A plant efficient genetic transformation vector based on rhizobium and its application

Abstract

The present invention relates to a plant efficient genetic transformation vector based on rhizobium rhizobium, which is modified based on the pTi vector, and its T-DNA left border pTi-LB and right border pTi-RB are replaced with the left border pRi-LB and the right border pRi-RB respectively; its T-DNA region is deleted. Hyg expression cassette, while in its original RUBY The front end of the expression cassette is connected to the target gene expression cassette; in its non-T-DNA region Ori Components and SmR Insert between components VirGN54D insert The present invention aims to enhance the efficient genetic transformation of exogenous plant genes in multiple species, with broad adaptability across a wide range of plant species, particularly in genotype-dependent and difficult-to-transform plant species such as cucumber, white radish, broad bean, cauliflower, nightshade, pokeweed, ginkgo, phalaenopsis, rose, and Chinese pine.

Description

Technical Field

[0001] The present invention belongs to the field of plant molecular biology and genetic engineering, and particularly relates to a plant efficient genetic transformation vector based on rhizobium rhizogenes and its application. Background Art

[0002] Plant genetic transformation, as a key technology for integrating exogenous genetic material into the host genome, is a core means to achieve crop trait improvement and functional research. Although emerging technologies such as CRISPR-Cas9 gene editing and synthetic biology have provided new tools for plant genetic manipulation, their application is still limited to a few model species. In contrast, Agrobacterium-mediated transformation ( Agrobacterium Agrobacterium-Mediated Transformation (AMT) remains the most widely used plant genetic transformation system due to its efficient T-DNA transfer mechanism and precise genomic integration. The molecular mechanism of AMT involves the coordinated action of multiple components: the VirD2 protein specifically recognizes and cleaves the transfer DNA (T-DNA) border sequence to form single-stranded T-DNA (ss-T-DNA), while the VirE2 protein non-covalently associates to form a protective nucleoprotein complex, preventing nuclease degradation and facilitating nuclear localization. This complex is transported across the membrane via the type IV secretion system (T4SS). Numerous studies have improved AMT efficiency in many plant species by optimizing induction conditions, Agrobacterium tumefaciens strain types, and virulence genes. However, this technique still has numerous limitations, particularly in genotype-dependent and difficult-to-transform plants. This leads to low efficiency or even ineffectiveness, making it difficult to obtain stable transgenic plants, and severely restricting the progress of genetic engineering. Therefore, the development of more efficient tools to optimize the AMT process remains a challenge. Summary of the Invention

[0003] The present invention relates to a plant high-efficiency genetic transformation vector based on rhizobium rhizobium, which is used to improve the genetic transformation efficiency of various plants and overcome many shortcomings of existing transformation technologies, such as vector structure redundancy, low T-DNA cleavage efficiency, poor stability, low verification accuracy, and difficulty in application in various gene-dependent and difficult-to-transform plant species.

[0004] The present invention adopts the following technical solutions:

[0005] A genetic transformation vector is obtained by modifying a pTi vector, specifically comprising: (1) replacing the left border pTi-LB and the right border pTi-RB of the T-DNA of the pTi vector with the left border pRi-LB and the right border pRi-RB respectively; (2) deleting the T-DNA region of the original pTi vector Hyg expression cassette, while in its original RUBYThe front end of the expression cassette is connected to the target gene expression cassette; (3) in its non-T-DNA region Ori Components and SmR Insert between components VirGN54D insert element; (4) the 106th position of the RepA protein coding gene sequence of pVS1 in its non-T-DNA region was mutated from an arginine coding sequence to a histidine coding sequence.

[0006] The nucleotide sequence of the pTi-LB is shown in SEQ ID No. 1, and the nucleotide sequence of the pTi-RB is shown in SEQ ID No. 2.

[0007] The nucleotide sequence of the pRi-LB is shown in SEQ ID No. 5, and the nucleotide sequence of the pRi-RB is shown in SEQ ID No. 6.

[0008] Among them, the VirGN54D insert The nucleotide sequence of the element is shown in SEQ ID No.7.

[0009] The nucleotide sequence of the mutated RepA protein encoding gene is shown in SEQ ID No.9.

[0010] An application of the above genetic transformation vector in genetic transformation of plants.

[0011] In the application, the plants include cucumber, white radish, broad bean, Chinese cabbage, Solanum nigrum, Phakolanum strychnos, Ginkgo biloba, Phalaenopsis orchid, Chinese rose and Pinus tabulaeformis.

[0012] The beneficial effects of the present invention are:

[0013] (1) The present invention makes multiple modifications to the plant binary overexpression vector pTi. Targeting the T-DNA region, an artificial super drive sequence is introduced into the left border (LB) and right border (RB) sequences to enhance the recognition and cleavage efficiency of the VirD2 protein on the border sequences, thereby significantly improving the transfer efficiency of T-DNA. In the non-T-DNA region, a super drive sequence is inserted downstream of the resistance gene. VirGN54D insert The pVS1 RepA element is a functional element that promotes high, constitutive expression of the toxic gene, independent of VirA or chemical inducers, providing more favorable conditions for plant genetic transformation. Furthermore, a targeted mutagenesis of amino acid position 106 (arginine Arg to histidine His) in the pVS1 RepA element significantly improves the replication stability of the vector in the host bacteria, further enhancing transformation efficiency.

[0014] (2) This vector system has demonstrated outstanding advantages in a variety of plants that are traditionally gene-dependent and difficult to transform transiently (cucumber, white radish, broad bean, broccoli, nightshade, pokeweed, ginkgo, phalaenopsis, rose, and Chinese pine). Its core advantages include high transformation efficiency, good stability, a wide range of applications, accurate verification, and simple operation. It provides an efficient and stable genetic transformation tool for plant gene function research, molecular breeding, and biotechnology applications, and is of great value in both scientific research and industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of different vector structures and sequence optimization for transformation.

[0016] Figure 2 Genetic transformation of tobacco mediated by different vectors for modification RUBY Timing comparison chart.

[0017] Figure 3 This is a diagram of the efficient genetic transformation of Pinus tabulaeformis roots mediated by the pRi2vGR106 vector.

[0018] Figure 4 Genetic transformation of tobacco mediated by different vectors RUBY Comparison chart of quantitative analysis of betalains.

[0019] Figure 5 This figure verifies the broad spectrum of pRi2vGR106 vector-mediated genetic transformation of multiple species. DETAILED DESCRIPTION

[0020] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions are within the scope of this application. Unless otherwise specified, the chemical reagents used in the examples are conventional commercial reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0021] Example 1 Modification of the original vector pTi

[0022] like Figure 1 As shown, the left border pTi-LB (SEQ ID No. 1) and the right border pTi-RB (SEQ ID No. 2) of the T-DNA of the original pTi vector were replaced with pRiA4T-LB (SEQ ID No. 3) and pRiA4T-RB (SEQ ID No. 4) of the Ri plasmid in the rhizobium rhizobium strain ArA4, respectively.

[0023] On this basis, the T-DNA region of the original pTi vector was modified. Hygexpression cassette, while in its original RUBY Front-end ligation of the expression cassette GUS Expression cassette 。 described GUS The expression cassette is CaMV 35S promoter- GUS Reporter gene-HA-6×His-AtHSP 18.2 Term terminator. The vector after the above transformation was named pRiA4T.

[0024] The pTi original vector and the pRiA4T recombinant vector were transformed into Escherichia coli competent cells DH5α (ATCC 53868) using the heat shock method to obtain E. coli cells containing the pTi original vector and the pRiA4T recombinant vector, respectively, and cultured for 12-16 h. The recombinant plasmids were extracted from the E. coli cells and transformed into Rhizobium rhizogenes competent cells K599 (NCPPB2659) using the chemical transformation method to obtain Rhizobium rhizogenes containing the recombinant vectors.

[0025] The bacterial suspensions of Rhizobium rhizobium containing the pTi original vector and the pRiA4T recombinant vector were subjected to 7,500 rpm The bacteria were collected by centrifugation for 10 min to allow them to adhere to the wall of the centrifuge tube and resuspended twice with a resuspension solution containing 10 mM 2-morpholineethanesulfonic acid (MES), 10 mM magnesium chloride (KOH-pH=5.2), and 200 mM acetosyringone to adjust the OD value of the working solution containing rhizobium to 0. 600 Between 0.8 and 1.2.

[0026] After infection, it was observed that the pRiA4T vector accumulated betalain faster than the original vector pTi in tobacco leaves (e.g. Figure 2 ).

[0027] Example 2: Transformation based on pRiA4T vector

[0028] The pRiA4T vector obtained in Example 1 was further modified. The left border pRiA4T-LB (SEQ ID No. 3) and the right border pRiA4T-RB (SEQ ID No. 4) of the T-DNA of the pRiA4T vector were replaced with the left border pRi-LB (SEQ ID No. 5) and the right border pRi-RB (SEQ ID No. 6) of the Ri plasmid from the Rhizobium rhizogenes strain K599, respectively. This vector was named pRi2 vector.

[0029] Based on the pRi2 vector, in its non-T-DNA region Ori Components and SmR Insert between components VirGN54D insertThe element (SEQ ID No.7) promotes the high expression of the toxic gene without relying on VirA or chemical inducers, promotes plant genetic transformation, and the vector is named pRi2vG vector.

[0030] On the pRi2vG vector, the arginine Arg at position 106 of the RepA protein of pVS1 in its non-T-DNA region was mutated to histidine His. The amino acid sequence of the mutated RepA protein is shown in SEQ ID No. 8, and its nucleotide sequence is shown in SEQ ID No. 9. The vector was named pRi2vGR106.

[0031] The pTi, pRiA4T, pRi2vG, and pRi2vGR106 vectors were transformed using a conventional tobacco transient transformation method, i.e., rhizobium was injected into tobacco leaves to infect them. The efficiency of target protein delivery in the tobacco transient transformation experiment was verified by observing the rate of RUBY protein accumulation at different observation time points (e.g., 12 h, 24 h, and 36 h). Figure 2 After infection, it was found that the area infected by the rhizobium containing the pRi2vGR106 vector first expressed the RUBY protein, while the original vector pTi expressed the slowest.

[0032] Example 3: pRi2vGR106 vector improves the genetic transformation efficiency of Pinus tabulaeformis roots

[0033] The pRi2vGR106 vector with the highest transient transformation efficiency in tobacco in Example 2 was used to carry out genetic transformation of Pinus tabulaeformis roots.

[0034] The recombinant vector pRi2vGR106 was transformed into rhizobium competent cells by chemical transformation. 8 to 10 positive single clones were picked and cultured in 400 μl TY+75 mg / L spectinomycin (Spec) liquid medium at 28°C and 200 rpm Then, 200 μl of bacterial solution was added to 20 ml of TY+ 75 mg / L Spec liquid medium and shaken vigorously to expand the bacterial solution to a concentration of OD 600 = 0.8~1.5, and then the bacterial solution was heated at room temperature at 7000 rpm Centrifuge for 10 min, remove the supernatant, resuspend the pellet in TY+75 mg / L Spec liquid medium, aspirate 350-400 μl of bacterial liquid and spread it on TY+75 mg / L Spec solid medium, and place it upside down in a 28°C incubator until a thick bacterial layer grows on the surface of the solid medium.

[0035] The TY liquid culture includes: 5 g / L peptone, 3 g / L yeast extract and 1 M calcium chloride; if solid culture medium is prepared, 15 g / L agar powder is added.

[0036] When the stems of cultured Pinus tabulaeformis seedlings exceed 3 cm in length, a blade is tilted at a 45-degree angle to cut off approximately 0.5–1.0 cm above or below the rhizome junction. The plant wound is then immersed in a resuspension of rhizobia. A vacuum is then applied to the plant at a pressure of 0.05 MPa for 8 minutes. The plant explants are removed from the resuspension, and the root wounds are then coated with rhizobia containing the pRi2vGR106 vector. The plant is then transplanted into a soil culture medium and cultured at 19–24°C for 30–45 days. Stably transformed adventitious roots grow from the hypocotyls of the Pinus tabulaeformis.

[0037] The soil culture medium is a coarse vermiculite soil medium, the particle size of the coarse vermiculite is preferably 5-8 mm, and the soil medium is preferably contained in a 50-hole tray with a length of 54 cm × a width of 28 cm × a depth of 4.5 cm. The humidity of the growth environment is 65% to 75%. The light intensity during the growth period is 150 μmo1·m -2 ·s -1 The photoperiod during the growth period was 16 h.

[0038] The rhizobium solution containing the pRi2vGR106 vector was used to transform the root system of Pinus tabulaeformis, and the original pTi vector was used as the control group. RUBY The expression of reporter genes was used to verify the transformation efficiency of transgenic Pinus tabulaeformis roots (e.g. Figure 3 After infection, it was found that the transgenic roots of Pinus tabulaeformis infected with rhizobium containing the pRi2vGR106 vector expressed obvious betacyanin pigment, which was the result of RUBY protein accumulation, indicating that the modified vector has a high genetic transformation efficiency.

[0039] Example 4 Expression efficiency of the vector system

[0040] To evaluate the expression efficiency of different vector systems, Agrobacterium rhizogenes transformed with four vector constructs, pRiA4T, pRi2, pRi2vG and pRi2vGR106, were used to transiently transform tobacco leaves. The expression efficiency of the leaves was evaluated by high performance liquid phase extraction and spectrophotometry. RUBY Betalain expressed by the reporter gene was quantitatively detected.

[0041] The specific experimental steps are as follows:

[0042] (1) Tobacco leaves transiently transformed with four different vectors were selected, and about 100 mg (fresh weight) of the color-developing part was cut out as the extraction material.

[0043] (2) Place the sample in liquid nitrogen for rapid freezing and grind it into powder using a mortar.

[0044] (3) Transfer the ground powder to a centrifuge tube, add 700 μl of 40% methanol solution (v / v) containing 50 mM sodium ascorbate, vortex thoroughly to mix, and then place in a constant temperature shaker for 20 min.

[0045] (4) The mixture was heated at 4°C at 10,000 rpm Centrifuge for 10 min and collect the supernatant into a centrifuge tube.

[0046] (5) Adjust the pH of the collected supernatant to 3–5 using 0.1 M HCl solution.

[0047] (6) Again at 4°C with 10,000 rpm Centrifuge for 10 min and discard the supernatant.

[0048] (7) Take 1 mL of the crude sample treated as above, add 4 mL of the composite organic reagent, vortex and shake for 10 min, and then let it stand for 5 min to allow the solution to separate.

[0049] (8) Again at 4°C with 10,000 rpm Centrifuge for 10 min and collect the supernatant.

[0050] (9) The absorbance was measured at a wavelength of 532 nm using a UV-visible spectrophotometer. All samples were subjected to three biological experiments and the average value was taken for determination. Pigments extracted from fresh tobacco leaves that had not been transiently transformed were used as blank controls to measure the concentration of betacyanin in different solutions.

[0051] The results of the quantitative spectrophotometric test showed that ( Figure 4 ), there are significant differences in the accumulation efficiency of betalains in different vector systems. Among them, the Agrobacterium rhizogenes infection group transformed with the pRi2vGR106 vector showed the highest betalain accumulation of 4.21 mg / 100mL, followed by pRi2vG with 3.76 mg / 100mL, the pigment production of pRi2 was 1.55 mg / 100mL, and the expression efficiency of pRiA4T was the lowest at 1.23 mg / 100mL. This data shows that the optimization of pRi2vGR106 significantly improved RUBY Reporter gene expression efficiency.

[0052] Example 5 Application of vectors in transient transformation of multiple species

[0053] The above vectors (pTi, pRi2vGR106) were transformed into Escherichia coli competent cells to obtain Escherichia coli cells containing the recombinant vector and cultured for 12-16 hours. The recombinant plasmid was extracted from the Escherichia coli cells and transformed into Rhizobium rhizogenes K599 competent cells to obtain Rhizobium rhizogenes containing the recombinant vector.

[0054] The bacterial solution of rhizobium was subjected to 7,500 rpm The bacteria were collected by centrifugation for 10 min to allow them to adhere to the wall of the centrifuge tube and resuspended twice with a resuspension solution containing 10 mM 2-morpholineethanesulfonic acid (MES), 10 mM magnesium chloride (KOH-pH=5.2), and 200 mM acetosyringone to adjust the OD value of the working solution containing rhizobium to 0. 600 Between 0.8 and 1.2.

[0055] The working solution containing the pTi original vector and the pRi2vGR106 vector was transiently transformed into the leaves of various plant species, such as cucumber, white radish, broad bean, cauliflower, nightshade, pokeweed, ginkgo, phalaenopsis, and rose. The culture conditions after infection were: first dark treatment for 8 hours, then placed in light conditions for 24-40 hours, growth temperature of 19-23°C, humidity of 65%-75%, and light intensity of 150 μmol·m -2 ·s -1 .

[0056] The rhizobium solution containing the pTi vector was injected into leaves of various species as blank controls (e.g. Figure 5 After 8 hours of dark treatment, betalain accumulation was observed after 40 hours of light culture. The results showed that, compared with the control under the same time and space conditions, plant leaves infected with rhizobia containing the pRi2vGR106 vector showed higher T-DNA transfer efficiency and faster RUBY protein accumulation.

Claims

1. A genetic transformation vector, characterized in that The pTi vector was modified, specifically including: (1) replacing the left border pTi-LB and the right border pTi-RB of the T-DNA of the pTi vector with the left border pRi-LB and the right border pRi-RB respectively; (2) deleting the T-DNA region of the original pTi vector Hyg expression cassette, while in its original RUBY The front end of the expression cassette is connected to the target gene expression cassette; (3) in its non-T-DNA region Ori Components and SmR Insert between components VirGN54D insert element; (4) the 106th position of the RepA protein coding gene sequence of pVS1 in its non-T-DNA region was mutated from an arginine coding sequence to a histidine coding sequence; The nucleotide sequence of the pRi-LB is shown in SEQ ID No. 5, and the nucleotide sequence of the pRi-RB is shown in SEQ ID No. 6; VirGN54D insert The nucleotide sequence of the element is shown in SEQ ID No. 7; the nucleotide sequence of the mutated RepA protein encoding gene is shown in SEQ ID No.

9.

2. Use of the genetic transformation vector according to claim 1 in genetic transformation of plants.

3. The use according to claim 2, characterized in that The plants include cucumber, white radish, broad bean, Chinese cabbage, Solanum nigrum, Chinese pokeweed, ginkgo, Phalaenopsis, Chinese rose and Chinese pine.

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