Agrobacterium rhizogenes-mediated wild jujube seedling hypocotyl in-vivo transgenosis method

Abstract

The invention discloses an agrobacterium rhizogenes-mediated wild jujube seedling hypocotyl in-vivo transgenosis method, and belongs to the technical field of woody plant genetic engineering. The method mainly comprises the following four steps: cultivating wild jujube seedlings, preparing agrobacterium rhizogenes infection liquid containing a GUS label and a ZjMAPKK4 gene, scratching hypocotyl, smearing the agrobacterium rhizogenes infection liquid on a wound to induce hairy roots, and identifying and screening transgenic positive hairy roots. According to the method, wild jujube seedlings serve as materials, agrobacterium rhizogenes serve as mediation, the hypocotyl position is selected to be scratched and infected, and a transgenic material specifically expressed at the root can be rapidly obtained. The method is easy to operate, short in period and low in cost, the process of wild jujube gene function verification can be greatly accelerated, a new method is provided for wild jujube gene function verification, and the method has important significance on wild jujube gene functions and genetic engineering breeding.

Description

Technical Field

[0001] The invention relates to the technical field of woody plant genetic engineering, in particular to an in vivo genetic modification method for hypocotyls of wild jujube seedlings mediated by Agrobacterium rhizogenes. Background Art

[0002] Ziziphus jujuba Ziziphus acidojujuba Cheng et Liu) is native to my country and belongs to the genus Ziziphus (Rhamnaceae). Ziziphus Mill.) plant, which is a traditional dominant and characteristic dry fruit tree species in my country. Ziziphus jujuba Mill.) and the most commonly used rootstock, a commonly used sedative and hypnotic Chinese medicine and ecological construction tree species. The sour jujube has strong resistance to drought, barrenness and salinity, and is one of the pioneer tree species in the arid and barren areas of northern my country. It has a wide range of adaptability and a well-developed root system. It plays a very important role in soil and water conservation, windbreak and sand fixation, and greening of barren mountains. The sour jujube itself also has very high economic value. The fruit can be eaten fresh or processed into juice. The vitamin C content in the pulp is very high. The seed kernel has a significant sedative and hypnotic effect, and the dosage is large and the price is high.

[0003] There are few studies on the genetic transformation system of sour jujube, and most of them focus on the use of Agrobacterium tumefaciens to mediate sour jujube callus and leaves, but the cycle is long, the operation is complicated and the cost is high. With the development of plant genetic transformation research, researchers have found that using plant in vivo tissues (non-ex vivo living tissues) as receptor materials, without tissue culture to establish Agrobacterium rhizogenes-induced transgenic hairy roots, and regenerating and transforming plants from hairy roots, has high transformation efficiency and short cycle, which is conducive to gene function verification, but whether it is suitable for sour jujube transgenic research needs further verification and optimization.

[0004] Therefore, establishing an in vivo transgenic method mediated by Agrobacterium rhizogenes on sour jujube is of great significance for the genetic engineering breeding of sour jujube and jujube and the study of related gene functions. Summary of the invention

[0005] The purpose of the present invention is to provide a simple and practical new method for in vivo genetic modification of sour jujube mediated by Agrobacterium rhizogenes that is independent of tissue culture, so as to provide technical support for genetic engineering breeding of sour jujube and jujube and research on related gene functions.

[0006] To achieve the above objectives, after a large number of experimental studies and explorations, the present invention provides a new method for in vivo genetic modification of hypocotyls of jujube seedlings mediated by Agrobacterium rhizogenes, and has been successfully applied to ZjMAPKK4 Genetic transformation of the mitogen-activated protein kinase kinase 4 (mitogen-activated protein kinase kinase 4, which has multiple biological functions). The steps are as follows:

[0007] S1. sowing seeds of Solanum jujube and cultivating Solanum jujube seedlings to be infected.

[0008] S2. The recombinant plasmid of the exogenous target gene is transferred into the competent cells of Agrobacterium rhizogenes. After plating, the cells are cultured in a constant temperature incubator at 28°C for 2-3 days. A single circular plaque is selected and placed in a TY liquid medium with corresponding resistance. The cells are shaken at 28°C for 12-16 hours to obtain an activated transgenic Agrobacterium rhizogenes bacterial solution.

[0009] S3. Verify the target vector by PCR analysis of Agrobacterium rhizogenes in the above bacterial solution 35S::GUS-ZjMAPKK4 The Agrobacterium rhizogenes has been successfully transformed, and glycerol equal to the volume of bacterial solution has been added and stored at -20℃;

[0010] S4, spreading the bacterial solution stored at -20°C onto TY solid culture medium, and culturing at 28°C for 1-2 days to obtain a transgenic Agrobacterium rhizogenes bacterial layer, referred to as a solid infection bacterial layer;

[0011] S5, selecting the well-growing sour jujube seedlings cultured in step S1, scratching the hypocotyl below the cotyledons with a blade, and applying the solid infection bacterial layer obtained in step S4 to the wound site with a sterile disposable coating stick for infection;

[0012] S6. After the infection, cover the infected sour jujube seedlings with a transparent plastic cover and incubate them in the dark overnight, and then incubate them at 26°C with a 16 / 8 h light-dark cycle;

[0013] S7. After 7-14 days of cultivation, callus tissue is generated in the infected hypocotyl of the sour jujube seedlings, and hairy roots are generated on the callus tissue after 7-10 days. The hairy roots are stained with GUS histochemistry to identify the transgenic status of the hairy roots, and transgenic hairy root materials are obtained. After growing for 1-2 weeks, samples are taken again for GUS tag identification and quantitative detection of the target gene to obtain in vivo transgenic sour jujube hairy roots.

[0014] S8. After confirming that the transgenic jujube hairy roots are obtained, cultivate the hairy roots until they are sufficient to support the entire plant, then cut off the original non-transgenic main root and leave only the transgenic hairy roots. At this time, the plant is a transgenic composite plant. Water it regularly, observe the root growth status, and change the pot in time.

[0015] 2. The method according to claim 1 is characterized in that after the wild jujube kernels are soaked in clean water for 18 hours, they need to be cleaned with distilled water, and the cleaned kernels are evenly placed in a sterile culture dish covered with moist gauze, and placed in a constant temperature incubator at 28°C for 4-5 days (16 hours of light and 8 hours of darkness). When the gauze becomes dry every day, water is sprayed in time to keep the gauze moist but not too wet. The gauze is not squeezed out of water as the standard, and seeds with a radicle length of about 2 cm are selected for sowing, and cultured for 2 weeks at 26°C and 16 / 8 h light-dark cycle.

[0016] 3. The method according to claim 1, characterized in that, in step S2, the recombinant plasmid of the exogenous target gene contains the exogenous target gene ZjMAPKK4 The recombinant plasmid expression vector is pCAMBIA1391 , having kanamycin sulfate resistance; the Agrobacterium rhizogenes competent cells are Agrobacterium rhizogenes K599; the TY liquid culture medium contains 50 mg / L kanamycin sulfate and 50 mg / L streptomycin sulfate.

[0017] 4. The method according to claim 1, in step S4, the amount of bacterial solution with an OD value of 0.8 applied is 200-300 μL, and the culture is inverted.

[0018] 5. The method according to claim 1, wherein in step S5, the blade is tilted at an angle of 45° to scratch the hypocotyl below the cotyledons of the seedlings downward; the bacterial layer should completely cover the wound site.

[0019] 6. The method according to claim 1, wherein in step S7, after callus tissue is generated at the wound of the hypocotyl, the callus tissue is buried in soil to induce hairy roots; and the root tips of the hairy roots are taken for PCR and GUS histochemical staining detection.

[0020] The specific technical effects of the present invention are as follows:

[0021] The present invention provides a method for utilizing GUS The transgenic method of infecting the hypocotyls of sour jujube seedlings with Agrobacterium rhizogenes containing reporter genes does not require tissue culture and aseptic operation, is simple and easy to operate, and can obtain transgenic plants with stable root expression in a short period of time. The obtained transgenic hairy roots can be used to identify the expression of related genes and applied to the functional identification of sour jujube and jujube genes, providing a practical and efficient new method for genetic engineering breeding of sour jujube and jujube. The present invention is the first to establish a new in vivo transgenic method for sour jujube seedling hypocotyls mediated by Agrobacterium rhizogenes, and for the first time obtain overexpression ZjMAPKK4 Genetic modified material of Jujube jujube.

[0022] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings used in the description of the embodiments of the present invention. The drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0024] Figure 1 It is the seedlings of sour jujube to be infected;

[0025] Figure 2 Is prepared ZjMAPKK4 Flow chart of genetically modified root materials of sour jujube with genes;

[0026] Figure 3 It is the callus tissue produced after 3 pieces of infection;

[0027] Figure 4 It is the morphology of transgenic roots at different periods;

[0028] Figure 5 This is the GUS staining result of the positive hairy roots of transgenic jujube;

[0029] Figure 6 The positive hairy roots of transgenic jujube GUS Gene PCR test results;

[0030] Figure 7 The positive hairy roots of transgenic jujube GUS and ZjMAPKK4 qRT-PCR test results; DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0032] In order to make the purpose, technical scheme and advantages of the present application more clear, thorough and complete, the technical scheme of the present invention is described in detail below through the accompanying drawings and examples. The following detailed description is the description of the embodiments, which is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0033] The reagents, materials, kits and instruments used in the examples are all common commercial products and can be purchased on the market. Example 1

[0034] Prepare the culture medium as follows:

[0035] Prepare 100 mL TY medium, accurately weigh 0.5 g tryptone and 0.3 g yeast extract powder, add 100 mL double distilled water, stir to fully dissolve the components, sterilize in a high-temperature and high-pressure autoclave at 121 ℃ for 20 min, add 1 M CaCl2 aqueous solution after sterilization (high-temperature sterilization at 121 ℃ for 20 min), add 1 mL 1M CaCl2 aqueous solution to every 100 mL of medium, and add 1.5 g agar powder to TY solid medium.

[0036] Example 2

[0037] Explant preparation: Select the seeds of jujube with uniform size, full body and bright dark red color. Soak the seeds in water for 18 hours, place the seeds evenly in a culture dish covered with moist gauze, and culture them in a 28℃ constant temperature incubator for 4-5 days, with 16 hours of light and 8 hours of darkness. Select seeds with a radicle length of about 2 cm for sowing, and culture them at 26℃, 16 hours of light and 8 hours of darkness. When the seedlings are 2 weeks old, scratch the hypocotyl (rhizome) below the cotyledons to obtain the material to be infected, see Figure 1 . Example 3

[0038] Prepare the Agrobacterium rhizogenes infection layer as follows:

[0039] (1) The recombinant plasmid that has been sequenced correctly ZjMAPKK4 Add Agrobacterium rhizogenes K599 competent cells, mix quickly, let stand on ice for 5 min, liquid nitrogen for 5 min, 37°C water bath for 5 min, ice bath for 5 min, add 700 μl TY liquid medium without antibiotics, shake and culture at 28°C for 2-3 h, centrifuge at 6000 rpm for 1 min to collect the bacteria, keep 100 μl supernatant to resuspend the bacteria and spread it on TY plates containing kanamycin sulfate and streptomycin sulfate, culture in a 28°C incubator for 2-3 d, select a single circular plaque, pick the bacteria into TY liquid medium with streptomycin and kanamycin sulfate resistance, and shake the bacteria at 28°C for 12-16 h.

[0041] (2) Take 1 mL of the cultured bacterial liquid and store it at -20℃. When infecting, activate the bacterial liquid to an OD value of 0.8. Take 200-300 μl and spread it on TY solid culture medium. Invert and place it in a 28℃ incubator for 1-2 days. When a bacterial layer appears, the infected bacterial layer is obtained. Example 4

[0042] The method of infecting the sour jujube seedlings with the hypocotyls is as follows:

[0043] Select healthy 2-week-old sour jujube seedlings, scratch the hypocotyl below the cotyledon of the seedlings with a blade at an angle of 45°, and apply the solid infection layer obtained by culture to the wound site with a sterile disposable coating stick; after the infection, cover the sour jujube seedlings with a transparent plastic cover, culture them in the dark overnight, and then culture them at 26℃, 16 / 8h light and dark cycle; culture for about 7-14 days, callus tissue is generated at the infected site, and the callus tissue growth morphology is shown in Figure 3 ; After 7-10 days, the callus tissue generates hairy roots, the flow chart is shown in Figure 2 ; After 3 weeks, the hairy roots grow to 4-5 cm. The growth process of the hairy roots can be seen in Figure 4 . Example 5

[0044] The positive identification of transgenic hairy roots is as follows: The hairy roots produced in Example 4 were sampled and identified. The cut root tip was placed in a 1.5 mL centrifuge tube, and GUS dye solution was poured in to completely immerse the root tip. The centrifuge tube was wrapped with tin foil and placed in a 37°C incubator for 12-24 h. The uninfected seedlings were used as negative controls. The staining results are shown in FIG. Figure 5 .

[0045] The positive hairy roots produced in Example 4 were sampled again, and the cut hairy roots were placed in a sterilized 1.5 mL centrifuge tube and fully ground. The DNA and RNA of the hairy roots were extracted using the CTAB method and RNA extraction kit, respectively. The DNA and RNA of uninfected roots were extracted using the same method as a control. The obtained RNA was reverse transcribed into cDNA using a reverse transcription kit, and the extracted DNA was used as a template for GUS tag detection. The primers and program were respectively GUS -F:5'-ATGTTACGTCCTGTAGAAACCCCA-3', GUS -R: 5'-TCAAAGACGCGGTGATACATAT-3', 94℃ 4min; (94℃ 50s, 56℃ 30s, 72℃ 70s), 35 cycles; using the reverse transcribed cDNA as a template, the target gene and GUS The expression level of genes was detected by primers and procedures. ZjMAPKK4 -qPCR-F:5'-CCACCATCCCCATCAACT-3', ZjMAPKK4 -qPCR-R: 5'-GACCGTGCCACCATTTCC-3', 95℃ for 15min; (95℃ for 5s, 61℃ for 30s, 72℃ for 20s), 40 cycles; stored at 4℃; GUS -qPCR-F:5′-AAGAGCGATGAGCACGAGTA-3′ GUS -qPCR-R: 5'-AGAAATCAAACAACCCACGA-3', 95℃ 5min; (95℃ 15s, 60℃ 30s, 72℃ 30s), 45 cycles, stored at 4℃, results are shown in Figure 6 .

[0046] Results Analysis Figure 5 The results of GUS histochemical staining showed that the hairy roots were obviously stained blue, while the negative control was not stained blue. The transgenic roots 2 and 3 in the figure were both stained blue. The results showed that the in vivo transgenic method for the hypocotyl of jujube seedlings mediated by Agrobacterium rhizogenes provided by the present invention successfully obtained the overexpression ZjMAPKK4 Genetic material of sour jujube, Figure 4 The morphology of the transgenic roots of the sour jujube seedlings at different stages.

[0047] Depend on Figure 6 It can be seen that the detection GUS The tag successfully amplified the target band. Figure 7 It can be seen that the target gene and GUS The gene expression level was significantly higher than that in the roots of wild-type plants, indicating that the in vivo transgenic method of the hypocotyls of jujube seedlings mediated by Agrobacterium rhizogenes provided by the present invention was successfully obtained. ZjMAPKK4 Overexpression of genes in transgenic root materials.

[0048] Therefore, the in vivo genetic transformation method of the hypocotyl of jujube seedlings mediated by Agrobacterium rhizogenes provided by the present invention is simple and easy to operate, takes a short time, and transgenic materials can be obtained in 5-6 weeks; the present invention discloses for the first time the overexpression method mediated by Agrobacterium rhizogenes. ZjMAPKK4 Gene, obtain transgenic material.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions and implementation effects of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for in vivo transgenic of hypocotyls of jujube seedlings mediated by Agrobacterium rhizogenes, characterized in that: Here are the steps: S1. sowing seeds of Solanum jujube and cultivating Solanum jujube seedlings to be infected. 2.S2, transfer the recombinant plasmid of the exogenous target gene into the competent cells of Agrobacterium rhizogenes, culture them in a 28℃ constant temperature incubator for 2-3 days after plating, select a single circular plaque, pick it into TY liquid medium with kanamycin and streptomycin resistance, shake the bacteria at 28℃ for 12-16 hours to obtain the activated transgenic Agrobacterium rhizogenes bacterial liquid; S3. Verify the target vector by PCR analysis of Agrobacterium rhizogenes in the above bacterial solution 35S::GUS-ZjMAPKK4 The culture has been successfully transformed into Agrobacterium rhizogenes. Add an equal volume of glycerol to the bacterial solution and store it at -20℃; S4, spreading the bacterial solution stored at -20°C onto TY solid culture medium, and culturing at 28°C for 1-2 days to obtain a transgenic Agrobacterium rhizogenes bacterial layer, referred to as a solid infection bacterial layer; S5, selecting the well-growing sour jujube seedlings cultured in step S1, scratching the hypocotyl below the cotyledons with a blade, and applying the solid infection bacterial layer obtained in step S4 to the wound site with a sterile disposable coating stick for infection; S6. After the infection, cover the infected sour jujube seedlings with a transparent plastic cover and incubate them in the dark overnight, and then incubate them at 26°C with a 16 / 8 h light-dark cycle; S7. After 7-14 days of cultivation, callus tissue is generated in the infected hypocotyl of the sour jujube seedlings, and hairy roots are generated on the callus tissue after 7-10 days. The hairy roots are subjected to GUS histochemical staining to identify the transgenic status of the hairy roots, and transgenic hairy root materials are obtained. After 1-2 weeks of growth, samples are taken again for GUS tag identification and quantitative detection of the target gene to obtain in vivo transgenic sour jujube hairy roots. 3.S8. After confirming that the transgenic jujube hairy roots are obtained, cultivate the hairy roots until they are sufficient to support the entire plant, then cut off the original non-transgenic main root and leave only the transgenic hairy roots. At this time, the plant is a transgenic composite plant. Water it regularly, observe the root growth status, and change the pot in time.

4. The method according to claim 1, characterized in that In step S1, after the sour jujube kernels are soaked in clean water for 18 hours, they need to be cleaned with distilled water, and the cleaned kernels are evenly placed in a sterile culture dish covered with moist gauze, and placed in a 28°C constant temperature incubator for 4-5 days (16 hours of light and 8 hours of darkness). When the gauze becomes dry every day, water is sprayed in time to keep the gauze moist but not too wet. The gauze is not able to squeeze out water as the standard, and seeds with a radicle length of about 2 cm are selected for sowing, and cultured at 26°C and 16 / 8 h light-dark cycle for 2 weeks.

5. The method according to claim 1, characterized in that In step S2, the recombinant plasmid of the exogenous target gene contains the exogenous target gene ZjMAPKK4 (mitogen-activated protein kinase kinase 4), the recombinant plasmid expression vector is pCAMBIA1391 , having kanamycin sulfate resistance; the Agrobacterium rhizogenes competent cells are Agrobacterium rhizogenes K599; the TY liquid culture medium contains 50 mg / L kanamycin sulfate and 50 mg / L streptomycin sulfate.

6. The method according to claim 1, wherein in step S4, the amount of bacterial solution with an OD value of 0.8 applied is 200-300 μL, and the culture is inverted.

7. The method according to claim 1, wherein in step S5, the blade is tilted at an angle of 45° to scratch the hypocotyl below the cotyledons of the seedlings downward; the bacterial layer should completely cover the wound site.

8. The method according to claim 1, wherein in step S7, after callus tissue is generated at the wound of the hypocotyl, the callus tissue is buried in soil to induce hairy roots; and the root tips of the hairy roots are taken for PCR and GUS histochemical staining detection.

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