Method for efficiently and rapidly obtaining stable transgenic jujube plant based on agrobacterium rhizogenes

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a method for efficiently and rapidly obtaining a stable transgenic jujube plant based on agrobacterium rhizogenes. The invention provides a method for efficiently and rapidly obtaining a stable transgenic jujube plant based on agrobacterium rhizogenes, which comprises the following steps: by taking a leaf as an explant, infecting a leaf disc by using the agrobacterium rhizogenes to directly obtain a transgenic leaf, inducing the generation of adventitious buds of the leaf, and culturing the adventitious buds to obtain the transgenic plant. The method disclosed by the invention is simple to operate and shorter in period, and the obtained transgenic plant is good in repeatability and high in transformation efficiency. The research on the agrobacterium rhizogenes-mediated genetic transformation of the jujube has important significance. By continuously optimizing the technology and the research method, the genetic transformation efficiency and the stability of the jujubes are expected to be improved, and a firmer foundation is provided for application in the fields of rapid genetic improvement, functional gene research and the like of the jujubes.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly relates to a method for efficiently and rapidly obtaining stable transgenic jujube plants based on Agrobacterium rhizogenes. Background Art

[0002] Jujube (Ziziphus jujuba Mill.) is a unique fruit tree in China, with rich nutritional value and various health care effects. Wild jujube (Ziziphus jujuba Mill. var. spinosa), as the wild species of jujube, has strong stress resistance and is tolerant to barrenness, and can be used as the rootstock of cultivated jujube. Its fruit has a unique flavor and a high kernel rate, and has extensive application value in the food and pharmaceutical fields. At present, the Agrobacterium-mediated method is a relatively mature and effective genetic transformation method, and the leaf disc method is one of the most common methods. The genetic transformation research of jujube mainly focuses on the way mediated by Agrobacterium tumefaciens, but its genetic transformation efficiency and stability still need to be improved.

[0003] The Agrobacterium-mediated method is the main method for plant gene transformation. As an ideal material for the genetic transformation of plant exogenous genes, leaves are widely sourced and easy to obtain, so they are widely used in the genetic improvement of fruit trees at the cell level. For the genetic improvement of jujube, a regeneration system for jujube anthers, leaves, etc. has been established, providing a basis for genetic transformation research. However, compared with other fruit trees and crops, the genetic transformation research of jujube lags behind, and large-scale transgenic plants with application value have not been obtained. This is mainly affected by the complex genetic background of jujube and the difficulty of tissue culture regeneration, resulting in problems of difficult genetic transformation and low efficiency. Although some jujube genetic transformation systems have been established, they still face problems such as low efficiency and poor repeatability, which restrict the application of this technology in jujube trees.

[0004] In Agrobacterium-mediated genetic transformation, Agrobacterium tumefaciens and Agrobacterium rhizogenes are the main types. The Agrobacterium plasmid system has the function of DNA transfer and integration, among which Ti plasmid and Ri plasmid are the most commonly used plasmid types. The Ti plasmid exists in Agrobacterium tumefaciens, while the Ri plasmid exists in Agrobacterium rhizogenes. The two plasmids have similarities in structure and function, but each has its own characteristics. Through the T-DNA region carried by the plasmid, Agrobacterium can integrate foreign genes into the plant receptor genome to achieve stable expression of foreign genes. Infection of plants by Agrobacterium rhizogenes can induce the formation of hairy roots, and the Ri plasmid plays a key role in this process. For most plant genetic transformation studies, the Agrobacterium tumefaciens-mediated method is still the main way. However, obtaining transgenic hairy roots mediated by Agrobacterium rhizogenes also has the potential to develop into plants. By introducing Agrobacterium rhizogenes into plant stems and other receptor materials, transgenic hairy roots can be obtained, providing new ideas for genetic transformation research. However, the current method can only obtain transgenic hairy roots and cannot achieve stable genetic transgenic plants. In order to obtain stable genetic transformation plants, further research on the in vitro tissue culture and regeneration technology of hairy roots is still needed, which makes the cycle of obtaining transformed plants longer and the steps more cumbersome. Therefore, it is very important to develop an efficient and rapid method for obtaining stable transgenic jujube plants. Summary of the Invention

[0005] The present invention provides a method for efficiently and rapidly obtaining stable transgenic jujube plants based on Agrobacterium rhizogenes. Using the method of the present invention to prepare transgenic jujube plants, with leaves as explants, stable genetic transformation plants of jujube can be rapidly obtained, the operation is simple, the cycle of obtaining transgenic plants is shorter, the repeatability is good, and the transformation efficiency is high.

[0006] In order to solve the above technical problems, the present invention proposes the following technical solutions:

[0007] The present invention provides a method for efficiently and rapidly obtaining stable transgenic jujube plants based on Agrobacterium rhizogenes, comprising the following steps:

[0008] Mix the leaves of jujube tissue culture seedlings with the Agrobacterium rhizogenes infection solution for infection and co-culture to obtain infected jujube leaves;

[0009] Culture the infected jujube leaves to obtain transgenic jujube plants.

[0010] Preferably, the leaves of the jujube tissue culture seedlings include the 3rd to 5th leaves counted from the top bud of the tissue culture seedlings.

[0011] Preferably, before taking the leaves of the jujube tissue culture seedlings, subculture of the jujube tissue culture seedlings is also included. The time of the subculture is 14 - 60 d. The composition of the subculture medium includes: using MS as the basic medium, and also including 0.8 - 1.0 mg / L 6 - BA, 0.1 - 0.3 mg / L IBA, 0.005 - 0.015 mg / L TDZ, 25 - 35 g / L sucrose and 5 - 7 g / L agar, with pH = 5.6 - 5.8;

[0012] The temperature of the subculture is 25 ± 2 °C, the light intensity is 8000 - 20000 lux, and the light cycle is 12 - 16 h / d of light culture and 8 - 12 h / d of dark culture.

[0013] Preferably, the preparation method of the Agrobacterium rhizogenes infection solution includes: mixing the Agrobacterium rhizogenes cells containing the recombinant expression vector with the resuspension solution to obtain the infection solution; the Agrobacterium rhizogenes cells are obtained by centrifuging the bacterial solution, and the volume ratio of the bacterial solution to the resuspension solution is 1 - 2:1.

[0014] Preferably, the resuspension solution contains 10 mM 2-(N - morpholino)ethanesulfonic acid monohydrate, 10 mM magnesium chloride and 100 μM acetosyringone.

[0015] Preferably, the recombinant expression vector includes, but is not limited to, the over - expression vector pCY - 35gk - GFP vector and the silencing expression vector pFGC5941.

[0016] Preferably, the time of the co - culture is 2 - 3 d; the co - culture is dark culture; the temperature of the co - culture is 25 ± 2 °C; the composition of the co - culture medium includes: using MS as the basic medium, and also including 0.2 - 0.4 mg / L IBA, 1.0 - 3.0 mg / L TDZ, 30 g / L sucrose, 6 g / L agar, 80 - 200 μM acetosyringone and 1 - 1.5 mM betaine.

[0017] Preferably, the culture includes sequentially performing induction culture, screening, proliferation culture, subculture and rooting culture of adventitious buds to obtain transgenic jujube plants.

[0018] Preferably, the medium used for the induction culture of adventitious buds includes the induction medium. The composition of the induction medium includes: using MS as the basic medium, and also including: 0.2 - 0.4 mg / L IBA, 1.0 - 3.0 mg / L TDZ, 0.5 - 5.0 mg / L AgNO 3, carbenicillin 300 - 500 mg / L, 25 - 35 g / L sucrose and 5 - 7 g / L agar, pH = 5.6 - 5.8; or, the composition of the induction medium includes: using MS as the basic medium, and further including: 0.2 - 0.4 mg / L IBA, 1.0 - 3.0 mg / L TDZ, 0.5 - 5.0 mg / L AgNO 3 , 300 - 500 mg / L carbenicillin, screening agent, 25 - 35 g / L sucrose and 5 - 7 g / L agar, pH = 5.6 - 5.8;

[0019] The medium used for proliferation culture includes a proliferation medium, and the composition of the proliferation medium includes: using MS as the basic medium, and further including: 0.8 - 1.5 mg / L 6 - BA, 0.1 - 0.3 mg / L IBA, 0.005 - 0.015 mg / L TDZ, 25 - 35 g / L sucrose and 5 - 7 g / L agar; or, the composition of the induction medium includes: using MS as the basic medium, and further including: 0.8 - 1.5 mg / L 6 - BA, 0.1 - 0.3 mg / L IBA, 0.005 - 0.015 mg / L TDZ, 300 - 500 mg / L carbenicillin, screening agent and 25 - 35 g / L sucrose and 5 - 7 g / L agar;

[0020] The medium used for rooting culture includes a rooting medium, and the composition of the rooting medium includes: using 1 / 2MS as the basic medium, and further including: 0.1 - 0.5 mg / L IBA, 0 - 0.2 mg / L NAA, 0 - 0.5 g / L activated carbon, 15 - 20 g / L sucrose, 5 - 7 g / L agar, pH = 5.6 - 5.8.

[0021] Preferably, the light intensity for the induction culture of adventitious buds is 8000 - 20000 lux, the photoperiod is 12 - 16 h / d for light culture and 8 - 12 h / d for dark culture, the temperature is 25 ± 2 °C, and the time is 28 - 56 d.

[0022] Advantages of the present invention: The present invention provides a method for efficiently and rapidly obtaining stable transgenic jujube plants based on Agrobacterium rhizogenes. Using leaves as explants, transgenic leaves are directly obtained by infecting leaf discs with Agrobacterium rhizogenes, and adventitious buds are induced from the leaves, and then the adventitious buds are cultured to obtain transgenic plants. In the prior art, when genetic transformation is carried out using Agrobacterium rhizogenes, transgenic hairy roots are mainly obtained by inducing adventitious roots, and transgenic plants can only be obtained after the transgenic hairy roots are redifferentiated. Different from the method of obtaining hairy roots by infecting plants with Agrobacterium rhizogenes, the method of the present invention can skip the induction of hairy roots and directly obtain adventitious buds of wild jujube with stable genetic transformation by infecting leaf discs with Agrobacterium rhizogenes. Using this method, stable genetically transformed plants of wild jujube can be obtained quickly. The method of the present invention omits the step of inducing adventitious buds from transgenic hairy roots, is simple to operate, has a shorter cycle, and the obtained transgenic plants have good repeatability and high transformation efficiency. The research on Agrobacterium rhizogenes-mediated genetic transformation of jujube in the present invention is of great significance. By continuously optimizing the technology and research methods, it is expected to improve the genetic transformation efficiency and stability of jujube, and provide a more solid foundation for the application in the fields of rapid genetic improvement of jujube and functional gene research, etc. Brief Description of the Drawings

[0023] Figure 1 Shows the growth state of the subcultured tissue culture seedlings of wild jujube used in the present invention;

[0024] Figure 2 Shows the vector map of pFGC5941-35sEGFP used in Example 1 of the present invention;

[0025] Figure 3 Shows the growth state of wild jujube leaves on the selection medium after infection;

[0026] Figure 4 Shows the fluorescence image of wild jujube leaves on the selection medium identified by the LUYOR-3415RG dual-wavelength fluorescent protein excitation light source after infection;

[0027] Figure 5 Shows the growth state of adventitious buds on the selection and proliferation medium;

[0028] Figure 6 Shows the fluorescence image of transgenic wild jujube plants and wild-type wild jujube plants identified by the LUYOR-3415RG dual-wavelength fluorescent protein excitation light source;

[0029] Figure 7 Shows the agarose gel electrophoresis pattern of PCR amplification detection of the plants screened by fluorescence;

[0030] Figure 8For the growth status of rooted seedlings and after transplantation, A shows the state of rooted seedlings under natural light; B shows the fluorescence image of rooted seedlings identified under the LUYOR-3415RG dual-wavelength fluorescent protein excitation light source; C shows the state of the transplanted plants under natural light; D shows the fluorescence image of the transplanted plants identified under the LUYOR-3415RG dual-wavelength fluorescent protein excitation light source; on the left side of A, B, C, and D are transgenic plants, and on the right side are wild-type plants;

[0031] Figure 9 This is the map of the pCY-35gk-GFP vector used in Example 2 of the present invention. Detailed implementation manners

[0032] The present invention provides a method for efficiently and rapidly obtaining stable transgenic jujube plants based on Agrobacterium rhizogenes, comprising the following steps:

[0033] Mix the leaves of jujube tissue culture seedlings with the Agrobacterium rhizogenes infection solution for infection and co-culture to obtain the infected jujube leaves;

[0034] Culture the infected jujube leaves to obtain transgenic jujube plants.

[0035] In the present invention, the leaves of jujube tissue culture seedlings are mixed with the Agrobacterium rhizogenes infection solution for infection and co-culture to obtain the infected jujube leaves. As an optional implementation manner, the jujube in the present invention is wild jujube; in the examples of the present invention, the asexual tissue culture seedlings of Qingjian wild jujube (Zizphus jujuba var. Spinosa 'Qingjiansuanzao') are used for effect verification. The asexual line in the present invention is a clonal population with a single genetic background produced by tissue culture. The asexual tissue culture seedlings of jujube in the present invention are preferably sterile tissue culture seedlings obtained by using the current-year branches as explants through disinfection and culture. The present invention has no special limitation on the composition of the culture medium applied to the jujube tissue culture seedlings and the size of the tissue culture seedlings, as long as they are cultured into conventional tissue culture seedlings.

[0036] As an alternative embodiment, the leaves of the tissue-cultured jujube seedlings in the present invention include the 3rd to 6th leaves counted downward from the apical bud of the tissue-cultured seedlings, or can also be the 3rd to 5th leaves. As an alternative embodiment, before taking the leaves, the tissue-cultured jujube seedlings are subcultured. In the present invention, the tissue-cultured jujube seedlings are inoculated in a subculture medium for subculture. The composition of the subculture medium in the present invention preferably includes: taking MS as the basic medium, and also including 0.8 - 1.0 mg / L 6-BA, 0.1 - 0.3 mg / L IBA, 0.005 - 0.015 mg / L TDZ, 25 - 35 g / L sucrose and 5 - 7 g / L agar, with pH = 5.6 - 5.8. The concentration of 6-BA in the subculture medium provided by the present invention can be 0.8 - 1.0 mg / L, more preferably 1 mg. The concentration of IBA in the subculture medium provided by the present invention can be 0.1 - 0.3 mg / L, more preferably 0.2 mg / L. The concentration of TDZ in the subculture medium provided by the present invention can be 0.005 - 0.015 mg / L, more preferably 0.006 mg / L. The concentration of sucrose in the subculture medium provided by the present invention can be 25 - 35 g / L, more preferably 30 g / L. The concentration of agar in the subculture medium provided by the present invention can be 5 - 7 g / L, more preferably 6 g / L. The function of the subculture medium is to obtain tissue-cultured jujube seedlings with strong growth. Compared with the proliferated seedlings, the leaves of the subcultured seedlings are larger, in better condition and easier to operate. As an alternative embodiment, the temperature of the subculture is 23 - 27 °C, or can also be 25 °C; the time of the subculture is 14 - 60 d, or can also be 14 - 30 d, more preferably 14 d; the subculture is light-dark alternating culture, the light time can be 12 - 16 h / d, or can also be 12 - 14 h / d, the dark time can be 8 - 12 h / d, or can also be 10 - 12 h / d, and the light intensity in the present invention can be 8000 - 20000 lux, or can also be 15000 - 20000 lux.

[0037] After the subculture is completed, the present invention obtains tissue-cultured jujube seedlings with strong growth, and the leaves are taken from the tissue-cultured seedlings obtained by subculture as the donor to be infected.

[0038] As an alternative embodiment, the method for preparing the Agrobacterium rhizogenes infection solution of the present invention includes: mixing the Agrobacterium rhizogenes cells containing the recombinant expression vector with a resuspension solution to obtain the infection solution; the Agrobacterium rhizogenes cells are obtained by centrifuging the bacterial solution, and the volume ratio of the bacterial solution to the resuspension solution is 1-2:1, more preferably 1:1. As an alternative embodiment, the present invention does not have special limitations on the source of the Agrobacterium rhizogenes, and conventional products can be used. The Agrobacterium rhizogenes of the present invention is K599 or MSU440; in the examples of the present invention, Agrobacterium rhizogenes K599 is used for effect verification. The method for preparing the Agrobacterium rhizogenes cells containing the recombinant expression vector of the present invention includes: transforming the recombinant expression vector into Agrobacterium rhizogenes, picking the transformed positive single colonies and performing enlarged culture using a screening medium to obtain a positive bacterial solution; the culture is carried out until the OD 600 ≈1.0. The present invention centrifuges the positive bacterial solution to obtain Agrobacterium rhizogenes cells. As an alternative embodiment, the composition of the screening medium includes: 5 g / L tryptone, 3 g / L yeast extract, 1.1 g / L anhydrous calcium chloride, 50 mg / L streptomycin, and 50 mg / L kanamycin. The temperature of the enlarged culture of the present invention is 28 °C, and the rotation speed is 220 rpm. As an alternative embodiment, the rotation speed of the centrifugation is 4000 rpm, and the centrifugation time is 10 min. The present invention does not have special limitations on the method for transforming the recombinant expression vector into Agrobacterium rhizogenes, and conventional methods can be used. The present invention does not have special limitations on the preparation method of the recombinant expression vector, and conventional methods can be used. The vectors in the recombinant expression vectors of the present invention include, but are not limited to, the overexpression vector pCY-35gk-GFP vector and the silencing expression vector pFGC5941. The pCY-35gk-GFP of the present invention is a commercially purchased vector, purchased from Xuchang Cangyu Biotechnology Co., Ltd., and pFGC5941-35sEGFP is a vector obtained by inserting 35s::EGFP into the pFGC5941 (pFGC5941 is purchased from Wuhan Miaoling Biotechnology Co., Ltd.) vector.

[0039] As an alternative embodiment, the present invention mixes the leaves obtained after the subculture with the Agrobacterium rhizogenes infection solution for infection and co-culture. As an alternative embodiment, the infection is completed under vacuum conditions, which is vacuum negative pressure infection. The infection time is 8-10 min, the vacuum degree of the infection is 0.06 Mpa, and water stains will appear at the edges of the infected leaves.

[0040] In the present invention, the infected leaves are blotted with sterile filter paper to remove the excess liquid, and then inoculated into the co-culture medium for co-culture. The co-culture in the present invention is dark culture, and the time of the co-culture is 3 d; the temperature of the co-culture can be 23-27 °C or 25 °C. As an alternative embodiment, the composition of the co-culture medium used in the co-culture of the present invention includes: using MS as the basic medium, and further including: 0.2-0.4 mg / L IBA, 1.0-3.0 mg / L TDZ, 30 g / L sucrose, 6 g / L agar, 80-200 μM acetosyringone and 1-1.5 mM betaine. The pH of the co-culture medium in the present invention is 5.8. The concentration of IBA in the co-culture medium provided by the present invention can be 0.2-0.4 mg / L, more preferably 0.3 mg / L. The concentration of TDZ in the co-culture medium provided by the present invention can be 1.0-3.0 mg / L, more preferably 2.5 mg / L. The concentration of betaine in the co-culture medium provided by the present invention is 1-1.5 mM, more preferably 1 mM. The concentration of acetosyringone in the co-culture medium provided by the present invention is 80-200 μM, more preferably 100 μM. The function of the co-culture medium is to activate the expression of vir genes and drive the cleavage and transfer of T-DNA into the jujube genome.

[0041] After the co-culture is completed, in the present invention, the leaves obtained from the co-culture are transferred to the adventitious bud induction medium for adventitious bud induction culture to obtain adventitious buds. The composition of the adventitious bud induction medium in the present invention includes: using MS as the basic medium, and further including: 0.2-0.4 mg / L IBA, 1.0-3.0 mg / L TDZ, 0.5-5.0 mg / L AgNO 3 , carbenicillin 300-500 mg / L, 25-35 g / L sucrose and 5-7 g / L agar.

[0042] In the induction medium provided by the present invention, the concentration of IBA can be 0.2 - 0.4 mg / L, more preferably 0.3 mg / L. The concentration of TDZ in the induction medium provided by the present invention can be 1.0 - 3.0 mg / L, more preferably 2.5 mg / L. The concentration of silver nitrate in the induction medium provided by the present invention is 0.5 - 5.0 mg / L, and can also be 1.5 - 3.5 mg / L. The concentration of carbenicillin in the induction medium provided by the present invention is 300 - 500 mg / L, more preferably 400 mg / L. The function of carbenicillin (Carb) in the present invention is to kill Agrobacterium rhizogenes. The induction medium of the present invention preferably further includes a screening agent, and the screening agent can be adjusted according to the gene screening marker carried in the vector. In the specific embodiment provided by the present invention, the pFGC5941 vector used carries the BlpR gene, and the BlpR gene is a screening marker and is a herbicide Basta resistance gene. Therefore, the screening agent in the induction medium provided by the present invention is Basta, and the concentration of Basta is 1 mg / L. Adding Basta to the medium can screen out positive adventitious buds containing the recombinant expression vector. The concentration of sucrose in the induction medium provided by the present invention is 25 - 35 g / L, and can also be 30 g / L. The concentration of agar in the induction medium provided by the present invention is 5 - 7 g / L, and can also be 6 g / L. The pH of the adventitious bud induction medium of the present invention is 5.6 - 5.8.

[0043] As an alternative embodiment, the temperature of the induction culture can be 23 - 27 °C, and can also be 25 °C; the time of the induction culture is 28 - 56 d, and can also be 34 - 45 d; the induction culture is a light-dark alternating culture, the light time can be 12 - 16 h / d, and can also be 12 - 14 h / d, the dark time can be 8 - 12 h / d, and can also be 10 - 12 h / d. The light intensity of the present invention can be 8000 - 20000 lux, and can also be 16000 - 20000 lux.

[0044] Since the vector pFGC5941-35sEGFP used in the present invention contains the EGFP green fluorescent protein, the transformed positive adventitious buds emit green fluorescence. After the induction culture is completed, the present invention preferably screens the obtained product to obtain adventitious buds with green fluorescence. In the embodiment of the present invention, the recombinant expression vector pFGC5941-35sEGFP is used during infection. Since the recombinant expression vector pFGC5941-35sEGFP contains the fluorescent tag EGFP, the positive adventitious buds have green fluorescence after irradiation.

[0045] After screening out the positive adventitious buds, the present invention transfers the seedlings containing the positive adventitious buds to a proliferation medium for proliferation culture, subculture and rooting culture to obtain transgenic jujube plants.

[0046] As an alternative embodiment, the composition of the proliferation medium includes: using MS as the basic medium, and further including: 0.8 - 1.5 mg / L 6 - BA, 0.1 - 0.3 mg / L IBA, 0.005 - 0.015 mg / L TDZ, 25 - 35 g / L sucrose, and 5 - 7 g / L agar, with the pH adjusted to 5.6 - 5.8. In the proliferation medium provided by the present invention, the concentration of IBA can be 0.1 - 0.3 mg / L, more preferably 0.2 mg / L. In the proliferation medium provided by the present invention, the concentration of TDZ can be 0.005 - 0.015 mg / L, more preferably 0.012 mg / L. In the proliferation medium provided by the present invention, the concentration of 6 - BA is 0.8 - 1.5 mg / L, and can also be 1.0 mg / L. In the proliferation medium provided by the present invention, the concentration of sucrose is 25 - 35 g / L, and can also be 30 g / L. In the proliferation medium provided by the present invention, the concentration of agar is 5 - 7 g / L, and can also be 6 g / L. In the proliferation medium provided by the present invention, the concentration of carbenicillin is 300 - 500 mg / L, more preferably 400 mg / L. The function of carbenicillin (Carb) in the present invention is to kill Agrobacterium rhizogenes. The proliferation medium of the present invention preferably further includes a screening agent, and the screening agent can be adjusted according to the gene screening marker carried in the vector. As an alternative embodiment, the temperature of the proliferation culture can be 23 - 27 °C, and can also be 25 °C; the time of the proliferation culture is 14 - 28 d, and can also be 22 - 26 d; the proliferation culture is a light - dark alternating culture, the light time can be 12 - 16 h / d, and can also be 12 - 14 h / d, the dark time can be 8 - 12 h / d, and can also be 10 - 12 h / d. The light intensity of the present invention can be 8000 - 20000 lux, and can also be 16000 - 20000 lux.

[0047] As an alternative embodiment, the medium used in the subculture is the subculture medium; the selection of the subculture medium used is the same as that of the subculture medium for cultivating tissue - cultured seedlings described above, and will not be elaborated here.

[0048] As an alternative embodiment, the medium used in the rooting culture is the rooting medium. The composition of the rooting medium includes using 1 / 2MS as the basic medium, and further including: 0.1 - 0.5 mg / L IBA, 0 - 0.2 mg / L NAA, 0 - 0.5 g / L activated carbon, 15 - 20 g / L sucrose, and 5 - 7 g / L agar, with pH = 5.6 - 5.8.

[0049] In the rooting medium provided by the present invention, the concentration of IBA can be 0.1 - 0.5 mg / L, more preferably 0.2 - 0.4 mg / L. The concentration of NAA in the rooting medium provided by the present invention can be 0 - 2.0 mg / L, more preferably 1.0 mg / L. The concentration of activated carbon in the rooting medium provided by the present invention is 0 - 0.5 g / L, and can also be 0.3 g / L. The concentration of sucrose in the rooting medium provided by the present invention is 15 - 20 g / L, and can also be 15 g / L. The concentration of agar in the rooting medium provided by the present invention is 5 - 7 g / L, and can also be 6 g / L. As an alternative embodiment, the temperature for the rooting culture can be 23 - 27 °C, and can also be 25 °C; the time for the rooting culture is 14 - 28 d, and can also be 20 - 26 d; the rooting culture is a light-dark alternating culture, the light time can be 12 - 16 h / d, and can also be 12 - 14 h / d, the dark time can be 8 - 12 h / d, and can also be 10 - 12 h / d, and the light intensity of the present invention can be 8000 - 20000 lux, and can also be 16000 - 20000 lux.

[0050] The method for genetic transformation of jujube mediated by Agrobacterium rhizogenes provided by the present invention uses the leaves after subculture of jujube clone tissue culture seedlings as the donor to be infected. The leaves of jujube are co-cultured with Agrobacterium rhizogenes, and the Agrobacterium rhizogenes containing the recombinant expression vector is used to infect the leaves with the infection solution; the leaves after co-culture are subjected to adventitious bud induction culture, screening, proliferation culture, subculture and rooting culture to obtain transgenic jujube plants. The method of the present invention is simple to operate, has a short cycle, good repeatability and high transformation efficiency.

[0051] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0053] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0054] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which will be apparent to those skilled in the art. Other embodiments obtained from the description of the present invention will be apparent to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0055] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0056] In the following examples, the tissue culture seedlings of the Qingjian wild jujube clone 'SX' were cultured in this laboratory, and pFGC5941-35sEGFP was constructed in this laboratory; the competent cells of Agrobacterium rhizogenes K599 were purchased from Shanghai Weidi Biotechnology Co., Ltd. The tissue culture seedlings of the Qingjian wild jujube clone 'SX' used in the following examples were the tissue culture seedlings of the Qingjian wild jujube clone 'SX' obtained from the same batch of culture.

[0057] The composition of TY liquid medium is: 5 g / L tryptone, 3 g / L yeast extract, and 1.1 g / L anhydrous calcium chloride.

[0058] The composition of TY solid medium is: 5 g / L tryptone, 3 g / L yeast extract, 1.1 g / L anhydrous calcium chloride (CaCl 2 ) and 15 - 20 g / L agar.

[0059] Example 1

[0060] 1. Material preparation:

[0061] (1) Preparation of tissue culture seedlings of the Qingjian wild jujube clone 'SX': Sterile tissue culture seedlings obtained by using current-year branches as explants through disinfection and culture.

[0062] (2) Using the leaves of the tissue culture seedlings of the Ziziphus jujuba var. Spinosa 'Qingjiansuanzao' clone 'SX' as materials, the preparation method of the leaves is as follows: Subculture the tissue culture seedlings of the Ziziphus jujuba var. Spinosa 'Qingjiansuanzao' clone 'SX' on the subculture medium for 14 days. Before infection, select the tissue culture seedlings with strong growth, and take the 3rd to 5th leaves counted from the top bud as the donor to be infected. The composition of the subculture medium is: based on the MS basic medium, supplemented with exogenous hormones TDZ 0.006 mg / L, IBA 0.2 mg / L, 6-BA 1 mg / L, 30 g / L sucrose and 6 g / L agar powder, and adjust the pH to 5.8. The growth status of the subcultured tissue culture seedlings of Ziziphus jujuba can be seen in Figure 1 . The temperature for subculture is 25 ± 2 °C, the light intensity is 20000 lux, and the photoperiod is 16 h / d of light culture and 8 h / d of dark culture.

[0063] 2. Vector construction: It is recommended to use a plasmid containing a fluorescent tag as the vector. In this example, the pFGC5941-35sEGFP plasmid is used. This plasmid is obtained by digesting pFGC5941 with EcoRI restriction endonuclease and then recombining the 35s::EGFP expression cassette using homologous recombinase. The vector map can be seen in Figure 2 . The pFGC5941 plasmid itself contains the reporter gene BlpR. On this basis, 35s::EGFP is inserted into the vector. It can be considered that EGFP is the inserted target gene, and any gene can be used to prepare transgenic jujube plants by this method.

[0064] 3. Transformation of Agrobacterium rhizogenes: The competent cells of Agrobacterium rhizogenes K599 were purchased from Shanghai Weidi Biotechnology Co., Ltd. Mix 0.5 μg of the pFGC5941-35sEGFP plasmid with the K599 competent cells in a solid-liquid mixed state and place them on ice for 5 min. The addition amount of the pFGC5941-35sEGFP plasmid does not exceed 1 / 10 of the volume of the competent cells. Then place them in liquid nitrogen for 5 min, then in a 37 °C water bath for 5 min, and then on ice for 5 min. Finally, add 700 μl of TY liquid medium and resuscitate at 28 °C for 2 - 3 h. Spread the resuscitated K599 bacterial solution on TY solid medium containing 50 mg / L streptomycin and 50 mg / L kanamycin and culture at 28 °C for 2 - 3 days. Pick a single colony, and after colony verification, use 30 mL of TY liquid medium containing 50 mg / L streptomycin and 50 mg / L kanamycin for large-scale culture. When the OD of the cultured bacterial solution 600When OD600 = 1.0, the bacteria were harvested by centrifugation at 5000 rpm for 5 min using a centrifuge. The supernatant was discarded, and the obtained bacterial cells were resuspended with a resuspension solution to obtain an infection solution. The volume of the resuspension solution was the same as the volume of the bacterial solution before centrifugation. The resuspension solution contained 10 mM MES, 10 mM magnesium chloride, and 100 μM acetosyringone.

[0065] 4. Infection and co-culture: Soak the Ziziphus jujuba Mill. var. spinosa leaves prepared in step 1 in the infection solution obtained in step 3, and use a vacuum pump to infect for 10 min at a vacuum degree of 0.06 Mpa. At this time, water stains will appear at the edges of the infected leaves. Blot the infected leaves with sterile filter paper to remove excess liquid, place the leaves face down on the co-culture medium, and incubate in the dark for 3 d. After dark incubation, the Ziziphus jujuba Mill. var. spinosa leaves on the selection medium are seen Figure 3 . The composition of the co-culture medium is: using MS as the basic medium, supplemented with 2.5 mg / L TDZ, 0.2 mg / L IBA, 30 g / L sucrose, 6 g / L agar, 1 mM betaine, and 100 μM acetosyringone.

[0066] 5. Induction of resistant adventitious buds: Transfer the leaves after co-culture in step 4 to the adventitious bud induction medium, change the medium every 14 d, and adventitious buds can be obtained after 4 weeks of culture, as shown in Figure 4 . The composition of the adventitious bud induction medium is: using MS as the basic medium, supplemented with 2.5 mg / L TDZ, 0.2 mg / L IBA, 30 g / L sucrose, 6 g / L agar, 400 mg / L carbenicillin (Carb), 5 mg / L AgNO 3 3 and 1 mg / L glufosinate-ammonium Basta. The adventitious bud induction culture is carried out under the conditions of a light intensity of 20000 lux, a photoperiod of 16 h / d light and 8 h / d dark, and a temperature of 25 ± 2 °C.

[0067] 6. Screening and identification of adventitious buds: Use a LUYOR-3415RG dual-wavelength fluorescent protein excitation light source for fluorescence screening ( Figure 4 ), number the obtained fluorescent adventitious buds, and 12 adventitious buds with green fluorescence are obtained, numbered 1-12. Transfer the tissue culture seedlings of the screened adventitious buds with green fluorescence to a proliferation medium containing Basta and Carb for proliferation culture for 14 d. After the proliferation culture is completed, see Figure 5, after a certain number of adventitious buds are obtained, subculture is carried out for 14 days, and finally rooting culture is carried out for 14 days. The composition of the proliferation medium is: using MS as the basic medium, supplemented with 0.012 mg / L TDZ, 0.2 mg / L IBA, 1.0 mg / L 6-BA, 400 mg / L carbenicillin (Carb), 1 mg / L Basta, 30 g / L sucrose and 6 g / L agar; the composition of the subculture medium is: using MS as the basic medium, supplemented with 0.006 mg / L TDZ, 0.2 mg / L IBA, 1 mg / L 6-BA, 30 g / L sucrose and 6 g / L agar powder, and the pH is adjusted to 5.8; the composition of the rooting medium is: using 1 / 2MS as the basic medium, supplemented with 0.2 mg / L IBA, 0.1 mg / L NAA, 0.3 g / L activated carbon, 15 g / L sucrose and 6 g / L agar. The culture conditions for proliferation, subculture and rooting are 25 °C, 20000 lux, and a light / dark cycle of 16 / 8 h.

[0068] 7. Hardening off and transplanting: The adventitious buds grow in the rooting medium for about 1 month. When the adventitious roots grow vigorously, the tissue culture bottle cap can be opened to half cover the bottle mouth and placed for 3 days. Wait until the water vapor in the bottle disappears, pour out the tissue culture seedlings from the bottle, and gently wash the medium around the roots. Transplant the washed tissue culture seedlings into flower pots containing peat, vermiculite, and perlite. The volume ratio of peat, vermiculite, and perlite is 2:1:1. After watering thoroughly, place them in a seedling tray with a transparent plastic cover, and maintain the humidity for slow seedling acclimation. After one week, slowly reduce the humidity in the seedling tray, and the plastic cover can be removed when it reaches the indoor humidity.

[0069] 8. Identification of transgenic plants: After the transplanting is completed, screening of transgenic plants is carried out. Screening is carried out by irradiating the plants with a LUYOR-3415RG dual-wavelength fluorescent protein excitation light source. The results are shown in Figure 6 . According to Figure 6 , it can be known that wild-type plants will emit red fluorescence under the excitation light due to the autofluorescence of chloroplasts, while transgenic plants, because they contain EGFP, will emit green fluorescence in parts with fewer chloroplasts and orange fluorescence in parts with more chloroplasts due to fluorescence superposition. The wild-type plants were obtained by directly subculturing and rooting the tissue culture seedlings of the Qingjian wild jujube clone 'SX' that had not been transformed. The composition of the subculture medium is: using MS basic medium, supplemented with exogenous hormones 0.006 mg / L TDZ, 0.2 mg / L IBA, 1 mg / L 6-BA, 30 g / L sucrose and 6 g / L agar powder, and the pH is adjusted to 5.8; the composition of the rooting medium is: using 1 / 2MS basic medium, supplemented with exogenous hormones 0.2 mg / L IBA, 0.1 mg / L NAA, 0.3 g / L activated carbon, 15 g / L sucrose and 6 g / L agar powder, and the pH is adjusted to 5.8; the culture conditions for subculture and rooting are the same as above.

[0070] Extract DNA from fluorescent plants and perform PCR detection, or use Bar resistance gene test paper for detection. Design upstream and downstream primers based on the sequence of BlpR gene on the vector (BlpR gene is the screening marker on the pFGC5941 vector): F-CACCATCGTCAACCACTAC (SEQ ID NO.1); R-CAGTTCCCGTGCTTGAAG (SEQ ID NO.2), and the PCR program is: pre-denaturation at 98℃ for 3min; then denaturation at 98℃ for 10s, annealing at 60℃ for 10s, extension at 72℃ for 10s, and cycle 35 times; extension at 72℃ for 5min. The theoretical size of the amplified product is 426bp. Analyze by agarose gel electrophoresis experiment, the results are shown in Figure 7 The template added to the negative control (N) PCR was ddH 2 O, the wild-type Qingjian sour jujube clone 'SX' DNA (WT) did not amplify a band of about 426bp, while the PCR detection bands of strains 1 to 12 with GFP fluorescence were consistent with the pFGC5941-35sEGFP plasmid PCR (positive control, P) band results, with a positive rate of 100%. Figure 8 The phenotypic difference between the transgenic plants and the wild-type Qingjian sour jujube clone 'SX' plants under the excitation light source is shown. It can be seen that the positive transformation success rate of the plants of the method of the present invention can reach 100%. The leaf disc transformation experiment of Example 1 set up 3 parallel experiments, each parallel experiment had two discs of leaves, and each disc had 25 leaves.

[0071] Example 2

[0072] The vector pFGC5941-35sEGFP used in Example 1 was replaced with pCY-35gk-GFP (see the pCY-35sgk-GFP vector map for details). Figure 9 ), and the rest of the operations were the same as in Example 1. In Example 2, 3 parallel experiments were set up, each with two plates of leaves, and each plate had 25 leaves to obtain transgenic plants. The transgenic plants were identified according to the detection and identification methods in Example 1, and the transformation rate and the positive rate of transformed plants were counted. The results are shown in Table 1.

[0073] Table 1 Positive rate and transformation rate of transformed plants in Example 2

[0074] Number of experiments Number of leaf discs Number of fluorescent adventitious buds Conversion rate Positive rate 1 50 20 40％ 100％ 2 50 23 46％ 100％ 3 50 25 50％ 100％

[0075] In summary, the present invention provides a method for genetic transformation of jujube mediated by Agrobacterium rhizogenes. The leaves after subculture of jujube clone tissue culture seedlings are used as the donor to be infected. The leaves of jujube are co-cultured with Agrobacterium rhizogenes, and the Agrobacterium rhizogenes containing the recombinant expression vector is used to infect the leaves with the infection solution. The leaves after co-culture are subjected to adventitious bud induction culture, screening, proliferation culture, subculture and rooting culture to obtain transgenic jujube plants. The method of the present invention is simple to operate, and it only takes 2 months at the fastest from transformation to screening to obtain transgenic plants. It has a short cycle, good repeatability and a 100% positive rate of plants, with high transformation efficiency.

[0076] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for efficiently and quickly obtaining stable transgenic jujube plants based on Agrobacterium rhizogenes, characterized in that: The following steps are involved: Mixing the leaves of the jujube tissue culture seedlings with the infection solution of Agrobacterium rhizogenes for infection and co-cultivation to obtain infected jujube leaves; The infected jujube leaves are cultured to obtain transgenic jujube plants.

2. The method according to claim 1, characterized in that The leaves of the jujube tissue culture seedlings include the third to fifth leaves counted downward from the top bud of the tissue culture seedlings.

3. The method according to claim 1 or 2, characterized in that: Before taking the leaves of the jujube tissue culture seedlings, the jujube tissue culture seedlings are subjected to subculture, and the subculture time is 14 to 60 days; the composition of the subculture medium includes: MS as the basic medium, and also includes 0.8 to 1.0 mg / L 6-BA, 0.1 to 0.3 mg / L IBA, 0.005 to 0.015 mg / L TDZ, 25 to 35 g / L sucrose and 5 to 7 g / L agar, pH = 5.6 to 5.8; The temperature of the subculture is 25±2° C., the light intensity is 8000-20000 lux, and the photoperiod is 12-16 h / d for light culture and 8-12 h / d for dark culture.

4. The method according to claim 1, characterized in that: The preparation method of the Agrobacterium rhizogenes infection liquid comprises: mixing the Agrobacterium rhizogenes cells containing the recombinant expression vector with a resuspended liquid to obtain the infection liquid; the Agrobacterium rhizogenes cells are obtained by centrifuging the bacterial liquid, and the volume ratio of the bacterial liquid to the resuspended liquid is 1-2:

1.

5. The method according to claim 4, characterized in that The resuspension contained 10 mM 2-(N-morpholino)ethanesulfonic acid monohydrate, 10 mM magnesium chloride and 100 μM acetosyringone.

6. The method according to claim 4, characterized in that The recombinant expression vectors include but are not limited to the overexpression vector pCY-35gk-GFP vector and the silent expression vector pFGC5941.

7. The method according to claim 1, characterized in that The co-cultivation time is 2 to 3 days; the co-cultivation is dark culture; the co-cultivation temperature is 25±2°C; the co-cultivation culture medium composition includes: MS as the basic culture medium, and also includes 0.2 to 0.4 mg / L IBA, 1.0 to 3.0 mg / L TDZ, 30 g / L sucrose, 6 g / L agar, 80 to 200 μM acetosyringone and 1 to 1.5 mM betaine.

8. The method according to claim 1, characterized in that The culture comprises sequentially carrying out induction culture, screening, proliferation culture, subculture culture and rooting culture of adventitious buds to obtain transgenic jujube plants.

9. The method according to claim 8, characterized in that The culture medium used for the induction culture of the adventitious buds includes an induction culture medium, wherein the composition of the induction culture medium includes: MS as a basic culture medium, and further includes: 0.2-0.4 mg / L IBA, 1.0-3.0 mg / L TDZ, 0.5-5.0 mg / LAgNO3, 300-500 mg / L carbenicillin, 25-35 g / L sucrose and 5-7 g / L agar, and the pH value is 5.6-5.8; or, the composition of the induction culture medium includes: MS as a basic culture medium, and further includes: 0.2-0.4 mg / L IBA, 1.0-3.0 mg / LTDZ, 0.5-5.0 mg / LAgNO3, 300-500 mg / L carbenicillin, a screening agent, 25-35 g / L sucrose and 5-7 g / L agar, and the pH value is 5.6-5.8; The culture medium used in the proliferation culture includes a proliferation culture medium, wherein the composition of the proliferation culture medium includes: MS as a basic culture medium, and further includes: 0.8-1.5 mg / L 6-BA, 0.1-0.3 mg / L IBA, 0.005-0.015 mg / L TDZ, 25-35 g / L sucrose and 5-7 g / L agar; or, the composition of the induction culture medium includes: MS as a basic culture medium, and further includes: 0.8-1.5 mg / L 6-BA, 0.1-0.3 mg / L IBA, 0.005-0.015 mg / L TDZ, 300-500 mg / L carbenicillin, a selection agent, 25-35 g / L sucrose and 5-7 g / L agar; The culture medium used for the rooting culture comprises a rooting culture medium, and the composition of the rooting culture medium comprises: 1 / 2MS as a basic culture medium, and further comprises: 0.1-0.5 mg / L IBA, 0-0.2 mg / L NAA, 0-0.5 g / L activated carbon, 15-20 g / L sucrose, 5-7 g / L agar, and pH=5.6-5.

8.

10. The method according to claim 8 or 9, characterized in that: The light intensity of the adventitious bud induction culture is 8000-20000 lux, the light cycle is 12-16 h / d light culture and 8-12 h / d dark culture, the temperature is 25±2° C., and the time is 28-56 days.

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