Efficient plant genetic transformation vector based on rhizobium rhizogenes and application of efficient plant genetic transformation vector

Abstract

The invention relates to an efficient plant genetic transformation vector based on rhizobium rhizogenes, which is modified on the basis of a pTi vector, and a left boundary pTi-LB and a right boundary pTi-RB of T-DNA of the vector are respectively replaced by a left boundary pRi-LB and a right boundary pRi-RB; deleting a Hyg expression cassette in the T-DNA region, and connecting a target gene expression cassette to the front end of the original RUBY expression cassette; a VirGN54D insert element is inserted between an Ori element and an SmR element in a non-T-DNA (Transfer-Deoxyribose Nucleic Acid) region; the 106th site of a RepA protein coding gene sequence in pVS1 of a non-T-DNA region is mutated into a histidine coding sequence from an arginine coding sequence. The invention aims to enhance efficient genetic transformation of plant exogenous genes in multiple species, has broad-spectrum adaptability to plant species, and particularly has obvious effects in multiple plant species which depend on genotypes and are difficult to transform, such as cucumbers, white radishes, broad beans, Chinese little greens, black nightshade, pokeberry roots, gingko, phalaenopsis, Chinese roses, Chinese pines and the like.

Description

Technical Field

[0001] The present invention belongs to the fields of plant molecular biology and genetic engineering, and particularly relates to a high-efficiency plant 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 the 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 applications are still limited to a few model species. In contrast, Agrobacterium-mediated transformation ( Agrobacterium -Mediated Transformation, AMT) still remains the most widely used plant genetic transformation system at present due to its efficient T-DNA transfer mechanism and precise genome integration characteristics. The molecular mechanism of AMT involves the synergistic 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 forms a protective nucleoprotein complex through non-covalent binding to prevent nuclease degradation and assist nuclear localization. These complexes complete transmembrane transport through the type IV secretion system (T4SS). Based on this, a large number of studies have improved the AMT efficiency of many plant species by optimizing induction conditions, Agrobacterium tumefaciens strain types, and virulence genes. However, this technology still has many limitations. Especially for genotype-dependent and difficult-to-transform plants, the efficiency is low or even ineffective, and it is difficult to obtain stable transgenic plants, severely restricting the process of genetic engineering. Therefore, more efficient tools still need to be developed to optimize the AMT process. Summary of the Invention

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

[0004] The present invention adopts the following technical solutions: A genetic transformation vector obtained by modifying the pTi vector, 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 Hyg expression cassette in the T-DNA region of the original pTi vector, and at the same time in its original RUBYThe front end of the expression cassette is connected to the target gene expression cassette; (3) Insert between the Ori element and the SmR element in its non-T-DNA region the VirGN54D insert element; (4) Mutate the 106th position of the RepA protein coding gene sequence of pVS1 in its non-T-DNA region from the arginine coding sequence to the histidine coding sequence. Ori element and SmR insert between elements VirGN54D insert element; (4) Mutate the 106th position of the RepA protein coding gene sequence of pVS1 in its non-T-DNA region from the arginine coding sequence to the histidine coding sequence.

[0005] Among them, 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.

[0006] Among them, 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.

[0007] Among them, the VirGN54D insert element has a nucleotide sequence shown in SEQ ID No.7.

[0008] Among them, the nucleotide sequence of the mutated RepA protein coding gene is shown in SEQ ID No.9.

[0009] An application of the above genetic transformation vector in the genetic transformation of plants.

[0010] In the application, the plants include cucumber, white radish, broad bean, Chinese flowering cabbage, black nightshade, pokeweed, ginkgo, phalaenopsis, Chinese rose, and Chinese pine.

[0011] The beneficial effects of the present invention are as follows: (1) The present invention makes multiple modifications to the plant binary overexpression vector pTi. For the T-DNA region, a super-driving sequence is artificially introduced into the left border (LB) and right border (RB) sequences to enhance the recognition and cleavage efficiency of the VirD2 protein for the border sequences, thereby significantly improving the transfer efficiency of T-DNA. In the non-T-DNA region, insert the VirGN54D insert functional element downstream of the resistance gene to promote the constitutive high expression of the virulence gene, and this expression does not depend on VirA or chemical inducers, providing more favorable conditions for plant genetic transformation. At the same time, a directed mutation (arginine Arg mutated to histidine His) is made to the 106th amino acid of the pVS1 RepA element, significantly improving the replication stability of the vector in the host bacterium and further enhancing the transformation efficiency. VirGN54D insert functional element, promoting the constitutive high expression of the virulence gene, and this expression does not depend on VirA or chemical inducers, providing more favorable conditions for plant genetic transformation. At the same time, a directed mutation (arginine Arg mutated to histidine His) is made to the 106th amino acid of the pVS1 RepA element, significantly improving the replication stability of the vector in the host bacterium and further enhancing the transformation efficiency.

[0012] (2) This vector system has shown outstanding advantages in a variety of traditional gene-dependent and difficult-to-transiently-transform plants (cucumber, white radish, broad bean, Chinese flowering cabbage, Solanum nigrum, Phytolacca acinosa, Ginkgo biloba, Phalaenopsis aphrodite, Rosa chinensis, and Chinese pine). It has core advantages such as high transformation efficiency, good stability, wide applicability, accurate verification, and simple operation, providing an efficient and stable genetic transformation tool for plant gene function research, molecular breeding, and biotechnology applications, and having important value in both scientific research and industrial fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagrams of different modified vector structures and sequence optimizations.

[0014] Figure 2 Temporal comparison diagrams of tobacco genetic transformation mediated by different modified vectors RUBY

[0015] Figure 3 High-efficiency genetic transformation diagram of Chinese pine roots mediated by the pRi2vGR106 vector.

[0016] Figure 4 Betacyanin quantitative analysis comparison diagrams of tobacco genetic transformation mediated by different vectors RUBY

[0017] Figure 5 Broad-spectrum verification diagram of multi-species genetic transformation mediated by the pRi2vGR106 vector. DETAILED DESCRIPTION OF THE INVENTION

[0018] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps, or conditions of the present invention all fall within the scope of this application. If not specifically specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0019] Example 1 Modification of the original vector pTi As Figure 1 shown, the left border pTi-LB (SEQ ID No.1) and right border pTi-RB (SEQ ID No.2) of the T-DNA of the original pTi vector were respectively replaced with the pRiA4T-LB (SEQ ID No.3) and pRiA4T-RB (SEQ ID No.4) of the Ri plasmid in the rhizobium strain ArA4.

[0020] On this basis, the T-DNA region of the original pTi vector was modified. The Hyg expression cassette in the T-DNA region of the original pTi vector was deleted, and at the same time, at its originalRUBY Front-end connection of the expression cassette GUS Expression cassette 。 The GUS expression cassette is the CaMV 35S promoter - GUS reporter gene - HA - 6×His - AtHSP 18.2 Term terminator. The vector modified as above was named pRiA4T.

[0021] The pTi original vector and the pRiA4T recombinant vector were respectively transformed into the Escherichia coli competent cell DH5α (ATCC 53868) by heat shock method to obtain Escherichia coli cells containing the pTi original vector and the pRiA4T recombinant vector, and cultured for 12 - 16 h; the recombinant plasmid was extracted from the Escherichia coli cells and transformed into the Agrobacterium rhizogenes competent cell K599 (NCPPB2659) by chemical transformation method to obtain Agrobacterium rhizogenes containing the recombinant vector.

[0022] The bacterial solutions of Agrobacterium rhizogenes containing the pTi original vector and the pRiA4T recombinant vector were respectively centrifuged at 7,500 rpm for 10 min to collect the bacteria, so that the bacteria adhered to the centrifuge tube wall, and resuspended twice with a resuspension solution containing 10 mM 2 - morpholinoethanesulfonic acid (MES), 10 mM magnesium chloride (KOH - pH = 5.2), and 200 mM acetosyringone, so that the OD of the working solution containing Agrobacterium rhizogenes 600 was between 0.8 and 1.2.

[0023] After infection, it was found by observation that the pRiA4T vector accumulated betacyanin faster than the original vector pTi in tobacco leaves (such as Figure 2 ).

[0024] Example 2 Modification based on the pRiA4T vector Based on the pRiA4T vector obtained in Example 1, further modification was carried out. 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 respectively 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 in the Agrobacterium rhizogenes strain K599, and this vector was named pRi2 vector.

[0025] On the basis of the pRi2 vector, between its non - T - DNA region Ori elements and SmR elements, insert VirGN54D insertElement (SEQ ID No.7), which promotes the constitutive overexpression of virulence genes and is independent of VirA or chemical inducers, promotes plant genetic transformation. This vector is named the pRi2vG vector.

[0026] On the pRi2vG vector, the arginine (Arg) at position 106 of the RepA protein of pVS1 in its non-T-DNA region was further 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. This vector is named pRi2vGR106.

[0027] For the pTi, pRiA4T, pRi2vG, and pRi2vGR106 vectors, the conventional tobacco transient transformation method was used, that is, Agrobacterium rhizogenes was injected into tobacco leaves. By setting different observation time points (such as 12 h, 24 h, 36 h), the rate of RUBY protein accumulation was observed to verify the delivery efficiency of the target protein in the tobacco transient transformation experiment (such as Figure 2 ). After infection, it was found that the area infected by Agrobacterium rhizogenes containing the pRi2vGR106 vector expressed the RUBY protein first, and the original vector pTi expressed the slowest.

[0028] Example 3: The pRi2vGR106 vector improves the genetic transformation efficiency of Pinus tabuliformis roots Using the pRi2vGR106 vector with the highest tobacco transient transformation efficiency in Example 2, the genetic transformation of Pinus tabuliformis roots was carried out.

[0029] The recombinant vector pRi2vGR106 was transformed into Agrobacterium rhizogenes competent cells by the chemical transformation method. 8 - 10 positive monoclonal colonies were picked. First, each tube was cultured overnight in a small shaker at 28°C with 400 μl of TY + 75 mg / L spectinomycin (Spec) liquid medium at 200 rpm rpm. Then, 200 μl of the bacterial solution was added to 20 ml of TY + 75 mg / L Spec liquid medium for large-scale culture. After the bacterial solution concentration reached OD 600 = 0.8 - 1.5, the bacterial solution was centrifuged at 7000 rpm rpm for 10 min at room temperature. The supernatant was removed, and the precipitate was resuspended in TY + 75 mg / L Spec liquid medium. 350 - 400 μl of the bacterial solution was pipetted and spread on TY + 75 mg / L Spec solid medium, and then inverted in an incubator at 28°C until a thick bacterial layer grew on the surface of the solid medium.

[0030] The TY liquid culture medium includes: 5 g / L peptone, 3 g / L yeast extract, and 1 M calcium chloride; if preparing solid medium, add 15 g / L agar powder.

[0031] When the stem length of the cultured Chinese pine seedlings exceeds 3 cm, cut off about 0.5 - 1.0 cm above or below the root-stem junction with a blade tilted at 45°. Immerse the plant wound in the resuspended solution of Rhizobium rhizogenes, and then apply vacuum to the plant. The vacuum pressure value is 0.05 MPa, and the duration is 8 min. Take out the plant explant from the resuspended solution, wrap the root wound of the plant with the Rhizobium rhizogenes cells containing the pRi2vGR106 vector, and then transplant it into the soil culture substrate. Culture it at 19 - 24°C for 30 - 45 days, and stable transgenic adventitious roots will grow at the hypocotyl of Chinese pine.

[0032] Among them, the soil culture substrate 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% - 75%. The light intensity during the growth period is 150 μmo1·m -2 ·s -1 , and the photoperiod during the growth period is 16 h.

[0033] Perform genetic transformation of the Chinese pine root system with the above-mentioned Rhizobium rhizogenes solution containing the pRi2vGR106 vector. Use the pTi original vector as a control group and observe RUBY the expression of the reporter gene to verify the transformation efficiency of the transgenic roots of Chinese pine (such as Figure 3 ). After infection, it was found that the transgenic roots of Chinese pine infected with Rhizobium rhizogenes containing the pRi2vGR106 vector expressed obvious betacyanin pigments, which was the result of the accumulation of RUBY protein, indicating that the modified vector has high genetic transformation efficiency.

[0034] Example 4 Expression Efficiency of the Vector System To evaluate the expression efficiency of different vector systems, select the Agrobacterium rhizogenes liquid transformed by four vector constructs of pRiA4T, pRi2, pRi2vG, and pRi2vGR106 to transiently transform tobacco leaves, and perform quantitative detection of the betacyanin expressed by the reporter gene through high-performance liquid extraction and spectrophotometry. RUBY

[0035] The specific experimental steps are as follows: (1) Select the tobacco leaves transiently transformed by four different vectors, and cut about 100 mg (fresh weight) of the colored part as the extraction material.

[0036] (2) Place the sample in liquid nitrogen and quickly freeze it, and grind it into a powder with a mortar.

[0037] (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 it on a thermostatic shaker and shake for 20 min.

[0038] (4) Centrifuge the mixture at 4 °C at 10,000 rpm for 10 min, and collect the supernatant into a centrifuge tube.

[0039] (5) Adjust the pH value of the collected supernatant to 3 - 5 using 0.1 M HCl solution.

[0040] (6) Centrifuge again at 4 °C at 10,000 rpm for 10 min, and discard the supernatant.

[0041] (7) Take 1 mL of the crude sample treated above, add 4 mL of the composite organic reagent, vortex and shake for 10 min, and then let it stand for 5 min to wait for the solution to separate.

[0042] (8) Centrifuge again at 4 °C at 10,000 rpm for 10 min, and collect the supernatant.

[0043] (9) Measure the absorbance at a wavelength of 532 nm using a UV - visible spectrophotometer. All the measured samples were averaged after three biological test replicates. The pigments extracted from fresh tobacco leaves that were not transiently transformed were used as a blank control to detect the concentration of betacyanin in different solutions.

[0044] The results of quantitative detection by spectrophotometry showed (as Figure 4 ) that there were significant differences in the accumulation efficiency of betacyanin among different vector systems. Among them, the hairy root agrobacterium infection group transformed with the pRi2vGR106 vector showed the highest betacyanin accumulation of 4.21 mg / 100 mL, followed by pRi2vG with 3.76 mg / 100 mL, the pigment yield of pRi2 was 1.55 mg / 100 mL, and the expression efficiency of pRiA4T was the lowest at 1.23 mg / 100 mL. This data indicates that the optimization of pRi2vGR106 significantly improved the RUBY expression efficiency of the reporter gene.

[0045] Example 5 Application of the vector in multi - species transient transformation Transform the above vectors (pTi, pRi2vGR106) into Escherichia coli competent cells to obtain Escherichia coli cells containing the recombinant vector, and culture for 12 - 16 h; extract the recombinant plasmid from the Escherichia coli cells and transform it into the competent cells of Rhizobium rhizogenes K599 to obtain Rhizobium rhizogenes containing the recombinant vector.

[0046] Centrifuge the bacterial liquid of Rhizobium rhizogenes at 7,500 rpm for 10 min to collect the bacteria, making the bacteria adhere to the centrifuge tube wall, and resuspend twice with a resuspension solution containing 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 10 mM magnesium chloride (KOH - pH = 5.2), and 200 mM acetosyringone, so that the OD of the working solution containing Rhizobium rhizogenes 600 is between 0.8 and 1.2.

[0047] Transiently transform the working solutions containing the pTi original vector and the pRi2vGR106 vector into the leaves of multiple species of plants such as cucumber, white radish, broad bean, Chinese flowering cabbage, Solanum nigrum, Phytolacca acinosa, Ginkgo biloba, Phalaenopsis aphrodite, Rosa chinensis, etc. The culture conditions after infection are: first, dark treatment for 8 h, then placed under light conditions for 24 - 40 h, the growth temperature is 19 - 23°C, the humidity is 65% - 75%, and the light intensity during growth is 150 μmol·m -2 ·s -1 .

[0048] Inject the bacterial liquid of Rhizobium rhizogenes containing the pTi vector into the leaves of multiple species as a blank control (such as Figure 5 ). After 8 h of dark treatment, observe the accumulation of betacyanin after 40 h of light culture. The final results show that compared with the control under the same time and space conditions, the plant leaves infected with Rhizobium rhizogenes containing the pRi2vGR106 vector all showed the characteristics of higher transfer efficiency of T-DNA and faster accumulation of RUBY protein.

Claims

1. A genetic transformation vector, characterized in that, Modify the pTi vector, specifically including: (1) Replace 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) Delete the Hyg expression cassette in the T-DNA region of the original pTi vector, and at the same time connect the target gene expression cassette to the front end of its original RUBY expression cassette; (3) Insert Ori element and SmR element between VirGN54D insert elements; (4) Mutate the 106th position of the RepA protein coding gene sequence of pVS1 in its non-T-DNA region from the arginine coding sequence to the histidine coding sequence.

2. The genetic transformation vector according to claim 1, characterized in that The nucleotide sequence of the said pRi-LB is shown as SEQ ID No.5, and the nucleotide sequence of the said pRi-RB is shown as SEQ ID No.

6.

3. The genetic transformation vector according to claim 1, characterized in that, The VirGN54D insert nucleotide sequence of the element is shown in SEQ ID No.

7.

4. The genetic transformation vector according to claim 1, characterized in that, The nucleotide sequence of the mutated RepA protein coding gene is shown as SEQ ID No.

9.

5. Use of a genetic transformation vector as described in any one of claims 1 to 4 in the genetic transformation of plants.

6. The application according to claim 5, wherein The said plants include cucumber, white radish, broad bean, Chinese flowering cabbage, black nightshade, pokeweed, ginkgo, phalaenopsis, Chinese rose and Chinese pine.

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