EHA105, or Agrobacterium LBA4404; among them, Agrobacterium LBA4404 has the best transformation efficiency.

[0009] Furthermore, in the aforementioned in vitro transient transformation method for mulberry trees, the OD of the Agrobacterium-infected solution... 600 The value is 0.5~1.0; the preferred OD is... 600 =0.5.

[0010] Furthermore, in the above-mentioned method for transient transformation of mulberry in vitro, the Agrobacterium tumefaciens infiltration solution used in step 2 to infect mulberry in vitro tissue also contains Silwet L-77 surfactant, and its concentration is preferably 0.02%.

[0011] Furthermore, in the above-mentioned method for instantaneous transformation of mulberry trees in vitro, the conditions for ultrasonic treatment can be: ultrasonic treatment in a 50 kHz ultrasonic instrument for 10-30 s, with 20 s being the optimal time.

[0012] Furthermore, in the above-mentioned mulberry tree in vitro instantaneous transformation method, the vacuum impregnation conditions are: 0.7 MPa vacuum conditions for 20 min of infiltration and impregnation.

[0013] Furthermore, in the above-mentioned method for the instantaneous transformation of mulberry trees in vitro, when the mulberry tree tissue in vitro is a tissue culture seedling, the method also includes step 3: inoculating the infected tissue culture seedling into a subculture medium for co-culture, and then washing the tissue culture seedling with bacteria and inoculating it into a sterile subculture medium for continued culture, with 7 days of culture being optimal.

[0014] Furthermore, in a specific embodiment of the present invention, the transformation effect of mulberry tissue culture seedlings with genotypes of 'Taiguo', 'Yaosang', 'Aoyu', and '8632' was verified, with 'Taiguo' being the best.

[0015] Furthermore, the subculture medium used for the tissue culture seedlings was DKW + ZT 2.0 mg / L + 6-BA 2.0 mg / L + IAA 0.1 mg / L + sucrose 30 g / L + agar 6.5 g / L, and the culture time of the tissue culture seedlings in step 3 was 4~10 days.

[0016] The beneficial effects of this invention are as follows: Based on the Agrobacterium vacuum penetration infection method combined with ultrasonic treatment, this invention overcomes the problem of low injection infection efficiency caused by leaf vein tissue, and establishes a rapid, high-throughput in vitro instantaneous transformation system for mulberry seedlings. Furthermore, the instantaneous transformation efficiency of mulberry tissue culture seedlings is further optimized by adjusting the concentration of acetylsyleugenone, Agrobacterium strain type, Agrobacterium bacterial solution concentration, ultrasonic treatment time, and culture time. Moreover, the system provided by this invention has been successfully used in… MaANS and MaDFR Functional verification was performed by transiently transforming isolated mulberries. Therefore, the method established in this invention enables rapid, efficient, and large-scale functional verification of mulberry genes, providing technical support for advancing the post-genomic era of mulberry. Attached Figure Description

[0017] Figure 1A comparative graph showing the effects of different mulberry genotypes (A, B), ultrasonic treatment time (C), acetylsyl syringone concentration (D), Agrobacterium tumefaciens bacterial solution concentration (E), culture time (F), and Agrobacterium tumefaciens strain type (G) on the instantaneous transformation efficiency of mulberry tissue culture seedlings.

[0018] Figure 2 In vitro instantaneous transformation of mulberry fruit in Example 10 MaANS and MaDFR The results of the overexpression function analysis are shown in the figure. In the figure, A is a comparison of the appearance color of mulberry fruit at different times after infiltration infection, B is a comparison of the appearance color of mulberry fruit 7 days after infiltration infection, and C is a comparison of the changes in anthocyanin content 7 days after infiltration infection. Detailed Implementation

[0019] To make the technical problems, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0020] Unless otherwise specified, the methods described in the following examples are conventional; the reagents and materials described are commercially available unless otherwise specified.

[0021] Example 1 This example uses 'Taiguo' mulberry genotype tissue culture seedlings (subcultured for 25 days) as the test material. GFP This is a reporter gene. The transient conversion process is as follows: Will contain GFP A single clone of Agrobacterium GV3101 containing the gene was cultured in 1 ml of LB broth containing the corresponding antibiotic at 28°C with shaking (220 rpm) for 1 day. Then, it was added to 50 ml of LB broth containing the corresponding antibiotic and cultured overnight at 28°C with shaking (220 rpm) until OD (Organic Dose). 600 The bacterial concentration was 1.6-1.8 (the bacterial suspension was golden yellow). The cells were collected by centrifugation at 25°C, 4,000 × g for 10 min, resuspended in osmotic buffer, and cultured until the bacterial concentration reached OD100. 600 The concentration was 0.75, resulting in the Agrobacterium infection solution. The osmotic buffer (pH 5.6) contained 10 mM MES, 10 mM MgCl2, and 150 μmol / L AS.

[0022] Before infiltration, filter-sterilized Silwet L-77 surfactant was added to the Agrobacterium infection solution to a final concentration of 0.02%. The tissue culture seedlings were first immersed whole in the infiltration buffer and treated with a 50 kHz ultrasonic instrument for 20 seconds. Then, the seedlings were immersed whole in the Agrobacterium infection solution and infiltrated under a 0.7 MPa vacuum for 20 minutes. After inoculating the seedlings onto subculture medium and co-culturing for 2 days, the seedlings were washed with 500 mg / L sterile cephalosporin solution for 10 minutes, and finally inoculated onto sterile subculture medium for another 2 days. The subculture medium consisted of: DKW + ZT 2.0 mg / L + 6-BA 2.0 mg / L + IAA 0.1 mg / L + sucrose 30 g / L + agar 6.5 g / L.

[0023] Examples 2-4 Unlike Example 1, Examples 2-4 used tissue culture seedlings with different mulberry genotypes as test materials, as shown in the table below:

[0024] After the transformation was completed, the GFP protein content in the leaves of the tissue culture seedlings obtained in Examples 1-4 was measured, and the results are as follows: Figure 1 As shown in Figures A and B, the GFP protein content in the leaves of 'Taiguo' tissue culture seedlings was significantly higher than that of other genotypes, and the GFP fluorescence signal in the leaves was significantly stronger than that of other genotypes, indicating that 'Taiguo' had the highest transient transformation efficiency.

[0025] Example 5 Following the operating procedure of Example 1, this example explores the effects of ultrasonic processing times of 0, 10, 20, and 30 seconds at a 50 kHz frequency on... GFP The impact of instantaneous conversion efficiency.

[0026] After the transformation was completed, the GFP protein content in the leaves of tissue culture seedlings treated with ultrasound for different durations was measured. The results are as follows: Figure 1 As shown in Figure C, the tissue culture seedling leaves had the highest GFP protein content under 20 s ultrasonic treatment, indicating the highest instantaneous conversion efficiency.

[0027] Example 6 Following the procedure in Example 1, this example explores the effects of AS concentrations of 50, 100, 150, and 200 μmol / L in the osmotic buffer on... GFP The impact of instantaneous conversion efficiency.

[0028] After transformation, the GFP protein content in the leaves of tissue culture seedlings under different AS concentrations was measured, and the results are as follows: Figure 1As shown in Figure D, the GFP concentration in the leaves of tissue culture seedlings treated with 150 μmol / L AS was significantly higher than that of other concentrations, indicating that it had the highest instantaneous conversion efficiency.

[0029] Example 7 Following the procedure in Example 1, this example explores the bacterial concentration OD in the Agrobacterium-containing inoculum. 600 The values ​​are 0.5, 0.75, and 1.0 respectively. GFP The impact of instantaneous conversion efficiency.

[0030] After transformation, the GFP protein content in the leaves of tissue culture seedlings was measured at different bacterial concentrations, and the results are as follows: Figure 1 As shown in Figure E: bacterial concentration OD 600 The highest GFP protein content was observed in the leaves when the value was 0.5.

[0031] Example 8 Following the procedure in Example 1, this example explores the effects of different culture times of 4, 7, 10, and 15 days on... GFP The effect of instantaneous conversion efficiency; the specific culture time scheme is as follows: after the tissue culture seedlings are infiltrated in a vacuum, they are first co-cultured for 2 days, then washed with sterile cephalosporin water, and then inoculated into sterile subculture medium for subculture for 2 days, 5 days, 8 days, and 13 days.

[0032] After transformation, the GFP protein content in the leaves of tissue culture seedlings at different culture times was measured, and the results are as follows: Figure 1 As shown in Figure F, the highest GFP protein content was observed in the leaves of tissue culture seedlings cultured for 7 days after infiltration infection.

[0033] Example 9 Following the procedure in Example 1, this example explores the effects of different Agrobacterium strains GV3101, EHA105, and LBA4404 on... GFP The impact of instantaneous conversion efficiency.

[0034] After transformation, the GFP protein content in the leaves of tissue culture seedlings infected with different Agrobacterium strains was measured, and the results are as follows: Figure 1 As shown in Figure G, the transient transformation mediated by Agrobacterium LBA4404 resulted in the highest GFP protein content.

[0035] Example 10 This example uses the detached fruit of the mulberry variety 'Zijing' 10 days after flowering as the test material. MaANS and MaDFR Genes were transiently transformed into mulberries for overexpression functional verification, with an empty vector serving as a control. The specific process was as follows: Will contain MaANS Gene or MaDFRA single clone of Agrobacterium LBA4404 containing the gene was cultured in 1 ml of LB broth containing the corresponding antibiotic at 28°C with shaking (220 rpm) for 1 day. Then, it was added to 50 ml of LB broth containing the corresponding antibiotic and cultured overnight at 28°C with shaking (220 rpm) until OD (Organic Dose). 600 The bacterial concentration was 1.6-1.8 (the bacterial suspension was golden yellow). The cells were collected by centrifugation at 25°C, 4,000 × g for 10 min, resuspended in osmotic buffer, and cultured until the bacterial concentration reached OD100. 600 The concentration was 0.75, resulting in the Agrobacterium infection solution. The osmotic buffer (pH 5.6) contained 10 mM MES, 10 mM MgCl2, and 150 μmol / L AS.

[0036] Before infiltration, filter-sterilized Silwet L-77 surfactant was added to the Agrobacterium infection solution to a final concentration of 0.02 wt%. The fruit was first immersed in the infiltration buffer and treated with a 50 kHz ultrasonic instrument for 20 s. Then, the fruit was immersed in the Agrobacterium infection solution and infiltrated under a vacuum of 0.7 MPa for 20 min.

[0037] The infected mulberries were placed in petri dishes and incubated at 28 ℃ with sterile filter paper in the dishes. The dishes were moistened with sterile double-distilled water containing 30 g / L sucrose. After 5 days, some mulberries began to accumulate pigment, while the control group showed no significant changes. The coloring of the mulberries was particularly noticeable after 7 days of infection, with anthocyanin content significantly higher than the control group (see...). Figure 2 ).

[0038] In summary, the in vitro transient transformation technology of mulberry provided by this invention is feasible and efficient for verifying the function of mulberry genes, and provides technical support for promoting the development of basic research on mulberry.

[0039] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for in vitro transient transformation of mulberry trees based on vacuum permeation mediated by Agrobacterium tumefaciens, characterized in that, Specifically: Step 1: After shaking culture of Agrobacterium tumefaciens containing the target gene, collect the bacterial cells and then resuspend them in osmotic buffer to obtain Agrobacterium infection solution; Step 2: First, immerse the mulberry tissue culture seedlings in a permeation buffer solution for ultrasonic treatment, and then immerse the mulberry tissue culture seedlings in Agrobacterium infection solution for vacuum permeation infection; The permeation buffer solution is specifically: sterile double-distilled water containing 10mM MES, 10mM MgCl2, 50-200μmol / L acetylsylgenone; and pH 5.6; The OD600 of the Agrobacterium infection solution is 0.5-1.0, and the Agrobacterium infection solution also contains 0.02% Silwet L-77 surfactant; The conditions for ultrasonic treatment are: 50KHz, ultrasonic treatment for 10-30s; The conditions for vacuum penetration contamination are: penetration contamination under vacuum conditions for 20 minutes.

2. The method for in vitro transient transformation of mulberry trees based on vacuum permeation mediated by Agrobacterium tumefaciens according to claim 1, characterized in that, The Agrobacterium tumefaciens is Agrobacterium GV3101, Agrobacterium EHA105, or Agrobacterium LBA4404.

3. The method for in vitro transient transformation of mulberry trees based on vacuum permeation mediated by Agrobacterium tumefaciens according to claim 1, characterized in that, The method also includes: Step 3: Inoculate the infected tissue culture seedlings into a subculture medium for co-culture, then wash the seedlings and inoculate them into a sterile subculture medium for continued culture.

4. The method for in vitro transient transformation of mulberry trees based on vacuum permeation mediated by Agrobacterium tumefaciens according to claim 3, characterized in that, The subculture medium consisted of DKW + ZT 2.0 mg / L + 6-BA 2.0 mg / L + IAA 0.1 mg / L + sucrose 30 g / L + agar 6.5 g / L.

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