Application of tumor cell inhibitors in improving wheat genetic transformation efficiency

Abstract

This invention discloses the use of tumor cell inhibitors to improve the efficiency of genetic transformation in wheat, belonging to the field of mutation or genetic engineering. The technical problem addressed by this invention is how to improve the efficiency of genetic transformation in plants. The tumor cell inhibitor disclosed in this invention is at least one of a histone modification inhibitor and a serine / threonine protein kinase inhibitor. The histone modification inhibitor is the histone deacetylase inhibitor tucidinostat, and the serine / threonine protein kinase inhibitor is Torin2. The present invention discovers that treating plant genetic transformation calli with tumor cell inhibitors promotes callus differentiation into more regenerated buds, thereby improving the efficiency of genetic transformation in plants.

Description

Technical Field

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

[0002] wheat( Triticum aestivum Wheat (L.) is one of the world's most important food crops, its yield and quality directly impacting food security and sustainable agricultural development. With the challenges of population growth and climate change, traditional breeding methods are no longer able to meet the demand for high-yield and highly resistant wheat varieties. Therefore, genetic transformation technology, as a highly effective means of plant improvement, has garnered widespread attention.

[0003] Genetic transformation technologies primarily include genetic engineering and transgenic techniques. They improve wheat traits by directly introducing foreign genes into wheat cells. This technology can introduce beneficial genes for disease resistance, stress tolerance, and yield enhancement, overcoming the time, cycle, and complexity inherent in traditional breeding methods. Research on wheat genetic transformation began in the 1980s, but transformation efficiency has been low due to factors such as the complexity of the wheat genome and its poor regeneration capacity.

[0004] Currently, the most commonly used genetic transformation methods for wheat are Agrobacterium-mediated transformation and gene gun transformation. Gene gun transformation involves physically injecting DNA particles directly into plant cells, and while suitable for a variety of species, it suffers from low transformation efficiency, uneven gene integration, and multiple copies. Agrobacterium-mediated transformation utilizes the natural transduction properties of Agrobacterium to integrate foreign genes into the wheat genome, resulting in higher transformation efficiency.

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

[0006] In short, wheat genetic transformation technology provides new ideas and methods for wheat improvement, which can accelerate the breeding process, improve wheat's stress resistance and yield, 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 problems, the present invention first provides the use of a tumor cell inhibitor in improving the efficiency of plant genetic transformation. The tumor cell inhibitor is at least one of a histone modification inhibitor and a serine / threonine protein kinase inhibitor.

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

[0010] Specifically, the histone deacetylase inhibitor may be tucidinostat.

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

[0012] The use of the tumor cell inhibitor in promoting plant callus regeneration, or in improving the regeneration ability of plant callus, also falls within the protection scope of the present invention.

[0013] The present invention also provides a plant genetic transformation method, which comprises: culturing plant callus tissue that has been subjected to genetic transformation treatment on a culture medium containing the tumor cell inhibitor to obtain treated callus tissue; and subjecting the treated callus tissue to differentiation culture and / or rooting culture, thereby achieving plant genetic transformation.

[0014] In the above method, the plant callus tissue treated with genetic transformation is the plant callus tissue treated with exogenous DNA fragments for genetic transformation, such as the callus tissue infected with Agrobacterium.

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

[0016] The culture medium containing the tumor cell inhibitor can be a screening medium used for screening, that is, screening and treatment of the tumor cell inhibitor can be performed simultaneously.

[0017] Specifically, the screening culture medium is a culture medium containing corresponding resistance and is used to screen for the target exogenous DNA fragment.

[0018] In one 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 4.33 g / L plant culture 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 MgCl2·6H2O, 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 / LAgNO3, 10 mM Tucidinostat, pH 5.8.

[0019] In one 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 4.33 g / L plant culture 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 MgCl2·6H2O, 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 AgNO3, 10 mM Tucidinostat, pH 5.8.

[0020] The 100×MS vitamins aqueous solution is composed of a solvent and a solute, 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), and 0.05 g / L nicotinic acid.

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

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

[0023] The culture medium also contains components for callus growth, regeneration or screening of the plant, such as plant culture medium M524, MS vitamins aqueous solution, 2,4-D, Picroram, maltose, glutamine, casein hydrolysate, MgCl2·6H2O, MES, ascorbic acid, carbenicillin, phosphinothricin, AgNO3, etc.

[0024] In one embodiment of the present invention, the culture medium is composed of a solvent and a solute, the solvent is water, and the solute and its concentration in the screening culture medium are 4.33 g / L plant culture medium M524, 10 ml / L 100×MSvitamins 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 MgCl2·6H2O, 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 / LAgNO3, 10 mM Tucidinostat, pH 5.8.

[0025] In one embodiment of the present invention, the culture medium is composed of a solvent and a solute, the solvent is water, and the solute and its concentration in the screening culture medium are 4.33 g / L plant culture medium M524, 10 ml / L 100×MSvitamins 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 MgCl2·6H2O, 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 AgNO3, 10 mM Tucidinostat, pH 5.8.

[0026] In the present invention, the plant may be M1) or M2) or M3):

[0027] M1) monocots or dicots;

[0028] M2) grasses;

[0029] M3) Wheat.

[0030] The present invention utilizes tumor cell inhibitors to treat plant genetic transformation callus, so that the callus differentiates into more regenerated buds, thereby improving the genetic transformation efficiency of the plant.

[0031] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The results of callus redifferentiation and budding after treatment in the blank control group (left) and the Tucidinostat experimental group (right).

[0033] Figure 2 The results of callus redifferentiation and budding after treatment in the blank control group (left) and the Torin2 experimental group (right).

[0034] Figure 3 This figure shows the genetic transformation efficiency (i.e., the ratio of positive seedlings to calli) of wheat calli screened using a screening medium containing tucidinostat and Torin2. CK represents the blank control. DETAILED DESCRIPTION

[0035] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, and instruments used in the following examples are all commercially available unless otherwise specified.

[0036] Tucidinostat (Cidamide, molecular formula: C 22 H 19 FN4O2, CAS: 1616493-44-7): Duopler, purity ≥98%, Tucidinostat is a potent and orally bioavailable HDAC inhibitor.

[0037] GSK126 (methyltransferase inhibitor, molecular formula: C 31 H 38 N6O2): Beijing Bailingwei Technology Co., Ltd., purity ≥98%.

[0038] GSK-J1 (demethylase inhibitor, molecular formula: C 22 H 23 N5O2): Abimat Pharmaceutical Technology (Shanghai) Co., Ltd., purity ≥99%.

[0039] Torin2 (Molecular formula: C 24H 15 F3N4O, CAS: 1223001-51-1): Merck Chemical, purity ≥98%, Torin2 is a serine / threonine protein kinase (mTOR) inhibitor.

[0040] 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.

[0041] Plant culture medium M524: Murashige & Skoog Bas Salt Mix (Beijing Bailingke Biotechnology Co., Ltd., M524).

[0042] 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 hydrochloride, 0.05 g / L pyridoxine-HCl, and 0.05 g / L nicotinic acid.

[0043] 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 hydrochloride, 0.05 g / L vitamin B6, and 0.05 g / L niacin.

[0044] Example 1: Tucidinostat and Torin2 can promote the redifferentiation of calli in wheat genetic transformation

[0045] This embodiment provides a method for improving wheat genetic transformation efficiency, the steps are as follows:

[0046] 1. Construction of recombinant vectors and recombinant bacteria

[0047] The GFP-F (5′-CTGCAGGTCGACTCTAGAGGATCCCGGGATGGTGAGCAAGGGCGAGG-3′ (SEQ ID No. 2)) and GFP-R (5′-TTCGAGCTCTCTAGAACTAGTTTAAGATCTGTACAGCTCGT-3′ (SEQ ID No. 3)) primers were used to amplify the GFP-R primers. GFP Genes will pWMB110 The vector was double-digested with Smal and Spel enzymes, and the amplified fragment was then cloned seamlessly. GFP Gene recombination to pWMB110 The recombinant vector pWMB110-GFP was obtained. The sequence of pWMB110-GFP is shown in SEQ ID No. 1.

[0048] pWMB110-GFP was introduced into Agrobacterium EHA105 (ZC142-1, Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd.) to obtain recombinant Agrobacterium.

[0049] 2. Genetic transformation of wheat

[0050] 1) Preparation of Agrobacterium infection fluid: Resuspend the cultured recombinant Agrobacterium in culture medium to obtain an Agrobacterium infection fluid with an OD value of 1.0. The solvent of the culture medium used was water, and the solutes and their concentrations were 0.433 g / L plant culture medium M524, 10 g / L sucrose, and 0.5 g / L 2-morpholineethanesulfonic acid (MES), respectively, at a pH of 5.8.

[0051] 2) 14-15 days after pollination, immature embryos of KN199 wheat were removed and infected with Agrobacterium infection solution for 10 minutes. The embryos were then cultured in co-cultivation medium (water solvent, 0.433 g / L plant medium M524, 10 g / L sucrose, 0.5 g / L 2-morpholineethanesulfonic acid (MES), 0.85 mg / L AgNO₃, 1.25 mg / L CuSO₄·5H₂O, 8 g / L agarose, pH 5.8) at 23°C in a dark incubator for 2 days.

[0052] 3) After excising the embryo, transfer the embryo to recovery medium (the solvent of the medium is water, and the solutes and their concentrations are 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 Didicamba, 40 g / L maltose, 0.5 g / L glutamine, 0.1 g / L casein hydrolysate, 0.75 g / L MgCl2·6H2O, 1.95 g / L MES, 5 g / L agarose, 100 mg / L ascorbic acid, 250 mg / L carbenicillin, 0.85 mg / L AgNO3, pH 5.8) and culture in a dark incubator at 25°C for 5-6 days to induce callus;

[0053] 4) The resulting calli were cultured on a selection medium containing 10 mM tucidinostat (water as the solvent, 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 MgCl2·6H2O, 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 AgNO3, 10 mM tucidinostat, pH 5.8) at 25°C in a dark incubator for 14 days.

[0054] 5) The calli were transferred again to a secondary selection medium containing 10 mM tucidinostat (the solvent of the medium was water; the solutes and their concentrations were 4.33 g / L plant medium M524, 10 ml / L 100× MS vitamin aqueous solution, 0.5 mg / L 2,4-D, 2.2 mg / L picroram, 40 g / L maltose, 0.5 g / L glutamine, 0.1 g / L casein hydrolysate, 0.75 g / L MgCl2·6H2O, 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 AgNO3, 10 mM tucidinostat, pH 5.8) and cultured in a 25°C incubator in the dark for 21 days.

[0055] 6) The calli were then transferred to redifferentiation medium (the solvent of the medium was water, and the solutes and their concentrations were 4.33 g / L plant medium M524, 10 ml / L 100×LS vitamin aqueous solution, 2 mg / L zeatin, 20 g / L sucrose, 0.5 g / L MES, 2.5 mg / L CuSO4·5H2O, 3 g / L plant gelatin, 100 mg / L cefotaxime, 250 mg / L carbenicillin, and 5 mg / L phosphinothricin) and induced to differentiate into shoots for 10 days (25°C, light).

[0056] 7) Finally, transfer the differentiated shoots to rooting medium (the solvent of the medium is water, and the solutes and their concentrations are 4.33 g / L plant culture medium M524, 10 ml / L 100×LS vitamin aqueous solution, 0.2 mg / L IBA, 15 g / L sucrose, 0.5 g / L MES, 2.5 mg / L CuSO4·5H2O, 3 g / L plant gelatin, 250 mg / L carbenicillin, and 5 mg / L phosphinothricin) and induce root formation at 25°C in the light. Count the number of positive shoots.

[0057] According to the method of steps 1) to 7) above, the "screening medium containing 10 mM tucidinostat" was replaced with a screening medium not containing tucidinostat (except for not containing tucidinostat, the other components of this medium were the same as the "screening medium containing 10 mM tucidinostat"), and the "secondary screening medium containing 10 mM tucidinostat" was replaced with a secondary screening medium not containing tucidinostat (except for not containing tucidinostat, the other components of this medium were the same as the "secondary screening medium containing 10 mM tucidinostat"), and other steps remained unchanged as blank controls.

[0058] According to the method of the above steps 1)-7), the "screening medium containing 10mM tucidinostat" was replaced with the "screening medium containing 10mM GSK126" (this medium is the medium obtained by replacing 10mM tucidinostat in the "screening medium containing 10mM tucidinostat" with 10mM GSK126), and the "secondary screening medium containing 10mM tucidinostat" was replaced with the "secondary screening medium containing 10mM GSK126" (this medium is the medium obtained by replacing 10mM tucidinostat in the "secondary screening medium containing 10mM tucidinostat" with 10mM GSK126). Other steps remained unchanged and this was used as the GSK126 experimental group.

[0059] According to the method of the above steps 1)-7), the "screening medium containing 10mM tucidinostat" was replaced with the "screening medium containing 10mM GSK-J1" (this medium is the medium obtained by replacing 10mM tucidinostat in the "screening medium containing 10mM tucidinostat" with 10mM GSK-J1), and the "secondary screening medium containing 10mM tucidinostat" was replaced with the "secondary screening medium containing 10mM GSK-J1" (this medium is the medium obtained by replacing 10mM tucidinostat in the "secondary screening medium containing 10mM tucidinostat" with 10mM GSK-J1). Other steps remained unchanged and the cells were used as the GSK-J1 experimental group.

[0060] According to the method of the above steps 1)-7), the "screening medium containing 10mM tucidinostat" was replaced with the "screening medium containing 10mM Torin2" (this medium is the medium obtained by replacing 10mM tucidinostat in the "screening medium containing 10mM tucidinostat" with 10mM Torin2), and the "secondary screening medium containing 10mM tucidinostat" was replaced with the "secondary screening medium containing 10mM Torin2" (this medium is the medium obtained by replacing 10mM tucidinostat in the "secondary screening medium containing 10mM tucidinostat" with 10mM Torin2). Other steps remained unchanged, and this was used as the Torin2 experimental group.

[0061] The callus redifferentiation and budding in the Tucidinostat experimental group were as follows: Figure 1 As shown in Figure 2, the callus redifferentiation and budding in the Torin2 experimental group are as follows: Figure 2 shown.

[0062] The results of positive seedling numbers are shown in Table 1 and Figure 3 When the screening medium contained Tucidinostat and Torin2, the ratio of the number of positive seedlings to the number of calli was significantly higher than that of the blank control group, while when the screening medium contained GSK126 and GSK-J1, the ratio of the number of positive seedlings to the number of calli was significantly lower than that of the blank control group. This shows that Tucidinostat and Torin2 can promote the redifferentiation of callus tissue in wheat genetic transformation and improve the genetic transformation efficiency of wheat. They can both be used for wheat genetic transformation and for the redifferentiation of wheat callus tissue in non-genetic transformation.

[0063]

[0064] The genetic transformation experiments were repeated and the data were processed using SPSS11.5 statistical software. One-way ANOVA test was used. In the "Significance Analysis" column, there were significant differences between the "ratio of positive seedlings to callus numbers" data with different letters, and there were no significant differences between the data with the same letters.

[0065] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Application of a tumor cell inhibitor in improving the efficiency of wheat genetic transformation, characterized in that: The tumor cell inhibitor is Torin2.

2. Use of the tumor cell inhibitor according to claim 1 in promoting callus regeneration in wheat genetic transformation, or in improving callus regeneration capacity in wheat genetic transformation.

3. A wheat genetic transformation method, characterized in that: The method comprises: culturing genetically transformed wheat callus on a culture medium containing the tumor cell inhibitor according to claim 1 to obtain treated callus; and subjecting the treated callus to differentiation culture and / or rooting culture, thereby achieving genetic transformation of wheat.

4. Use of the tumor cell inhibitor according to claim 1 in wheat genetic transformation.

Images