Application of tumor cell inhibitor in improvement of wheat genetic transformation efficiency

Abstract

The invention discloses application of a tumor cell inhibitor to improvement of wheat genetic transformation efficiency, and belongs to the field of variation or genetic engineering. The technical problem to be solved by the invention is how to improve the genetic transformation efficiency of plants. The tumor cell inhibitor disclosed by the invention is at least one of a histone modification inhibitor and a serine / threonine protein kinase inhibitor, the histone modification inhibitor is a histone deacetylase inhibitor chidamide (Tucidinostat), and the serine / threonine protein kinase inhibitor is Torin 2. According to the invention, the tumor cell inhibitor is used for treating the plant genetic transformation callus, so that more regenerated buds are differentiated from the callus, and the genetic transformation efficiency of the plant is improved.

Description

Technical Field

[0001] The present invention belongs to the field of mutation or genetic engineering, and specifically relates to the application of tumor cell inhibitors in improving the genetic transformation efficiency of wheat. Background Art

[0002] Wheat ( Triticum aestivum L.) is one of the important food crops globally, and its yield and quality directly affect human food safety and agricultural sustainable development. With the challenges of population growth and climate change, traditional breeding methods have been difficult to meet the demand for high-yield and high-resistant wheat varieties. Therefore, genetic transformation technology, as an efficient plant improvement method, has received extensive attention.

[0003] Genetic transformation technology mainly includes genetic engineering and transgenic technology, which can improve wheat traits by directly introducing foreign genes into wheat cells. This technology can introduce beneficial genes such as disease resistance, stress resistance, and increased yield, overcoming the problems of long time, long cycle, and complexity faced in traditional breeding methods. The research on wheat genetic transformation began in the 1980s, but due to factors such as the complexity of the wheat genome and poor regeneration ability, the transformation efficiency has been relatively low.

[0004] Currently, the commonly used wheat genetic transformation methods mainly include Agrobacterium-mediated transformation and particle bombardment. Particle bombardment directly shoots DNA particles into plant cells by physical methods, which is applicable to different species, but has low transformation efficiency, uneven gene integration, and multiple copies. Agrobacterium-mediated transformation utilizes the natural transduction characteristics of Agrobacterium to integrate foreign genes into the wheat genome, with relatively high transformation efficiency.

[0005] With the development of genome editing technologies (such as CRISPR / Cas9 technology), the research on wheat genetic transformation has entered a new stage. These technologies not only improve the accuracy and efficiency of gene modification but also effectively reduce the regulatory barriers for transgenic products, promoting the rapid improvement of wheat varieties.

[0006] In summary, wheat genetic transformation technology provides new ideas and methods for wheat improvement, can accelerate the breeding process, improve the stress resistance and yield of wheat, and provide strong support for global food security. Summary of the Invention

[0007] The technical problem to be solved by the present invention is how to improve the genetic transformation efficiency of plants.

[0008] To solve the above technical problem, the present invention first provides the application of tumor cell inhibitors in improving the genetic transformation efficiency of plants, and the tumor cell inhibitor is at least one of histone modification inhibitors and serine / threonine protein kinase inhibitors.

[0009] In the above application, the histone modification inhibitor may be a histone deacetylase inhibitor.

[0010] Specifically, the histone deacetylase inhibitor may be Chidamide (Tucidinostat).

[0011] In the above application, the serine / threonine protein kinase inhibitor may be Torin2.

[0012] The application of the tumor cell inhibitor in promoting the regeneration of plant callus or in improving the regeneration ability of plant callus also belongs to the protection scope of the present invention.

[0013] The present invention also provides a method for plant genetic transformation, which includes: culturing the genetically transformed plant callus on a medium containing the tumor cell inhibitor to obtain the treated callus; differentiating and / or rooting the treated callus to achieve the genetic transformation of the plant.

[0014] In the above method, the genetically transformed plant callus is the plant callus treated with an exogenous DNA fragment for genetic transformation, such as the callus infected by Agrobacterium.

[0015] In the above method, the concentration of the tumor cell inhibitor in the medium can be adjusted according to actual needs or different plants, as long as it can promote the regeneration of callus. In an embodiment of the present invention, the concentration of the tumor cell inhibitor is 10 mM.

[0016] The medium containing the tumor cell inhibitor may be a screening medium for screening. That is, the screening can be carried out synchronously with the treatment of the tumor cell inhibitor.

[0017] Specifically, the screening medium is a medium containing corresponding resistance for screening the medium containing the target exogenous DNA fragment.

[0018] In an embodiment of the present invention, the screening medium is composed of a solvent and a solute. The solvent is water, and the solute and its concentration in the screening medium are respectively 4.33 g / L of plant medium M524, 10 ml / L of 100×MS vitamins aqueous solution, 0.5 mg / L of 2,4-D, 2.2 mg / L of Picloram, 40 g / L of maltose, 0.5 g / L of glutamine, 0.1 g / L of casein hydrolysate, 0.75 g / L of MgCl 2 ·6H 2O, 1.95 g / L MES, 5 g / L agarose, 100 mg / L ascorbic acid, 250 mg / L carbenicillin, 5 mg / L phosphinothricin, 0.85 mg / L AgNO 3 , 10 mM Tucidinostat, pH 5.8.

[0019] In one embodiment of the present invention, the screening medium is composed of a solvent and solutes. The solvent is water, and the solutes and their concentrations in the screening medium are 4.33 g / L plant medium M524, 10 ml / L 100×MS vitamins aqueous solution, 0.5 mg / L 2,4-D, 2.2 mg / L Picloram, 40 g / L maltose, 0.5 g / L glutamine, 0.1 g / L casein hydrolysate, 0.75 g / L MgCl 2 ·6H 2 O, 1.95 g / L MES, 5 g / L agarose, 100 mg / L ascorbic acid, 250 mg / L carbenicillin, 10 mg / L phosphinothricin, 0.85 mg / L AgNO 3 , 10 mM Tucidinostat, pH 5.8.

[0020] Among them, the 100×MS vitamins aqueous solution is composed of a solvent and solutes. The solvent is water, and the solutes and their concentrations in the 100×MS vitamins aqueous solution are 10 g / L inositol, 0.2 g / L glycine, 0.1 g / L thiamine hydrochloride (Thiamine-HCl), 0.05 g / L vitamin B6 (Pyridoxine-HCl), 0.05 g / L nicotinic acid.

[0021] The application of the tumor cell inhibitor in plant genetic transformation also belongs to the protection scope of the present invention.

[0022] The present invention also provides a medium for culturing plant callus, and the medium contains the tumor cell inhibitor.

[0023] The medium also contains components for the growth, regeneration or screening of the plant callus, such as plant medium M524, MS vitamins aqueous solution, 2,4-D, Picloram, maltose, glutamine, casein hydrolysate, MgCl 2 ·6H 2O, MES, ascorbic acid, carbenicillin, phosphinothricin, AgNO 3 etc.

[0024] In one embodiment of the present invention, the culture medium consists of a solvent and solutes. The solvent is water, and the solutes and their concentrations in the screening medium are respectively 4.33 g / L of plant medium M524, 10 ml / L of 100×MS vitamins aqueous solution, 0.5 mg / L of 2,4-D, 2.2 mg / L of Picloram, 40 g / L of maltose, 0.5 g / L of glutamine, 0.1 g / L of casein hydrolysate, 0.75 g / L of MgCl 2 ·6H 2 O, 1.95 g / L of MES, 5 g / L of agarose, 100 mg / L of ascorbic acid, 250 mg / L of carbenicillin, 5 mg / L of phosphinothricin, 0.85 mg / L of AgNO 3 , 10 mM Tucidinostat, pH 5.8.

[0025] In one embodiment of the present invention, the culture medium consists of a solvent and solutes. The solvent is water, and the solutes and their concentrations in the screening medium are respectively 4.33 g / L of plant medium M524, 10 ml / L of 100×MS vitamins aqueous solution, 0.5 mg / L of 2,4-D, 2.2 mg / L of Picloram, 40 g / L of maltose, 0.5 g / L of glutamine, 0.1 g / L of casein hydrolysate, 0.75 g / L of MgCl 2 ·6H 2 O, 1.95 g / L of MES, 5 g / L of agarose, 100 mg / L of ascorbic acid, 250 mg / L of carbenicillin, 10 mg / L of phosphinothricin, 0.85 mg / L of AgNO 3 , 10 mM Tucidinostat, pH 5.8.

[0026] In the present invention, the plant can be M1) or M2) or M3): M1) monocotyledonous plants or dicotyledonous plants; M2) gramineous plants; M3) wheat.

[0027] The present invention uses a tumor cell inhibitor to treat plant genetic transformation callus, enabling the callus to differentiate into more regenerated buds, thereby improving the genetic transformation efficiency of plants.

[0028] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It shows the situation of callus redifferentiation and budding after treatment with the blank control group (left figure) and the Tucidinostat experimental group (right figure).

[0030] Figure 2 It shows the situation of callus redifferentiation and budding after treatment with the blank control group (left figure) and the Torin2 experimental group (right figure).

[0031] Figure 3 It shows the results of the genetic transformation efficiency (i.e., the ratio of the number of positive seedlings to the number of calli) of wheat calli screened with a screening medium containing Tucidinostat and Torin2. CK represents the blank control group. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0033] Tucidinostat (Chidamide, molecular formula: C 22 H 19 FN 4 O 2 , CAS: 1616493-44-7): Duopule, purity ≥ 98%. Tucidinostat is an effective HDAC inhibitor with high oral bioavailability.

[0034] GSK126 (methyltransferase inhibitor, molecular formula: C 31 H 38 N 6 O 2 ): Beijing Bailingwei Technology Co., Ltd., purity ≥ 98%.

[0035] GSK-J1 (demethylase inhibitor, molecular formula: C 22 H 23 N 5 O 2 ): Abmart Pharmaceutical Technology (Shanghai) Co., Ltd., purity ≥ 99%.

[0036] Torin2 (molecular formula: C24 H 15 F 3 N 4 O, CAS: 1223001-51-1): Merck Chemicals, purity ≥ 98%, Torin2 is a serine / threonine protein kinase (mTOR) inhibitor.

[0037] Among them, Tucidinostat, GSK126, GSK-J1, and Torin2 can all inhibit the growth of tumor cells or induce tumor cell death. Tucidinostat can inhibit the growth and spread of tumor cells, and Torin2 can inhibit the proliferation and survival of tumor cells.

[0038] Plant medium M524: MURASHIGE&SKOOG BAS SALT MIX (Beijing Bailingke Biotechnology Co., Ltd., M524).

[0039] 100× MS vitamins aqueous solution: The solvent is water, and the solutes and their concentrations are 10 g / L myo-inositol, 0.2 g / L glycine, 0.1 g / L thiamine-HCl, 0.05 g / L pyridoxine-HCl, and 0.05 g / L nicotinic acid respectively.

[0040] 100× LS vitamins aqueous solution: The solvent is water, and the solutes and their concentrations are 10 g / L myo-inositol, 0.1 g / L thiamine-HCl, 0.05 g / L pyridoxine-HCl, and 0.05 g / L nicotinic acid respectively.

[0041] Example 1. Tucidinostat and Torin2 can promote the redifferentiation of callus in wheat genetic transformation This example provides a method for improving the efficiency of wheat genetic transformation, and the steps are as follows: 1. Construction of recombinant vectors and recombinant bacteria Use primers GFP-F (5′-CTGCAGGTCGACTCTAGAGGATCCCGGGATGGTGAGCAAGGGCGAGG-3′ (SEQ ID No.2)) and GFP-R (5′-TTCGAGCTCTCTAGAACTAGTTTAAGATCTGTACAGCTCGT-3′ (SEQ ID No.3)) to amplify the GFP gene, and pWMB110 Digest the vector with Smal and Spel double enzymes, and then perform seamless cloning on the amplifiedGFP The gene was recombined into pWMB110 the vector to obtain the recombinant vector pWMB110-GFP, and the sequence of pWMB110-GFP is shown in SEQ ID No.1.

[0042] pWMB110-GFP was introduced into Agrobacterium tumefaciens EHA105 (ZC142-1, Beijing Zhuangmeng International Biotechnology Co., Ltd.) to obtain recombinant Agrobacterium tumefaciens.

[0043] 2. Genetic transformation of wheat 1) Preparation of Agrobacterium tumefaciens infection solution: The cultured recombinant Agrobacterium tumefaciens was resuspended in the medium to obtain the Agrobacterium tumefaciens infection solution, and the OD value of the Agrobacterium tumefaciens infection solution was 1.0; among them, the solvent of the used medium was water, and the solutes and their concentrations were 0.433 g / L plant medium M524, 10 g / L sucrose, 0.5 g / L 2-(N-morpholino)ethanesulfonic acid (MES), and pH 5.8.

[0044] 2) At 14-15 days after pollination of KN199 wheat, the young embryos were dissected, infected with the Agrobacterium tumefaciens infection solution for 10 minutes, and then co-cultured on the co-culture medium (the solvent of this medium was water, and the solutes and their concentrations were 0.433 g / L plant medium M524, 10 g / L sucrose, 0.5 g / L 2-(N-morpholino)ethanesulfonic acid (MES), 0.85 mg / L AgNO 3 , 1.25 mg / L CuSO 4 ·5H 2 O, 8 g / L agarose, pH 5.8) in the dark at 23°C for 2 days; 3) After removing the germ, it was transferred to the recovery medium (the solvent of this medium was water, and the solutes and their concentrations were 4.33 g / L plant medium M524, 10 ml / L 100×MS vitamins aqueous solution, 0.5 mg / L 2,4-D, 2.5 mg / L Dicamba, 40 g / L maltose, 0.5 g / L glutamine, 0.1 g / L casein hydrolysate, 0.75 g / LMgCl 2 ·6H 2 O, 1.95 g / L MES, 5 g / L agarose, 100 mg / L ascorbic acid, 250 mg / L carbenicillin, 0.85 mg / L AgNO 3 , pH 5.8) and cultured in the dark at 25°C for 5-6 days to induce callus; 4) Culture the obtained callus on a screening medium containing 10 mM Tucidinostat (the solvent of this medium is water, and the solutes and their concentrations are respectively 4.33 g / L plant medium M524, 10 ml / L 100×MS vitamins aqueous solution, 0.5 mg / L 2,4-D, 2.2 mg / L Picloram, 40 g / L maltose, 0.5 g / L glutamine, 0.1 g / L casein hydrolysate, 0.75 g / L MgCl 2 ·6H 2 O, 1.95 g / L MES, 5 g / L agarose, 100 mg / L ascorbic acid, 250 mg / L carbenicillin, 5 mg / L phosphinothricin, 0.85 mg / L AgNO 3 , 10 mM Tucidinostat, pH 5.8) in the dark at 25 °C in an incubator for 14 days; 5) Transfer the callus again to a secondary screening medium containing 10 mM Tucidinostat (the solvent of this medium is water, and the solutes and their concentrations are respectively 4.33 g / L plant medium M524, 10 ml / L 100×MS vitamins aqueous solution, 0.5 mg / L 2,4-D, 2.2 mg / L Picloram, 40 g / L maltose, 0.5 g / L glutamine, 0.1 g / L casein hydrolysate, 0.75 g / L MgCl 2 ·6H 2 O, 1.95 g / L MES, 5 g / L agarose, 100 mg / L ascorbic acid, 250 mg / L carbenicillin, 10 mg / L phosphinothricin, 0.85 mg / L AgNO 3 , 10 mM Tucidinostat, pH 5.8) in the dark at 25 °C in an incubator for 21 days; 6) Then transfer the callus to a regeneration medium (the solvent of this medium is water, and the solutes and their concentrations are respectively 4.33 g / L plant medium M524, 10 ml / L 100×LS vitamins aqueous solution, 2 mg / L zeatin, 20 g / L sucrose, 0.5 g / L MES, 2.5 mg / L CuSO 4 ·5H 2On the medium (3 g / L phytoagar, 100 mg / L cefotaxime, 250 mg / L carbenicillin, 5 mg / L phosphinothricin), induce bud differentiation for 10 days (25 °C, light). 7) Finally, transfer the differentiated buds to the rooting medium (the solvent of this medium is water, and the solutes and their concentrations are respectively 4.33 g / L plant medium M524, 10 ml / L 100×LS vitamins aqueous solution, 0.2 mg / L IBA, 15 g / L sucrose, 0.5 g / L MES, 2.5 mg / L CuSO 4 ·5H 2 O, 3 g / L phytoagar, 250 mg / L carbenicillin, 5 mg / L phosphinothricin), induce root formation at 25 °C under light, and count the number of positive seedlings.

[0045] According to the methods of steps 1)-7) above, replace the "screening medium containing 10 mM Tucidinostat" with a screening medium without Tucidinostat (except for not containing Tucidinostat, the other components of this medium are the same as those of the "screening medium containing 10 mM Tucidinostat"), and replace the "secondary screening medium containing 10 mM Tucidinostat" with a secondary screening medium without Tucidinostat (except for not containing Tucidinostat, the other components of this medium are the same as those of the "secondary screening medium containing 10 mM Tucidinostat"), and keep the other steps unchanged as a blank control.

[0046] According to the methods of steps 1)-7) above, replace the "screening medium containing 10 mM Tucidinostat" with a "screening medium containing 10 mM GSK126" (this medium is obtained by replacing 10 mM Tucidinostat in the "screening medium containing 10 mM Tucidinostat" with 10 mM GSK126), and replace the "secondary screening medium containing 10 mM Tucidinostat" with a "secondary screening medium containing 10 mM GSK126" (this medium is obtained by replacing 10 mM Tucidinostat in the "secondary screening medium containing 10 mM Tucidinostat" with 10 mM GSK126), and keep the other steps unchanged as the GSK126 experimental group.

[0047] According to the method of the above steps 1)-7), replace the "screening medium containing 10 mM Tucidinostat" with "screening medium containing 10 mM GSK-J1" (this medium is obtained by replacing 10 mM Tucidinostat in the "screening medium containing 10 mM Tucidinostat" with 10 mM GSK-J1), replace the "secondary screening medium containing 10 mM Tucidinostat" with "secondary screening medium containing 10 mM GSK-J1" (this medium is obtained by replacing 10 mM Tucidinostat in the "secondary screening medium containing 10 mM Tucidinostat" with 10 mM GSK-J1), and keep the other steps unchanged, which is used as the GSK-J1 experimental group.

[0048] According to the method of the above steps 1)-7), replace the "screening medium containing 10 mM Tucidinostat" with "screening medium containing 10 mM Torin2" (this medium is obtained by replacing 10 mM Tucidinostat in the "screening medium containing 10 mM Tucidinostat" with 10 mM Torin2), replace the "secondary screening medium containing 10 mM Tucidinostat" with "secondary screening medium containing 10 mM Torin2" (this medium is obtained by replacing 10 mM Tucidinostat in the "secondary screening medium containing 10 mM Tucidinostat" with 10 mM Torin2), and keep the other steps unchanged, which is used as the Torin2 experimental group.

[0049] The callus redifferentiation and budding situation of the Tucidinostat experimental group is as Figure 1 shown, and the callus redifferentiation and budding situation of the Torin2 experimental group is as Figure 2 shown.

[0050] The results of the number of positive seedlings are shown in Table 1 and Figure 3 , when Tucidinostat and Torin2 are contained in the screening medium, the ratio of the number of positive seedlings to the number of calli is significantly higher than that of the blank control group, while when GSK126 and GSK-J1 are contained in the screening medium, the ratio of the number of positive seedlings to the number of calli is significantly lower than that of the blank control group, indicating that both Tucidinostat and Torin2 can promote the redifferentiation of callus in wheat genetic transformation, improve the genetic transformation efficiency of wheat, can be used for wheat genetic transformation, and can also be used for the redifferentiation of wheat callus in non-genetic transformation.

[0051]

[0052] For the genetic transformation experiments, repeated experiments were carried out. The SPSS 11.5 statistical software was used to process the data, and the One-way ANOVA test was adopted. Among the data of the "ratio of the number of positive seedlings to the number of calluses" in the "significance analysis" column, there were significant differences between the data with different letters, and there were no significant differences between the data with the same letter.

[0053] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application and are made by using conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. The use of a tumor cell inhibitor in improving the efficiency of plant genetic transformation, characterized in that: The tumor cell inhibitor is at least one of a histone modification inhibitor and a serine / threonine protein kinase inhibitor.

2. The use according to claim 1, characterized in that: The histone modification inhibitor is a histone deacetylase inhibitor.

3. The use according to claim 2, characterized in that: The histone deacetylase inhibitor is chidamide.

4. The use according to claim 1, characterized in that: The serine / threonine protein kinase inhibitor is Torin2.

5. Use of the tumor cell inhibitor according to any one of claims 1 to 4 in promoting plant callus regeneration, or in improving plant callus regeneration ability.

6. The use according to any one of claims 1 to 5, characterized in that: The plant is M1) or M2) or M3): M1) monocots or dicots; M2) Gramineae; M3) Wheat.

7. A method for plant genetic transformation, characterized in that: The method comprises: culturing plant callus treated with genetic transformation on a culture medium containing the tumor cell inhibitor described in any one of claims 1 to 4 to obtain treated callus; and subjecting the treated callus to differentiation culture and / or rooting culture, thereby achieving genetic transformation of the plant.

8. The method according to claim 7, characterized in that: The plant is M1) or M2) or M3): M1) monocots or dicots; M2) Gramineae; M3) Wheat.

9. Use of the tumor cell inhibitor according to any one of claims 1 to 4 in plant genetic transformation.

10. A culture medium for culturing plant callus, characterized in that: The culture medium contains the tumor cell inhibitor according to any one of claims 1-4.

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